**Meet the editors**

Professor Dr Gabor B. Racz is a Grover Murray professor and Chairman Emeritus of the Department of Anesthesiology at Texas Tech University Health Science Center in Lubbock Texas, USA. He is a founder of the International Pain Institute at Texas Tech, the World Institute of Pain, and the Texas Pain Society. He is an international leader in the field of pain.

Professor Dr Carl E. Noe trained under Dr Racz, and he is a professor of Anesthesiology and Pain Management at the University of Texas Southwestern Medical Center in Dallas, Texas. He is the Medical Director of the Eugene McDermott Center for Pain Management, Division Director, Medical Director of the Parkland Memorial Hospital Pain Clinic, and Director of the Pain Fellowship Program.

Contents

**Preface XI** 

Jen-Kun Cheng

Pradeep K. Dhal,

Chapter 3 **Molecular Aspects of Opioid** 

Igor Ukrainets

Chapter 6 **Reduced Antinociceptive** 

Matthew S. Alkaitis,

Chapter 7 **Applied Radiologic Science** 

Kevin L. Wininger

Chapter 1 **Intrathecal Studies on Animal Pain Models 3** 

Diego A. Gianolio and Robert J. Miller

Chapter 4 **Creation of New Local Anesthetics Based** 

Chapter 5 **Neuroprotection and Pain Management 81** 

**Effect of Repeated Treatment with a Cannabinoid Receptor Type 2 Agonist in Cannabinoid-Tolerant** 

Christian Ndong, Russell P. Landry III,

Kambiz Hassanzadeh and Esmael Izadpanah

**Rats Following Spinal Nerve Transection 101** 

Joyce A. DeLeo and E. Alfonso Romero-Sandoval

**in the Treatment of Pain: Interventional Pain Medicine 123** 

**Therapies for the Treatment of Chronic Pain 27** 

**Receptors and Opioid Receptor Painkillers 43**  Austin B. Yongye and Karina Martínez-Mayorga

**on Quinoline Derivatives and Related Heterocycles 63** 

**Part 1 Pain Science 1** 

Chapter 2 **Polymer Based** 

### Contents

#### **Preface XIII**


**in the Treatment of Pain: Interventional Pain Medicine 123**  Kevin L. Wininger

### **Part 2 Acute Pain 159**  Chapter 8 **Local Anesthetic Agents in Arthroscopy 161**  Joseph Baker Chapter 9 **Multimodal Analgesia for Postoperative Pain Management 177**  G. Ulufer Sivrikaya Chapter 10 **The Effect of General Anesthesia and General Anesthesia Plus Epidural Levobupivacaine or Bupivacaine on Hemodynami Stress Response and Postoperative Pain 211**  Semra Calimli, Ahmet Topal, Atilla Erol, Aybars Tavlan and Seref Otelcioglu Chapter 11 **Propofol and Postoperative Pain: Systematic Review and Meta-Analysis 223**  Antigona Hasani, Hysni Jashari, Valbon Gashi and Albion Dervishi Chapter 12 **Efficacy of Continuous Femoral Nerve Block with Stimulating Catheters Versus Nonstimulating Catheters - A Systematic-Narrative Review 243**  Mario Dauri, Ludovica Celidonio, Sarit Nahmias, Eleonora Fabbi, Filadelfo Coniglione and Maria Beatrice Silvi Chapter 13 **Regional Anesthesia for the Trauma Patient 261**  Stephen D. Lucas, Linda Le-Wendling and F. Kayser Enneking

Contents VII

**Part 4 Chronic Pain 335** 

Chapter 18 **Chronic Pain in People with** 

Chapter 19 **The Role of Peripheral** 

Chapter 21 **PsychologicalStrategies**

**Part 5 Cancer Pain 467**

FuZhou Wang

Chapter 22 **Radiation Mucositis 469**  P. S. Satheesh Kumar

Yurdanur Demir

**Part 7 Nursing and Pain 517** 

Chapter 25 **When Theoretical Knowledge** 

Chapter 24 **Overview of Collateral** 

Kathryn Nicholson Perry

**Physically Disabling Conditions:**

**Nerve Blocks in the Interdisciplinary Care of Children with Chronic Pain:** 

Gillian R. Lauder and Nicholas West

Chapter 20 **Risk Factors in Opioid Treatment of Chronic** 

**Part 6 Non Pharmacological Treatments 483**

**Adhesions and Percutaneous Neuroplasty 337**  Gabor B. Racz, Miles R. Day, James E. Heavner,

**A Case Series and Review of the Literature 395**

Renata Ferrari, Michela Capraro and Marco Visentin

Chapter 23 **Non-Pharmacological Therapies in Pain Management 485** 

**A Modified Formulated Chinese Acupuncture 503**  Chih-Shung Wong, Chun-Chang Yeh and Shan-Chi Ko

**Explanatory Model on Nurse's Pain Management 519** 

**Meridian Therapy in Pain Management:** 

**Is Not Enough: Introduction of an** 

Katrin Blondal and Sigridur Halldorsdottir

**Non-Cancer Pain: A Multidisciplinary Assessment 419**

**in Pain Management: Optimizing Procedures in Clinics 459**

Jeffrey P. Smith, Jared Scott, Carl E. Noe, Laslo Nagy and Hana Ilner

**A Review of the Application of Biopsychosocial Models 371** 

Chapter 17 **Epidural Lysis of** 

#### **Part 3 Opioids 285**

Chapter 14 **Opioid Analgesics 287**  Maree T. Smith and Wei H. Goh

Chapter 15 **Pain Management and Costs of a Combination of Oxycodone + Naloxone in Low Back Pain Patients 307**  R. Rychlik, K. Viehmann, D. Daniel, P. Kiencke and J. Kresimon

Chapter 16 **The Role of Opioid Analgesics in the Treatment of Pain in Cancer Patients 321**  Wojciech Leppert

#### **Part 4 Chronic Pain 335**

VI Contents

**Part 2 Acute Pain 159**

Joseph Baker

Chapter 9 **Multimodal Analgesia** 

Chapter 10 **The Effect of General** 

Chapter 12 **Efficacy of Continuous** 

Stephen D. Lucas,

**Part 3 Opioids 285** 

Chapter 14 **Opioid Analgesics 287** 

Chapter 15 **Pain Management and** 

G. Ulufer Sivrikaya

Chapter 8 **Local Anesthetic Agents in Arthroscopy 161**

**for Postoperative Pain Management 177** 

**Plus Epidural Levobupivacaine or Bupivacaine on** 

**Hemodynami Stress Response and Postoperative Pain 211**

**Anesthesia and General Anesthesia** 

Atilla Erol, Aybars Tavlan and Seref Otelcioglu

**Systematic Review and Meta-Analysis 223**

**Stimulating Catheters Versus Nonstimulating Catheters - A Systematic-Narrative Review 243**

Filadelfo Coniglione and Maria Beatrice Silvi

Chapter 13 **Regional Anesthesia for the Trauma Patient 261**

Linda Le-Wendling and F. Kayser Enneking

**Oxycodone + Naloxone in Low Back Pain Patients 307** 

**in the Treatment of Pain in Cancer Patients 321**

Semra Calimli, Ahmet Topal,

Antigona Hasani, Hysni Jashari, Valbon Gashi and Albion Dervishi

**Femoral Nerve Block with** 

Mario Dauri, Ludovica Celidonio, Sarit Nahmias, Eleonora Fabbi,

Maree T. Smith and Wei H. Goh

**Costs of a Combination of** 

D. Daniel, P. Kiencke and J. Kresimon

R. Rychlik, K. Viehmann,

Chapter 16 **The Role of Opioid Analgesics**

Wojciech Leppert

Chapter 11 **Propofol and Postoperative Pain:** 

	- **Part 5 Cancer Pain 467**
	- **Part 6 Non Pharmacological Treatments 483**
	- **Part 7 Nursing and Pain 517**

XII Contents

#### **Part 8 Complex Regional Pain Syndrome and Reflex Sympathetic Dystrophy 543**

Chapter 26 **Complex Regional Pain Syndrome 545**  Gabor B. Racz and Carl E. Noe

### Preface

Recently, the Institute of Medicine in the United States assembled a panel to look at the issue of pain and its treatment. The comprehensive report has outlined that there is a great deal of suffering, and an excessive amount of money is being spent on many ineffective treatments for treating pain, and the associated costs with the disability that is the consequence of poorly‐treated pain is even more staggering.

Despite this, significant gains have indeed been achieved over the past 20 years in the treatment of pain by interventional pain and other pain specialists. The evidence is accumulating, and the etiologies of painful conditions are also better understood.

Policy makers need to include input from physicians recognized in interventional pain training and experience, as well as other practicing physicians, in order to avoid excluding data from studies regarding one camp of pain practitioners which may be unknown to another camp of practitioners. Interventional pain management should not be isolated from interdisciplinary and pharmacological pain management camps, and the field should evolve to a point where patients are evaluated and treated using the best of all worlds.

The reader must always be careful and maintain personal selectiveness for incorporating those areas that are indeed in the best interest of the patient that suffers from pain. The material contained within this book has been assembled more for quality rather than completeness. One may wish to have other and different areas covered, but there is only so much that can be accomplished in a limited amount of time and space. There is a significant amount covered in the various parts on opioids which must be looked at, as treatment of pain in many parts of the world is heavily opioid dependent.

It is interesting to note that in the United States over the last couple of years, the mortality rate from prescribed opioids has exceeded that for motor vehicle‐induced mortality. When opioids are used, the nature of the medication is such that it is subject to diversion and abuse. Disciplining patients should not be the responsibility of the physician, but verifying the use interestingly helps to maintain the appropriate use. For example, urine tests two to six times per year, qualitative and quantitative is followed by a one‐third reduction in the inappropriate use of the medication. Physicians need to look at whatever is in the best interest of the patient, and clearly the unintended overuse is not.

#### XII Preface

Dr Noe and myself go back for many years in the arena of interventional pain evaluation, research, teaching, and treatment.The clear recognition is that we could not have fulfilled the rolls that one has to fulfill without the complete devotion to our partners, Laura, Dr Noe's devoted wife, and Enid, my beautiful wife of 50 years this year. After a very brief discussion between my friend, Dr Carl Noe, and myself, we felt that the most appropriate way for us to express our gratitude is by dedicating this book to them.

We hope that the readers will find information that will make them think and improve their outlook on a fair and balanced vision of pain and its treatment, regardless of which branch of medicine they practice.

The purpose of this project is to bring the best minds together from around the world quickly and on an ongoing basis to make a world with less pain, hence the title *Painless*. The electronic format will leverage technology to allow for rapid additions of new material and updates of this initial presentation. We look forward to this global conversation with you.

> **Gabor B. Racz, MD, FIPP, ABIPP** Grover E. Murray Professor Professor and Chairman Emeritus Texas Tech University Health Sciences Center USA

XII Preface

book to them.

conversation with you.

which branch of medicine they practice.

Dr Noe and myself go back for many years in the arena of interventional pain evaluation, research, teaching, and treatment.The clear recognition is that we could not have fulfilled the rolls that one has to fulfill without the complete devotion to our partners, Laura, Dr Noe's devoted wife, and Enid, my beautiful wife of 50 years this year. After a very brief discussion between my friend, Dr Carl Noe, and myself, we felt that the most appropriate way for us to express our gratitude is by dedicating this

We hope that the readers will find information that will make them think and improve their outlook on a fair and balanced vision of pain and its treatment, regardless of

The purpose of this project is to bring the best minds together from around the world quickly and on an ongoing basis to make a world with less pain, hence the title *Painless*. The electronic format will leverage technology to allow for rapid additions of new material and updates of this initial presentation. We look forward to this global

> **Gabor B. Racz, MD, FIPP, ABIPP** Grover E. Murray Professor Professor and Chairman Emeritus

> > USA

Texas Tech University Health Sciences Center

**Part 1** 

**Pain Science** 

## **Part 1**

**Pain Science** 

**1** 

Jen-Kun Cheng

*Taiwan* 

**Intrathecal Studies on Animal Pain Models** 

Spinal and epidural anesthesia have been widely used in clinical settings for the management of peri-operative, neuropathic and cancer pain (Dureja et al., 2010; Hong, 2010; Mercadante, 1999). They provide another route for the analgesic administration in addition to oral or systemic absorption. Since the pain pathway initiate with primary and secondary neurons located in dorsal root ganglion and spinal cord, respectively, the *intrathecal* (spinal) route may provide an effective alternative for less drug dosage and fewer side

In recent decades, many animal pain models have been developed to explore the possible mechanisms involved in the pathogenesis of clinically relevant pain statuses, such as postoperative (Brennan et al., 1996), neuropathic (Kim & Chung, 1992), inflammatory (Wheeler-Aceto et al., 1990) and cancer pain (Clohisy & Mantyh, 2003). These studies not only help to extent our understanding on pain mechanisms but also provide novel promising agents or targets for the management of different pain situations (Mogil et al., 2010). In this chapter, we present various animal pain models, emphasizing on *intrathecal* studies, and potential therapeutic molecular targets and analgesics found in latest years. In addition, the

The first mentioned *intrathecal* study using rat animal model was reported by Yaksh, beginning with the study of *intrathecal* morphine (Yaksh et al., 1977). For *intrathecal* drug administration, a polyethylene catheter is inserted *intrathecally* in rats during inhalation anesthesia (LoPachin et al., 1981). The catheter is passed caudally from the cisterna magnum to the level of lumbar enlargement. Since the development of *intrathecal* catheterization, lots of studies explored the pharmacology and pain pathways using *intrathecal* space as a route of drug administration, either in basic researches or clinical studies. The *intrathecal* studies on various pain models provide a lot of promising analgesics for the management of

The postoperative or incisional pain model was proposed by Brennan in 1996 (Brennan et al., 1996). A 1-cm longitudinal incision is made through skin, fascia and muscle of the plantar aspect of the hindpaw in anesthetized rats. The lesion produced reliable and

related neurotoxicity studies and morphine-induced tolerance will be mentioned.

**1. Introduction** 

effects, compared with systemic administration.

**2. Intrathecal animal pain studies** 

different pain statuses.

**2.1 Postoperative pain model** 

*Mackay Memorial Hospital/Mackay Medical College* 

## **Intrathecal Studies on Animal Pain Models**

### Jen-Kun Cheng

*Mackay Memorial Hospital/Mackay Medical College Taiwan* 

#### **1. Introduction**

Spinal and epidural anesthesia have been widely used in clinical settings for the management of peri-operative, neuropathic and cancer pain (Dureja et al., 2010; Hong, 2010; Mercadante, 1999). They provide another route for the analgesic administration in addition to oral or systemic absorption. Since the pain pathway initiate with primary and secondary neurons located in dorsal root ganglion and spinal cord, respectively, the *intrathecal* (spinal) route may provide an effective alternative for less drug dosage and fewer side effects, compared with systemic administration.

In recent decades, many animal pain models have been developed to explore the possible mechanisms involved in the pathogenesis of clinically relevant pain statuses, such as postoperative (Brennan et al., 1996), neuropathic (Kim & Chung, 1992), inflammatory (Wheeler-Aceto et al., 1990) and cancer pain (Clohisy & Mantyh, 2003). These studies not only help to extent our understanding on pain mechanisms but also provide novel promising agents or targets for the management of different pain situations (Mogil et al., 2010). In this chapter, we present various animal pain models, emphasizing on *intrathecal* studies, and potential therapeutic molecular targets and analgesics found in latest years. In addition, the related neurotoxicity studies and morphine-induced tolerance will be mentioned.

#### **2. Intrathecal animal pain studies**

The first mentioned *intrathecal* study using rat animal model was reported by Yaksh, beginning with the study of *intrathecal* morphine (Yaksh et al., 1977). For *intrathecal* drug administration, a polyethylene catheter is inserted *intrathecally* in rats during inhalation anesthesia (LoPachin et al., 1981). The catheter is passed caudally from the cisterna magnum to the level of lumbar enlargement. Since the development of *intrathecal* catheterization, lots of studies explored the pharmacology and pain pathways using *intrathecal* space as a route of drug administration, either in basic researches or clinical studies. The *intrathecal* studies on various pain models provide a lot of promising analgesics for the management of different pain statuses.

#### **2.1 Postoperative pain model**

The postoperative or incisional pain model was proposed by Brennan in 1996 (Brennan et al., 1996). A 1-cm longitudinal incision is made through skin, fascia and muscle of the plantar aspect of the hindpaw in anesthetized rats. The lesion produced reliable and

Intrathecal Studies on Animal Pain Models 5

The L5/6 spinal nerve ligation neuropathic pain model was reported by Kim and Chung in 1992 (Kim & Chung, 1992). This model involves a tight ligation of L5 and L6 spinal nerves of animals under anesthesia. The nociceptive behavioral assessments also consist of von Frey hair test (Chaplan et al., 1994) and radiant heat test (Hargreaves et al., 1988) for the quantification of mechanical allodynia and thermal hyperalgesia, respectively, on the affected hindpaw. Compared with postoperative pain model and formalin inflammatory pain model, this model induced chronic nociceptive behaviors lasting for several weeks. This chronic pain model helps to reveal the possible mechanisms involved in the development and maintenance of nerve injury-induced pain, either the neuronal

Spared nerve injury pain model was developed by Decosterd and Woolf in 2000 (Decosterd & Woolf, 2000). An adaptation of spared nerve injury surgery was later developed in the mouse (Bourquin et al., 2006). This model involves a lesion of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact. The spared nerve injury model differs from the L5/6 spinal ligation pain model in that the co-mingling of distal intact axons with degenerating axons is restricted, and it permits behavioral testing of the non-injured skin territories adjacent to the denervated areas. The mechanical (von Frey and pinprick) sensitivity and thermal (hot and

Cancer pain significantly affects the diagnosis, quality of life and survival of patients with cancer. Tumor growth may produce inflammation in tumor bearing tissues, which will release inflammatory mediators to stimulate nociceptors. Tumor growth may also compress the peripheral nerves in tumor bearing tissues, inducing nerve injury. Therefore, cancer pain is likely to share mechanisms of inflammatory pain and neuropathic pain, although this pain may have distinct mechanisms (Ghilardi et al., 2010). Whether inflammation or nerve injury dominates during tumor growth may depend on the interactions between tumor cells

In recent years, several laboratories have developed cancer pain models by inoculation of tumor cells into a hindpaw of mouse (Constantin et al., 2008). Animals inoculated with melanoma cells into the plantar of the hindpaw show marked pain hypersensitivity and peripheral nerve degeneration (Gao et al., 2009a). We have used this melanoma cancer pain model to test the anti-tumor growth and analgesic effects of JNK inhibitor (Gao et al., 2009a). Other cancer pain models include breast, prostate and bone cancer pain models (Bloom et al., 2011; Ghilardi et al., 2010; Jimenez-Andrade et al., 2010). These cancer pain models may possess different pathophysiologies for pain induction. For example, intramedullary injection of breast cancer cells could induce periosteal sprouting of CGRP(+) sensory fibers and pain, both of which could be blocked by anti-nerve growth factor (NGF) (Bloom et al., 2011). Inhibitor of NGF receptor TrkA has been shown to attenuate bone cancer pain and tumor-induced sprouting of sensory nerve fibers (Ghilardi et al., 2010). Similarly, NGF also plays an important role in the induction of prostate cancer-induced sensory fiber sprouting

cold) responsiveness is increased in the ipsilateral sural territory.

components or glial components.

**2.4 Cancer pain model**

and surrounding tissues (Cain et al., 2001).

and bone pain (Jimenez-Andrade et al., 2010).

**3. Potential therapeutic molecular targets for pain management** 

Voltage-gated ion channels and glial cells have all been found to be promising therapeutic targets for pain management. Voltage-gated ion channels are a class of transmembrane ion

quantifiable mechanical allodynia and thermal hyperalgesia around the wound and spontaneous nociceptive behaviors for about one week, which mimics the clinical course of postoperative pain. Selective denervations of the rat hindpaw prior to foot incision reveal both the sural and tibial nerves are responsible for the nociception transmission from the incision. This model helps to better understand mechanisms of sensitization caused by surgery and provide promising therapeutics for postoperative pain management (Kang & Brennan, 2009).

#### **2.2 Inflammatory formalin pain model**

The formalin test involves subcutaneous injection of 5% formaldehyde (50 l) at the plantar surface of the rat hindpaw, using a 27-gauge needle. After injection, the rat displays characteristic nociceptive behaviors, flinching, shaking, biting and licking of the injected paw. Two phases of nociceptive behaviors are observed after formalin injection as described previously (Abbott et al., 1995). Phase 1 is initiated within seconds after injection and it lasts for about 5–10min. After several minutes quiescent, a second phase of flinching occurs and peaks at 25–35 min after injection.

The formalin-induced nociceptive response in rats is believed to be an inflammatory pain and involves central sensitization in the spinal cord (Abbott et al., 1995). The hindpaw injection of formalin induces tissue injury leading to acute (phase 1) and facilitated (phase 2) states of pain. The phase 2 response is believed to be a persistent input-induced nociceptive behavior mediated through central sensitization (Coderre & Melzack, 1992). LTP of C-fiberevoked field potentials in the spinal superficial dorsal horn has been reported in the formalin-injected rats (Sandkuhler & Liu, 1998). *Intrathecal* injection of T-type Ca2+ channel blockers (mibefradil and Ni2+) has been reported to attenuate formalin-induced pain behaviours, either phase 1 or 2, indicating the important role of T-type Ca2+ channel in the spinal central sensitization (Cheng et al., 2007). Other chemical irritants, such as complete Freund's adjuvant (CFA), carrageenan or capsaicin, could also be used to be injected subcutaneously into the plantar surface of rat hindpaw to induce pain behaviors (Duarte et al., 2011; Thorpe et al., 2011; Yu et al., 2011).

#### **2.3 Nerve injury-induced neuropathic pain model**

Nerve injuries due to trauma, chemotherapy, diabetic mellitus or tumor invasion may induce neuropathic pain, which is usually refractory to conventional analgesic agents, including opioids and non-steroid anti-inflammatory agents. For the past decades, several animal models have been developed to mimic the clinical conditions and explore the possible mechanisms underlying neuropathic pain. Among these neuropathic pain models, nerve injury-induced neuropathic pain (NINP) models, such as spinal nerve ligation, spared nerve injury and chronic constriction injury, are most often studied (Ji & Strichartz, 2004 ).

Several targets have been proposed to be involved in the pathogenesis of NINP, such as NMDA receptors (Szekely et al., 2002) and ion channels (Rogers et al., 2006). Recently, new molecules have been emerging as promising targets for the treatment of NINP, such as purinergic receptors (Donnelly-Roberts et al., 2008), cannabinoid receptors (Lynch & Campbell, 2011), transient receptor potential V1 (TRPV1) receptor (Facer et al., 2007), chemokine receptors (White et al., 2007), acid-sensing ion channel (Mazzuca et al., 2007; Poirot et al., 2006), annexin 2 light chain p11 (Foulkes et al., 2006) and matrix metalloproteinase (Kawasaki et al., 2008a).

quantifiable mechanical allodynia and thermal hyperalgesia around the wound and spontaneous nociceptive behaviors for about one week, which mimics the clinical course of postoperative pain. Selective denervations of the rat hindpaw prior to foot incision reveal both the sural and tibial nerves are responsible for the nociception transmission from the incision. This model helps to better understand mechanisms of sensitization caused by surgery and provide promising therapeutics for postoperative pain management (Kang &

The formalin test involves subcutaneous injection of 5% formaldehyde (50 l) at the plantar surface of the rat hindpaw, using a 27-gauge needle. After injection, the rat displays characteristic nociceptive behaviors, flinching, shaking, biting and licking of the injected paw. Two phases of nociceptive behaviors are observed after formalin injection as described previously (Abbott et al., 1995). Phase 1 is initiated within seconds after injection and it lasts for about 5–10min. After several minutes quiescent, a second phase of flinching occurs and

The formalin-induced nociceptive response in rats is believed to be an inflammatory pain and involves central sensitization in the spinal cord (Abbott et al., 1995). The hindpaw injection of formalin induces tissue injury leading to acute (phase 1) and facilitated (phase 2) states of pain. The phase 2 response is believed to be a persistent input-induced nociceptive behavior mediated through central sensitization (Coderre & Melzack, 1992). LTP of C-fiberevoked field potentials in the spinal superficial dorsal horn has been reported in the formalin-injected rats (Sandkuhler & Liu, 1998). *Intrathecal* injection of T-type Ca2+ channel blockers (mibefradil and Ni2+) has been reported to attenuate formalin-induced pain behaviours, either phase 1 or 2, indicating the important role of T-type Ca2+ channel in the spinal central sensitization (Cheng et al., 2007). Other chemical irritants, such as complete Freund's adjuvant (CFA), carrageenan or capsaicin, could also be used to be injected subcutaneously into the plantar surface of rat hindpaw to induce pain behaviors (Duarte et

Nerve injuries due to trauma, chemotherapy, diabetic mellitus or tumor invasion may induce neuropathic pain, which is usually refractory to conventional analgesic agents, including opioids and non-steroid anti-inflammatory agents. For the past decades, several animal models have been developed to mimic the clinical conditions and explore the possible mechanisms underlying neuropathic pain. Among these neuropathic pain models, nerve injury-induced neuropathic pain (NINP) models, such as spinal nerve ligation, spared nerve injury and chronic constriction injury, are most often studied (Ji & Strichartz, 2004 ). Several targets have been proposed to be involved in the pathogenesis of NINP, such as NMDA receptors (Szekely et al., 2002) and ion channels (Rogers et al., 2006). Recently, new molecules have been emerging as promising targets for the treatment of NINP, such as purinergic receptors (Donnelly-Roberts et al., 2008), cannabinoid receptors (Lynch & Campbell, 2011), transient receptor potential V1 (TRPV1) receptor (Facer et al., 2007), chemokine receptors (White et al., 2007), acid-sensing ion channel (Mazzuca et al., 2007; Poirot et al., 2006), annexin 2 light chain p11 (Foulkes et al., 2006) and matrix

Brennan, 2009).

**2.2 Inflammatory formalin pain model** 

peaks at 25–35 min after injection.

al., 2011; Thorpe et al., 2011; Yu et al., 2011).

metalloproteinase (Kawasaki et al., 2008a).

**2.3 Nerve injury-induced neuropathic pain model**

The L5/6 spinal nerve ligation neuropathic pain model was reported by Kim and Chung in 1992 (Kim & Chung, 1992). This model involves a tight ligation of L5 and L6 spinal nerves of animals under anesthesia. The nociceptive behavioral assessments also consist of von Frey hair test (Chaplan et al., 1994) and radiant heat test (Hargreaves et al., 1988) for the quantification of mechanical allodynia and thermal hyperalgesia, respectively, on the affected hindpaw. Compared with postoperative pain model and formalin inflammatory pain model, this model induced chronic nociceptive behaviors lasting for several weeks. This chronic pain model helps to reveal the possible mechanisms involved in the development and maintenance of nerve injury-induced pain, either the neuronal components or glial components.

Spared nerve injury pain model was developed by Decosterd and Woolf in 2000 (Decosterd & Woolf, 2000). An adaptation of spared nerve injury surgery was later developed in the mouse (Bourquin et al., 2006). This model involves a lesion of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact. The spared nerve injury model differs from the L5/6 spinal ligation pain model in that the co-mingling of distal intact axons with degenerating axons is restricted, and it permits behavioral testing of the non-injured skin territories adjacent to the denervated areas. The mechanical (von Frey and pinprick) sensitivity and thermal (hot and cold) responsiveness is increased in the ipsilateral sural territory.

#### **2.4 Cancer pain model**

Cancer pain significantly affects the diagnosis, quality of life and survival of patients with cancer. Tumor growth may produce inflammation in tumor bearing tissues, which will release inflammatory mediators to stimulate nociceptors. Tumor growth may also compress the peripheral nerves in tumor bearing tissues, inducing nerve injury. Therefore, cancer pain is likely to share mechanisms of inflammatory pain and neuropathic pain, although this pain may have distinct mechanisms (Ghilardi et al., 2010). Whether inflammation or nerve injury dominates during tumor growth may depend on the interactions between tumor cells and surrounding tissues (Cain et al., 2001).

In recent years, several laboratories have developed cancer pain models by inoculation of tumor cells into a hindpaw of mouse (Constantin et al., 2008). Animals inoculated with melanoma cells into the plantar of the hindpaw show marked pain hypersensitivity and peripheral nerve degeneration (Gao et al., 2009a). We have used this melanoma cancer pain model to test the anti-tumor growth and analgesic effects of JNK inhibitor (Gao et al., 2009a). Other cancer pain models include breast, prostate and bone cancer pain models (Bloom et al., 2011; Ghilardi et al., 2010; Jimenez-Andrade et al., 2010). These cancer pain models may possess different pathophysiologies for pain induction. For example, intramedullary injection of breast cancer cells could induce periosteal sprouting of CGRP(+) sensory fibers and pain, both of which could be blocked by anti-nerve growth factor (NGF) (Bloom et al., 2011). Inhibitor of NGF receptor TrkA has been shown to attenuate bone cancer pain and tumor-induced sprouting of sensory nerve fibers (Ghilardi et al., 2010). Similarly, NGF also plays an important role in the induction of prostate cancer-induced sensory fiber sprouting and bone pain (Jimenez-Andrade et al., 2010).

#### **3. Potential therapeutic molecular targets for pain management**

Voltage-gated ion channels and glial cells have all been found to be promising therapeutic targets for pain management. Voltage-gated ion channels are a class of transmembrane ion

Intrathecal Studies on Animal Pain Models 7

involvement of Na+ channel β2 subunit in neuropathic and inflammatory pain has been

In addition to changes in protein expression, phosphorylation-induce change of conductance or gating property of Na+ channels may also lead to enhanced neuronal excitability and NINP (Aurilio et al., 2008). The activation of presynaptic delta-opioid receptor by enkephalin has been reported to prevent the increase in neuronal NaV1.7 in DRG through inhibition of PKC and p38 (Chattopadhyay et al., 2008). Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine involved in NINP formation (Schafers et al., 2003), was found to enhance TTX-resistant Na+ currents in isolated DRG neurons *via* a TNF receptor 1- and p38-dependent mechanism (Jin & Gereau, 2006). The Na+ currents of isolated sensory neurons can be enhanced by protein kinase A and protein kinase C (Gold et al., 1998; Mo et al., 2011), both of which are involved in NINP (Gao et al., 2005; Song et al., 2006). Phosphorylation of TTX-S and TTX-R sodium channels involving both serine/threonine and tyrosine sites has been reported to contribute to painful diabetic neuropathy (Hong et al., 2004). Further studies are required to reveal the exact role of Na+

Voltage-gated Ca2+ channels are involved in neuron excitability, neurotransmitter release, synaptic transmission and gene expression (Dolmetsch et al., 2001). Ca2+ channels are constituted by the pore-forming α-subunit and auxiliary subunits, β- and α2δ�subunits. They are classified into Cav1, Cav2 and Cav3 families based on their structure homology, but are categorized as L- (Cav1.1, Cav1.2 and Cav1.3), P/Q- (Cav2.1), N- (Cav2.2), R- (Cav2.3), and T- (Cav3.1, Cav3.2 and Cav3.3) type based on their sensitivity to specific blockers, activation/inactivation characteristics and current conductance (Catterall et al., 2002). Various Ca2+ channel blockers have been tested in the postoperative, inflammatory and neuropathic pain models (Cheng et al., 2007). The potential use of Ca2+ channel blockers for neuropathic pain treatment and roles of Ca2+ channels in ascending pain pathway have

N-type Ca2+ channels are distributed in the dorsal root ganglia and spinal dorsal horn. It is generally believed that N-type Ca2+ channels are involved in the neurotransmitter release of spinal dorsal horn (Smith et al., 2002). Substance P, one of the neurotransmitter of primary sensory neurons, has been found to be mostly co-localized with N-type Ca2+ channels in the

Several lines of evidence indicate that N-type Ca2+ channels play an important role in NINP. Mice lacking N-type Ca2+ channels exhibit reduced signs of neuropathic pain after spinal nerve ligation (Saegusa et al., 2001). *Intrathecal* small interference RNA knockdown of Ntype Ca2+ channels reversed sciatic nerve constriction-induced tactile allodynia and thermal

New non-peptide compounds with N-type Ca2+ channel blocking property have been recently developed in pharmaceutical companies for the treatment of neuropathic pain (Knutsen et al., 2007). A highly reversible ω-conotoxin FVIA, a potent N-type Ca2+ channel blocker with fewer side effects, was found to possess analgesic effect in the formalin test and neuropathic pain models (Lee et al., 2010). Recent findings suggest that diminished Ca2+

extensively reviewed (Brackenbury & Isom, 2008).

channel phosphorylation in the pathogenesis of NINP.

been well reviewed (Yaksh, 2006; Zamponi et al., 2009).

spinal dorsal horn (Westenbroek et al., 1998).

**3.2 Voltage-gated Ca2+ channels** 

**3.2.1 N-type Ca2+ channels** 

hyperalgesia (Altier et al., 2007).

channels that are activated by changes in membrane potential; these types of ion channels are especially critical in excitable cells, including neuronal, cardiac and skeletal cells (Szu-Yu Ho & Rasband, 2011), or even cancer cell migration (Cuddapah & Sontheimer, 2011). Since voltage-gated ion channels are important for neuronal excitability, conduction and transmission, they have long been the targets of interest in the field of pain research.

#### **3.1 Voltage-gated Na<sup>+</sup> channels**

Voltage-gated Na+ channels are essential for the initiation of action potentials which are crucial for nerve conduction. Their activation and inactivation are strongly gated by the membrane potential of neuronal cells, but their properties can also be modulated by Gproteins or protein kinases (Kakimura et al., 2010). Voltage-gated Na+ channels are constituted by the pore-forming α−subunit and auxiliary β-subunits. Up to now, nine α−subunits (Nav1.1-1.9) and four β-subunits (β1-4) have been identified (Catterall et al., 2005). The Na+ channels can be either sensitive (Nav1.1, Nav1.2, Nav1.3, Nav1.6) or resistant (Nav1.4, Nav1.5, Nav1.7, Nav1.8, Nav1.9) to tetrodotoxin (TTX), a toxin found in the liver of puffer fish. Neuronal cells contain most of the Na+ channel subtypes but Nav1.4 and Nav1.5, respectively, are mainly in skeletal and cardiac muscles (Jarecki et al., 2010). Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 have been found in adult dorsal root ganglion (DRG) sensory neurons and these isoforms can be important for the firing properties of sensory neurons (Hunanyan et al., 2011). After spared nerve injury in rats, altered neuronal electrogenesis in DRG neurons, such as accelerated re-priming of TTX-sensitive Na+ currents, was observed and may be due to a complex regulation of voltage-gated Na+ channels (Berta et al., 2008; Wang et al., 2011).

Several lines of evidence indicate that Nav1.7, and Nav1.8 are involved in pain regulation, especially NINP (Lampert et al., 2010). Nav1.7 and Nav1.8 channels have been shown to accumulate in neuroma endings in humans with neuropathic pain (Kretschmer et al., 2002). This accumulation may be due to a loss of myelin inhibition or target determined transfer of Na+ channels (Aurilio et al., 2008). Loss of Nav1.7 function may lead to complete insensitivity to pain in humans (Cox et al., 2010). Compounds possessing Nav1.7 blocking effects have been reported to reverse nerve injury-induced mechanical allodynia (Tyagarajan et al., 2010). Nav1.8 is increased in sciatic nerve after nerve injury and *intrathecal*  antisense oligoneucleotide directed against Nav1.8 is effective in neuropathic pain models (Joshi et al., 2006). A μΩ-conotoxin MrVIB was found to be a preferential Nav1.8 blocker and could reverse partial sciatic nerve ligation-induced mechanical allodynia and thermal hyperalgesia, when given *intrathecally* (Ekberg et al., 2006). Intraperitoneal administration of A-803467, a selective Nav1.8 blocker, has been reported to attenuate nerve injury-induced mechanical allodynia (Jarvis et al., 2007). Nonetheless, Nassar et al. found that mice lacking Nav1.7 and Nav1.8 still develop neuropathic pain after spinal nerve ligation (Nassar et al., 2005). Recent studies also revealed a role of Nav1.3 (Mo et al., 2011) and Nav1.9 (Leo et al., 2010) in the development of neuropathic pain. For normal nerve conduction, Nav1.1 family is involved (Catterall et al., 2010). Therefore, the selective Nav1.3, Nav1.7, Nav1.8 and Nav1.9 channel blockers will have clinical potential in the treatment of neuropathic pain since they do not affect normal neuronal conduction.

Besides the pore-forming α-subunit, β2 subunit was reported to be up-regulated in injured and non-injured sensory neurons after peripheral nerve injuries (Pertin et al., 2005) and the development of spared nerve injury-induced mechanical allodynia is attenuated in β2-null mice (Lopez-Santiago et al., 2006), suggesting the important role of β2 subunit in NINP. The

channels that are activated by changes in membrane potential; these types of ion channels are especially critical in excitable cells, including neuronal, cardiac and skeletal cells (Szu-Yu Ho & Rasband, 2011), or even cancer cell migration (Cuddapah & Sontheimer, 2011). Since voltage-gated ion channels are important for neuronal excitability, conduction and

Voltage-gated Na+ channels are essential for the initiation of action potentials which are crucial for nerve conduction. Their activation and inactivation are strongly gated by the membrane potential of neuronal cells, but their properties can also be modulated by Gproteins or protein kinases (Kakimura et al., 2010). Voltage-gated Na+ channels are constituted by the pore-forming α−subunit and auxiliary β-subunits. Up to now, nine α−subunits (Nav1.1-1.9) and four β-subunits (β1-4) have been identified (Catterall et al., 2005). The Na+ channels can be either sensitive (Nav1.1, Nav1.2, Nav1.3, Nav1.6) or resistant (Nav1.4, Nav1.5, Nav1.7, Nav1.8, Nav1.9) to tetrodotoxin (TTX), a toxin found in the liver of puffer fish. Neuronal cells contain most of the Na+ channel subtypes but Nav1.4 and Nav1.5, respectively, are mainly in skeletal and cardiac muscles (Jarecki et al., 2010). Nav1.1, Nav1.3, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 have been found in adult dorsal root ganglion (DRG) sensory neurons and these isoforms can be important for the firing properties of sensory neurons (Hunanyan et al., 2011). After spared nerve injury in rats, altered neuronal electrogenesis in DRG neurons, such as accelerated re-priming of TTX-sensitive Na+ currents, was observed and may be due to a complex regulation of voltage-gated Na+

Several lines of evidence indicate that Nav1.7, and Nav1.8 are involved in pain regulation, especially NINP (Lampert et al., 2010). Nav1.7 and Nav1.8 channels have been shown to accumulate in neuroma endings in humans with neuropathic pain (Kretschmer et al., 2002). This accumulation may be due to a loss of myelin inhibition or target determined transfer of Na+ channels (Aurilio et al., 2008). Loss of Nav1.7 function may lead to complete insensitivity to pain in humans (Cox et al., 2010). Compounds possessing Nav1.7 blocking effects have been reported to reverse nerve injury-induced mechanical allodynia (Tyagarajan et al., 2010). Nav1.8 is increased in sciatic nerve after nerve injury and *intrathecal*  antisense oligoneucleotide directed against Nav1.8 is effective in neuropathic pain models (Joshi et al., 2006). A μΩ-conotoxin MrVIB was found to be a preferential Nav1.8 blocker and could reverse partial sciatic nerve ligation-induced mechanical allodynia and thermal hyperalgesia, when given *intrathecally* (Ekberg et al., 2006). Intraperitoneal administration of A-803467, a selective Nav1.8 blocker, has been reported to attenuate nerve injury-induced mechanical allodynia (Jarvis et al., 2007). Nonetheless, Nassar et al. found that mice lacking Nav1.7 and Nav1.8 still develop neuropathic pain after spinal nerve ligation (Nassar et al., 2005). Recent studies also revealed a role of Nav1.3 (Mo et al., 2011) and Nav1.9 (Leo et al., 2010) in the development of neuropathic pain. For normal nerve conduction, Nav1.1 family is involved (Catterall et al., 2010). Therefore, the selective Nav1.3, Nav1.7, Nav1.8 and Nav1.9 channel blockers will have clinical potential in the treatment of neuropathic pain

Besides the pore-forming α-subunit, β2 subunit was reported to be up-regulated in injured and non-injured sensory neurons after peripheral nerve injuries (Pertin et al., 2005) and the development of spared nerve injury-induced mechanical allodynia is attenuated in β2-null mice (Lopez-Santiago et al., 2006), suggesting the important role of β2 subunit in NINP. The

transmission, they have long been the targets of interest in the field of pain research.

 **channels** 

channels (Berta et al., 2008; Wang et al., 2011).

since they do not affect normal neuronal conduction.

**3.1 Voltage-gated Na<sup>+</sup>**

involvement of Na+ channel β2 subunit in neuropathic and inflammatory pain has been extensively reviewed (Brackenbury & Isom, 2008).

In addition to changes in protein expression, phosphorylation-induce change of conductance or gating property of Na+ channels may also lead to enhanced neuronal excitability and NINP (Aurilio et al., 2008). The activation of presynaptic delta-opioid receptor by enkephalin has been reported to prevent the increase in neuronal NaV1.7 in DRG through inhibition of PKC and p38 (Chattopadhyay et al., 2008). Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine involved in NINP formation (Schafers et al., 2003), was found to enhance TTX-resistant Na+ currents in isolated DRG neurons *via* a TNF receptor 1- and p38-dependent mechanism (Jin & Gereau, 2006). The Na+ currents of isolated sensory neurons can be enhanced by protein kinase A and protein kinase C (Gold et al., 1998; Mo et al., 2011), both of which are involved in NINP (Gao et al., 2005; Song et al., 2006). Phosphorylation of TTX-S and TTX-R sodium channels involving both serine/threonine and tyrosine sites has been reported to contribute to painful diabetic neuropathy (Hong et al., 2004). Further studies are required to reveal the exact role of Na+ channel phosphorylation in the pathogenesis of NINP.

#### **3.2 Voltage-gated Ca2+ channels**

Voltage-gated Ca2+ channels are involved in neuron excitability, neurotransmitter release, synaptic transmission and gene expression (Dolmetsch et al., 2001). Ca2+ channels are constituted by the pore-forming α-subunit and auxiliary subunits, β- and α2δ�subunits. They are classified into Cav1, Cav2 and Cav3 families based on their structure homology, but are categorized as L- (Cav1.1, Cav1.2 and Cav1.3), P/Q- (Cav2.1), N- (Cav2.2), R- (Cav2.3), and T- (Cav3.1, Cav3.2 and Cav3.3) type based on their sensitivity to specific blockers, activation/inactivation characteristics and current conductance (Catterall et al., 2002). Various Ca2+ channel blockers have been tested in the postoperative, inflammatory and neuropathic pain models (Cheng et al., 2007). The potential use of Ca2+ channel blockers for neuropathic pain treatment and roles of Ca2+ channels in ascending pain pathway have been well reviewed (Yaksh, 2006; Zamponi et al., 2009).

#### **3.2.1 N-type Ca2+ channels**

N-type Ca2+ channels are distributed in the dorsal root ganglia and spinal dorsal horn. It is generally believed that N-type Ca2+ channels are involved in the neurotransmitter release of spinal dorsal horn (Smith et al., 2002). Substance P, one of the neurotransmitter of primary sensory neurons, has been found to be mostly co-localized with N-type Ca2+ channels in the spinal dorsal horn (Westenbroek et al., 1998).

Several lines of evidence indicate that N-type Ca2+ channels play an important role in NINP. Mice lacking N-type Ca2+ channels exhibit reduced signs of neuropathic pain after spinal nerve ligation (Saegusa et al., 2001). *Intrathecal* small interference RNA knockdown of Ntype Ca2+ channels reversed sciatic nerve constriction-induced tactile allodynia and thermal hyperalgesia (Altier et al., 2007).

New non-peptide compounds with N-type Ca2+ channel blocking property have been recently developed in pharmaceutical companies for the treatment of neuropathic pain (Knutsen et al., 2007). A highly reversible ω-conotoxin FVIA, a potent N-type Ca2+ channel blocker with fewer side effects, was found to possess analgesic effect in the formalin test and neuropathic pain models (Lee et al., 2010). Recent findings suggest that diminished Ca2+

Intrathecal Studies on Animal Pain Models 9

isoforms (α2δ-1~4) were identified (Qin et al., 2002). The α2δ-1 subunit is up-regulated in dorsal root ganglion and dorsal spinal cord after peripheral nerve injury (Li et al., 2004). *Intrathecal* injection of α2δ-1 antisense oligonucleotide could block this up-regulation in spinal dorsal horn and diminish injury-induced tactile allodynia (Li et al., 2004). Over expression of α2δ-1 in spinal dorsal horn neurons could enhance Ca2+ currents, exaggerate dorsal horn neuronal responses to external stimuli and increase the nociceptive responses in

α2δ subunit is the specific binding site in the central nervous system of gabapentin and its analogue pregabalin (Klugbauer et al., 2003), both of which have been shown to be effective in preclinical and clinical studies of neuropathic pain (Cheng & Chiou, 2006). Gabapentin was first designed as a chemical analogue of γ-aminobutyric acid, an inhibitory neurotransmitter, to treat spasticity and was later found to have anticonvulsant and antinociceptive activities in various seizure and pain models. A point mutation of the arginine 217 of α2δ-1 subunit, which is critical for gabapentin binding (Wang et al., 1999), was found to cause a loss of gabapentin-induced analgesia (Field et al., 2006). Recently, chronic *intrathecal* infusion of gabapentin was found to prevent nerve ligation-induced mechanical allodynia and thermal hyperalgesia without causing obvious neuropathological

Gabapentin has been found to attenuate morphine-induced tolerance (Lin et al., 2005) and this finding may encourage the combined use of gabapentin with morphine in the treatment of neuropathic pain. It is interesting to note that α2δ-1 subunit was identied to be a receptor involved in excitatory synapse formation and gabapentin may act by blocking new

The opening of K+ channel may lead to cell repolarization and make the neuron less excitable and down-regulation of K+ channel in nociceptive neurons may decrease pain threshold. There are 12 different families of voltage-gated K+ channels (Kv1 to Kv12) and all Kv channels are tetramers of α subunits (Ocana et al., 2004). A-type K+ channel (A-channels) is a group of Kv channels that are activated transiently and inactivated rapidly. Five Achannels Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 were found in mammals (Chien et al., 2007; Mienville et al., 1999; Serodio et al., 1996). Except for Kv3.4 with high-voltage activation, the other four are activated at low voltages (Coetzee et al., 1999). Kv1.4 proteins in the somata of DRG neurons are greatly reduced in the L5/6 spinal nerve ligation pain model (Rasband et al., 2001). The expression of Kv1.4 is also reduced in the small-/medium sized (A-/C-) trigeminal ganglion neurons after temporomandibular joint inflammation (Takeda et al., 2008). Gene expressions of Kv1.2, Kv1.4, and Kv4.2 are down-regulated in the DRG following sciatic nerve transection (Park et al., 2003). Recent study also revealed the Kv1.2 expression is decreased in DRG neurons from rats with irritable bowel syndrome, a visceral pain model (Luo et al., 2011). The expression of Kv3.4 and Kv4.3 in DRG neurons were found to be also decreased after spinal nerve ligation and *intrathecal* injections of antisense oligodeoxynucleotides against Kv3.4 or Kv4.3 in naïve rats could induce mechanical hypersensitivity (Chien et al., 2007). New compounds with A-type K+ channel opening activity, such as KW-7158 (Sculptoreanu et al., 2004), may prove to be effective for the

The Kv7 channel (also known as KCNQ) opener retigabine has been reported to be effective in sciatic chronic constrict injury (Blackburn-Munro & Jensen, 2003) and L5 spinal nerve

neuropathic pain models (Li et al., 2006).

synapse formation (Eroglu et al., 2009).

**3.3 Voltage-gated K+**

treatment of NINP.

changes in spinal cord and cauda equine (Chu et al., 2011).

 **channels** 

influx through N-type Ca2+ channels may contribute to sensory neuron dysfunction and pain after nerve injury (McCallum et al., 2011).

#### **3.2.2 T-type Ca2+ channels**

T-type Ca2+ channels are low-voltage activated Ca2+ channels. It can serve as an initiator to trigger the opening of high-voltage activated ion channels. In spinal dorsal horn, it may be involved in spontaneous neurotransmitter release and long term potentiation (LTP) (Ikeda et al., 2003). LTP, a form of synaptic plasticity, in the spinal dorsal horn is believed to contribute to the central sensitization of pain transmission (Ji et al., 2003), a wiring phenomenon usually observed in neuropathic pain (Romanelli & Esposito, 2004).

Among three subtypes of T-type Ca2+ channels, CaV3.1, CaV3.2 and CaV3.3, CaV3.2 mRNAs are mostly abundant in the spinal dorsal horn and are limited to the superficial layers (Talley et al., 1999). *Intrathecal* injection of the antisense oligonucleotide targeted to the α1 subunit of CaV3.2, but not CaV3.3 or CaV3.1, produced analgesic effect in both acute and neuropathic pain states (Bourinet et al., 2005), suggesting that CaV3.2 is much more involved in spinal nociceptive pathway than CaV3.1 and CaV3.3.

Subtype-specific blockers of T-type Ca2+ channels are not commercially available. However, mibefradil, a non-selective T-type Ca2+ channel blocker, when given systemically or intraplantarly, can reverse mechanical allodynia and thermal hyperalgesia induced by L5/6 spinal nerve ligation (Dogrul et al., 2003). Our recent work on *intrathecal* T-type Ca2+ channel blockers (mibefradil or Ni2+) revealed their effectiveness in the second phase of formalin test (Cheng et al., 2007). In these years, small molecules with potent blocking effect on T-type Ca2+ channels, such as KYS05090, have been developed (Doddareddy et al., 2007; Seo et al., 2007). Recent studies revealed spinal T-type Ca2+ (Cav3.2 and Cav3.3 but not Cav3.1) channels may play an important role in the pathogenesis of chronic compression of DRGinduced neuropathic pain (Wen et al., 2010). In addition, Cav3.2-dependent activation of extracellular signal-regulated kinase in the anterior nucleus of paraventricular thalamus was found to contribute to the development of acid-induced chronic mechanical hyperalgesia (Chen et al., 2010).

#### **3.2.3 P/Q- and R-type Ca2+ channels**

Compared with N-type Ca2+ channel, it seems P/Q type is much less important in NINP. Only one study using transgenic mice revealed its involvement in chronic constriction injury-induced mechanical allodynia (Luvisetto et al., 2006). The hypoalgesic behaviors of P/Q-type Ca2+ channel mutant mouse suggest P/Q-type Ca2+ channel has a pro-nociceptive role (Fukumoto et al., 2009). As for R-type Ca2+ channel, its blocker SNX-482 could inhibit Cfiber and Aδ-fiber-mediated neuronal responses after L5/6 spinal nerve ligation, when administered *intrathecally* (Matthews et al., 2007). Moreover, the responses to innocuous mechanical and thermal stimuli were more sensitive to SNX-482 in nerve-ligated rats than control animals (Matthews et al., 2007). These findings suggest spinal R-type Ca2+ channel could be a potential therapeutic target for NINP. Blocking the R-type Ca2+ channel has been reported to enhance morphine analgesia and reduce morphine-induced tolerance (Yokoyama et al., 2004).

#### **3.2.4 2 subunit of Ca2+ channels**

α2δ subunit is one of the modulatory subunits of Ca2+ channels, which could modulate the membrane targeting and conductance of α1 subunit of Ca2+ channel (Felix, 1999). Four

influx through N-type Ca2+ channels may contribute to sensory neuron dysfunction and

T-type Ca2+ channels are low-voltage activated Ca2+ channels. It can serve as an initiator to trigger the opening of high-voltage activated ion channels. In spinal dorsal horn, it may be involved in spontaneous neurotransmitter release and long term potentiation (LTP) (Ikeda et al., 2003). LTP, a form of synaptic plasticity, in the spinal dorsal horn is believed to contribute to the central sensitization of pain transmission (Ji et al., 2003), a wiring

Among three subtypes of T-type Ca2+ channels, CaV3.1, CaV3.2 and CaV3.3, CaV3.2 mRNAs are mostly abundant in the spinal dorsal horn and are limited to the superficial layers (Talley et al., 1999). *Intrathecal* injection of the antisense oligonucleotide targeted to the α1 subunit of CaV3.2, but not CaV3.3 or CaV3.1, produced analgesic effect in both acute and neuropathic pain states (Bourinet et al., 2005), suggesting that CaV3.2 is much more involved

Subtype-specific blockers of T-type Ca2+ channels are not commercially available. However, mibefradil, a non-selective T-type Ca2+ channel blocker, when given systemically or intraplantarly, can reverse mechanical allodynia and thermal hyperalgesia induced by L5/6 spinal nerve ligation (Dogrul et al., 2003). Our recent work on *intrathecal* T-type Ca2+ channel blockers (mibefradil or Ni2+) revealed their effectiveness in the second phase of formalin test (Cheng et al., 2007). In these years, small molecules with potent blocking effect on T-type Ca2+ channels, such as KYS05090, have been developed (Doddareddy et al., 2007; Seo et al., 2007). Recent studies revealed spinal T-type Ca2+ (Cav3.2 and Cav3.3 but not Cav3.1) channels may play an important role in the pathogenesis of chronic compression of DRGinduced neuropathic pain (Wen et al., 2010). In addition, Cav3.2-dependent activation of extracellular signal-regulated kinase in the anterior nucleus of paraventricular thalamus was found to contribute to the development of acid-induced chronic mechanical hyperalgesia

Compared with N-type Ca2+ channel, it seems P/Q type is much less important in NINP. Only one study using transgenic mice revealed its involvement in chronic constriction injury-induced mechanical allodynia (Luvisetto et al., 2006). The hypoalgesic behaviors of P/Q-type Ca2+ channel mutant mouse suggest P/Q-type Ca2+ channel has a pro-nociceptive role (Fukumoto et al., 2009). As for R-type Ca2+ channel, its blocker SNX-482 could inhibit Cfiber and Aδ-fiber-mediated neuronal responses after L5/6 spinal nerve ligation, when administered *intrathecally* (Matthews et al., 2007). Moreover, the responses to innocuous mechanical and thermal stimuli were more sensitive to SNX-482 in nerve-ligated rats than control animals (Matthews et al., 2007). These findings suggest spinal R-type Ca2+ channel could be a potential therapeutic target for NINP. Blocking the R-type Ca2+ channel has been reported to enhance morphine analgesia and reduce morphine-induced tolerance

α2δ subunit is one of the modulatory subunits of Ca2+ channels, which could modulate the membrane targeting and conductance of α1 subunit of Ca2+ channel (Felix, 1999). Four

phenomenon usually observed in neuropathic pain (Romanelli & Esposito, 2004).

pain after nerve injury (McCallum et al., 2011).

in spinal nociceptive pathway than CaV3.1 and CaV3.3.

**3.2.2 T-type Ca2+ channels**

(Chen et al., 2010).

(Yokoyama et al., 2004).

**3.2.4 2 subunit of Ca2+ channels**

**3.2.3 P/Q- and R-type Ca2+ channels** 

isoforms (α2δ-1~4) were identified (Qin et al., 2002). The α2δ-1 subunit is up-regulated in dorsal root ganglion and dorsal spinal cord after peripheral nerve injury (Li et al., 2004). *Intrathecal* injection of α2δ-1 antisense oligonucleotide could block this up-regulation in spinal dorsal horn and diminish injury-induced tactile allodynia (Li et al., 2004). Over expression of α2δ-1 in spinal dorsal horn neurons could enhance Ca2+ currents, exaggerate dorsal horn neuronal responses to external stimuli and increase the nociceptive responses in neuropathic pain models (Li et al., 2006).

α2δ subunit is the specific binding site in the central nervous system of gabapentin and its analogue pregabalin (Klugbauer et al., 2003), both of which have been shown to be effective in preclinical and clinical studies of neuropathic pain (Cheng & Chiou, 2006). Gabapentin was first designed as a chemical analogue of γ-aminobutyric acid, an inhibitory neurotransmitter, to treat spasticity and was later found to have anticonvulsant and antinociceptive activities in various seizure and pain models. A point mutation of the arginine 217 of α2δ-1 subunit, which is critical for gabapentin binding (Wang et al., 1999), was found to cause a loss of gabapentin-induced analgesia (Field et al., 2006). Recently, chronic *intrathecal* infusion of gabapentin was found to prevent nerve ligation-induced mechanical allodynia and thermal hyperalgesia without causing obvious neuropathological changes in spinal cord and cauda equine (Chu et al., 2011).

Gabapentin has been found to attenuate morphine-induced tolerance (Lin et al., 2005) and this finding may encourage the combined use of gabapentin with morphine in the treatment of neuropathic pain. It is interesting to note that α2δ-1 subunit was identied to be a receptor involved in excitatory synapse formation and gabapentin may act by blocking new synapse formation (Eroglu et al., 2009).

#### **3.3 Voltage-gated K+ channels**

The opening of K+ channel may lead to cell repolarization and make the neuron less excitable and down-regulation of K+ channel in nociceptive neurons may decrease pain threshold. There are 12 different families of voltage-gated K+ channels (Kv1 to Kv12) and all Kv channels are tetramers of α subunits (Ocana et al., 2004). A-type K+ channel (A-channels) is a group of Kv channels that are activated transiently and inactivated rapidly. Five Achannels Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 were found in mammals (Chien et al., 2007; Mienville et al., 1999; Serodio et al., 1996). Except for Kv3.4 with high-voltage activation, the other four are activated at low voltages (Coetzee et al., 1999). Kv1.4 proteins in the somata of DRG neurons are greatly reduced in the L5/6 spinal nerve ligation pain model (Rasband et al., 2001). The expression of Kv1.4 is also reduced in the small-/medium sized (A-/C-) trigeminal ganglion neurons after temporomandibular joint inflammation (Takeda et al., 2008). Gene expressions of Kv1.2, Kv1.4, and Kv4.2 are down-regulated in the DRG following sciatic nerve transection (Park et al., 2003). Recent study also revealed the Kv1.2 expression is decreased in DRG neurons from rats with irritable bowel syndrome, a visceral pain model (Luo et al., 2011). The expression of Kv3.4 and Kv4.3 in DRG neurons were found to be also decreased after spinal nerve ligation and *intrathecal* injections of antisense oligodeoxynucleotides against Kv3.4 or Kv4.3 in naïve rats could induce mechanical hypersensitivity (Chien et al., 2007). New compounds with A-type K+ channel opening activity, such as KW-7158 (Sculptoreanu et al., 2004), may prove to be effective for the treatment of NINP.

The Kv7 channel (also known as KCNQ) opener retigabine has been reported to be effective in sciatic chronic constrict injury (Blackburn-Munro & Jensen, 2003) and L5 spinal nerve

Intrathecal Studies on Animal Pain Models 11

Changes in the expression and function of voltage-gated ion channels in the pain pathway may contribute to the development and maintenance of NINP. Manipulations aiming at voltage-gated ion channels may provide novel strategies for the treatment of NINP. In addition to ion channel modulators, recent studies also reveal the promising roles of glial

During the last decade, the neuroimmune system, such as spinal glial cells, has been found to be critical for the development and maintenance of nerve injury-induced neuropathic pain (Watkins et al., 2007). Nerve injury not only induces morphological changes of microglia but also biochemical changes to induce pain. Nerve injury results in a upregulation of P2X4 receptor (Tsuda et al., 2003) and CX3CR1 receptor in spinal cord microglia (Verge et al., 2004; Zhuang et al., 2007). *Intrathecal* blockade of P2X4 and CX3CR1 signaling attenuates NINP (Tsuda et al., 2003; Zhuang et al., 2007). The chemokine receptor CCR2 and the Toll-like recepotor-4 (TLR4) are also important for the formation of neuropathic pain *via* microglial activation (Abbadie et al., 2003; Tanga et al., 2005). Phosphorylation of p38 in microglia via activation of P2X4 receptor could increase the synthesis and release of the neurotrophin BDNF and pro-inflammatory cytokines (IL-1, IL-6, and TNF-, all of which could enhance nociceptive transmission in the spinal cord (Coull

Our study using continuous *intrathecal* infusion of minocycline, a microglia inhibitor, revealed its effectiveness in attenuating the development of nerve injury-induced pain and no obvious spinal neurotoxicity was observed after the infusion (Lin et al., 2007). Other glial modulators, such as AV-411 (Ledeboer et al., 2006) and pentoxifylline (Mika et al., 2007), also possessed analgesic effect in NINP models. In addition to glial activation, compliment activation was recently found to participate in spinal nerve ligation-induced pain (Levin et al., 2008). Similar with gabapentin, minocycline could also attenuate morphine-induced tolerance (Cui et al., 2008) and this made itself a promising drug to be co-administered with morphine in the treatment of neuropathic pain. It is worthwhile to note that the attenuation effect of minocycline on morphine-induced tolerance is associated with inhibition of p38 activation in spinal microglia caused by chronic

In contrast to microglia, which is important for the development phase of NINP (Ji & Suter, 2007), astrocytes activation was critical for the maintenance phase of NINP (Zhuang et al., 2006). JNK-induced MCP-1 production and JAK-STAT3 pathway in spinal cord astrocytes was found to contribute to the maintenance of NINP (Gao et al., 2009b; Tsuda et al., 2011). The role of astrocyte activation and kinases involved in glial activation after nerve injury

Morphine is the main drug used in pain clinics, especially in cancer pain. Recent animal studies also revealed the effectiveness of morphine in NINP models (Mika et al., 2007; Zhang et al., 2005). However, acute and chronic use of morphine can induce hyperalgesia and analgesia tolerance (Mao et al., 1994), which often lead to increased drug consumption

inhibitors, such as minocycline, and morphine in the management of NINP.

et al., 2005; Ji & Suter, 2007; Kawasaki et al., 2008b; Wang et al., 2010)

morphine (Cui et al., 2008).

and unwanted side-effects.

have been well reviewed (Gao & Ji, 2010; Ji et al., 2009).

**4. Morphine in nerve injury-induced neuropathic pain** 

**3.5 Microglia and astrocyte activation in nerve injury-induced neuropathic pain** 

ligation (Dost et al., 2004) pain models. It is important to note that the antiallodynic effect of retigabine could be inhibited by linopirdine, a selective KCNQ channel blocker, indicating the involvement of KCNQ channel opening in the effect of retigabine (Dost et al., 2004). When directly applied to the spinal cord, retigabine inhibited the A and C fiber-mediated response of dorsal horn neurons to noxious stimuli (Passmore et al., 2003). Recently, the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib was found to enhance Kv7.2-7.4, Kv7.2/7.3 and Kv7.3/7.5 currents expressed in HEK 293 cells, providing a novel mechanism for its antinociceptive effect (Du et al., 2011b). Based on these reports, further efforts may be needed to develop subtype-specific K+ channel openers and to test their effects in NINP models.

Just as voltage-gated Na+ channels, K+ channels could also be modulated by phosphorylation (Sergeant et al., 2005). The Kv4.2 current of spinal dorsal horn neurons could be inhibited by extracellular signal-regulated kinase (ERK)-induced phosphorylation (Hu et al., 2003). Genetic elimination of Kv4.2 increases excitability of dorsal horn neurons and sensitivity to tactile and thermal stimuli (Hu et al., 2006). This modulation of Kv4.2 by ERK may underlie the induction of central sensitization, a cellular mechanism of NINP (Ji et al., 2003). The role of Kv channels in different trigeminal neuropathic and inflammatory pain models was recently reviewed (Takeda et al., 2011).

#### **3.4 Other K<sup>+</sup> channels**

In addition to Kv channels, there are other K+ channels that are important for pain modulation, such as G-protein coupled inwardly rectifying (GIRK or Kir3), ATP-sensitive (KATP or Kir6), Ca2+-activated (KCa) and two-pore (K2P) K+ channels (Gutman et al., 2003). Activation of KATP channels was recently found to antagonize nociceptive behavior and hyper-excitability of DRG neurons from rats (Du et al., 2011a). Following partial sciatic nerve ligation, elevated tyrosine phosphorylation (pY12) of Kir3.1 was observed in the spinal superficial dorsal horn of wild type, but not Kir3.1 knock-out, mice (Ippolito et al., 2005). This phosphorylation may suppress channel conductance and accelerate channel deactivation (Ippolito et al., 2002), leading to enhanced neuronal excitability and could possibly contribute to the genesis of NINP. It is interesting to note that induced expression of Kir2.1 in chronically compressed DRG neurons can effectively suppress the neuronal excitability and, if induced at the beginning of the chronic compression, prevent the development of compression-induced hyperalgesia (Ma et al., 2010).

The TREK-1 channel is a member of mechano-gated K2P family, one of the targets of inhalation anesthetics (Patel et al., 1999). TREK-1 is highly expressed in small sensory neurons and extensively co-localized with TRPV1 (Alloui et al., 2006). Mice with a disrupted TREK-1 gene are more sensitive to painful heat and low threshold mechanical stimuli and display an increased thermal and mechanical hyperalgesia in conditions of inflammation (Alloui et al., 2006). On the other hand, the TREK-1 null mice showed decreased sensitivity to acetone (less cold allodynia) after sciatic nerve ligation (Alloui et al., 2006). The chemotherapy drug oxaliplatin, which induces cold hypersensitivity, could lower the expression of TREK-1 (Descoeur et al., 2011). Future studies are needed to elucidate the role of TREK-1 channels in NINP. Similar as TREK-1, TREK-2 is also a member of the K2P family. TREK-2 provide the major background K+ conductance in cell body of small to mediumsized DRG neurons (Mathie, 2007), which are the major component of nociceptors. Based on these findings, it is also intriguing to investigate the role of TREK-2 in NINP (Huang & Yu, 2008).

ligation (Dost et al., 2004) pain models. It is important to note that the antiallodynic effect of retigabine could be inhibited by linopirdine, a selective KCNQ channel blocker, indicating the involvement of KCNQ channel opening in the effect of retigabine (Dost et al., 2004). When directly applied to the spinal cord, retigabine inhibited the A and C fiber-mediated response of dorsal horn neurons to noxious stimuli (Passmore et al., 2003). Recently, the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib was found to enhance Kv7.2-7.4, Kv7.2/7.3 and Kv7.3/7.5 currents expressed in HEK 293 cells, providing a novel mechanism for its antinociceptive effect (Du et al., 2011b). Based on these reports, further efforts may be needed to develop subtype-specific K+ channel openers and to test their effects in NINP

Just as voltage-gated Na+ channels, K+ channels could also be modulated by phosphorylation (Sergeant et al., 2005). The Kv4.2 current of spinal dorsal horn neurons could be inhibited by extracellular signal-regulated kinase (ERK)-induced phosphorylation (Hu et al., 2003). Genetic elimination of Kv4.2 increases excitability of dorsal horn neurons and sensitivity to tactile and thermal stimuli (Hu et al., 2006). This modulation of Kv4.2 by ERK may underlie the induction of central sensitization, a cellular mechanism of NINP (Ji et al., 2003). The role of Kv channels in different trigeminal neuropathic and inflammatory

In addition to Kv channels, there are other K+ channels that are important for pain modulation, such as G-protein coupled inwardly rectifying (GIRK or Kir3), ATP-sensitive (KATP or Kir6), Ca2+-activated (KCa) and two-pore (K2P) K+ channels (Gutman et al., 2003). Activation of KATP channels was recently found to antagonize nociceptive behavior and hyper-excitability of DRG neurons from rats (Du et al., 2011a). Following partial sciatic nerve ligation, elevated tyrosine phosphorylation (pY12) of Kir3.1 was observed in the spinal superficial dorsal horn of wild type, but not Kir3.1 knock-out, mice (Ippolito et al., 2005). This phosphorylation may suppress channel conductance and accelerate channel deactivation (Ippolito et al., 2002), leading to enhanced neuronal excitability and could possibly contribute to the genesis of NINP. It is interesting to note that induced expression of Kir2.1 in chronically compressed DRG neurons can effectively suppress the neuronal excitability and, if induced at the beginning of the chronic compression, prevent the

The TREK-1 channel is a member of mechano-gated K2P family, one of the targets of inhalation anesthetics (Patel et al., 1999). TREK-1 is highly expressed in small sensory neurons and extensively co-localized with TRPV1 (Alloui et al., 2006). Mice with a disrupted TREK-1 gene are more sensitive to painful heat and low threshold mechanical stimuli and display an increased thermal and mechanical hyperalgesia in conditions of inflammation (Alloui et al., 2006). On the other hand, the TREK-1 null mice showed decreased sensitivity to acetone (less cold allodynia) after sciatic nerve ligation (Alloui et al., 2006). The chemotherapy drug oxaliplatin, which induces cold hypersensitivity, could lower the expression of TREK-1 (Descoeur et al., 2011). Future studies are needed to elucidate the role of TREK-1 channels in NINP. Similar as TREK-1, TREK-2 is also a member of the K2P family. TREK-2 provide the major background K+ conductance in cell body of small to mediumsized DRG neurons (Mathie, 2007), which are the major component of nociceptors. Based on these findings, it is also intriguing to investigate the role of TREK-2 in NINP (Huang & Yu,

pain models was recently reviewed (Takeda et al., 2011).

development of compression-induced hyperalgesia (Ma et al., 2010).

 **channels** 

models.

**3.4 Other K<sup>+</sup>**

2008).

Changes in the expression and function of voltage-gated ion channels in the pain pathway may contribute to the development and maintenance of NINP. Manipulations aiming at voltage-gated ion channels may provide novel strategies for the treatment of NINP. In addition to ion channel modulators, recent studies also reveal the promising roles of glial inhibitors, such as minocycline, and morphine in the management of NINP.

#### **3.5 Microglia and astrocyte activation in nerve injury-induced neuropathic pain**

During the last decade, the neuroimmune system, such as spinal glial cells, has been found to be critical for the development and maintenance of nerve injury-induced neuropathic pain (Watkins et al., 2007). Nerve injury not only induces morphological changes of microglia but also biochemical changes to induce pain. Nerve injury results in a upregulation of P2X4 receptor (Tsuda et al., 2003) and CX3CR1 receptor in spinal cord microglia (Verge et al., 2004; Zhuang et al., 2007). *Intrathecal* blockade of P2X4 and CX3CR1 signaling attenuates NINP (Tsuda et al., 2003; Zhuang et al., 2007). The chemokine receptor CCR2 and the Toll-like recepotor-4 (TLR4) are also important for the formation of neuropathic pain *via* microglial activation (Abbadie et al., 2003; Tanga et al., 2005). Phosphorylation of p38 in microglia via activation of P2X4 receptor could increase the synthesis and release of the neurotrophin BDNF and pro-inflammatory cytokines (IL-1, IL-6, and TNF-, all of which could enhance nociceptive transmission in the spinal cord (Coull et al., 2005; Ji & Suter, 2007; Kawasaki et al., 2008b; Wang et al., 2010)

Our study using continuous *intrathecal* infusion of minocycline, a microglia inhibitor, revealed its effectiveness in attenuating the development of nerve injury-induced pain and no obvious spinal neurotoxicity was observed after the infusion (Lin et al., 2007). Other glial modulators, such as AV-411 (Ledeboer et al., 2006) and pentoxifylline (Mika et al., 2007), also possessed analgesic effect in NINP models. In addition to glial activation, compliment activation was recently found to participate in spinal nerve ligation-induced pain (Levin et al., 2008). Similar with gabapentin, minocycline could also attenuate morphine-induced tolerance (Cui et al., 2008) and this made itself a promising drug to be co-administered with morphine in the treatment of neuropathic pain. It is worthwhile to note that the attenuation effect of minocycline on morphine-induced tolerance is associated with inhibition of p38 activation in spinal microglia caused by chronic morphine (Cui et al., 2008).

In contrast to microglia, which is important for the development phase of NINP (Ji & Suter, 2007), astrocytes activation was critical for the maintenance phase of NINP (Zhuang et al., 2006). JNK-induced MCP-1 production and JAK-STAT3 pathway in spinal cord astrocytes was found to contribute to the maintenance of NINP (Gao et al., 2009b; Tsuda et al., 2011). The role of astrocyte activation and kinases involved in glial activation after nerve injury have been well reviewed (Gao & Ji, 2010; Ji et al., 2009).

#### **4. Morphine in nerve injury-induced neuropathic pain**

Morphine is the main drug used in pain clinics, especially in cancer pain. Recent animal studies also revealed the effectiveness of morphine in NINP models (Mika et al., 2007; Zhang et al., 2005). However, acute and chronic use of morphine can induce hyperalgesia and analgesia tolerance (Mao et al., 1994), which often lead to increased drug consumption and unwanted side-effects.

Intrathecal Studies on Animal Pain Models 13

*Intrathecal* space has been a route for spinal anesthesia and analgesics. This space also provides us a way to explore the possible mechanisms involved in pain transmission. Since pain is a major world-wide issue in clinical settings, more and more *intrathecal* animal studies have been undertaken to explore the possible mechanisms involved in the formation of different pain statuses and help to develop promising analgesics to alleviate the suffering of pain patients. These efforts will eventually help to provide better pain managements in

This chapter was supported by a John J. Bonica Trainee Fellowship from the International Association for the Study of Pain (IASP), a grant of NSC 98-2314-B-195-002-MY3 from National Science Council, Taipei, Taiwan and grants MMH 10015 and 10044 from Mackay

Abbadie, C., Lindia, J.A., Cumiskey, A.M., Peterson, L.B., Mudgett, J.S., Bayne, E.K.,

Abbott, F.V., Franklin, K.B. & Westbrook, R.F. (1995). The formalin test: scoring properties of

Alloui, A., Zimmermann, K., Mamet, J., Duprat, F., Noel, J., Chemin, J., Guy, N., Blondeau,

Altier, C., Dale, C.S., Kisilevsky, A.E., Chapman, K., Castiglioni, A.J., Matthews, E.A., Evans,

Aurilio, C., Pota, V., Pace, M.C., Passavanti, M.B. & Barbarisi, M. (2008). Ionic channels and

Bennett, G., Deer, T., Du Pen, S., Rauck, R., Yaksh, T. & Hassenbusch, S.J. (2000). Future

Berta, T., Poirot, O., Pertin, M., Ji, R.R., Kellenberger, S. & Decosterd, I. (2008).

Blackburn-Munro, G. & Jensen, B.S. (2003). The anticonvulsant retigabine attenuates

pain perception. *EMBO J*, Vol.25, No.11, pp. 2368-2376.

DeMartino, J.A., MacIntyre, D.E. & Forrest, M.J. (2003). Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. *Proc Natl Acad Sci U S A*,

the first and second phases of the pain response in rats. *Pain*, Vol.60, No.1, pp. 91-

N., Voilley, N., Rubat-Coudert, C., Borsotto, M., Romey, G., Heurteaux, C., Reeh, P., Eschalier, A. & Lazdunski, M. (2006). TREK-1, a K+ channel involved in polymodal

R.M., Dickenson, A.H., Lipscombe, D., Vergnolle, N. & Zamponi, G.W. (2007). Differential role of N-type calcium channel splice isoforms in pain. *J Neurosci*,

neuropathic pain: physiopathology and applications. *J Cell Physiol*, Vol.215, No.1,

directions in the management of pain by intraspinal drug delivery. *J Pain Symptom* 

Transcriptional and functional profiles of voltage-gated Na(+) channels in injured and non-injured DRG neurons in the SNI model of neuropathic pain. *Mol Cell* 

nociceptive behaviours in rat models of persistent and neuropathic pain. *Eur J* 

**6. Conclusion** 

clinical settings.

**8. References** 

102.

pp. 8-14.

**7. Acknowledgment**

Memorial Hospital, Taipei, Taiwan to J.K.C.

Vol.100, No.13, pp. 7947-7952.

Vol.27, No.24, pp. 6363-6373.

*Manage*, Vol.20, No.2, pp. S44-50.

*Neurosci*, Vol.37, No.2, pp. 196-208.

*Pharmacol*, Vol.460, No.2-3, pp. 109-116.

#### **4.1 Glial non-opioid/p38 pathway in morphine-induced analgesia and tolerance**

Using the tail flick test, Tseng's group has shown that morphine could induce anti-analgesia, which could be prevented by *levo*-, *dextro*naloxone **(a non-opioid ligand)** and p38 inhibitor *via* a glial non-opioid mechanism (Wu et al., 2006a; Wu et al., 2006b; Wu et al., 2005). From the works of Tseng's group, it could be summarized that 1) both *dextro*- and *levo*-morphine and lipopolysaccharide (LPS), a toll-like receptor (TLR)-4 agonist, could induce antianalgesia, which could be prevented by *dextro*-, *levo*-naloxone and p38 inhibitor; 2) the antianalgesia-inducing potency is: *dextro*-morphine > *levo*-morphine, and the reversal potency is: *levo*-naloxone > *dextro*-naloxone, which may imply the different binding affinities of *dextro*/*levo*- morphine and naloxone to the putative non-opioid receptor or TLR-4 (Hutchinson et al., 2007).

Inspired by the studies of Hong's group showing naloxone could attenuate LPS-induced microglial activation and neuronal damage (Liu et al., 2000), Watkin's group further tested the possible involvement of the putative nonopioid/TLR-4 pathway in NINP. They found *dextro*-naloxone, *levo-*naltrexone, and LPS-antagonist possess analgesic effects in chronic constriction neuropathic pain model (Hutchinson et al., 2007). Taken together with the role of glial p38 activation in NINP (Jin et al., 2003) and morphine-induced tolerance (Cui et al., 2006), it is possible that the putative glia non-opioid/TLR-4 pathway is important for the development of NINP and morphine-induced tolerance (Cui et al., 2006).

#### **4.2 Intrathecal studies on morphine tolerance**

Morphine has long been used *intrathecally* in the management of cancer and non-cancer chronic pain (Plummer et al., 1991; Roberts et al., 2001). However, the long-term use of morphine is associated with severe side-effects and tolerance (Osenbach & Harvey, 2001). Recently, many studies have revealed that *intrathecal* morphine could induce glial activation and neuro-inflammation in the spinal cord (Muscoli et al., 2010; Zhang et al., 2011). Several therapeutic targets have been found, including cytokine receptors, kappa-opioid receptors, N-methyl-D-aspartate receptors, and Toll-like receptors (Hameed et al., 2010; Lewis et al., 2010). Recently, tumor necrosis factor (TNF)-α antagonist etanercept was found to reverse morphine-induced tolerance and block morphine-induced neuroinflammation in the microglia (Shen et al., 2011). *Intrathecal* gabapentin and minocycline could also enhance the antinociceptive effects of morphine and attenuate morphine-induced tolerance (Habibi-Asl et al., 2009; Hutchinson et al., 2008; Lin et al., 2005). These promising agents may be coadministered with *intrathecal* morphine to improve the pain management for cancer patients (Christo & Mazloomdoost, 2008; Mercadante et al., 2004).

#### **5. Intrathecal neurotoxicity studies**

For a drug to be tested *intrathecally* in clinical trials, it is imperative to examine its neurotoxic effects rst in animals (Bennett et al., 2000; Smith et al., 2008). For instance, *intrathecal* lidocaine has been found to induce neuropathological changes in the spinal cord and cauda equina (Kirihara et al., 2003). Other analgesics, such as adenosine, sufentanil, alfentanil and morphine have all been tested *intrathecally* in animal studies to examine their potential neurotoxicity (Chiari et al., 1999; Sabbe et al., 1994; Westin et al., 2010). Recently, chronic *intrathecal* infusion of minocycline or gabapentin has been reported to cause no grossly neurotoxicity in animal studies (Chu et al., 2011; Lin et al., 2007), supporting the *intrathecal* use of these agents for pain management.

#### **6. Conclusion**

12 Pain Management – Current Issues and Opinions

Using the tail flick test, Tseng's group has shown that morphine could induce anti-analgesia, which could be prevented by *levo*-, *dextro*naloxone **(a non-opioid ligand)** and p38 inhibitor *via* a glial non-opioid mechanism (Wu et al., 2006a; Wu et al., 2006b; Wu et al., 2005). From the works of Tseng's group, it could be summarized that 1) both *dextro*- and *levo*-morphine and lipopolysaccharide (LPS), a toll-like receptor (TLR)-4 agonist, could induce antianalgesia, which could be prevented by *dextro*-, *levo*-naloxone and p38 inhibitor; 2) the antianalgesia-inducing potency is: *dextro*-morphine > *levo*-morphine, and the reversal potency is: *levo*-naloxone > *dextro*-naloxone, which may imply the different binding affinities of *dextro*/*levo*- morphine and naloxone to the putative non-opioid receptor or TLR-4

Inspired by the studies of Hong's group showing naloxone could attenuate LPS-induced microglial activation and neuronal damage (Liu et al., 2000), Watkin's group further tested the possible involvement of the putative nonopioid/TLR-4 pathway in NINP. They found *dextro*-naloxone, *levo-*naltrexone, and LPS-antagonist possess analgesic effects in chronic constriction neuropathic pain model (Hutchinson et al., 2007). Taken together with the role of glial p38 activation in NINP (Jin et al., 2003) and morphine-induced tolerance (Cui et al., 2006), it is possible that the putative glia non-opioid/TLR-4 pathway is important for the

Morphine has long been used *intrathecally* in the management of cancer and non-cancer chronic pain (Plummer et al., 1991; Roberts et al., 2001). However, the long-term use of morphine is associated with severe side-effects and tolerance (Osenbach & Harvey, 2001). Recently, many studies have revealed that *intrathecal* morphine could induce glial activation and neuro-inflammation in the spinal cord (Muscoli et al., 2010; Zhang et al., 2011). Several therapeutic targets have been found, including cytokine receptors, kappa-opioid receptors, N-methyl-D-aspartate receptors, and Toll-like receptors (Hameed et al., 2010; Lewis et al., 2010). Recently, tumor necrosis factor (TNF)-α antagonist etanercept was found to reverse morphine-induced tolerance and block morphine-induced neuroinflammation in the microglia (Shen et al., 2011). *Intrathecal* gabapentin and minocycline could also enhance the antinociceptive effects of morphine and attenuate morphine-induced tolerance (Habibi-Asl et al., 2009; Hutchinson et al., 2008; Lin et al., 2005). These promising agents may be coadministered with *intrathecal* morphine to improve the pain management for cancer patients

For a drug to be tested *intrathecally* in clinical trials, it is imperative to examine its neurotoxic effects rst in animals (Bennett et al., 2000; Smith et al., 2008). For instance, *intrathecal* lidocaine has been found to induce neuropathological changes in the spinal cord and cauda equina (Kirihara et al., 2003). Other analgesics, such as adenosine, sufentanil, alfentanil and morphine have all been tested *intrathecally* in animal studies to examine their potential neurotoxicity (Chiari et al., 1999; Sabbe et al., 1994; Westin et al., 2010). Recently, chronic *intrathecal* infusion of minocycline or gabapentin has been reported to cause no grossly neurotoxicity in animal studies (Chu et al., 2011; Lin et al., 2007), supporting the *intrathecal*

development of NINP and morphine-induced tolerance (Cui et al., 2006).

**4.2 Intrathecal studies on morphine tolerance** 

(Christo & Mazloomdoost, 2008; Mercadante et al., 2004).

**5. Intrathecal neurotoxicity studies** 

use of these agents for pain management.

**4.1 Glial non-opioid/p38 pathway in morphine-induced analgesia and tolerance**

(Hutchinson et al., 2007).

*Intrathecal* space has been a route for spinal anesthesia and analgesics. This space also provides us a way to explore the possible mechanisms involved in pain transmission. Since pain is a major world-wide issue in clinical settings, more and more *intrathecal* animal studies have been undertaken to explore the possible mechanisms involved in the formation of different pain statuses and help to develop promising analgesics to alleviate the suffering of pain patients. These efforts will eventually help to provide better pain managements in clinical settings.

#### **7. Acknowledgment**

This chapter was supported by a John J. Bonica Trainee Fellowship from the International Association for the Study of Pain (IASP), a grant of NSC 98-2314-B-195-002-MY3 from National Science Council, Taipei, Taiwan and grants MMH 10015 and 10044 from Mackay Memorial Hospital, Taipei, Taiwan to J.K.C.

#### **8. References**


Intrathecal Studies on Animal Pain Models 15

Chiari, A., Yaksh, T.L., Myers, R.R., Provencher, J., Moore, L., Lee, C.S. & Eisenach, J.C.

Chien, L.Y., Cheng, J.K., Chu, D., Cheng, C.F. & Tsaur, M.L. (2007). Reduced expression of

Christo, P.J. & Mazloomdoost, D. (2008). Interventional pain treatments for cancer pain. *Ann* 

Chu, L.C., Tsaur, M.L., Lin, C.S., Hung, Y.C., Wang, T.Y., Chen, C.C. & Cheng, J.K. (2011).

Clohisy, D.R. & Mantyh, P.W. (2003). Bone cancer pain. *Cancer*, Vol.97, No.3 Suppl, pp. 866-

Coderre, T.J. & Melzack, R. (1992). The contribution of excitatory amino acids to central

Coetzee, W.A., Amarillo, Y., Chiu, J., Chow, A., Lau, D., McCormack, T., Moreno, H., Nadal,

*Anesthesiology*, Vol.91, No.3, pp. 824-832.

*N Y Acad Sci*, Vol.1138, pp. 299-328.

*Neurosci*, Vol.12, No.9, pp. 3665-3670.

No.9, pp. E1670-1686.

Vol.22, No.1, pp. 114-123.

Migration. *Am J Physiol Cell Physiol*, pp.

873.

rats. *Br J Anaesth*, Vol.106, No.5, pp. 699-705.

hypersensitivity. *J Neurosci*, Vol.27, No.37, pp. 9855-9865.

(1999). Preclinical toxicity screening of intrathecal adenosine in rats and dogs.

A-type potassium channels in primary sensory neurons induces mechanical

Chronic intrathecal infusion of gabapentin prevents nerve ligation-induced pain in

sensitization and persistent nociception after formalin-induced tissue injury. *J* 

M.S., Ozaita, A., Pountney, D., Saganich, M., Vega-Saenz de Miera, E. & Rudy, B. (1999). Molecular diversity of K+ channels. *Ann N Y Acad Sci*, Vol.868, pp. 233-285. Constantin, C.E., Mair, N., Sailer, C.A., Andratsch, M., Xu, Z.Z., Blumer, M.J., Scherbakov,

N., Davis, J.B., Bluethmann, H., Ji, R.R. & Kress, M. (2008). Endogenous tumor necrosis factor alpha (TNFalpha) requires TNF receptor type 2 to generate heat hyperalgesia in a mouse cancer model. *J Neurosci*, Vol.28, No.19, pp. 5072-5081. Coull, J.A., Beggs, S., Boudreau, D., Boivin, D., Tsuda, M., Inoue, K., Gravel, C., Salter, M.W.

& De Koninck, Y. (2005). BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. *Nature*, Vol.438, No.7070, pp. 1017-1021. Cox, J.J., Sheynin, J., Shorer, Z., Reimann, F., Nicholas, A.K., Zubovic, L., Baralle, M., Wraige,

E., Manor, E., Levy, J., Woods, C.G. & Parvari, R. (2010). Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations. *Hum Mutat*, Vol.31,

mitogen-activated protein kinase in spinal microglia mediates morphine

A novel role of minocycline: attenuating morphine antinociceptive tolerance by inhibition of p38 MAPK in the activated spinal microglia. *Brain Behav Immun*,

Busserolles, J., Courteix, C., Noel, J., Lazdunski, M., Eschalier, A., Authier, N. & Bourinet, E. (2011). Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel expression in nociceptors. *EMBO Mol Med*, Vol.3, No.5, pp. 266-278.

Cuddapah, V.A. & Sontheimer, H. (2011). Ion Channels and the Control of Cancer Cell

Cui, Y., Chen, Y., Zhi, J.L., Guo, R.X., Feng, J.Q. & Chen, P.X. (2006). Activation of p38

Cui, Y., Liao, X.X., Liu, W., Guo, R.X., Wu, Z.Z., Zhao, C.M., Chen, P.X. & Feng, J.Q. (2008).

Decosterd, I. & Woolf, C.J. (2000). Spared nerve injury: an animal model of persistent

Descoeur, J., Pereira, V., Pizzoccaro, A., Francois, A., Ling, B., Maffre, V., Couette, B.,

antinociceptive tolerance. *Brain Res*, Vol.1069, No.1, pp. 235-243.

peripheral neuropathic pain. *Pain*, Vol.87, No.2, pp. 149-158.


Bloom, A.P., Jimenez-Andrade, J.M., Taylor, R.N., Castaneda-Corral, G., Kaczmarska, M.J.,

Bourinet, E., Alloui, A., Monteil, A., Barrere, C., Couette, B., Poirot, O., Pages, A., McRory, J.,

Bourquin, A.F., Suveges, M., Pertin, M., Gilliard, N., Sardy, S., Davison, A.C., Spahn, D.R. &

Brackenbury, W.J. & Isom, L.L. (2008). Voltage-gated Na+ channels: potential for beta

Brennan, T.J., Vandermeulen, E.P. & Gebhart, G.F. (1996). Characterization of a rat model of

Cain, D.M., Wacnik, P.W., Turner, M., Wendelschafer-Crabb, G., Kennedy, W.R., Wilcox,

Catterall, W.A., Kalume, F. & Oakley, J.C. (2010). NaV1.1 channels and epilepsy. *J Physiol*,

Catterall, W.A., Striessnig, J., Snutch, T.P. & Perez-Reyes, E. (2002). Voltage-gated calcium channels. *The IUPHAR compendium of voltage-gated ion channels*, pp. 32-56. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M. & Yaksh, T.L. (1994). Quantitative

Chattopadhyay, M., Mata, M. & Fink, D.J. (2008). Continuous delta-opioid receptor

Chen, W.K., Liu, I.Y., Chang, Y.T., Chen, Y.C., Chen, C.C., Yen, C.T. & Shin, H.S. (2010).

Cheng, J.K. & Chiou, L.C. (2006). Mechanisms of the antinociceptive action of gabapentin. *J* 

Cheng, J.K., Lin, C.S., Chen, C.C., Yang, J.R. & Chiou, L.C. (2007). Effects of intrathecal

murine model of cancer pain. *J Neurosci*, Vol.21, No.23, pp. 9367-9376. Catterall, W.A., Goldin, A.L. & Waxman, S.G. (2005). International Union of Pharmacology.

Sensory Nerve Fibers. *J Pain*, pp.

e11-14.

1203.

55-63.

No.26, pp. 6652-6658.

pp. 10360-10368.

nociception. *EMBO J*, Vol.24, No.2, pp. 315-324.

incisional pain. *Pain*, Vol.64, No.3, pp. 493-501.

channels. *Pharmacol Rev*, Vol.57, No.4, pp. 397-409.

Vol.588, No.Pt 11, pp. 1849-1859.

*Pharmacol Sci*, Vol.100, No.5, pp. 471-486.

*Pharmacol*, Vol.18, No.1, pp. 1-8.

Freeman, K.T., Coughlin, K.A., Ghilardi, J.R., Kuskowski, M.A. & Mantyh, P.W. (2011). Breast Cancer-Induced Bone Remodeling, Skeletal Pain and Sprouting of

Snutch, T.P., Eschalier, A. & Nargeot, J. (2005). Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in

Decosterd, I. (2006). Assessment and analysis of mechanical allodynia-like behavior induced by spared nerve injury (SNI) in the mouse. *Pain*, Vol.122, No.1-2, pp. 14

subunits as therapeutic targets. *Expert Opin Ther Targets*, Vol.12, No.9, pp. 1191-

G.L. & Simone, D.A. (2001). Functional interactions between tumor and peripheral nerve: changes in excitability and morphology of primary afferent fibers in a

XLVII. Nomenclature and structure-function relationships of voltage-gated sodium

assessment of tactile allodynia in the rat paw. *J Neurosci Methods*, Vol.53, No.1, pp.

activation reduces neuronal voltage-gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy. *J Neurosci*, Vol.28,

Ca(v)3.2 T-type Ca2+ channel-dependent activation of ERK in paraventricular thalamus modulates acid-induced chronic muscle pain. *J Neurosci*, Vol.30, No.31,

injection of T-type calcium channel blockers in the rat formalin test. *Behav* 


Intrathecal Studies on Animal Pain Models 17

Field, M.J., Cox, P.J., Stott, E., Melrose, H., Offord, J., Su, T.Z., Bramwell, S., Corradini, L.,

Fukumoto, N., Obama, Y., Kitamura, N., Niimi, K., Takahashi, E., Itakura, C. & Shibuya, I.

Gao, Y.J., Cheng, J.K., Zeng, Q., Xu, Z.Z., Decosterd, I., Xu, X. & Ji, R.R. (2009a). Selective

Gao, Y.J. & Ji, R.R. (2010). Targeting astrocyte signaling for chronic pain. *Neurotherapeutics*,

Gao, Y.J., Zhang, L., Samad, O.A., Suter, M.R., Yasuhiko, K., Xu, Z.Z., Park, J.Y., Lind, A.L.,

Ghilardi, J.R., Freeman, K.T., Jimenez-Andrade, J.M., Mantyh, W.G., Bloom, A.P.,

Gold, M.S., Levine, J.D. & Correa, A.M. (1998). Modulation of TTX-R INa by PKC and PKA

Gutman, G.A., Chandy, K.G., Adelman, J.P., Aiyar, J., Bayliss, D.A., Clapham, D.E.,

Habibi-Asl, B., Hassanzadeh, K. & Charkhpour, M. (2009). Central administration of

Hameed, H., Hameed, M. & Christo, P.J. (2010). The effect of morphine on glial cells as a

mouse, rolling mouse Nagoya. *Neuroscience*, Vol.160, No.1, pp. 165-173. Gao, X., Kim, H.K., Chung, J.M. & Chung, K. (2005). Enhancement of NMDA receptor

Vol.26, No.41, pp. 10499-10507.

146-155.

pp. 4096-4108.

Vol.7, No.4, pp. 482-493.

neuropathic rats. *Pain*, Vol.116, No.1-2, pp. 62-72.

and bone cancer pain. *Mol Pain*, Vol.6, pp. 87.

channels. *Pharmacol Rev*, Vol.55, No.4, pp. 583-586.

*Curr Pain Headache Rep*, Vol.14, No.2, pp. 96-104.

*Neurosci*, Vol.18, No.24, pp. 10345-10355.

*Analg*, Vol.109, No.3, pp. 936-942.

England, S., Winks, J., Kinloch, R.A., Hendrich, J., Dolphin, A.C., Webb, T. & Williams, D. (2006). Identification of the alpha2-delta-1 subunit of voltagedependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin. *Proc Natl Acad Sci U S A*, Vol.103, No.46, pp. 17537-17542. Foulkes, T., Nassar, M.A., Lane, T., Matthews, E.A., Baker, M.D., Gerke, V., Okuse, K.,

Dickenson, A.H. & Wood, J.N. (2006). Deletion of annexin 2 light chain p11 in nociceptors causes deficits in somatosensory coding and pain behavior. *J Neurosci*,

(2009). Hypoalgesic behaviors of P/Q-type voltage-gated Ca2+ channel mutant

phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in

inhibition of JNK with a peptide inhibitor attenuates pain hypersensitivity and tumor growth in a mouse skin cancer pain model. *Exp Neurol*, Vol.219, No.1, pp.

Ma, Q. & Ji, R.R. (2009b). JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain. *J Neurosci*, Vol.29, No.13,

Kuskowski, M.A. & Mantyh, P.W. (2010). Administration of a tropomyosin receptor kinase inhibitor attenuates sarcoma-induced nerve sprouting, neuroma formation

and their role in PGE2-induced sensitization of rat sensory neurons in vitro. *J* 

Covarriubias, M., Desir, G.V., Furuichi, K., Ganetzky, B., Garcia, M.L., Grissmer, S., Jan, L.Y., Karschin, A., Kim, D., Kuperschmidt, S., Kurachi, Y., Lazdunski, M., Lesage, F., Lester, H.A., McKinnon, D., Nichols, C.G., O'Kelly, I., Robbins, J., Robertson, G.A., Rudy, B., Sanguinetti, M., Seino, S., Stuehmer, W., Tamkun, M.M., Vandenberg, C.A., Wei, A., Wulff, H. & Wymore, R.S. (2003). International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium

minocycline and riluzole prevents morphine-induced tolerance in rats. *Anesth* 

potential therapeutic target for pharmacological development of analgesic drugs.


Doddareddy, M.R., Choo, H., Cho, Y.S., Rhim, H., Koh, H.Y., Lee, J.H., Jeong, S.W. & Pae,

Dogrul, A., Gardell, L.R., Ossipov, M.H., Tulunay, F.C., Lai, J. & Porreca, F. (2003). Reversal

Dolmetsch, R.E., Pajvani, U., Fife, K., Spotts, J.M. & Greenberg, M.E. (2001). Signaling to the

Donnelly-Roberts, D., McGaraughty, S., Shieh, C.C., Honore, P. & Jarvis, M.F. (2008). Painful purinergic receptors. *J Pharmacol Exp Ther*, Vol.324, No.2, pp. 409-415. Dost, R., Rostock, A. & Rundfeldt, C. (2004). The anti-hyperalgesic activity of retigabine is

Du, X., Wang, C. & Zhang, H. (2011a). Activation of ATP-sensitive potassium channels

Du, X., Zhang, X., Qi, J., An, H., Li, J., Wan, Y., Fu, Y., Gao, H., Gao, Z., Zhan, Y. & Zhang, H.

Duarte, D.B., Duan, J.H., Nicol, G.D., Vasko, M.R. & Hingtgen, C.M. (2011). Reduced

Dureja, G.P., Usmani, H., Khan, M., Tahseen, M. & Jamal, A. (2010). Efficacy of intrathecal

Ekberg, J., Jayamanne, A., Vaughan, C.W., Aslan, S., Thomas, L., Mould, J., Drinkwater, R.,

Eroglu, C., Allen, N.J., Susman, M.W., O'Rourke, N.A., Park, C.Y., Ozkan, E., Chakraborty,

Facer, P., Casula, M.A., Smith, G.D., Benham, C.D., Chessell, I.P., Bountra, C., Sinisi, M.,

Felix, R. (1999). Voltage-dependent Ca2+ channel 2 auxiliary subunit: structure, function

and regulation. *Receptors Channels*, Vol.6, No.5, pp. 351-362.

blockers. *Bioorg Med Chem*, Vol.15, No.2, pp. 1091-1105.

kinase pathway. *Science*, Vol.294, No.5541, pp. 333-339.

Vol.105, No.1-2, pp. 159-168.

*Pharmacol*, Vol.369, No.4, pp. 382-390.

potassium channels. *Br J Pharmacol*, pp.

*Sci U S A*, Vol.103, No.45, pp. 17030-17035.

Vol.139, No.2, pp. 380-392.

induced peripheral sensitization. *J Neurophysiol*, pp.

*Mol Pain*, Vol.7, No.1, pp. 35.

pp. 213-221.

11.

A.N. (2007). 3D pharmacophore based virtual screening of T-type calcium channel

of experimental neuropathic pain by T-type calcium channel blockers. *Pain*,

nucleus by an L-type calcium channel-calmodulin complex through the MAP

mediated by KCNQ potassium channel activation. *Naunyn Schmiedebergs Arch* 

antagonize nociceptive behavior and hyperexcitability of DRG neurons from rats.

(2011b). Characteristics and molecular basis of celecoxib modulation on Kv7

expression of SynGAP, a neuronal GTPase activating protein, enhances capsaicin-

midazolam with or without epidural methylprednisolone for management of postherpetic neuralgia involving lumbosacral dermatomes. *Pain Physician*, Vol.13, No.3,

Baker, M.D., Abrahamsen, B., Wood, J.N., Adams, D.J., Christie, M.J. & Lewis, R.J. (2006). muO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits. *Proc Natl Acad* 

C., Mulinyawe, S.B., Annis, D.S., Huberman, A.D., Green, E.M., Lawler, J., Dolmetsch, R., Garcia, K.C., Smith, S.J., Luo, Z.D., Rosenthal, A., Mosher, D.F. & Barres, B.A. (2009). Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. *Cell*,

Birch, R. & Anand, P. (2007). Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy. *BMC Neurol*, Vol.7, pp.


Intrathecal Studies on Animal Pain Models 19

Ji, R.R., Gereau, R.W.t., Malcangio, M. & Strichartz, G.R. (2009). MAP kinase and pain. *Brain* 

Ji, R.R., Kohno, T., Moore, K.A. & Woolf, C.J. (2003). Central sensitization and LTP: do pain

Ji, R.R. & Strichartz, G. (2004 ). Cell signaling and the genesis of neuropathic pain. *Sci STKE*,

Ji, R.R. & Suter, M.R. (2007). p38 MAPK, microglial signaling, and neuropathic pain. *Mol* 

Jimenez-Andrade, J.M., Bloom, A.P., Stake, J.I., Mantyh, W.G., Taylor, R.N., Freeman, K.T.,

Jin, S.X., Zhuang, Z.Y., Woolf, C.J. & Ji, R.R. (2003). p38 mitogen-activated protein kinase is

Jin, X. & Gereau, R.W.t. (2006). Acute p38-mediated modulation of tetrodotoxin-resistant

Joshi, S.K., Mikusa, J.P., Hernandez, G., Baker, S., Shieh, C.C., Neelands, T., Zhang, X.F.,

Kakimura, J., Zheng, T., Uryu, N. & Ogata, N. (2010). Regulation of the spontaneous

Kang, S. & Brennan, T.J. (2009). Chemosensitivity and mechanosensitivity of nociceptors from incised rat hindpaw skin. *Anesthesiology*, Vol.111, No.1, pp. 155-164. Kawasaki, Y., Xu, Z.Z., Wang, X., Park, J.Y., Zhuang, Z.Y., Tan, P.H., Gao, Y.J., Roy, K.,

Kawasaki, Y., Zhang, L., Cheng, J.K. & Ji, R.R. (2008b). Cytokine mechanisms of central

Kim, S.H. & Chung, J.M. (1992). An experimental model for peripheral neuropathy

superficial spinal cord. *J Neurosci*, Vol.28, No.20, pp. 5189-5194.

and PKC pathways. *Mar Drugs*, Vol.8, No.3, pp. 728-740.

and memory share similar mechanisms? *Trends Neurosci*, Vol.26, No.12, pp. 696-705.

Ghilardi, J.R., Kuskowski, M.A. & Mantyh, P.W. (2010). Pathological sprouting of adult nociceptors in chronic prostate cancer-induced bone pain. *J Neurosci*, Vol.30,

activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. *J Neurosci*,

sodium channels in mouse sensory neurons by tumor necrosis factor-alpha. *J* 

Niforatos, W., Kage, K., Han, P., Krafte, D., Faltynek, C., Sullivan, J.P., Jarvis, M.F. & Honore, P. (2006). Involvement of the TTX-resistant sodium channel Nav 1.8 in inflammatory and neuropathic, but not post-operative, pain states. *Pain*, Vol.123,

augmentation of Na(V)1.9 in mouse dorsal root ganglion neurons: effect of PKA

Corfas, G., Lo, E.H. & Ji, R.R. (2008a). Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. *Nat Med*, Vol.14, No.3,

sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the

produced by segmental spinal nerve ligation in the rat. *Pain*, Vol.50, No.3, pp. 355-

the rat. *Proc Natl Acad Sci U S A*, Vol.104, No.20, pp. 8520-8525.

*Res Rev*, Vol.60, No.1, pp. 135-148.

Vol.252 pp. reE14.

*Pain*, Vol.3, pp. 33.

No.1-2, pp. 75-82.

pp. 331-336.

363.

No.44, pp. 14649-14656.

Vol.23, No.10, pp. 4017-4022.

*Neurosci*, Vol.26, No.1, pp. 246-255.

McGaraughty, S., Chu, K., Roeloffs, R., Zhong, C., Mikusa, J.P., Hernandez, G., Gauvin, D., Wade, C., Zhu, C., Pai, M., Scanio, M., Shi, L., Drizin, I., Gregg, R., Matulenko, M., Hakeem, A., Gross, M., Johnson, M., Marsh, K., Wagoner, P.K., Sullivan, J.P., Faltynek, C.R. & Krafte, D.S. (2007). A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in


Hargreaves, K., Dubner, R., Brown, F., Flores, C. & Joris, J. (1988). A new and sensitive

Hong, R.W. (2010). Less is more: the recent history of neuraxial labor analgesia. *Am J Ther*,

Hong, S., Morrow, T.J., Paulson, P.E., Isom, L.L. & Wiley, J.W. (2004). Early painful diabetic

Hu, H.J., Carrasquillo, Y., Karim, F., Jung, W.E., Nerbonne, J.M., Schwarz, T.L. & Gereau,

Hu, H.J., Glauner, K.S. & Gereau, R.W.t. (2003). ERK integrates PKA and PKC signaling in

Huang, D. & Yu, B. (2008). Recent advance and possible future in TREK-2: a two-pore

Hunanyan, A.S., Alessi, V., Patel, S., Pearse, D.D., Matthews, G. & Arvanian, V.L. (2011).

Hutchinson, M.R., Bland, S.T., Johnson, K.W., Rice, K.C., Maier, S.F. & Watkins, L.R. (2007).

Hutchinson, M.R., Northcutt, A.L., Chao, L.W., Kearney, J.J., Zhang, Y., Berkelhammer, D.L.,

Ikeda, H., Heinke, B., Ruscheweyh, R. & Sandkuhler, J. (2003). Synaptic plasticity in spinal

Ippolito, D.L., Temkin, P.A., Rogalski, S.L. & Chavkin, C. (2002). N-terminal tyrosine

Ippolito, D.L., Xu, M., Bruchas, M.R., Wickman, K. & Chavkin, C. (2005). Tyrosine

Jarecki, B.W., Piekarz, A.D., Jackson, J.O., 2nd & Cummins, T.R. (2010). Human voltage-

Jarvis, M.F., Honore, P., Shieh, C.C., Chapman, M., Joshi, S., Zhang, X.F., Kort, M., Carroll,

memory impairment. *Med Hypotheses*, Vol.70, No.3, pp. 618-624.

analgesia. *Brain Behav Immun*, Vol.22, No.8, pp. 1248-1256.

*Biol Chem*, Vol.277, No.36, pp. 32692-32696.

No.1, pp. 77-88.

Vol.17, No.5, pp. 492-497.

Vol.279, No.28, pp. 29341-29350.

*Neuron*, Vol.50, No.1, pp. 89-100.

Vol.90, No.3, pp. 1671-1679.

Vol.105, No.3, pp. 1033-1044.

pp. 1237-1240.

41683-41693.

pp. 369-378.

method for measuring thermal nociception in cutaneous hyperalgesia. *Pain*, Vol.32,

neuropathy is associated with differential changes in tetrodotoxin-sensitive and resistant sodium channels in dorsal root ganglion neurons in the rat. *J Biol Chem*,

R.W.t. (2006). The kv4.2 potassium channel subunit is required for pain plasticity.

superficial dorsal horn neurons. I. Modulation of A-type K+ currents. *J Neurophysiol*,

potassium channel may involved in the process of NPP, brain ischemia and

Alterations of action potentials and the localization of Nav1.6 sodium channels in spared axons after hemisection injury of the spinal cord in adult rats. *J Neurophysiol*,

Opioid-induced glial activation: mechanisms of activation and implications for opioid analgesia, dependence, and reward. *ScientificWorldJournal*, Vol.7, pp. 98-111.

Loram, L.C., Rozeske, R.R., Bland, S.T., Maier, S.F., Gleeson, T.T. & Watkins, L.R. (2008). Minocycline suppresses morphine-induced respiratory depression, suppresses morphine-induced reward, and enhances systemic morphine-induced

lamina I projection neurons that mediate hyperalgesia. *Science*, Vol.299, No.5610,

residues within the potassium channel Kir3 modulate GTPase activity of Galphai. *J* 

phosphorylation of K(ir)3.1 in spinal cord is induced by acute inflammation, chronic neuropathic pain, and behavioral stress. *J Biol Chem*, Vol.280, No.50, pp.

gated sodium channel mutations that cause inherited neuronal and muscle channelopathies increase resurgent sodium currents. *J Clin Invest*, Vol.120, No.1,

W., Marron, B., Atkinson, R., Thomas, J., Liu, D., Krambis, M., Liu, Y.,

McGaraughty, S., Chu, K., Roeloffs, R., Zhong, C., Mikusa, J.P., Hernandez, G., Gauvin, D., Wade, C., Zhu, C., Pai, M., Scanio, M., Shi, L., Drizin, I., Gregg, R., Matulenko, M., Hakeem, A., Gross, M., Johnson, M., Marsh, K., Wagoner, P.K., Sullivan, J.P., Faltynek, C.R. & Krafte, D.S. (2007). A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. *Proc Natl Acad Sci U S A*, Vol.104, No.20, pp. 8520-8525.


Intrathecal Studies on Animal Pain Models 21

Liu, B., Du, L. & Hong, J.S. (2000). Naloxone protects rat dopaminergic neurons against

LoPachin, R.M., Rudy, T.A. & Yaksh, T.L. (1981). An improved method for chronic

Lopez-Santiago, L.F., Pertin, M., Morisod, X., Chen, C., Hong, S., Wiley, J., Decosterd, I. &

Luo, J.L., Qin, H.Y., Wong, C.K., Tsang, S.Y., Huang, Y. & Bian, Z.X. (2011). Enhanced

Luvisetto, S., Marinelli, S., Panasiti, M.S., D'Amato, F.R., Fletcher, C.F., Pavone, F. &

Lynch, M.E. & Campbell, F. (2011). Cannabinoids for Treatment of Chronic Non-Cancer Pain; a Systematic Review of Randomized Trials. *Br J Clin Pharmacol*, pp. Ma, C., Rosenzweig, J., Zhang, P., Johns, D.C. & LaMotte, R.H. (2010). Expression of

Mao, J., Price, D.D. & Mayer, D.J. (1994). Thermal hyperalgesia in association with the

Mathie, A. (2007). Neuronal two-pore-domain potassium channels and their regulation by G

Matthews, E.A., Bee, L.A., Stephens, G.J. & Dickenson, A.H. (2007). The Cav2.3 calcium

McCallum, J.B., Wu, H.E., Tang, Q., Kwok, W.M. & Hogan, Q.H. (2011). Subtype-specific

Mercadante, S., Villari, P. & Ferrera, P. (2004). Dialogues on complex analgesic strategies for difficult pain syndromes. *Support Care Cancer*, Vol.12, No.8, pp. 599-603.

therapeutic dilemmas. *Reg Anesth Pain Med*, Vol.24, No.1, pp. 74-83.

of chronic neuropathic pain. *Eur J Neurosci*, Vol.25, No.12, pp. 3561-3569. Mazzuca, M., Heurteaux, C., Alloui, A., Diochot, S., Baron, A., Voilley, N., Blondeau, N.,

P/Q-type Ca2+ channels. *Neuroscience*, Vol.142, No.3, pp. 823-832.

and protein kinase C. *J Neurosci*, Vol.14, No.4, pp. 2301-2312.

opioid mechanisms. *Nat Neurosci*, Vol.10, No.8, pp. 943-945.

protein-coupled receptors. *J Physiol*, Vol.578, No.Pt 2, pp. 377-385.

generation. *J Pharmacol Exp Ther*, Vol.293, No.2, pp. 607-617.

cord. *Brain Res*, Vol.1054, No.2 pp. 167-173.

to pain. *J Neurosci*, Vol.26, No.30, pp. 7984-7994.

of the spinal ganglion. *Mol Pain*, Vol.6, pp. 65.

559-561.

600-609.

suppression of morphine-evoked excitatory amino acid release in the rat spinal

inflammatory damage through inhibition of microglia activation and superoxide

catheterization of the rat spinal subarachnoid space. *Physiol Behav*, Vol.27, No.3, pp.

Isom, L.L. (2006). Sodium channel beta2 subunits regulate tetrodotoxin-sensitive sodium channels in small dorsal root ganglion neurons and modulate the response

Excitability and Down-Regulated Voltage-Gated Potassium Channels in Colonic DRG Neurons from Neonatal Maternal Separation Rats. *J Pain*, Vol.12, No.5, pp.

Pietrobon, D. (2006). Pain sensitivity in mice lacking the Ca(v)2.1alpha1 subunit of

inwardly rectifying potassium channels by an inducible adenoviral vector reduced the neuronal hyperexcitability and hyperalgesia produced by chronic compression

development of morphine tolerance in rats: roles of excitatory amino acid receptors

channel antagonist SNX-482 reduces dorsal horn neuronal responses in a rat model

Escoubas, P., Gelot, A., Cupo, A., Zimmer, A., Zimmer, A.M., Eschalier, A. & Lazdunski, M. (2007). A tarantula peptide against pain via ASIC1a channels and

reduction of voltage-gated calcium current in medium-sized dorsal root ganglion neurons after painful peripheral nerve injury. *Neuroscience*, Vol.179, pp. 244-255. Mercadante, S. (1999). Neuraxial techniques for cancer pain: an opinion about unresolved


Kirihara, Y., Saito, Y., Sakura, S., Hashimoto, K., Kishimoto, T. & Yasui, Y. (2003).

Klugbauer, N., Marais, E. & Hofmann, F. (2003). Calcium channel alpha2delta subunits:

Knutsen, L.J., Hobbs, C.J., Earnshaw, C.G., Fiumana, A., Gilbert, J., Mellor, S.L., Radford, F.,

Kretschmer, T., Happel, L.T., England, J.D., Nguyen, D.H., Tiel, R.L., Beuerman, R.W. &

Lampert, A., O'Reilly, A.O., Reeh, P. & Leffler, A. (2010). Sodium channelopathies and pain.

Ledeboer, A., Liu, T., Shumilla, J.A., Mahoney, J.H., Vijay, S., Gross, M.I., Vargas, J.A.,

Leo, S., D'Hooge, R. & Meert, T. (2010). Exploring the role of nociceptor-specific sodium

Levin, M.E., Jin, J.G., Ji, R.R., Tong, J., Pomonis, J.D., Lavery, D.J., Miller, S.W. & Chiang,

Li, C.Y., Song, Y.H., Higuera, E.S. & Luo, Z.D. (2004). Spinal dorsal horn calcium channel

Li, C.Y., Zhang, X.L., Matthews, E.A., Li, K.W., Kurwa, A., Boroujerdi, A., Gross, J., Gold,

Lin, C.S., Tsaur, M.L., Chen, C.C., Wang, T.Y., Lin, C.F., Lai, Y.L., Hsu, T.C., Pan, Y.Y., Yang,

Lin, J.A., Lee, M.S., Wu, C.T., Yeh, C.C., Lin, S.L., Wen, Z.H. & Wong, C.S. (2005).

models of neuropathic pain. *Neuron Glia Biol*, Vol.2, No.4, pp. 279-291. Lee, S., Kim, Y., Back, S.K., Choi, H.W., Lee, J.Y., Jung, H.H., Ryu, J.H., Suh, H.W., Na, H.S.,

conotoxin FVIA on N type Ca2+ channels. *Mol Pain*, Vol.6, pp. 97.

channel blockers. *Bioorg Med Chem Lett*, Vol.17, No.3, pp. 662-667.

*Anesthesiology*, Vol.99, No.4, pp. 961-968.

Vol.144, No.8, pp. 803-810; discussion 810.

*Pflugers Arch*, Vol.460, No.2, pp. 249-263.

*Res*, Vol.208, No.1, pp. 149-157.

pp. 20-34.

Vol.32, No.3, pp. 209-216.

*Neuroscience*, Vol.165, No.2, pp. 569-583.

allodynia. *J Neurosci*, Vol.24, No.39, pp. 8494-8499.

No.6, pp. 639-647.

Comparative neurotoxicity of intrathecal and epidural lidocaine in rats.

differential expression, function, and drug binding. *J Bioenerg Biomembr*, Vol.35,

Smith, N.J., Birch, P.J., Russell Burley, J., Ward, S.D. & James, I.F. (2007). Synthesis and SAR of novel 2-arylthiazolidinones as selective analgesic N-type calcium

Kline, D.G. (2002). Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry. *Acta Neurochir (Wien)*,

Sultzbaugh, L., Claypool, M.D., Sanftner, L.M., Watkins, L.R. & Johnson, K.W. (2006). The glial modulatory drug AV411 attenuates mechanical allodynia in rat

Kim, H.J., Rhim, H. & Kim, J.I. (2010). Analgesic effect of highly reversible omega-

channels in pain transmission using Nav1.8 and Nav1.9 knockout mice. *Behav Brain* 

L.W. (2008). Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain. *Pain*, Vol.137, No.1, pp. 182-201. Lewis, S.S., Hutchinson, M.R., Rezvani, N., Loram, L.C., Zhang, Y., Maier, S.F., Rice, K.C. &

Watkins, L.R. (2010). Evidence that intrathecal morphine-3-glucuronide may cause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1beta.

2-1 subunit upregulation contributes to peripheral nerve injury-induced tactile

M.S., Dickenson, A.H., Feng, G. & Luo, Z.D. (2006). Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation. *Pain*, Vol.125, No.1-2,

C.H. & Cheng, J.K. (2007). Chronic intrathecal infusion of minocycline prevents the development of spinal-nerve ligation-induced pain in rats. *Reg Anesth Pain Med*,

Attenuation of morphine tolerance by intrathecal gabapentin is associated with

suppression of morphine-evoked excitatory amino acid release in the rat spinal cord. *Brain Res*, Vol.1054, No.2 pp. 167-173.


Intrathecal Studies on Animal Pain Models 23

Qin, N., Yagel, S., Momplaisir, M.L., Codd, E.E. & D'Andrea, M.R. (2002). Molecular cloning

Rasband, M.N., Park, E.W., Vanderah, T.W., Lai, J., Porreca, F. & Trimmer, J.S. (2001).

Roberts, L.J., Finch, P.M., Goucke, C.R. & Price, L.M. (2001). Outcome of intrathecal opioids

Rogers, M., Tang, L., Madge, D.J. & Stevens, E.B. (2006). The role of sodium channels in

Romanelli, P. & Esposito, V. (2004). The functional anatomy of neuropathic pain. *Neurosurg* 

Sabbe, M.B., Grafe, M.R., Mjanger, E., Tiseo, P.J., Hill, H.F. & Yaksh, T.L. (1994). Spinal

Saegusa, H., Kurihara, T., Zong, S., Kazuno, A., Matsuda, Y., Nonaka, T., Han, W.,

Sandkuhler, J. & Liu, X. (1998). Induction of long-term potentiation at spinal synapses by noxious stimulation or nerve injury. *Eur J Neurosci*, Vol.10, No.7, pp. 2476-2480. Schafers, M., Svensson, C.I., Sommer, C. & Sorkin, L.S. (2003). Tumor necrosis factor-alpha

delivery of sufentanil, alfentanil, and morphine in dogs. Physiologic and

Toriyama, H. & Tanabe, T. (2001). Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel. *EMBO J*, Vol.20, No.10,

induces mechanical allodynia after spinal nerve ligation by activation of p38 MAPK

2-hydroxy-2-methyl-N-(5,5,10-trioxo-4,10-dihydro thieno[3,2-c][1]benzothiepin-9 yl)propanamide] enhances A-type K+ currents in neurons of the dorsal root

(2007). Discovery of potent T-type calcium channel blocker. *Bioorg Med Chem Lett*,

B., Sanders, K.M. & Koh, S.D. (2005). Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II. *Am J Physiol Cell Physiol*, Vol.288,

type K+ channels operating at subthreshold potentials with unique expression in

Etanercept restores the antinociceptive effect of morphine and suppresses spinal neuroinflammation in morphine-tolerant rats. *Anesth Analg*, Vol.112, No.2, pp. 454-

calcium channel blocker, AM336, produces potent dose-dependent antinociception

in chronic non-cancer pain. *Eur J Pain*, Vol.5, No.4, pp. 353-361.

neuropathic pain. *Semin Cell Dev Biol*, Vol.17, No.5, pp. 571-581.

toxicologic investigations. *Anesthesiology*, Vol.81, No.4, pp. 899-920.

in primary sensory neurons. *J Neurosci*, Vol.23, No.7, pp. 2517-2521. Sculptoreanu, A., Yoshimura, N. & de Groat, W.C. (2004). KW-7158 [(2S)-(+)-3,3,3-trifluoro-

ganglion of the adult rat. *J Pharmacol Exp Ther*, Vol.310, No.1, pp. 159-168. Seo, H.N., Choi, J.Y., Choe, Y.J., Kim, Y., Rhim, H., Lee, S.H., Kim, J., Joo, D.J. & Lee, J.Y.

Sergeant, G.P., Ohya, S., Reihill, J.A., Perrino, B.A., Amberg, G.C., Imaizumi, Y., Horowitz,

Serodio, P., Vega-Saenz de Miera, E. & Rudy, B. (1996). Cloning of a novel component of A-

Shen, C.H., Tsai, R.Y., Shih, M.S., Lin, S.L., Tai, Y.H., Chien, C.C. & Wong, C.S. (2011).

Smith, H.S., Deer, T.R., Staats, P.S., Singh, V., Sehgal, N. & Cordner, H. (2008). Intrathecal

Smith, M.T., Cabot, P.J., Ross, F.B., Robertson, A.D. & Lewis, R.J. (2002). The novel N-type

heart and brain. *J Neurophysiol*, Vol.75, No.5, pp. 2174-2179.

drug delivery. *Pain Physician*, Vol.11, No.2 Suppl, pp. S89-S104.

*Mol Pharmacol*, Vol.62, No.3, pp. 485-496.

Vol.98, No.23, pp. 13373-13378.

*Clin N Am*, Vol.15, No.3, pp. 257-268.

pp. 2349-2356.

Vol.17, No.21, pp. 5740-5743.

No.2, pp. C304-313.

459.

and characterization of the human voltage-gated calcium channel 2-4 subunit.

Distinct potassium channels on pain-sensing neurons. *Proc Natl Acad Sci U S A*,


Mienville, J.M., Maric, I., Maric, D. & Clay, J.R. (1999). Loss of IA expression and increased

Mika, J., Osikowicz, M., Makuch, W. & Przewlocka, B. (2007). Minocycline and

Mo, G., Grant, R., O'Donnell, D., Ragsdale, D.S., Cao, C.Q. & Seguela, P. (2011). Neuropathic

Mogil, J.S., Davis, K.D. & Derbyshire, S.W. (2010). The necessity of animal models in pain

Muscoli, C., Doyle, T., Dagostino, C., Bryant, L., Chen, Z., Watkins, L.R., Ryerse, J.,

Nassar, M.A., Levato, A., Stirling, L.C. & Wood, J.N. (2005). Neuropathic pain develops normally in mice lacking both Na(v)1.7 and Na(v)1.8. *Mol Pain*, Vol.1, pp. 24. Ocana, M., Cendan, C.M., Cobos, E.J., Entrena, J.M. & Baeyens, J.M. (2004). Potassium

Osenbach, R.K. & Harvey, S. (2001). Neuraxial infusion in patients with chronic intractable cancer and noncancer pain. *Curr Pain Headache Rep*, Vol.5, No.3, pp. 241-249. Park, S.Y., Choi, J.Y., Kim, R.U., Lee, Y.S., Cho, H.J. & Kim, D.S. (2003). Downregulation of

neurotrophins in rat dorsal root ganglia. *Mol Cells*, Vol.16, No.2, pp. 256-259. Passmore, G.M., Selyanko, A.A., Mistry, M., Al-Qatari, M., Marsh, S.J., Matthews, E.A.,

Patel, A.J., Honore, E., Lesage, F., Fink, M., Romey, G. & Lazdunski, M. (1999). Inhalational

Pertin, M., Ji, R.R., Berta, T., Powell, A.J., Karchewski, L., Tate, S.N., Isom, L.L., Woolf, C.J.,

Plummer, J.L., Cherry, D.A., Cousins, M.J., Gourlay, G.K., Onley, M.M. & Evans, K.H.

Poirot, O., Berta, T., Decosterd, I. & Kellenberger, S. (2006). Distinct ASIC currents are

pain: a retrospective study. *Pain*, Vol.44, No.3, pp. 215-220.

1303-1310.

No.2-3, pp. 142-149.

*Mol Pain*, Vol.7, pp. 14.

Vol.500, No.1-3, pp. 203-219.

Vol.23, No.18, pp. 7227-7236.

No.5, pp. 422-426.

No.47, pp. 10970-10980.

Vol.576, No.Pt 1, pp. 215-234.

15400-15408.

research. *Pain*, Vol.151, No.1, pp. 12-17.

excitability in postnatal rat Cajal-Retzius cells. *J Neurophysiol*, Vol.82, No.3, pp.

pentoxifylline attenuate allodynia and hyperalgesia and potentiate the effects of morphine in rat and mouse models of neuropathic pain. *Eur J Pharmacol*, Vol.560,

Nav1.3-mediated sensitization to P2X activation is regulated by protein kinase C.

Bieberich, E., Neumman, W. & Salvemini, D. (2010). Counter-regulation of opioid analgesia by glial-derived bioactive sphingolipids. *J Neurosci*, Vol.30, No.46, pp.

channels and pain: present realities and future opportunities. *Eur J Pharmacol*,

voltage-gated potassium channel alpha gene expression by axotomy and

Dickenson, A.H., Brown, T.A., Burbidge, S.A., Main, M. & Brown, D.A. (2003). KCNQ/M currents in sensory neurons: significance for pain therapy. *J Neurosci*,

anesthetics activate two-pore-domain background K+ channels. *Nat Neurosci*, Vol.2,

Gilliard, N., Spahn, D.R. & Decosterd, I. (2005). Upregulation of the voltage-gated sodium channel beta2 subunit in neuropathic pain models: characterization of expression in injured and non-injured primary sensory neurons. *J Neurosci*, Vol.25,

(1991). Long-term spinal administration of morphine in cancer and non-cancer

expressed in rat putative nociceptors and are modulated by nerve injury. *J Physiol*,


Intrathecal Studies on Animal Pain Models 25

Wang, W., Gu, J., Li, Y.Q. & Tao, Y.X. (2011). Are voltage-gated sodium channels on the

Wang, Z., Ma, W., Chabot, J.G. & Quirion, R. (2010). Calcitonin gene-related peptide as a

Watkins, L.R., Hutchinson, M.R., Ledeboer, A., Wieseler-Frank, J., Milligan, E.D. & Maier,

Wen, X.J., Xu, S.Y., Chen, Z.X., Yang, C.X., Liang, H. & Li, H. (2010). The roles of T-type

Westin, B.D., Walker, S.M., Deumens, R., Grafe, M. & Yaksh, T.L. (2010). Validation of a

Wheeler-Aceto, H., Porreca, F. & Cowan, A. (1990). The rat paw formalin test: comparison of

White, F.A., Jung, H. & Miller, R.J. (2007). Chemokines and the pathophysiology of neuropathic pain. *Proc Natl Acad Sci U S A*, Vol.104, No.51, pp. 20151-20158. Wu, H.E., Sun, H.S., Cheng, C.W., Terashvili, M. & Tseng, L.F. (2006a). dextro-Naloxone or

Wu, H.E., Sun, H.S., Cheng, C.W. & Tseng, L.F. (2006b). p38 mitogen-activated protein

Yaksh, T.L. (2006). Calcium channels as therapeutic targets in neuropathic pain. *J Pain*, Vol.7,

Yaksh, T.L., Kohl, R.L. & Rudy, T.A. (1977). Induction of tolerance and withdrawal in rats

Yokoyama, K., Kurihara, T., Saegusa, H., Zong, S., Makita, K. & Tanabe, T. (2004). Blocking

Yu, Y.Q., Zhao, F., Guan, S.M. & Chen, J. (2011). Antisense-Mediated Knockdown of

spinal cord. *J Pharmacol Exp Ther*, Vol.314, No.3, pp. 1101-1108.

morphine tolerance. *Eur J Neurosci*, Vol.20, No.12, pp. 3516-3519.

Vol.7, pp. 16.

analgesia. *Pain*, Vol.151, No.1, pp. 194-205.

*Immun*, Vol.21, No.2, pp. 131-146.

*Neurosci*, Vol.18, No.16, pp. 6319-6330.

*Anesthesiology*, Vol.113, No.1, pp. 183-199.

*Neurosci*, Vol.24, No.9, pp. 2575-2580.

No.1 Suppl 1, pp. S13-30.

pp. 275-284.

noxious agents. *Pain*, Vol.40, No.2, pp. 229-238.

dorsal root ganglion involved in the development of neuropathic pain? *Mol Pain*,

regulator of neuronal CaMKII-CREB, microglial p38-NFkappaB and astroglial ERK-Stat1/3 cascades mediating the development of tolerance to morphine-induced

S.F. (2007). Norman Cousins Lecture. Glia as the "bad guys": implications for improving clinical pain control and the clinical utility of opioids. *Brain Behav* 

calcium channel in the development of neuropathic pain following chronic compression of rat dorsal root ganglia. *Pharmacology*, Vol.85, No.5, pp. 295-300. Westenbroek, R.E., Hoskins, L. & Catterall, W.A. (1998). Localization of Ca2+ channel

subtypes on rat spinal motor neurons, interneurons, and nerve terminals. *J* 

preclinical spinal safety model: effects of intrathecal morphine in the neonatal rat.

levo-naloxone reverses the attenuation of morphine antinociception induced by lipopolysaccharide in the mouse spinal cord via a non-opioid mechanism. *Eur J* 

kinase inhibitor SB203580 reverses the antianalgesia induced by dextro-morphine or morphine in the mouse spinal cord. *Eur J Pharmacol*, Vol.550, No.1-3, pp. 91-94. Wu, H.E., Thompson, J., Sun, H.S., Terashvili, M. & Tseng, L.F. (2005). Antianalgesia:

stereoselective action of dextro-morphine over levo-morphine on glia in the mouse

receiving morphine in the spinal subarachnoid space. *Eur J Pharmacol*, Vol.42, No.3,

the R-type (Cav2.3) Ca2+ channel enhanced morphine analgesia and reduced

Na(V)1.8, but Not Na(V)1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat. *PLoS One*, Vol.6, No.5, pp. e19865.

after intrathecal dosing in rats and inhibits substance P release in rat spinal cord slices. *Pain*, Vol.96, No.1-2, pp. 119-127.


Song, X.J., Wang, Z.B., Gan, Q. & Walters, E.T. (2006). cAMP and cGMP contribute to

Szekely, J.I., Torok, K. & Mate, G. (2002). The role of ionotropic glutamate receptors in

Szu-Yu Ho, T. & Rasband, M.N. (2011). Maintenance of neuronal polarity. *Dev Neurobiol*,

Takeda, M., Tanimoto, T., Nasu, M. & Matsumoto, S. (2008). Temporomandibular joint

Takeda, M., Tsuboi, Y., Kitagawa, J., Nakagawa, K., Iwata, K. & Matsumoto, S. (2011).

Talley, E.M., Cribbs, L.L., Lee, J.H., Daud, A., Perez-Reyes, E. & Bayliss, D.A. (1999).

activated (T-type) calcium channels. *J Neurosci*, Vol.19, No.6, pp. 1895-1911. Tanga, F.Y., Nutile-McMenemy, N. & DeLeo, J.A. (2005). The CNS role of Toll-like receptor 4

Thorpe, L.B., Goldie, M. & Dolan, S. (2011). Central and Local Administration of Gingko

Tsuda, M., Shigemoto-Mogami, Y., Koizumi, S., Mizokoshi, A., Kohsaka, S., Salter, M.W. &

Tyagarajan, S., Chakravarty, P.K., Zhou, B., Taylor, B., Eid, R., Fisher, M.H., Parsons, W.H.,

Wang, M., Offord, J., Oxender, D.L. & Su, T.Z. (1999). Structural requirement of the calciumchannel subunit 2 for gabapentin binding. *Biochem J*, Vol.342 (Pt 2), pp. 313-320.

Rat Carrageenan Model. *Anesth Analg*, Vol.112, No.5, pp. 1226-1231. Tsuda, M., Kohro, Y., Yano, T., Tsujikawa, T., Kitano, J., Tozaki-Saitoh, H., Koyanagi, S.,

after nerve injury. *Nature*, Vol.424, No.6950, pp. 778-783.

slices. *Pain*, Vol.96, No.1-2, pp. 119-127.

inflammatory pain. *Mol Pain*, Vol.7, pp. 5.

Vol.102, No.16, pp. 5856-5861.

Vol.134, No.Pt 4, pp. 1127-1139.

*Neurosci*, Vol.20, No.5, pp. 1150-1160.

No.10, pp. 887-912.

pp. 189-195.

Vol.71, No.6, pp. 474-482.

compression. *J Neurophysiol*, Vol.95, No.1, pp. 479-492.

after intrathecal dosing in rats and inhibits substance P release in rat spinal cord

sensory neuron hyperexcitability and hyperalgesia in rats with dorsal root ganglia

nociception with special regard to the AMPA binding sites. *Curr Pharm Des*, Vol.8,

inflammation decreases the voltage-gated K+ channel subtype 1.4 immunoreactivity of trigeminal ganglion neurons in rats. *Eur J Pain*, Vol.12, No.2,

Potassium channels as a potential therapeutic target for trigeminal neuropathic and

Differential distribution of three members of a gene family encoding low voltage-

in innate neuroimmunity and painful neuropathy. *Proc Natl Acad Sci U S A*,

Biloba Extract EGb 761(R) Inhibits Thermal Hyperalgesia and Inflammation in the

Ohdo, S., Ji, R.R., Salter, M.W. & Inoue, K. (2011). JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats. *Brain*,

Inoue, K. (2003). P2X4 receptors induced in spinal microglia gate tactile allodynia

Wyvratt, M.J., Lyons, K.A., Klatt, T., Li, X., Kumar, S., Williams, B., Felix, J., Priest, B.T., Brochu, R.M., Warren, V., Smith, M., Garcia, M., Kaczorowski, G.J., Martin, W.J., Abbadie, C., McGowan, E., Jochnowitz, N., Weber, A. & Duffy, J.L. (2010). Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain. *Bioorg Med Chem Lett*, Vol.20, No.24, pp. 7479-7482. Verge, G.M., Milligan, E.D., Maier, S.F., Watkins, L.R., Naeve, G.S. & Foster, A.C. (2004).

Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions. *Eur J* 


**2** 

 *USA* 

**Polymer Based Therapies** 

*Drug and Biomaterial R&D* 

**for the Treatment of Chronic Pain** 

*Genzyme Corporation – A Sanofi Company, Waltham, MA,* 

Pradeep K. Dhal, Diego A. Gianolio and Robert J. Miller\*

Since chronic pain manifests functional limitation, it is the leading cause of longer term disability [1, 2]. In the US alone, an estimated 75 million people suffer from chronic pain [3]. In addition to chronic pain, proper management of postoperative acute pain impacts the clinical outcome of patients undergoing surgery [4]. Opioid family of analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are the main stays of current pharmacological agents available for the management of chronic pain [5, 6]. However, current therapies for pain management show modest efficacy and are associated with significant side effects. The major adverse effects of oral NSAIDs are gastrointestinal bleeding, gastric ulcer, renal failure and cardiovascular risks (in particular with selective COX-2 inhibitors) [7, 8]. The side effects of opioid family therapies include constipation, nausea, cognitive impairment and most importantly addiction [9, 10]. Thus, development of safer and effective treatment

In recent years numerous efforts have been made to develop long-acting opioid analgesics and NSAIDs to modulate their pharmacokinetic profiles. Some of these include sustained release formulations and topical gels [11, 12]. Biological agents such as antibody against nerve growth factor (NGF) have also been evaluated as therapies for chronic pain. The anti-NGF antibody acts by sequestering NGF and thus inhibits its interaction with the NGF-

Polymeric approach offers an attractive route to develop novel therapeutic agents for effective management of chronic pain. Interesting physical and chemical characteristics of synthetic and natural polymers enable them as promising materials for biomedical applications such as therapeutic agents, drug delivery carriers, and medical devices [14, 15]. A number of polymer derived therapies have been commercialized in the marketplace [16, 17]. The present article reviews the current state of research and development efforts to discover and develop biomedical polymer as therapeutic agents for the treatment of chronic pain. While use of polymer-derived agents for the treatment of different kinds of pains will be highlighted, the primary focus of the present article pertains to management of pain arising from osteoarthritis. Furthermore, role of polymers as intrinsically pain relieving agents either alone or as chemical conjugates of low molecular weight pain modulating agents are described in this article. The research and development efforts to develop control release formulations of low molecular weight pain therapies are outside the scope of this article. There are in fact a number of

interesting articles that describe this aspect of pain management therapies [18, 19].

of chronic pain is an important goal of current pharmaceutical research.

**1. Introduction** 

receptor on the sensory neurons [13].


### **Polymer Based Therapies for the Treatment of Chronic Pain**

Pradeep K. Dhal, Diego A. Gianolio and Robert J. Miller\* *Drug and Biomaterial R&D Genzyme Corporation – A Sanofi Company, Waltham, MA, USA* 

#### **1. Introduction**

26 Pain Management – Current Issues and Opinions

Zamponi, G.W., Lewis, R.J., Todorovic, S.M., Arneric, S.P. & Snutch, T.P. (2009). Role of

Zhang, Y., Conklin, D.R., Li, X. & Eisenach, J.C. (2005). Intrathecal morphine reduces

Zhang, Y., Li, H., Li, Y., Sun, X., Zhu, M., Hanley, G., Lesage, G. & Yin, D. (2011). Essential

Zhuang, Z.Y., Kawasaki, Y., Tan, P.H., Wen, Y.R., Huang, J. & Ji, R.R. (2007). Role of the

Zhuang, Z.Y., Wen, Y.R., Zhang, D.R., Borsello, T., Bonny, C., Strichartz, G.R., Decosterd, I.

development and maintenance. *J Neurosci*, Vol.26, No.13, pp. 3551-3560.

adenosine receptor. *Anesthesiology*, Vol.102, No.2, pp. 416-420.

*Neurosci Lett*, Vol.489, No.1, pp. 43-47.

*Behav Immun*, Vol.21, No.5, pp. 642-651.

No.1, pp. 84-89.

voltage-gated calcium channels in ascending pain pathways. *Brain Res Rev*, Vol.60,

allodynia after peripheral nerve injury in rats via activation of a spinal A1

role of toll-like receptor 2 in morphine-induced microglia activation in mice.

CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine. *Brain* 

& Ji, R.R. (2006). A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain

Since chronic pain manifests functional limitation, it is the leading cause of longer term disability [1, 2]. In the US alone, an estimated 75 million people suffer from chronic pain [3]. In addition to chronic pain, proper management of postoperative acute pain impacts the clinical outcome of patients undergoing surgery [4]. Opioid family of analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs) are the main stays of current pharmacological agents available for the management of chronic pain [5, 6]. However, current therapies for pain management show modest efficacy and are associated with significant side effects. The major adverse effects of oral NSAIDs are gastrointestinal bleeding, gastric ulcer, renal failure and cardiovascular risks (in particular with selective COX-2 inhibitors) [7, 8]. The side effects of opioid family therapies include constipation, nausea, cognitive impairment and most importantly addiction [9, 10]. Thus, development of safer and effective treatment of chronic pain is an important goal of current pharmaceutical research.

In recent years numerous efforts have been made to develop long-acting opioid analgesics and NSAIDs to modulate their pharmacokinetic profiles. Some of these include sustained release formulations and topical gels [11, 12]. Biological agents such as antibody against nerve growth factor (NGF) have also been evaluated as therapies for chronic pain. The anti-NGF antibody acts by sequestering NGF and thus inhibits its interaction with the NGFreceptor on the sensory neurons [13].

Polymeric approach offers an attractive route to develop novel therapeutic agents for effective management of chronic pain. Interesting physical and chemical characteristics of synthetic and natural polymers enable them as promising materials for biomedical applications such as therapeutic agents, drug delivery carriers, and medical devices [14, 15]. A number of polymer derived therapies have been commercialized in the marketplace [16, 17]. The present article reviews the current state of research and development efforts to discover and develop biomedical polymer as therapeutic agents for the treatment of chronic pain. While use of polymer-derived agents for the treatment of different kinds of pains will be highlighted, the primary focus of the present article pertains to management of pain arising from osteoarthritis. Furthermore, role of polymers as intrinsically pain relieving agents either alone or as chemical conjugates of low molecular weight pain modulating agents are described in this article. The research and development efforts to develop control release formulations of low molecular weight pain therapies are outside the scope of this article. There are in fact a number of interesting articles that describe this aspect of pain management therapies [18, 19].

Polymer Based Therapies for the Treatment of Chronic Pain 29

arranged in an alternate fashion along the polymer backbone (Fig. 1). HA is ubiquitous in nature and is produced by every tissue of higher organisms and some bacteria. The biopolymer is found in the extracellular matrices (particularly in soft connective tissues), synovial fluid, and cartilage. HA is endogenously synthesized by chondrocytes and synoviocytes [37-39]. After being released into the synovial space, HA accumulates on the surfaces of cartilage and ligament. Endogenously synthesized HA is generally of very high molecular weight (in the range of 3 -5 million Dalton) and its fully hydrated form assumes a globular shape [40]. Unique viscoelastic properties of HA enables it to maintain rheological homeostasis of the synovial fluid in the joints and plays a critical role in providing lubrication, elasticity, and shock absorption to joint tissues. Furthermore, by providing a coat on the surface of articular cartilage, HA protects the cartilage and blocks the loss of proteoglycan from the cartilage matrix into the synovial space [41]. In healthy joint of human knee, the normal concentration of HA in the synovial fluid is in the range of 2.5 - 4.0 mg/mL. However, under pathological conditions such as osteoarthritis, the concentration of HA is significantly reduced (estimated to be ~ 1 - 2 mg/mL) [42]. Furthermore, the biopolymer undergoes degradation under diseased conditions with substantial reduction in molecular weight. A combination of lowering in concentration and molecular weights leads to lowering in viscosity and elasticity of synovial fluid and consequently adverse impact on joint function. Thus, catabolic degradation of HA directly

In addition to acting as a lubricant for the joint, HA has been reported to impart antiinflammatory, anabolic, and chondroprotective effects [43, 44]. Since OA onset is attributed to degradation of high molecular weight HA and its concentration in the synovial fluid, increase in HA concentration either by increasing the rate of the proteoglycan biosynthesis or by incorporating HA exogenously to the joint space would improve joint function and relieve OA associated chronic pain. Therefore, the effect of intraarticular administration of exogenous HA to restore rheological properties of the synovial fluid have been extensively

In order to maintain desired viscoelastic property of synovial fluid, the exogenous HA needs to have high molecular weight. It has been observed that the frequency and the amount of exogenous HA injected can be lowered by increasing the molecular weight of HA based exogenous viscosupplement. Towards that end, a variety of synthetic approaches have been undertaken to engineer high molecular weight HA derivatives [46]. In general, the desired rheological properties of HA based viscosupplements are achieved by crosslinking of naturally occurring linear HA to produce higher molecular weight

correlates with the onset OA.

studied [45].

compounds.

Fig. 1. Chemical structure of hyaluronic acid.

**3.2 HA derivatives for the treatment of osteoarthritis** 

#### **2. Osteoarthritis pain**

Osteoarthritis (OA) is one of the most prevalent musculo-skeletal degenerative diseases [20]. Although OA affects joints of the knee, hip, hand, and spine, knee is the most affected joint [21]. As a result of pain and reduced mobility, OA leads to significant loss of quality of life. Since OA is generally considered to be a result of mechanical "wear and tear" of joints, it typically affects people over the age of 60. However, its onset can be expedited at younger age due to other factors including obesity, genetic factors, and joint injury [22, 23]. Approximately 10% of the world's adult population over the age of 60 has been affected by OA [24]. Therefore, the economic burden of this disease, which includes healthcare costs and loss of productivity, is significant. These expenditures are likely to escalate with aging population. At present approximately 27 million people in the US suffer from OA and it has been estimated that by the year 2030 25% of the US adult population (a third of which of working age) will be affected by OA [25].

Although OA manifests a broad clinical syndrome, its primary cause has been attributed to the progressive breakdown of articular cartilage and chondrocytes within the synovial joints. This degeneration leads to narrowing of the joint space, suchondral sclerosis, and synovial inflammation. Breakdown of the cartilage results in alternation in joint mechanics, which further exacerbates the disease [26, 27]. In OA, concentrations of a number of mediators of inflammation such as cytokines, chemokines, and proteolytic enzymes like matrix metalloproteinases (MMPs) as well as free radicals are elevated in the synovial fluid that catalyze further degradation of cartilage [28, 29]. This process results in a self-sustaining degenerative circle that hinders the natural process of cartilage repair.

In spite of years of intensive research in tissue engineering, there has been no breakthrough to regenerate physiologically viable articular cartilage [30]. Also, no therapeutic agent has been developed that demonstrates structure modifying efficacy in OA patients [31]. The current therapies for OA are largely symptomatic in alleviating the chronic pain. These agents largely include anti-inflammatory agents, NSAIDs, and opioid family of analgesics. The relative efficacies of these therapies to relieve OA associated chronic pain have been modest at best [32, 33]. As mentioned earlier, long term use of these pharmacological agents results in major side effects (see above). In order to minimize systemic side effects associated with oral NSAIDs, topical agents containing the active agents have been developed. These delivery systems are expected to deliver the drugs in high concentrations locally and would reduce systemic side effects [34]. However, efficacy of these topical therapies is modest. In recent years, other novel therapeutic approaches for the management of OA pain has been pursued that include antibodies targeting NGF and antagonist of **T**ransient **R**eceptor **P**otential **V**anilloid (TRPV) family of ion channels [35, 36]. One of the attractive therapeutic options for treating OA associated pain are polymer based viscosupplements. The following section describes the state of viscosupplement based treatments for OA pain.

#### **3. Hyaluronic acid derived viscosupplements**

#### **3.1 Hyaluronic acid and its biology**

Hyaluronic acid or hyaluronan (HA) is a polysaccharide that belongs to the glycosaminoglycan class of biological macromolecules. This highly viscous anionic biopolymer is composed of -1, 3-D-glucuronic acid and -1, 4-N-acetyl-D-glucosamine

Osteoarthritis (OA) is one of the most prevalent musculo-skeletal degenerative diseases [20]. Although OA affects joints of the knee, hip, hand, and spine, knee is the most affected joint [21]. As a result of pain and reduced mobility, OA leads to significant loss of quality of life. Since OA is generally considered to be a result of mechanical "wear and tear" of joints, it typically affects people over the age of 60. However, its onset can be expedited at younger age due to other factors including obesity, genetic factors, and joint injury [22, 23]. Approximately 10% of the world's adult population over the age of 60 has been affected by OA [24]. Therefore, the economic burden of this disease, which includes healthcare costs and loss of productivity, is significant. These expenditures are likely to escalate with aging population. At present approximately 27 million people in the US suffer from OA and it has been estimated that by the year 2030 25% of the US adult population (a third of which of

Although OA manifests a broad clinical syndrome, its primary cause has been attributed to the progressive breakdown of articular cartilage and chondrocytes within the synovial joints. This degeneration leads to narrowing of the joint space, suchondral sclerosis, and synovial inflammation. Breakdown of the cartilage results in alternation in joint mechanics, which further exacerbates the disease [26, 27]. In OA, concentrations of a number of mediators of inflammation such as cytokines, chemokines, and proteolytic enzymes like matrix metalloproteinases (MMPs) as well as free radicals are elevated in the synovial fluid that catalyze further degradation of cartilage [28, 29]. This process results in a self-sustaining

In spite of years of intensive research in tissue engineering, there has been no breakthrough to regenerate physiologically viable articular cartilage [30]. Also, no therapeutic agent has been developed that demonstrates structure modifying efficacy in OA patients [31]. The current therapies for OA are largely symptomatic in alleviating the chronic pain. These agents largely include anti-inflammatory agents, NSAIDs, and opioid family of analgesics. The relative efficacies of these therapies to relieve OA associated chronic pain have been modest at best [32, 33]. As mentioned earlier, long term use of these pharmacological agents results in major side effects (see above). In order to minimize systemic side effects associated with oral NSAIDs, topical agents containing the active agents have been developed. These delivery systems are expected to deliver the drugs in high concentrations locally and would reduce systemic side effects [34]. However, efficacy of these topical therapies is modest. In recent years, other novel therapeutic approaches for the management of OA pain has been pursued that include antibodies targeting NGF and antagonist of **T**ransient **R**eceptor **P**otential **V**anilloid (TRPV) family of ion channels [35, 36]. One of the attractive therapeutic options for treating OA associated pain are polymer based viscosupplements. The following

degenerative circle that hinders the natural process of cartilage repair.

section describes the state of viscosupplement based treatments for OA pain.

Hyaluronic acid or hyaluronan (HA) is a polysaccharide that belongs to the glycosaminoglycan class of biological macromolecules. This highly viscous anionic



**3. Hyaluronic acid derived viscosupplements** 

**3.1 Hyaluronic acid and its biology** 

biopolymer is composed of

**2. Osteoarthritis pain** 

working age) will be affected by OA [25].

arranged in an alternate fashion along the polymer backbone (Fig. 1). HA is ubiquitous in nature and is produced by every tissue of higher organisms and some bacteria. The biopolymer is found in the extracellular matrices (particularly in soft connective tissues), synovial fluid, and cartilage. HA is endogenously synthesized by chondrocytes and synoviocytes [37-39]. After being released into the synovial space, HA accumulates on the surfaces of cartilage and ligament. Endogenously synthesized HA is generally of very high molecular weight (in the range of 3 -5 million Dalton) and its fully hydrated form assumes a globular shape [40]. Unique viscoelastic properties of HA enables it to maintain rheological homeostasis of the synovial fluid in the joints and plays a critical role in providing lubrication, elasticity, and shock absorption to joint tissues. Furthermore, by providing a coat on the surface of articular cartilage, HA protects the cartilage and blocks the loss of proteoglycan from the cartilage matrix into the synovial space [41]. In healthy joint of human knee, the normal concentration of HA in the synovial fluid is in the range of 2.5 - 4.0 mg/mL. However, under pathological conditions such as osteoarthritis, the concentration of HA is significantly reduced (estimated to be ~ 1 - 2 mg/mL) [42]. Furthermore, the biopolymer undergoes degradation under diseased conditions with substantial reduction in molecular weight. A combination of lowering in concentration and molecular weights leads to lowering in viscosity and elasticity of synovial fluid and consequently adverse impact on joint function. Thus, catabolic degradation of HA directly correlates with the onset OA.

Fig. 1. Chemical structure of hyaluronic acid.

#### **3.2 HA derivatives for the treatment of osteoarthritis**

In addition to acting as a lubricant for the joint, HA has been reported to impart antiinflammatory, anabolic, and chondroprotective effects [43, 44]. Since OA onset is attributed to degradation of high molecular weight HA and its concentration in the synovial fluid, increase in HA concentration either by increasing the rate of the proteoglycan biosynthesis or by incorporating HA exogenously to the joint space would improve joint function and relieve OA associated chronic pain. Therefore, the effect of intraarticular administration of exogenous HA to restore rheological properties of the synovial fluid have been extensively studied [45].

In order to maintain desired viscoelastic property of synovial fluid, the exogenous HA needs to have high molecular weight. It has been observed that the frequency and the amount of exogenous HA injected can be lowered by increasing the molecular weight of HA based exogenous viscosupplement. Towards that end, a variety of synthetic approaches have been undertaken to engineer high molecular weight HA derivatives [46]. In general, the desired rheological properties of HA based viscosupplements are achieved by crosslinking of naturally occurring linear HA to produce higher molecular weight compounds.

Polymer Based Therapies for the Treatment of Chronic Pain 31

The precursor HA for these preparations are obtained either from avian sources or by biofermentation in bacteria. Table 1 summarizes some important features of representative HA based viscosupplements that are marketed for intraarticular injection in the knee to relieve OA pain [52]. Particularly, these products differ by their molecular weights, which influence their rheological properties and hence residence time in the

type

hyaluronate Avian N/A 600 – 1,200

hyaluronate Biofermentation N/A 2,400 – 3,600

hyaluronate Avian N/A 500 - 730

hyaluronate Biofermentation N/A 800 – 1, 200

hyaluronan Biofermentation Chemical

One of the most effective viscosupplement that has been approved for clinical use is Synvisc (Hylan G-F 20) and its single injection formulation, Synvisc-One [53]. The main components of Synvisc are HA lightly crosslinked with formaldehyde (Hylan A) and divinyl sulfone crosslinked hylan A (Hylan B). Synvisc contains 90% (v/v) of Hylan A and 10% (v/v) of hylan B and its chemical structure is shown in Figure 3. Synvisc has been approved for the treatment of pain associated with mild to moderate OA. In subsequent clinical studies it has been observed that intraarticular injection of Synvisc resulted in significant pain relief in the carpometacapal joint, temporomandibular joint and the hip [54]. These findings suggest that pain relief from the intraarticular injections of HA-derived viscosupplements is not limited to knee. Since, OA of the hip is the second most common form of arthritis after OA of the knee, additional clinical investigation of the role of viscosupplements in relieving chronic pain arising from hip arthritis is

The biological mechanisms underlying the pharmacological action of HA derived viscosupplements to relieve OA pain are not completely understood. It was initially thought that since there is a reduced level of HA in OA joints, intraarticular injection of exogenous HA restores the rheological properties of synovial fluid to the level present in healthy joints. However, while the half-life of exogenous HA in the synovial fluid is only

Synvisc Hylan G-F 20 Avian Cross-linked 6,000

Table 1. Representative examples of clinically approved hyaluronic acid (HA) based

Molecular weight (kDa)

Modification 1,100 – 2,900

Brand name Generic name HA source Modification

joint.

Artz/Supartz Sodium

Euflexxa Sodium

Hyalgan Sodium

Intragel Sodium

warranted.

Orthovisc High mol. Wt.

viscosupplementation products (reference 45).

Functional group richness of HA has rendered it to be an important precursor material for the design and synthesis of numerous biomaterials with tuned physicochemical and biological properties that have found broad applications in biomedicine and biosurgery [47, 48]. HA offers three kinds of functional groups that can be used for chemical modification: carboxylic acid, primary and secondary hydroxyl, and N-acetyl (after removal of acetyl group to generate primary amine). While carboxyl groups can be modified to introduce amide and ester bonds, the hydroxyl groups can be subjected to reaction with various electrophiles such as epoxides, alkyl halides, alkyl tosylates, vinyl sulfones, etc. However, since HA is unstable at low pH, the chemical reactions employed for its modification must be selected very carefully so that they are mild and compatible to HA. This is necessary to avoid undesired degradation of HA to lower molecular weight. Furthermore, the byproducts of these reactions must be benign for both short- and long-term uses. Over the years, a great deal of research efforts have been put forth to synthesize chemically modified HA [49].

In order to synthesize HA derived viscosupplements, linear HA has been subjected to crosslinking reactions with a number of bifunctional reagents such as diepoxides, divinyl sulfone, epichlorohydrin etc [50, 51]. Some representative examples of crosslinking chemistries that were carried out to prepare HA based hydrogels are shown in Figure 2.

Fig. 2. Representative examples of crosslinking chemistries used to prepare HA hydrogels.

Several factors need to be taken into consideration while optimizing HA derived viscosupplementation products. For example, the rheological properties need to be tuned so that they match with those of native synovial fluid. The hydrogels must be free from any reagents that could trigger an inflammatory response associated with an exogenous material. The molecular weight of the hydrogel is critical to obtain desired clinical benefits since HA is prone to degradation and this process is accelerated in a diseased joint. A variety of HA derived crosslinked viscosupplements have been approved for human use.

Functional group richness of HA has rendered it to be an important precursor material for the design and synthesis of numerous biomaterials with tuned physicochemical and biological properties that have found broad applications in biomedicine and biosurgery [47, 48]. HA offers three kinds of functional groups that can be used for chemical modification: carboxylic acid, primary and secondary hydroxyl, and N-acetyl (after removal of acetyl group to generate primary amine). While carboxyl groups can be modified to introduce amide and ester bonds, the hydroxyl groups can be subjected to reaction with various electrophiles such as epoxides, alkyl halides, alkyl tosylates, vinyl sulfones, etc. However, since HA is unstable at low pH, the chemical reactions employed for its modification must be selected very carefully so that they are mild and compatible to HA. This is necessary to avoid undesired degradation of HA to lower molecular weight. Furthermore, the byproducts of these reactions must be benign for both short- and long-term uses. Over the years, a great deal of research efforts have been put forth to synthesize chemically modified

In order to synthesize HA derived viscosupplements, linear HA has been subjected to crosslinking reactions with a number of bifunctional reagents such as diepoxides, divinyl sulfone, epichlorohydrin etc [50, 51]. Some representative examples of crosslinking chemistries that were carried out to prepare HA based hydrogels are shown in Figure 2.

Fig. 2. Representative examples of crosslinking chemistries used to prepare HA hydrogels. Several factors need to be taken into consideration while optimizing HA derived viscosupplementation products. For example, the rheological properties need to be tuned so that they match with those of native synovial fluid. The hydrogels must be free from any reagents that could trigger an inflammatory response associated with an exogenous material. The molecular weight of the hydrogel is critical to obtain desired clinical benefits since HA is prone to degradation and this process is accelerated in a diseased joint. A variety of HA derived crosslinked viscosupplements have been approved for human use.

HA [49].

The precursor HA for these preparations are obtained either from avian sources or by biofermentation in bacteria. Table 1 summarizes some important features of representative HA based viscosupplements that are marketed for intraarticular injection in the knee to relieve OA pain [52]. Particularly, these products differ by their molecular weights, which influence their rheological properties and hence residence time in the joint.


Table 1. Representative examples of clinically approved hyaluronic acid (HA) based viscosupplementation products (reference 45).

One of the most effective viscosupplement that has been approved for clinical use is Synvisc (Hylan G-F 20) and its single injection formulation, Synvisc-One [53]. The main components of Synvisc are HA lightly crosslinked with formaldehyde (Hylan A) and divinyl sulfone crosslinked hylan A (Hylan B). Synvisc contains 90% (v/v) of Hylan A and 10% (v/v) of hylan B and its chemical structure is shown in Figure 3. Synvisc has been approved for the treatment of pain associated with mild to moderate OA. In subsequent clinical studies it has been observed that intraarticular injection of Synvisc resulted in significant pain relief in the carpometacapal joint, temporomandibular joint and the hip [54]. These findings suggest that pain relief from the intraarticular injections of HA-derived viscosupplements is not limited to knee. Since, OA of the hip is the second most common form of arthritis after OA of the knee, additional clinical investigation of the role of viscosupplements in relieving chronic pain arising from hip arthritis is warranted.

The biological mechanisms underlying the pharmacological action of HA derived viscosupplements to relieve OA pain are not completely understood. It was initially thought that since there is a reduced level of HA in OA joints, intraarticular injection of exogenous HA restores the rheological properties of synovial fluid to the level present in healthy joints. However, while the half-life of exogenous HA in the synovial fluid is only

Polymer Based Therapies for the Treatment of Chronic Pain 33

repeated injection of these catabolic agents can have adverse effect [59]. In order to achieve fast and longer lasting pain relief while minimizing the side effects of steroids, combination therapy of HA and corticosteroids have been envisioned. Non-covalently bound admixtures of HA gel with steroids, where the steroid is dispersed within the HA hydrogel matrix have been investigated as combination therapy to treat OA pain. This approach allows sustained local delivery of the steroid at OA site and would overcome the side effects associated with steroid overdose. Figure 4 shows the structures of representative corticosteroids that have used to prepare HA derived drug-

Fig. 4. Corticosteroids used to prepare HA-steroid composite hydrogel

Preparation stable formulation of crosslinked HA hydrogel, Synvisc with triamcinolone hexaacetonide (TAH) (Figure 4, **1**) was investigated by dispersing Tween-80 stabilized TAH colloidal suspension within a swollen gel of Syvisc [60]. By optimizing the ratio of Synvisc to TAH in the formulation mixture, a stable composite was obtained. The rheological properties of Synvisc were not adversely affected by the presence of the hydrophobic corticosteroid and the composition was found to be stable in an accelerated

Another steroid-viscosupplement composite was prepared by crosslinking linear HA in the presence of triamcinolone acetonide (Figure 4, **2**). In this study, divinyl sulfone was allowed to react partially with HA to generate a linear HA structure with pendant vinyl sulfone group. To a solution of this vinyl sulfone functionalized HA was added a suspension of **2** and resulting reaction mixture was treated with ,-dithio polyethylene glycol (PEG) as the crosslinking agent. A crosslinked HA gel with relatively homogeneously distributed steroid particles within the gel matrix was obtained. The synthetic strategy adopted for the preparation of this dual-acting viscosupplement is shown in Figure 5 [61]. In a preliminary clinical study, this steroid-HA composite (Hydros-TA) showed faster pain relief compared to the corresponding native viscosupplement alone. Long term clinical study involving larger patient population needs to be carried out to demonstrate the clinical efficacy of such steroid-viscosupplement composites to treat OA associated chronic

viscosupplement composites.

viscosupplements.

shelf life test.

pain.

few days, its clinical effect in reducing OA pain has been found to be maintained for several months [55]. This indicates that mechanism of action of HA derived viscosupplements is of multifactorial nature and is a combination of physical and biological effects. A number of *in vitro* studies have been carried out to investigate the biological activities of HA [56]. The results of these studies suggest that HA exhibits chondroprotective and anti-inflammatory effects in the synoviocytes by preventing invasion of inflammatory cells to the joint space. Biological activity of HA has also been attributed to down regulation of the gene expression of various inflammatory cytokines and catabolic enzymes like aggrecanase. Furthermore, being a natural ligand of the cell surface receptor CD44, HA has been thought to impart its effect by modulating CD44 mediated metabolism. In another *in vitro* study, when synovial fibroblasts were cultured with high molecular weight HA, newly synthesized HA molecules were found. These biological effects of exogenous HA may result in overall cartilage protection [57, 58]. Thus, well controlled clinical studies would shed further light on the chondro-protection properties of exogenous HA like Synvisc and lead to the discovery of novel therapies with disease modifying properties.

Fig. 3. Structure of Hylan B component of Synvisc.

#### **3.3 HA-steroid combinations for the treatment of chronic pain**

One of the shortcomings of HA derived viscosupplements is their slower onset of action to reduce OA pain relative to low molecular weight drugs such as NSAIDs and steroids. Therefore, the viscosupplements are generally administered weekly over a course of three to five weeks. As described earlier, unlike traditional pain killers, pain relief from viscosupplements lasts much longer (up to several months). Although intraarticular injection of corticosteroids achieves maximum benefit within few days of injection,

few days, its clinical effect in reducing OA pain has been found to be maintained for several months [55]. This indicates that mechanism of action of HA derived viscosupplements is of multifactorial nature and is a combination of physical and biological effects. A number of *in vitro* studies have been carried out to investigate the biological activities of HA [56]. The results of these studies suggest that HA exhibits chondroprotective and anti-inflammatory effects in the synoviocytes by preventing invasion of inflammatory cells to the joint space. Biological activity of HA has also been attributed to down regulation of the gene expression of various inflammatory cytokines and catabolic enzymes like aggrecanase. Furthermore, being a natural ligand of the cell surface receptor CD44, HA has been thought to impart its effect by modulating CD44 mediated metabolism. In another *in vitro* study, when synovial fibroblasts were cultured with high molecular weight HA, newly synthesized HA molecules were found. These biological effects of exogenous HA may result in overall cartilage protection [57, 58]. Thus, well controlled clinical studies would shed further light on the chondro-protection properties of exogenous HA like Synvisc and lead to the discovery of novel therapies with

disease modifying properties.

Fig. 3. Structure of Hylan B component of Synvisc.

**3.3 HA-steroid combinations for the treatment of chronic pain** 

One of the shortcomings of HA derived viscosupplements is their slower onset of action to reduce OA pain relative to low molecular weight drugs such as NSAIDs and steroids. Therefore, the viscosupplements are generally administered weekly over a course of three to five weeks. As described earlier, unlike traditional pain killers, pain relief from viscosupplements lasts much longer (up to several months). Although intraarticular injection of corticosteroids achieves maximum benefit within few days of injection, repeated injection of these catabolic agents can have adverse effect [59]. In order to achieve fast and longer lasting pain relief while minimizing the side effects of steroids, combination therapy of HA and corticosteroids have been envisioned. Non-covalently bound admixtures of HA gel with steroids, where the steroid is dispersed within the HA hydrogel matrix have been investigated as combination therapy to treat OA pain. This approach allows sustained local delivery of the steroid at OA site and would overcome the side effects associated with steroid overdose. Figure 4 shows the structures of representative corticosteroids that have used to prepare HA derived drugviscosupplement composites.

Fig. 4. Corticosteroids used to prepare HA-steroid composite hydrogel viscosupplements.

Preparation stable formulation of crosslinked HA hydrogel, Synvisc with triamcinolone hexaacetonide (TAH) (Figure 4, **1**) was investigated by dispersing Tween-80 stabilized TAH colloidal suspension within a swollen gel of Syvisc [60]. By optimizing the ratio of Synvisc to TAH in the formulation mixture, a stable composite was obtained. The rheological properties of Synvisc were not adversely affected by the presence of the hydrophobic corticosteroid and the composition was found to be stable in an accelerated shelf life test.

Another steroid-viscosupplement composite was prepared by crosslinking linear HA in the presence of triamcinolone acetonide (Figure 4, **2**). In this study, divinyl sulfone was allowed to react partially with HA to generate a linear HA structure with pendant vinyl sulfone group. To a solution of this vinyl sulfone functionalized HA was added a suspension of **2** and resulting reaction mixture was treated with ,-dithio polyethylene glycol (PEG) as the crosslinking agent. A crosslinked HA gel with relatively homogeneously distributed steroid particles within the gel matrix was obtained. The synthetic strategy adopted for the preparation of this dual-acting viscosupplement is shown in Figure 5 [61]. In a preliminary clinical study, this steroid-HA composite (Hydros-TA) showed faster pain relief compared to the corresponding native viscosupplement alone. Long term clinical study involving larger patient population needs to be carried out to demonstrate the clinical efficacy of such steroid-viscosupplement composites to treat OA associated chronic pain.

Polymer Based Therapies for the Treatment of Chronic Pain 35

matrix. Bupivacaine was conjugated to HA through a hydrolysable imide bond (Figure 6A). On the other hand, opioid drugs such as morphine, naloxone analogs were conjugated via a hydrolysable ester bonds (Figure 6B). A number of conjugates were synthesized by varying the nature of the linker arm, spacer length, and the amount of the drug loading. A systematic evaluation of the release kinetics of the drugs from the HA gel was carried out under *in vitro* conditions to identify an optimum composition. The optimum drug-HA conjugate from each class was evaluated *in vivo* for its biological activity. These drug-conjugated HA hydrogels exhibited therapeutic benefits by prolonging pain relief and were more effective than the individual agents and their admixtures. These preclinical research findings suggest that development of HA based viscosupplements conjugated with traditional pain relieving agents might lead to a promising new generation of long acting therapies for the treatment of OA associated

In related work, conjugates of HA with methotrexate (MTX) were synthesized to achieve viscosupplementation and anti-inflammatory effect concurrently intraarticularly [66]. Increased levels of TNF- have been found in the synovium of OA affected joints that can be mitigated by oral administration of MTX [67]. However, systemic administration of MTX is associated with certain side effects such as pneumonitis and myelosuppression [68]. Therefore, by localizing MTX to target joint by delivering it as a polymer conjugate, the systemic side effect could be minimized. After a careful structure-activity study by screening various linker arms and enzyme target groups, an optimized HA conjugate of MTX was identified (Figure 7). A peptiditic linker was chosen as target for cathepsin enzymes, which are over-expressed in OA joints. The polyethyleneglycol (PEG) linker was chosen to enable the peptide target to be accessible to the cathepsin enzyme in the joint environment. *In vitro* and *in vivo* studies revealed that the HA-MTX conjugate is capable of reducing joint pain and swelling of the knee. On the other hand, admixture of HA and MTX

chronic pain.

Fig. 6. HA conjugated local analgesics (A) and opioids.

showed marginal efficacy.

Fig. 5. Crosslinking of HA in the presence of a dispersion of triamcinolone acetonide to prepare viscosupplment-steroid composite hydrogel

#### **3.4 Covalent conjugates of HA and low molecular pain killers**

Intrinsic biocompatibility and its versatility for chemical modification make HA an attractive biomaterial to synthesize conjugated drug delivery systems. Chemical modification of HA has allowed the preparation of an array HA-drug conjugates and HA-protein conjugates as sustained-release carriers for drugs and biotherapeutics [62, 63]. Covalent conjugates of HA containing hydrogels with pain relieving agents have been explored as dual acting agents to treat chronic pain. This approach would offer a number of potential clinical benefits, that include: i) retaining viscosupplementation property of soluble HA, ii) minimizing the systemic exposure of NSAIDs and opioid family pain killers by localizing administration to the target site, iii) modulating the duration of action of these pain killers by incorporating appropriate conjugation chemistry to control the rate of cleavage of the drug from the HA gel, and iv) minimizing the frequency of administration of viscosupplement and the pain killer in the clinic. These features of the HA-drug conjugates could lead to better patient compliance and improved quality of life.

A series of HA derived functional hydrogels conjugated with local analgesics (e.g. bupivacaine) and opioid drugs (e.g. morphine) were synthesized in our laboratories as long acting treatments for chronic pain [64, 65]. Divinyl sulfone crosslinked HA hydrogel (Hylan B) was used as the polymer matrix for the synthesis of these drug conjugates. Appropriate linker arms were designed to tether these pain relieving agents to the HA

Fig. 5. Crosslinking of HA in the presence of a dispersion of triamcinolone acetonide to

Intrinsic biocompatibility and its versatility for chemical modification make HA an attractive biomaterial to synthesize conjugated drug delivery systems. Chemical modification of HA has allowed the preparation of an array HA-drug conjugates and HA-protein conjugates as sustained-release carriers for drugs and biotherapeutics [62, 63]. Covalent conjugates of HA containing hydrogels with pain relieving agents have been explored as dual acting agents to treat chronic pain. This approach would offer a number of potential clinical benefits, that include: i) retaining viscosupplementation property of soluble HA, ii) minimizing the systemic exposure of NSAIDs and opioid family pain killers by localizing administration to the target site, iii) modulating the duration of action of these pain killers by incorporating appropriate conjugation chemistry to control the rate of cleavage of the drug from the HA gel, and iv) minimizing the frequency of administration of viscosupplement and the pain killer in the clinic. These features of the HA-drug conjugates could lead to better patient compliance and improved

A series of HA derived functional hydrogels conjugated with local analgesics (e.g. bupivacaine) and opioid drugs (e.g. morphine) were synthesized in our laboratories as long acting treatments for chronic pain [64, 65]. Divinyl sulfone crosslinked HA hydrogel (Hylan B) was used as the polymer matrix for the synthesis of these drug conjugates. Appropriate linker arms were designed to tether these pain relieving agents to the HA

prepare viscosupplment-steroid composite hydrogel

quality of life.

**3.4 Covalent conjugates of HA and low molecular pain killers** 

matrix. Bupivacaine was conjugated to HA through a hydrolysable imide bond (Figure 6A). On the other hand, opioid drugs such as morphine, naloxone analogs were conjugated via a hydrolysable ester bonds (Figure 6B). A number of conjugates were synthesized by varying the nature of the linker arm, spacer length, and the amount of the drug loading. A systematic evaluation of the release kinetics of the drugs from the HA gel was carried out under *in vitro* conditions to identify an optimum composition. The optimum drug-HA conjugate from each class was evaluated *in vivo* for its biological activity. These drug-conjugated HA hydrogels exhibited therapeutic benefits by prolonging pain relief and were more effective than the individual agents and their admixtures. These preclinical research findings suggest that development of HA based viscosupplements conjugated with traditional pain relieving agents might lead to a promising new generation of long acting therapies for the treatment of OA associated chronic pain.

Fig. 6. HA conjugated local analgesics (A) and opioids.

In related work, conjugates of HA with methotrexate (MTX) were synthesized to achieve viscosupplementation and anti-inflammatory effect concurrently intraarticularly [66]. Increased levels of TNF- have been found in the synovium of OA affected joints that can be mitigated by oral administration of MTX [67]. However, systemic administration of MTX is associated with certain side effects such as pneumonitis and myelosuppression [68]. Therefore, by localizing MTX to target joint by delivering it as a polymer conjugate, the systemic side effect could be minimized. After a careful structure-activity study by screening various linker arms and enzyme target groups, an optimized HA conjugate of MTX was identified (Figure 7). A peptiditic linker was chosen as target for cathepsin enzymes, which are over-expressed in OA joints. The polyethyleneglycol (PEG) linker was chosen to enable the peptide target to be accessible to the cathepsin enzyme in the joint environment. *In vitro* and *in vivo* studies revealed that the HA-MTX conjugate is capable of reducing joint pain and swelling of the knee. On the other hand, admixture of HA and MTX showed marginal efficacy.

Polymer Based Therapies for the Treatment of Chronic Pain 37

Fig. 8. Polymer modified naloxol derivatives to prevent opioid induced constipation.

agents offer a new perspective to develop novel treatments for chronic pain.

Fig. 9. Structure of salicylic acid-based poly(anhydride-esters) as polymeric anti-

Fig. 10. Structure of morphine-based poly(ester-anhydride).

inflammatory agents.

Another interesting approach to develop polymeric pain relievers has been reported that utilizes a polymerization chemistry to synthesize poly(anhydride-esters), where the bioactive drug becomes part of the polymer backbone [74]. The general structure of this class of polymeric pain relievers is shown in Figure 9. Following this strategy, they were able to incorporate significant amounts (~ 62%) of the deliverable drug to the polymer chain. Hydrolytic degradation of these poly(anhydride-ester) polymers under physiological pH conditions releases the drug in a controlled manner. As a result, the side effects associated with the native drug (if released immediately) can be minimized. Some of the polymeric pain relievers reported are poly(anhydride-esters) containing the anti-inflammatory agent, salicylic acid and the opioid drug, morphine (Figure 10). Although syntheses of polymeric pain relievers based on these poly(anhydride-ester) scaffolds have been studied extensively, there is limited information about the biological activities of these polymers as treatments for chronic pain [75, 76]. Nevertheless, these polymeric opioids and anti-inflammatory

Fig. 7. HA conjugate of methotrexate as an intraarticular combination therapy for the treatment of OA pain.

#### **4. Polymer-opioid conjugates and polymeric opioid derivatives**

Besides being a first-line analgesic therapy for acute pain, opioids have been found to be useful in treating chronic pain. However, the adverse effects associated with their long term use limit the therapeutic benefits of opioid analgesics, thus leading to discontinuation of the therapy. Constipation (opioid-induced bowel dysfunction (OBD)) is one of the significant side effects associated with opioid therapies. OBD affects up to 80% of the patients undergoing opioid therapy. While other side effects associated with chronic use of opioids resolve with time, constipation continues to persist [69].

Efforts have been made to utilize polymeric approach to design and develop new generation of opioid analogs as pain killers. These polymeric compounds enable the patients to overcome OBD without losing the benefits of opioid therapy by limiting drugs' systemic absorption. Pegylation chemistry was utilized to synthesize these macromolecular opioids. The technology of pegylation has been successfully utilized to improve pharmacokinetic properties of a number of (bio)pharmaceutical agents [70]. Two representative polymer conjugated opioid derivatives are shown in Figure 8. These compounds consist of naloxol analogs linked to PEG chains through hydrolytically stable ether linkage [71, 72]. In preclinical studies, these pegylated opioid derivatives were found to maintain their centrally mediated analgesia, while antagonizing peripherally mediated constipation. One of the key conjugates, NKTR-118 (Figure 8A, n = 7) has proceeded to advanced clinical trial. In the phase II clinical trial, patients receiving NKTR-118 exhibited significant increase in bowel movement compared to patients receiving native naloxol, without compromising the analgesic property of the opioid [73]. NKTR-118 is currently undergoing phase III clinical trial.

O

NHAc

O

O HO

m

O

H

Asn Phe Phe N

Peptide

Linker

O

HO

O

OH

OH

O

O

O

NHAc

n

O

O

resolve with time, constipation continues to persist [69].

N

N

N

NH2

treatment of OA pain.

trial.

N

H2N

N

HO

O

**4. Polymer-opioid conjugates and polymeric opioid derivatives** 

HO

OH

OH

Fig. 7. HA conjugate of methotrexate as an intraarticular combination therapy for the

Besides being a first-line analgesic therapy for acute pain, opioids have been found to be useful in treating chronic pain. However, the adverse effects associated with their long term use limit the therapeutic benefits of opioid analgesics, thus leading to discontinuation of the therapy. Constipation (opioid-induced bowel dysfunction (OBD)) is one of the significant side effects associated with opioid therapies. OBD affects up to 80% of the patients undergoing opioid therapy. While other side effects associated with chronic use of opioids

Efforts have been made to utilize polymeric approach to design and develop new generation of opioid analogs as pain killers. These polymeric compounds enable the patients to overcome OBD without losing the benefits of opioid therapy by limiting drugs' systemic absorption. Pegylation chemistry was utilized to synthesize these macromolecular opioids. The technology of pegylation has been successfully utilized to improve pharmacokinetic properties of a number of (bio)pharmaceutical agents [70]. Two representative polymer conjugated opioid derivatives are shown in Figure 8. These compounds consist of naloxol analogs linked to PEG chains through hydrolytically stable ether linkage [71, 72]. In preclinical studies, these pegylated opioid derivatives were found to maintain their centrally mediated analgesia, while antagonizing peripherally mediated constipation. One of the key conjugates, NKTR-118 (Figure 8A, n = 7) has proceeded to advanced clinical trial. In the phase II clinical trial, patients receiving NKTR-118 exhibited significant increase in bowel movement compared to patients receiving native naloxol, without compromising the analgesic property of the opioid [73]. NKTR-118 is currently undergoing phase III clinical

O

HO O HN O

HO O

H N

Fig. 8. Polymer modified naloxol derivatives to prevent opioid induced constipation.

Another interesting approach to develop polymeric pain relievers has been reported that utilizes a polymerization chemistry to synthesize poly(anhydride-esters), where the bioactive drug becomes part of the polymer backbone [74]. The general structure of this class of polymeric pain relievers is shown in Figure 9. Following this strategy, they were able to incorporate significant amounts (~ 62%) of the deliverable drug to the polymer chain. Hydrolytic degradation of these poly(anhydride-ester) polymers under physiological pH conditions releases the drug in a controlled manner. As a result, the side effects associated with the native drug (if released immediately) can be minimized. Some of the polymeric pain relievers reported are poly(anhydride-esters) containing the anti-inflammatory agent, salicylic acid and the opioid drug, morphine (Figure 10). Although syntheses of polymeric pain relievers based on these poly(anhydride-ester) scaffolds have been studied extensively, there is limited information about the biological activities of these polymers as treatments for chronic pain [75, 76]. Nevertheless, these polymeric opioids and anti-inflammatory agents offer a new perspective to develop novel treatments for chronic pain.

Fig. 9. Structure of salicylic acid-based poly(anhydride-esters) as polymeric antiinflammatory agents.

Fig. 10. Structure of morphine-based poly(ester-anhydride).

Polymer Based Therapies for the Treatment of Chronic Pain 39

[10] Merza, Z. (2010). Chronic Use of Opioids and Endocrine System. *Hormone Metabol. Res.*,

[11] Weiniger, C. F.; Golovanevski, M.; Sokolsky-Papkov, M. & Domb, A. J. (2010). Review

[12] Haroutiunian, S.; Drennan,D. A.; Lipman, A. G. (2010). Topical NSAID Therapy for

[13] Cattaneo, A. (2010). Tanezumab, a Recombinant Humanized mAb Against Nerve

[14] Nair, L.S.& Laurencin, C. T. (2006). Polymers as Biomaterial for Tissue Engineering and Controlled Drug Delivery, *Adv. Biochem. Eng. Biotechnol.*, Vol. 102, No. 1, pp47-90 [15] Dhal, P. K., Huval, C. C. Holmes-Farley, S. R. & Jozefiak, T. J. Polymers as Drugs. *Adv.* 

[16] Duncan, R. (2003). The Dawning Era of Polymer Therapeutics. *Nat. Rev. Drug. Discov*.,

[17] Dhal, P. K. Polomoscanik, S.C.; Avila, L. Z.; Holmes-Farley, S.R. & Miller, R. J. (2009).

[18] Manjanna, K. M.; Shivakumar, B. & Kumar, T. M. P. (2010). Microencapsulation: An

[19] Rainsfold, K. D.; Kean, W. F. & Ehrlich, G. E. (2008). Review of the Pharmaceutical

[20] Felson, D. T. (2004). An Update on the Pathogenesis and Epidemiology of

[21] Kean, W. F. & Buchanan, W. F. (2004). Osteoarthritis: Symptoms, Sign, and Source of

[22] Sharma, L.; Kapoor, D. & Issa, S. (2006). Epidemiology of Osteoarthritis: An Update.

[23] Spector, T. D. & MacGregor, A. J. (2004). Risk Factors for Osteoarthritis: Genetics.

[24] WHO Scientific Group (2003). The Burden of Musculoskeletal Condition at the Start of the New Millennium. *World Health Organ. Tech. Rep. Ser.*, Vol. 919, pp 1-218 [25] Centers for Disease Control and Prevention (2010). Arthritis-related Statistics, August

http://www.cdc.gov/arthritis/data\_statistics/ arthritis\_related\_stats.htm [26] Wieland, H. A.; Michaelis, M.; Kirschbaum, B. J. & Rudolphi, K. A. (2010).

[27] Felson, D. T. & Neogi, T. (2004). Osteoarthritis: Is It a Disease of Cartilage or Bone?

[28] Roach, H. I.; Aigner, T.; Soder, S.; Haag, J.; Welkerling, H. (2007). Pathobiology of

Osteoarthritis: An Untreatable Disease. *Nat. Rev. Drug. Discov.*, Vol. 4, No. 4, pp.

Osteoarthritis: Pathomechanisms and Potential Therapeutic Targets. *Cur. Drug.* 

Epolamine. *Cur. Med. Res. Opin.*, Vol. 24, No.10, pp. 2967-2992

Osteoarthritis. *Radiol. Clin. N. Am.* , Vol. 42, No. 1, pp. 1-9

Pain. *Inflamm. Pharmacol.*, Vol. 12, No. 1, pp. 3-31

*Cur. Opin. Rheumatol.*, Vol. 18, No. 2, pp. 147- 156

*Osteoarthr. Cartil.*, Vol. 12, pp. S39-S44

*Arthrit Rheumatol.*, Vol. 50, pp 341-344

*Targets,* Vol.8, No. 2, pp. 271- 282

2010. Available from:

331-345

Functional Polymers as Therapeutic Agents: Concept to Marketplace. *Adv. Drug* 

Acclaimed Novel Drug-Delivery System for NSAIDs in Arthritis. *Crit. Rev. Therap.* 

Properties and Clinical Effects of the Topical NSAID Formulation, Diclofenac

Musculoskeletal Pain. *Pain. Med.*, Vol. 11, No. 4, pp.535-549

of Prolonged Local Anesthetic Action. *Expert. Opin. Drug. Deliv.*, Vol. 7, No.6,

Growth Factor for the Treatment of Acute and Chronic Pain. *Cur. Opin. Mol. Ther.*,

Vol. 42, No. 9, pp. 621-626.

Vol. 12, No.1, pp.94-106

Vol. 2, No. pp. 347-360

*Polym. Sci*., Vol. 192, No.1, pp 9-58 (2006)

*Deliv. Rev.*, Vol. 61, No.13, pp1121-1130

*Drug Carr. Sys*., Vol. 27, No.6, pp. 509-545

pp.737-752.

#### **5. Conclusion**

Because of its clinical relevance, development of novel pharmacologic agents for effective management of chronic pain continues to be an important goal of pharmaceutical research. Although numerous therapeutic agents with different modes of action have been developed to treat chronic pain, no single agent exhibits the most desired profile. For example, while opioids and NSAIDs remain the main stay of therapeutic options, concerns over their associated side effects have begun to limit their use. Polymeric approach offers a variety of options to develop a new generation of pain relievers, which include intrinsically bioactive polymers to different delivery systems for traditional pain killers. HA derived viscosupplements offer an attracting option to treat chronic pain due to excellent biocompatibility and various biological functions of HA. The ability to trigger various biological functions makes HA based viscosupplements as promising agents to not only relieve symptomatic effects of chronic OA pain, but also to bring about potentially disease modifying effects. Therapies comprising of polymers in combination with traditional pain killers (either as conjugates or as stable non-covalent formulations) have been found to minimize the side effects of the latter. By targeting the disease via different mechanisms of actions, these combination agents could become superior therapeutic options to treat chronic pain. With increasing understanding of the pathobiology of chronic pain and intense research in biomedical polymers, it will be possible to develop novel polymer based therapies in the near future that are safe and could act as structure modifying treatments for chronic pain.

#### **6. References**


Because of its clinical relevance, development of novel pharmacologic agents for effective management of chronic pain continues to be an important goal of pharmaceutical research. Although numerous therapeutic agents with different modes of action have been developed to treat chronic pain, no single agent exhibits the most desired profile. For example, while opioids and NSAIDs remain the main stay of therapeutic options, concerns over their associated side effects have begun to limit their use. Polymeric approach offers a variety of options to develop a new generation of pain relievers, which include intrinsically bioactive polymers to different delivery systems for traditional pain killers. HA derived viscosupplements offer an attracting option to treat chronic pain due to excellent biocompatibility and various biological functions of HA. The ability to trigger various biological functions makes HA based viscosupplements as promising agents to not only relieve symptomatic effects of chronic OA pain, but also to bring about potentially disease modifying effects. Therapies comprising of polymers in combination with traditional pain killers (either as conjugates or as stable non-covalent formulations) have been found to minimize the side effects of the latter. By targeting the disease via different mechanisms of actions, these combination agents could become superior therapeutic options to treat chronic pain. With increasing understanding of the pathobiology of chronic pain and intense research in biomedical polymers, it will be possible to develop novel polymer based therapies in the near future that are safe and could act as structure modifying treatments for

[1] Smith B. H & Torrance N. (2011). Management of Chronic Pain in Primary Care. *Cur.* 

[2] Teets, R.Y.; Dahmer, S. & Scott, E. (2010). Integrative Medicine Approach to Chronic

[3] National Center for Health Statistics (2006), *Health, United States, 2006 with Chartbook on* 

[4] Vadivelu, N.; Mitra, S. & Narayan, D. (2010). Recent Advances in Postoperative Pain

[5] Turk, D. C.; Wilson, H.D. & Cahana, A. (2011). Treatment of Chronic Non-cancer Pain.

[6] Chan, B. K. B.; Tam, L. K.; Wat, C. Y.; Chung, Y. F. ; Tsui, S. L. & Cheung, C. W. (2011).

[7] Scanzello, C. R.; Moskowitz, N. K. & Gibofsky, A. (2010). The Post-NSAID Era: What to

[8] Roth, S. H. (2011). Nonsteroidal Anti-Inflammatory Drug Gastropathy: New Avenues for

[9] Crofford, L. J. (2010). Adverse Effects of Chronic Opioid Therapy for Chronic Musculoskeletal Pain. *Nat. Rev. Rheumatol.* Vol. 6, No. 4, pp. 191-197

*Trends in the Health of Americans*. Hyattsville, MD: Dept. of Health and Human

Opioids in Chronic Non-cancer Pain. *Expert. Opin. Pharmacother.* , Vol. 12, No.5,

Use Now for the Pharmacologic Treatment of Pain and Inflammation in

*Opin. Suppor. Palliat. Care*, Vol. 5, No.2, pp. 137-142

Services, Centers for Disease Control and Prevention.

Management. *Yale J. Biol. Med.* Vol.83, No.1, pp.11-25

Osteoarthritis. *Cur. Rheomatol. Rep.*, Vol. 10, No.1, pp49-56

Safety. *Clin. Interven. Aging.*, Vol. 6, pp 125-131

Pain. *Primary Care*, vol.37, No.2, 407-421.

*Lancet*, Vol. 377, No.9784, pp. 2226-2235

**5. Conclusion** 

chronic pain.

**6. References** 

pp.705-720


http://www.cdc.gov/arthritis/data\_statistics/ arthritis\_related\_stats.htm


Polymer Based Therapies for the Treatment of Chronic Pain 41

Cowman, M. K. & Hales, C. A. (Eds.), Elsevier, Amsterdam, pp. 333- 357 [48] Leonelli, F.; La Bella, A.; Migneco, L. M.& Bettelo, R. M. (2007). Design, Synthesis and

[49] Schante, C. E.; Zuber, G.; Herlin, C. & Vandamme, T. F. (2011). Chemical Modification

[51] Allison, D. D. & Grande-Allen, K. J. (2006). Hyaluronan: A Powerful Tissue

[52] Abate, M.; Pulcini, D.; Di Iorio, A.& Schiavone, C. (2010). Viscosupplementa-tion with

[53] Stitik, T. P.; Kazi, A. & Kim, J.-H. (2008). Synvisc in Knee Osteoarthritis. *Future* 

[54] Migliore, A.; Giovannangeli, F.; Granta, M. & Lagana, B. (2010). Hylan G-F 20: Review

[55] Bagga, H.; Burkhardt, D.; Sambrook, P. & March, L. (2006). Long-term Effects of

[56] Moreland, L. W. (2002). Intra-articular Hyaluronan and Hylans for the Treatment of Osteoarthritis: Mechanisms of Action. *Arthr. Res. Ther.*, Vol. 5, No. 2, pp. 54 – 67 [57] Waddell, D. D. (2007). Viscosupplementation with Hyaluronan for Osteoarthritis of

[58] Altman, R. D. (2010).Non-avian-derived Hyaluronan for the Treatment of Osteoarthritis of the Knee. *Exper. Rev. Clin. Immunol.*, Vol. 6, No.1, pp. 21- 27 [59] Gossec, L.; Dougados, M. (2006). Do Intra-articular Therapies Work and Who Will Benefit Most? *Best Pract. Res. Clin. Rheumatol.*, Vol. 20, No. 1, pp 131 – 144 [60] Chang, G.; Voschin, E.; Yu, L.-P. & Skrabut, E. (2011). Stable Hyaluronan/Steroid Formulation. *United States Patent Application*, No.2011/00559918 A1 [61] Gravett, D. M; Daniloff, G. Y. & He, P. (2010). Modified Hyaluronic Acid Compositions

[62] Varghese, O.P.; Sun, W.; Hilborn, J. & Ossipov, D. A. (2009). In-situ Crosslinkable High

[63] Sun, L. T.; Buchholz, K. S.; Lotze, M. T. & Washburn, N. R. (2010). Cytokine Binding by Polysaccharide-Antibody Conjugates. *Mol. Pharm.*, Vol. 7, No. 5, pp. 1769 – 1777 [64] Gianolio, D. A.; Philbrook, M.; Avila, L. Z.; MacGreggor, H.; Duan, S. X.; Bernasconi,

Molecular Weight Hyaluronan-bisphosphonate Conjugates for Localized Delivery and Cell-specific Targeting: A Hydrogel Linked Prodrug Approach. *J. Am. Chem.* 

R.; Slavsky, M.; Dethlefsen, S.; Jarrett, P. K. & Miller, R. J. (2005). Synthesis and Evaluation of Hydrolyzable Hyaluronan-Tethered Bupivacaine Delivery Systems.

Biomedical Applications. *Carbohy. Polym.*, Vol. 85, No.3, pp.469- 489 [50] Balazs, E. A. (2009). Therapeutic Use of Hyaluronan. *Struct. Chem.*, Vol. Vol.20, No. 2,

Engineering Tool. *Tissue. Eng.*, Vol. 12, No. 8, pp. 2131- 2140

*Med. Insights: Arthr. Musculo. Disord.*, Vol. 3, No. 1, pp 55-68

and Related Methods. *Unites States Patent*, No. 7,829, 118 B1

*Cur. Pharm. Des.*, Vol. 16, No. 6, pp. 331- 340.

*Rheumatol.*, Vol. 3, No. 3, pp. 215- 222

*Rheumatol.*, Vol. 33, No. 5, pp. 946- 950

*Soc*., Vol. 131, No. 25, pp. 8781 – 8783

*Bioconj. Chem.,* Vol. 16, No. 6, pp. 1512 - 1518

360 -378

pp. 341-349

pp. 629- 642

Hyaluronan. In *Carbohydrate Chemistry, Biology and Medical Applications*. Garg, H.G.;

Applications of Hyaluronic acid- Paclitaxel Bioconjugates. *Molecules*, Vol. 13, pp.

of Hyaluronic Acid for the Synthesis of Derivatives for a Broad Range of

Intra-articular Hyaluronic Acid for the Treatment of Osteoarthritis in the Elderly.

of Its Safety and Efficacy in the Management of Joint Pain in Osteoarthritis. *Clin.* 

Intrarticular Hyaluronan on Synovial Fluids in Osteoarthritis of the Knee. *J.* 

Knee: Clinical Efficacy and Economic Implications. *Drug & Aging,* Vol. 24, No. 8,


[29] Burrage, P.S.; Mix, K. S. & Brinckerhiff, C. E. (2006). Matrix Metalloproteinases : Role in

[30] Becerra, J.; Andrades, J. A.; Guerado, E.; Zamora-Navas, P.; Lopez-Puertas, J. M. &

[31] Altman, R. D. (2005). Structure-/Disease-modifying Agents for Osteoarthritis. *Semin.* 

[32] Kroenke, K.; Krebbs, E. E. & Bair, M. J. (2009). Pharmacotherapy of Chronic Pain: A

[33] Manchikanti, L & Singh, A. (2008). Therapeutic Opioids: A Ten-year Perspective on the

[34] Schuelert, N.; Russell, F. A. & McDougall, J. J. (2011). *Ortho. Res. Rev.*, Vol. 3, No. 1, pp.

[35] Seidel, M. F.; Herguijuela, M.; Forkert, R. & Otten, U. (2010). Nerve Growth Factor in Rheumatic Diseases. Semi. *Arthritis. Rheumatol.*, Vol. 40, No.2, pp. 109- 126 [36] Wong, G. Y. & Gavva, N. R. (2009). Therapeutic Potential of Vanilloid Receptor TRPV1

[37] Laurent, T. C. & Fraser, J. R. (1992). Hyaluronan. *FASEB J.*, Vol. 6, No. &, pp. 2397- 2404 [38] Yamada, T. & Kawasaki, T. (2005). Microbial Synthesis of Hyaluronan and Chtin: New

[39] Balazs, E. (1982). The Physical Properties of Synovial Fluid and the Specific Role of

[40] Scott, J. E. & Heatley, F. (1999). Hyaluronan Forms Specific Stable Tertiary Structures in

[41] Shen, B.; Wei, A.; Bhargav, D., Kishen, T. & Diwan, A. D. (2010). Hyaluronan: Its

[42] Watterson, J. R. & Esdaile, J. M. (2000). Viscosupplementation: Therapeutic

[43] Volpi, N.; Schiller, J. & Stern, R. (2009). Role, Metabolism, Chemical Modification, and Applications of Hyaluronan. *Cur. Med. Chem*., Vol. 16, No. 14, pp. 1718- 1745 [44] Bastow, E. R., Byers, S. & Golub, S. B. (2008). Hyaluronan Synthesis and Degradation in

[45] Gigante, A. & Callegari, L. (2011). The Role of Intra-articular Hyaluronan in the

[46] Burdick, J. A. & Prestwich, G. D. (2011). Hyaluronic Hydrogels for Biomedical

[47] Avila, L. Z.; Gianolio, D. A.; Konowicz, P. A.; Philbrook, M.; Santos, M. R. & Miller, R.

J. (2008). Drug Delivery and Medical Applications of Chemically Modified

Cartilage and Bone. *Cell Mol. Life Sci.*, Vol. 65, No. 3, pp. 395- 413

Treatment of Osteoarthritis. *Rheumatol. Int.*, Vol. 31, pp 427 -444

Applications. *Adv. Mater.*, Vol. 23, No.12, pp. H41-H56

Reddi, A. H. (2010). Articular Cartilage: Structure and Regeneration. *Tissue Eng.* 

Synthesis of Recommendation from Systematic *Reviews. Gen. Hosp. Psychiatry*, Vol.

Complexities and Complications of the Escalating Use, Abuse, and Nonmedical

Agonists and Antagonists as Analgesics: Recent Advances and Setbacks. *Brain Res.* 

Hyaluronic Acid. In *Disorders of the Knee*, Helfert A. J. (Ed), J. B. Lippincott,

Aqueous Solution: A 13C NMR Study. *Proc. Natl. Acad. Sci. USA*, Vol. 96, No. 9, pp.

Potential Application in Intervertebral Disc Regeneration. *Ortho. Res. Rev.*, Vol. 2,

Mechanisms and Clinical Potential in Osteoarthritis of the Knee. *J. Am. Acad.* 

Arthritis. *Front. Biosci.*, Vol. 11, No. 1, pp 529-543

*Rheumatol. Arthritis*, Vol. 34, No. 6 (suppl. 2), pp. 3 -5

Use of Opioids. *Pain Physician*, Vol.11, No.2, pp. S63-S88

Approaches. *J. Biosci. Bioeng.*, Vol. 99, No. 6, pp. 521- 528

*Part B*., Vol. 16, No. 6, pp. 617- 627

*Rev.*, Vol. 60, No. 1, pp. 267- 277

*Orthop. Surg.*, Vol. 8, No. 5, pp. 277- 284

Philadelphia, pp. 61-74

4850-4855

No. 1, pp. 17-26

31, 206 -219

1-8

Hyaluronan. In *Carbohydrate Chemistry, Biology and Medical Applications*. Garg, H.G.; Cowman, M. K. & Hales, C. A. (Eds.), Elsevier, Amsterdam, pp. 333- 357


**3** 

*USA* 

**Molecular Aspects of Opioid** 

Austin B. Yongye and Karina Martínez-Mayorga

*Torrey Pines Institute for Molecular Studies, Port Saint Lucie, FL,* 

**Receptors and Opioid Receptor Painkillers** 

The unpleasant sensation of pain is experienced by all human beings at a given point in life. When pain gets severe and/or chronic it requires medical treatment. For over a thousand years, opioid agonists have been employed therapeutically to treat pain, with the first reports of such use involving the alkaloid morphine dated to the second century B.C.(Waldhoer, Bartlett et al. 2004) The term *opioid* refers to any substance with opium-like activity. Opium is extracted from the juice of the poppy plant *Papaver somniferum*. Opium contains in excess of 20 different alkaloids, and for centuries its crude form was used for pain management and for its psychological effects. In 1806 the German pharmacist Sertürner isolated a pure substance from opium, which he called morphine after the Greek god of dreams, Morpheus. Thereafter other alkaloids such as codeine (1832) and papaverine (1848) were isolated.(Reisine and Pasternak 1996) These discoveries paved the way for the use of pure alkaloids as opposed to crude opium in the medical profession. It became apparent that these alkaloids had a high potential for abuse and addiction. However, it was not until 1973 that the first descriptions of the pharmacological properties of morphine, along with other agonists and antagonists, at the level of the receptor were reported.(Pert, Pasternak et

Opioid receptors are of therapeutic relevance because they constitute the primary targets in the clinical treatment of both acute and chronic pain. They are members of the superfamily of seven helix transmembrane (TM) proteins known as G-protein coupled receptors (GPCRs); so-called because they are coupled in the cytoplasmic side to a group of Gi/Go hetero-trimeric proteins called G-proteins: Gα, G<sup>β</sup> and Gγ.(Eguchi M 2004) Currently four types of opioid receptors have been identified: (mu for morphine), (kappa for ketocyclazocine), (delta for deferens given that it was originally discovered in the vas deferens of mice)(Waldhoer, Bartlett et al. 2004) and orphan opioid receptor-like 1. They are in turn sub-divided into additional subtypes on the basis of their ligand binding and pharmacological profiles: 1-2, 1-3, and 1-2.(Pasternak 1993; Blakeney, Reid et al. 2007) The , and main types are the most studied, each playing a different role in pain sedation: the -receptor generates the most profound analgesia, but is also associated with constipation, respiratory depression, euphoria, tolerance, dependence and addition;(Schmauss and Yaksh 1984; Cowan, Zhu et al. 1988) the -receptor is involved in pain relief from thermal sources,(Mansour, Khachaturian et al. 1988) but like the μ-receptor, it is also associated with respiratory depression and addiction;(Abdelhamid, Sultana et al.

**1. Introduction** 

al. 1973)


### **Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers**

Austin B. Yongye and Karina Martínez-Mayorga *Torrey Pines Institute for Molecular Studies, Port Saint Lucie, FL, USA* 

#### **1. Introduction**

42 Pain Management – Current Issues and Opinions

[65] Gianolio, D. A.; Philbrook, M.; Avila, L. Z.; Young, L. E.; Plate, L.; Santos, M. R.;

[66] Homma, A.; Sato, H.; Okamachi, A. et. al. (2009).Novel Hyaluronic Acid- Methotrexate

[67] Bondeson, J. (2010). Activated Synovial Macrophages as Targets for Osteoarthritis

[68] Hamstra, D. A.; Page, M.; Maybuam, J. & Rehemtulla, A. (2000). Expression of

[69] Asai, T. & Power, I. (1999). Naloxone Inhibits Gastric Emptying in Rats. *Anesth. Analg*.,

[70] Harris, J. M. & Chess, R.B. (2003). Effect of Pegylation on Pharmaceuticals. *Nat. Rev.* 

[71] Jude-Fishburn, C. S.; Riley, T. A.; Zacarias, A. N. & Gursahani, H. (2011). Pegylated

[72] Diego, L.; Atayee, R.; Helmons, P.; Hsiao, G & von Gunten, C.F. (2011). Novel Opioid

[73] Hipkin, R. W. & Dolle, R. E. (2010). Opioid Receptor Antagonists for Gastrointestinal

[74] Schemltzer, R. C. ; Johnson, M; Griffin, J. & Uhrich, K. (2008). Comparison of Salicylate

Polymerization. *J. Biomat. Sci. Polym. Edn.*, Vol. 19, No. 10, pp. 1295- 1306 [75] Feng, W. Yu, L. & Uhrich, K. E. (2008). Opioid-Based Poly(anhydride-esters): New Approach to Preventing Drug Abuse. *Polym. Prepr*., Vol. 49, No. 2, pp. 454 – 455 [76] Rosario-Melendez, Roselin; Delgado-Rivera, Roberto; Yu, Lei & Uhrich, K. E. (2011).

Poly(anhydride- ester). *Polym. Mater. Sci. Eng.*, Vol. 105, No. 2, pp 833 - 835

Dysfunction. *Ann. Rep. Med. Chem.* Vol. 45, No. 1, 143 – 155

*In Vitro* and *In vivo*. *Bioconj. Chem.,* Vol. 19, No. 9, pp. 1767 – 1774

Drug Therapy. *Cur. Drug. Tar.*, Vol. 11, No. 5, pp. 576 - 585

4656

657.

Vol. 88, No. 1, pp. 204 – 208

Vol. 20, No. 8, pp. 1047- 1056

WO 2011088140 A1

*Drug. Discov.*, Vol. 2, No.3, pp. 214 - 221

Bernasconi, R.; Liu H.; Ahn, S., Sun, W. Jarrett, P. K. & Miller, R. J. (2008). Hyaluronan-Conjugated Opioid Depots: Synthetic Strategies and Release Kinetics

Conjugates for Osteoarthritis Treatment. *Bioorg. Med. Chem.*, Vol. 17, No. pp. 4647 –

Endogenously Activated Secreted or Cell Surface Carboxypeptidase A Sensitizes Tumor Cells to Methotrexate--Peptide Prodrugs. *Cancer Res.*, Vol. 60, No. 3, pp

Opioids with Low Potential for Abuse and Side Effects. *PCT Int. Appl.*,

Antagonists for Opioid-Induced Bowel Dysfunction. *Exper. Opin. Investig. Drugs*,

Based Poly(anhydride-esters) formed via Melt Condensation versus Solution

Synthesis, Characterization, and *In Vitro* Studies of a Morphine-Based

The unpleasant sensation of pain is experienced by all human beings at a given point in life. When pain gets severe and/or chronic it requires medical treatment. For over a thousand years, opioid agonists have been employed therapeutically to treat pain, with the first reports of such use involving the alkaloid morphine dated to the second century B.C.(Waldhoer, Bartlett et al. 2004) The term *opioid* refers to any substance with opium-like activity. Opium is extracted from the juice of the poppy plant *Papaver somniferum*. Opium contains in excess of 20 different alkaloids, and for centuries its crude form was used for pain management and for its psychological effects. In 1806 the German pharmacist Sertürner isolated a pure substance from opium, which he called morphine after the Greek god of dreams, Morpheus. Thereafter other alkaloids such as codeine (1832) and papaverine (1848) were isolated.(Reisine and Pasternak 1996) These discoveries paved the way for the use of pure alkaloids as opposed to crude opium in the medical profession. It became apparent that these alkaloids had a high potential for abuse and addiction. However, it was not until 1973 that the first descriptions of the pharmacological properties of morphine, along with other agonists and antagonists, at the level of the receptor were reported.(Pert, Pasternak et al. 1973)

Opioid receptors are of therapeutic relevance because they constitute the primary targets in the clinical treatment of both acute and chronic pain. They are members of the superfamily of seven helix transmembrane (TM) proteins known as G-protein coupled receptors (GPCRs); so-called because they are coupled in the cytoplasmic side to a group of Gi/Go hetero-trimeric proteins called G-proteins: Gα, G<sup>β</sup> and Gγ.(Eguchi M 2004) Currently four types of opioid receptors have been identified: (mu for morphine), (kappa for ketocyclazocine), (delta for deferens given that it was originally discovered in the vas deferens of mice)(Waldhoer, Bartlett et al. 2004) and orphan opioid receptor-like 1. They are in turn sub-divided into additional subtypes on the basis of their ligand binding and pharmacological profiles: 1-2, 1-3, and 1-2.(Pasternak 1993; Blakeney, Reid et al. 2007) The , and main types are the most studied, each playing a different role in pain sedation: the -receptor generates the most profound analgesia, but is also associated with constipation, respiratory depression, euphoria, tolerance, dependence and addition;(Schmauss and Yaksh 1984; Cowan, Zhu et al. 1988) the -receptor is involved in pain relief from thermal sources,(Mansour, Khachaturian et al. 1988) but like the μ-receptor, it is also associated with respiratory depression and addiction;(Abdelhamid, Sultana et al.

Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 45

(see Figure1). The experimental studies include: Li *et al.* (Li, Han et al. 2007) employing agonists and inverse-agonists of the muscarinic acetylcholine GPCR; Xu *et al.* (Xu, Sanz et al. 2008) identified inter-residue interaction differences between the active and inactive states for the -opioid receptor. From the computational side, molecular dynamics simulations studies suggest that -opioid receptor agonists and antagonists bind to the receptor with a set of interactions that are specific to each class.(Kolinski and Filipek 2008) In addition, MD simulations have been utilized to elucidate an increase in solvent exposure of the intracellular domains between helices 3 and 6, and different interactions between the arginine of the E/DRY motif for active and inactive GPCRs.(Fanelli and De

To describe the mechanism of activation and action of opioid receptors it suffices to describe the cellular assembly of these receptors. Opioid receptors comprise three domains: an extracellular N-terminus, seven transmembrane -helices and an intracellular C-terminus, Figure 1. The 7TM helices are arranged sequentially in a counter-clockwise manner when viewed from the extracellular side, and are linked by loops called EL1, EL2, EL3, IL1, IL2 and IL3. EL and IL denote extracellular loop and intracellular loop, respectively. Across the receptors the intracellular loops share the highest sequence homology (90%), followed by TM domains (70%), while the extracellular loops, the N- and C-termini show the greatest diversity.(Knapp, Malatynska et al. 1995) Coupling between the receptors and G-proteins

Activation and signaling from opioid receptors by different classes of ligands are regulated by a highly conserved mechanism.(Finn and Whistler 2001; Eguchi 2004) They are activated naturally by endogenous peptides, but also by exogenous opiates. Agonist-dependent opioid receptor activation induces conformational changes in the receptor, which promote exchange of Gα-bound GDP for unbound GTP, followed by dissociation of the G-proteins from the receptor. The Gα unit further dissociates from the Gβγ units. Signal transduction occurs via GTP-bound Gα inhibiting adenylate cyclase, responsible for producing cyclic adenosine monophosphate (cAMP). Down-regulation of cAMP results in the reduction of voltage-dependent current and neurotransmitter release.(Eguchi 2004) Moreover, the threshold of voltage-dependent ion channels becomes more negative, decreasing inward flow of current responsible for spontaneous neuronal activity resulting in a drop in cellular excitability. cAMP reduction also leads to a decrease in neurotransmitter release by cAMPdependent protein kinase. The G<sup>β</sup> and Gγ subunits also play key roles in decreasing cell excitability by inhibiting voltage-gated Ca2+ channels, hyperpolarizing the membrane and up-regulating the conduction of potassium.(Eguchi 2004) These combined decreases in neurotransmitter release and excitability are manifested as analgesia. Finally, the inactive state is re-constituted when Gα-bound GTP is hydrolyzed to GDP, re-association with Gβγ

Numerous experimental approaches have been utilized to investigate GPCR structure and activation including: solution and solid-state NMR, fluorescence, IR and UV spectroscopy, spin-labeling, site-directed mutagenesis, substituted cysteine accessibility, disulphide crosslinking, engineering metal-binding sites, and identification of constitutively active mutants.(Gether 2000; Meng and Bourne 2001; Parnot, Miserey-Lenkei et al. 2002; Decaillot, Befort et al. 2003; Hubbell, Altenbach et al. 2003; Struts, Salgado et al. 2011) Experimentally and computationally, the importance of the lipid membrane should be recognized. It is well

Benedetti 2006)

**2.2 Mechanism of activation of opioid receptors** 

occurs via the pertussis toxin sensitive Gα unit.

and recoupling with the receptor.

1991; Maldonado, Negus et al. 1992) the -receptor mediates pain originating from chemical stimuli,(Leighton, Johnson et al. 1987; Wollemann, Benyhe et al. 1993) but it promotes dysphoria, diuresis and sedation.(von Voigtlander, Lahti et al. 1983; Lahti, Mickelson et al. 1985) There is also evidence that opioid receptors exist as homo- or hetero-oligomeric complexes and that their pharmacological responses may be cross-modulated.(Zhu, King et al. 1999; Rutherford, Wang et al. 2008) For instance, Waldhoer M et al. used 6'-GNTI to demonstrate the existence of a δ-κ hetero-dimer *in vivo*.(Waldhoer, Fong et al. 2005) Furthermore, δ-opioid antagonists suppress some of the side effects of -opioid agonists such as dependence and tolerance while retaining their analgesic properties.(Ananthan 2006) The realization of this potential for cross-modulation generated interests in developing so-called bivalent ligands of opioid receptors.(Dietis, Guerrini et al. 2009; Balboni, Salvadori et al. 2011) One therapeutic relevance of opioid receptors worth mentioning is that opioid receptors antagonists such as naloxone are utilized clinically in the treatment of morphine and heroin addiction and overdose.(Blakeney, Reid et al. 2007) In this chapter, we summarize structural aspects of opioid receptors and opioid receptor ligands, with special emphasis on the -opioid receptor. The importance of the combined use of experimental information and computational models is highlighted.

Fig. 1. (A) The three domains of the μ-opioid receptor. Intracellular serine, threonine and tyrosine residues are shown in red. (B) Extracellular perspective: the seven transmembrane helices are arranged sequentially in a counterclockwise direction. The modeled active and inactive structures are shown in cyan and tan, respectively. A substantial structural difference between the two states can be seen at TM6. (C) Hypothesized outcome of degree of ligand-induced receptor endocytosis. Homology models from Pogozheva, I. D., A. L. Lomize, et al. (1998), Fowler, C. B. et al. (2004).

#### **2. Biochemical and biophysical characterization of the μ-opioid receptor**

#### **2.1 Structural studies of the -opioid receptor**

The notion of preferential stabilization of distinct conformational states by agonists and non-agonists has been established experimentally and also demonstrated computationally

1991; Maldonado, Negus et al. 1992) the -receptor mediates pain originating from chemical stimuli,(Leighton, Johnson et al. 1987; Wollemann, Benyhe et al. 1993) but it promotes dysphoria, diuresis and sedation.(von Voigtlander, Lahti et al. 1983; Lahti, Mickelson et al. 1985) There is also evidence that opioid receptors exist as homo- or hetero-oligomeric complexes and that their pharmacological responses may be cross-modulated.(Zhu, King et al. 1999; Rutherford, Wang et al. 2008) For instance, Waldhoer M et al. used 6'-GNTI to demonstrate the existence of a δ-κ hetero-dimer *in vivo*.(Waldhoer, Fong et al. 2005) Furthermore, δ-opioid antagonists suppress some of the side effects of -opioid agonists such as dependence and tolerance while retaining their analgesic properties.(Ananthan 2006) The realization of this potential for cross-modulation generated interests in developing so-called bivalent ligands of opioid receptors.(Dietis, Guerrini et al. 2009; Balboni, Salvadori et al. 2011) One therapeutic relevance of opioid receptors worth mentioning is that opioid receptors antagonists such as naloxone are utilized clinically in the treatment of morphine and heroin addiction and overdose.(Blakeney, Reid et al. 2007) In this chapter, we summarize structural aspects of opioid receptors and opioid receptor ligands, with special emphasis on the -opioid receptor. The importance of the combined use of experimental

Fig. 1. (A) The three domains of the μ-opioid receptor. Intracellular serine, threonine and tyrosine residues are shown in red. (B) Extracellular perspective: the seven transmembrane helices are arranged sequentially in a counterclockwise direction. The modeled active and inactive structures are shown in cyan and tan, respectively. A substantial structural

difference between the two states can be seen at TM6. (C) Hypothesized outcome of degree of ligand-induced receptor endocytosis. Homology models from Pogozheva, I. D., A. L.

**2. Biochemical and biophysical characterization of the μ-opioid receptor** 

The notion of preferential stabilization of distinct conformational states by agonists and non-agonists has been established experimentally and also demonstrated computationally

information and computational models is highlighted.

Lomize, et al. (1998), Fowler, C. B. et al. (2004).

**2.1 Structural studies of the -opioid receptor** 

(see Figure1). The experimental studies include: Li *et al.* (Li, Han et al. 2007) employing agonists and inverse-agonists of the muscarinic acetylcholine GPCR; Xu *et al.* (Xu, Sanz et al. 2008) identified inter-residue interaction differences between the active and inactive states for the -opioid receptor. From the computational side, molecular dynamics simulations studies suggest that -opioid receptor agonists and antagonists bind to the receptor with a set of interactions that are specific to each class.(Kolinski and Filipek 2008) In addition, MD simulations have been utilized to elucidate an increase in solvent exposure of the intracellular domains between helices 3 and 6, and different interactions between the arginine of the E/DRY motif for active and inactive GPCRs.(Fanelli and De Benedetti 2006)

#### **2.2 Mechanism of activation of opioid receptors**

To describe the mechanism of activation and action of opioid receptors it suffices to describe the cellular assembly of these receptors. Opioid receptors comprise three domains: an extracellular N-terminus, seven transmembrane -helices and an intracellular C-terminus, Figure 1. The 7TM helices are arranged sequentially in a counter-clockwise manner when viewed from the extracellular side, and are linked by loops called EL1, EL2, EL3, IL1, IL2 and IL3. EL and IL denote extracellular loop and intracellular loop, respectively. Across the receptors the intracellular loops share the highest sequence homology (90%), followed by TM domains (70%), while the extracellular loops, the N- and C-termini show the greatest diversity.(Knapp, Malatynska et al. 1995) Coupling between the receptors and G-proteins occurs via the pertussis toxin sensitive Gα unit.

Activation and signaling from opioid receptors by different classes of ligands are regulated by a highly conserved mechanism.(Finn and Whistler 2001; Eguchi 2004) They are activated naturally by endogenous peptides, but also by exogenous opiates. Agonist-dependent opioid receptor activation induces conformational changes in the receptor, which promote exchange of Gα-bound GDP for unbound GTP, followed by dissociation of the G-proteins from the receptor. The Gα unit further dissociates from the Gβγ units. Signal transduction occurs via GTP-bound Gα inhibiting adenylate cyclase, responsible for producing cyclic adenosine monophosphate (cAMP). Down-regulation of cAMP results in the reduction of voltage-dependent current and neurotransmitter release.(Eguchi 2004) Moreover, the threshold of voltage-dependent ion channels becomes more negative, decreasing inward flow of current responsible for spontaneous neuronal activity resulting in a drop in cellular excitability. cAMP reduction also leads to a decrease in neurotransmitter release by cAMPdependent protein kinase. The G<sup>β</sup> and Gγ subunits also play key roles in decreasing cell excitability by inhibiting voltage-gated Ca2+ channels, hyperpolarizing the membrane and up-regulating the conduction of potassium.(Eguchi 2004) These combined decreases in neurotransmitter release and excitability are manifested as analgesia. Finally, the inactive state is re-constituted when Gα-bound GTP is hydrolyzed to GDP, re-association with Gβγ and recoupling with the receptor.

Numerous experimental approaches have been utilized to investigate GPCR structure and activation including: solution and solid-state NMR, fluorescence, IR and UV spectroscopy, spin-labeling, site-directed mutagenesis, substituted cysteine accessibility, disulphide crosslinking, engineering metal-binding sites, and identification of constitutively active mutants.(Gether 2000; Meng and Bourne 2001; Parnot, Miserey-Lenkei et al. 2002; Decaillot, Befort et al. 2003; Hubbell, Altenbach et al. 2003; Struts, Salgado et al. 2011) Experimentally and computationally, the importance of the lipid membrane should be recognized. It is well

Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 47

the ligands disrupt the hydrogen-bonding network, while the hydrophobic portion compete with the hydrophobic residues. Finally, disrupting the interactions in group 4 residues results in the separation of TM6 and TM7 in the intracellular side and possibly destabilizing

Insights about the conformation of the activated state of the -opioid receptor are based on modeling experimental distance constraints derived from site-directed mutagenesis, interhelix H-bonds, disulphide bonds, and engineered Zn2+ binding sites between the -opioid receptor and analogues of the receptor-bound conformation of a cyclic tetra-peptidomimetic, JOM6.(Fowler, Pogozheva et al. 2004) Structural data for the active state were also derived from disulphide bonds between TM5 and TM6 in the ACM3 muscarinic receptor,(Ward SD, JBC 2002) intrinsic allosteric Zn2+ binding sites in TM5 and TM6 of the β2-adrenergic receptor,(Swaminath, Lee et al. 2003) engineered activating metal-coordination center akin to those between TM3 and TM7 in the β2-adrenergic(Elling, Thirstrup et al. 1999) and tachykinin(Holst, Elling et al. 2000) receptors, between TM2 and TM3 of the MC4 melanocortin receptor. Finally one hydrogen bond constraint from the δ-opioid receptor(Decaillot, Befort et al. 2003) was introduced. A comparison between the modeled structures of the active and inactive states of the -opioid receptor is shown in Figure 1. A noticeable difference is seen in

interactions with Gα and exposure to other secondary protein effectors.

TM6 highlighting the rigid-body movement described for the -opioid receptor.

recycled to the cell surface, preventing the development of tolerance.

The formation of an opioid ligand-receptor complex results in structural changes at the extracellular and transmembrane domains, which are propagated to the intracellular

**2.3 Internalization of opioid receptors and changes in downstream signaling** 

Signal transduction by the -opioid receptor is determined by properties of the ligand such as affinity, potency, efficacy, bio-availability and half-life, collectively defined as 'relative activity' or RA.(Martini and Whistler 2007) In addition, the length of time the receptorligand complex remains coupled to the G-protein, is controlled by receptor desensitization, endocytosis and to an extent the pharmacokinetic properties of the ligand. It has been noted that ligand activity and endocytosis do not have a linear relationship.(Martini and Whistler 2007) Hence, an interplay of relative activity versus endocytosis (RAVE) for each ligand determines the magnitude of the signal transduced. Thus each ligand-receptor complex has an associated RAVE value. As highlighted by Martini L et al.(Martini and Whistler 2007) endogenous peptides have good RA values at the μ-opioid receptor, and also induce significant desensitization and endocytosis. Based on this reasoning, the good balance between their RA and VE values explain why they do not induce tolerance. Another example is methadone. Methadone has comparable potency with encephalin and is also an equally good receptor internalizer.(Whistler, Chuang et al. 1999) Nonetheless, it has a longer half-life compared to other opioids, and consequently a larger RA value giving rise to a moderately higher RAVE value. The extension of the RAVE analysis to morphine is more complicated and invokes secondary protein effectors and region-selective differences in receptor endocytosis.(Martini and Whistler 2007) In general, agonists such as morphine with high RAVE values are more likely to induce tolerance. It has been demonstrated that the development of -opioid tolerance is inversely related to the ability of an agonist to promote receptor endocytosis or internalization.(Whistler, Chuang et al. 1999; Finn and Whistler 2001) This theory distinguishes two types of agonists based on their ability to stabilize different receptor conformational states, resulting in phosphorylation by different kinases. Depending on the type of kinase the receptor can be rapidly endocytosed, resensitized and

documented that membrane composition affects receptor function.(Botelho, Gibson et al. 2002; Botelho, Huber et al. 2006) From the computational side, molecular dynamics simulations showed that the modification of the original positioning of the lipids in the membrane influences the dynamics of the protein.(Lau, Grossfield et al. 2007) In addition, water flux though the transmembrane helices, has been proposed to affect rhodopsin activation. (Grossfield, Pitman et al. 2008) Lastly, the time scale involved in the activation of GPCRs is a challenging task. However, the combined use of computer power and experimental information allows for the generation of detailed structural information. For instance, 2000 ns molecular dynamics simulations and solid-state 2H-NMR data were combined to elucidate the protonation state of key residues directly involved in rhodopsin activation.(Martinez-Mayorga, Pitman et al. 2006) This exemplifies how computational models can provide detailed structural information not available otherwise.

Advances in crystallography and molecular engineering have provided the three-dimensional structures of a few GPCR's: rhodopsin,(Palczewski, Kumasaka et al. 2000; Ridge and Palczewski 2007; Choe, Kim et al. 2011) -adrenergic receptor,(Kobilka and Schertler 2008) and adenosine receptor.(Jaakola, Griffith et al. 2008) In the absence of experimental structures of opioid receptors, the 2.6-Å resolution crystal structure of bovine rhodopsin(Palczewski, Kumasaka et al. 2000) has served as a template for generating homology models of these receptors,(Pogozheva, Lomize et al. 1998; Fowler, Pogozheva et al. 2004; Fowler, Pogozheva et al. 2004; Pogozheva, Przydzial et al. 2005) Like rhodopsin, opioid receptors belong to class A of the GPCR superfamily. The crystallographic structure of the active state of rhodopsin is now available (Choe, Kim et al. 2011) and can be contrasted with the large body of literature that suggests a common active conformation among the class-A GPCRs. (Karnik, Gogonea et al. 2003) In the activated state TM6 undergoes outward rigid-body translation toward TM5, but away from TM3 and TM7. As a result, a cavity opens up in the intracellular domain in contact with G-proteins. Similar movements have been also suggested for TM1-3 and TM7.(Lin and Sakmar 1996; Gether, Lin et al. 1997; Altenbach, Cai et al. 2001) A better understanding of these activation mechanisms at the molecular level could lead to new drugs geared towards the therapeutic regulation of their functions.

Decaillot FM et al. applied mutagenesis to study the mechanism of activation of the human -opioid receptor.(Decaillot, Befort et al. 2003) By analyzing 30 constitutively active mutants of this receptor, mutations hypothesized to produce distinct active conformations were grouped into four abutting areas of the receptor from the extracellular (group I) to the intracellular (group IV) domain. Details about the residues that form each group can be found in Decaillot FM. A sequential binding mechanism was proposed to activate the receptor.(Decaillot, Befort et al. 2003) Sequential binding in GPCRs is not uncommon. A similar mechanism has been postulated for the β2-adrenergic receptors.(Swaminath, Xiang et al. 2004) In the case of the -opioid receptor agonists bind to residues in group I comprising a hydrophobic region in EL3, weakening interactions with TM6 and TM7 in the extracellular domain thus initiating a signal. Next, the ligand enters the binding pocket disrupting interactions in groups II and III. Group II residues form a molecular switch that controls movements of TM3. Group III residues are closest to the binding site, consist of patches of hydrophilic and hydrophobic residues and form a network of interactions between residues derived from TM3, 6 and 7. The disruption of these interactions results in a receptor state that is susceptible to activation and helps propagate signals to the intracellular side. It was hypothesized that the amphiphilic nature of opiates and opioid ligands makes them complementary to residues in group III, i.e., the hydrophilic portion of

documented that membrane composition affects receptor function.(Botelho, Gibson et al. 2002; Botelho, Huber et al. 2006) From the computational side, molecular dynamics simulations showed that the modification of the original positioning of the lipids in the membrane influences the dynamics of the protein.(Lau, Grossfield et al. 2007) In addition, water flux though the transmembrane helices, has been proposed to affect rhodopsin activation. (Grossfield, Pitman et al. 2008) Lastly, the time scale involved in the activation of GPCRs is a challenging task. However, the combined use of computer power and experimental information allows for the generation of detailed structural information. For instance, 2000 ns molecular dynamics simulations and solid-state 2H-NMR data were combined to elucidate the protonation state of key residues directly involved in rhodopsin activation.(Martinez-Mayorga, Pitman et al. 2006) This exemplifies how computational

Advances in crystallography and molecular engineering have provided the three-dimensional structures of a few GPCR's: rhodopsin,(Palczewski, Kumasaka et al. 2000; Ridge and Palczewski 2007; Choe, Kim et al. 2011) -adrenergic receptor,(Kobilka and Schertler 2008) and adenosine receptor.(Jaakola, Griffith et al. 2008) In the absence of experimental structures of opioid receptors, the 2.6-Å resolution crystal structure of bovine rhodopsin(Palczewski, Kumasaka et al. 2000) has served as a template for generating homology models of these receptors,(Pogozheva, Lomize et al. 1998; Fowler, Pogozheva et al. 2004; Fowler, Pogozheva et al. 2004; Pogozheva, Przydzial et al. 2005) Like rhodopsin, opioid receptors belong to class A of the GPCR superfamily. The crystallographic structure of the active state of rhodopsin is now available (Choe, Kim et al. 2011) and can be contrasted with the large body of literature that suggests a common active conformation among the class-A GPCRs. (Karnik, Gogonea et al. 2003) In the activated state TM6 undergoes outward rigid-body translation toward TM5, but away from TM3 and TM7. As a result, a cavity opens up in the intracellular domain in contact with G-proteins. Similar movements have been also suggested for TM1-3 and TM7.(Lin and Sakmar 1996; Gether, Lin et al. 1997; Altenbach, Cai et al. 2001) A better understanding of these activation mechanisms at the molecular level could lead to new drugs geared towards the

Decaillot FM et al. applied mutagenesis to study the mechanism of activation of the human -opioid receptor.(Decaillot, Befort et al. 2003) By analyzing 30 constitutively active mutants of this receptor, mutations hypothesized to produce distinct active conformations were grouped into four abutting areas of the receptor from the extracellular (group I) to the intracellular (group IV) domain. Details about the residues that form each group can be found in Decaillot FM. A sequential binding mechanism was proposed to activate the receptor.(Decaillot, Befort et al. 2003) Sequential binding in GPCRs is not uncommon. A similar mechanism has been postulated for the β2-adrenergic receptors.(Swaminath, Xiang et al. 2004) In the case of the -opioid receptor agonists bind to residues in group I comprising a hydrophobic region in EL3, weakening interactions with TM6 and TM7 in the extracellular domain thus initiating a signal. Next, the ligand enters the binding pocket disrupting interactions in groups II and III. Group II residues form a molecular switch that controls movements of TM3. Group III residues are closest to the binding site, consist of patches of hydrophilic and hydrophobic residues and form a network of interactions between residues derived from TM3, 6 and 7. The disruption of these interactions results in a receptor state that is susceptible to activation and helps propagate signals to the intracellular side. It was hypothesized that the amphiphilic nature of opiates and opioid ligands makes them complementary to residues in group III, i.e., the hydrophilic portion of

models can provide detailed structural information not available otherwise.

therapeutic regulation of their functions.

the ligands disrupt the hydrogen-bonding network, while the hydrophobic portion compete with the hydrophobic residues. Finally, disrupting the interactions in group 4 residues results in the separation of TM6 and TM7 in the intracellular side and possibly destabilizing interactions with Gα and exposure to other secondary protein effectors.

Insights about the conformation of the activated state of the -opioid receptor are based on modeling experimental distance constraints derived from site-directed mutagenesis, interhelix H-bonds, disulphide bonds, and engineered Zn2+ binding sites between the -opioid receptor and analogues of the receptor-bound conformation of a cyclic tetra-peptidomimetic, JOM6.(Fowler, Pogozheva et al. 2004) Structural data for the active state were also derived from disulphide bonds between TM5 and TM6 in the ACM3 muscarinic receptor,(Ward SD, JBC 2002) intrinsic allosteric Zn2+ binding sites in TM5 and TM6 of the β2-adrenergic receptor,(Swaminath, Lee et al. 2003) engineered activating metal-coordination center akin to those between TM3 and TM7 in the β2-adrenergic(Elling, Thirstrup et al. 1999) and tachykinin(Holst, Elling et al. 2000) receptors, between TM2 and TM3 of the MC4 melanocortin receptor. Finally one hydrogen bond constraint from the δ-opioid receptor(Decaillot, Befort et al. 2003) was introduced. A comparison between the modeled structures of the active and inactive states of the -opioid receptor is shown in Figure 1. A noticeable difference is seen in TM6 highlighting the rigid-body movement described for the -opioid receptor.

#### **2.3 Internalization of opioid receptors and changes in downstream signaling**

Signal transduction by the -opioid receptor is determined by properties of the ligand such as affinity, potency, efficacy, bio-availability and half-life, collectively defined as 'relative activity' or RA.(Martini and Whistler 2007) In addition, the length of time the receptorligand complex remains coupled to the G-protein, is controlled by receptor desensitization, endocytosis and to an extent the pharmacokinetic properties of the ligand. It has been noted that ligand activity and endocytosis do not have a linear relationship.(Martini and Whistler 2007) Hence, an interplay of relative activity versus endocytosis (RAVE) for each ligand determines the magnitude of the signal transduced. Thus each ligand-receptor complex has an associated RAVE value. As highlighted by Martini L et al.(Martini and Whistler 2007) endogenous peptides have good RA values at the μ-opioid receptor, and also induce significant desensitization and endocytosis. Based on this reasoning, the good balance between their RA and VE values explain why they do not induce tolerance. Another example is methadone. Methadone has comparable potency with encephalin and is also an equally good receptor internalizer.(Whistler, Chuang et al. 1999) Nonetheless, it has a longer half-life compared to other opioids, and consequently a larger RA value giving rise to a moderately higher RAVE value. The extension of the RAVE analysis to morphine is more complicated and invokes secondary protein effectors and region-selective differences in receptor endocytosis.(Martini and Whistler 2007) In general, agonists such as morphine with high RAVE values are more likely to induce tolerance. It has been demonstrated that the development of -opioid tolerance is inversely related to the ability of an agonist to promote receptor endocytosis or internalization.(Whistler, Chuang et al. 1999; Finn and Whistler 2001) This theory distinguishes two types of agonists based on their ability to stabilize different receptor conformational states, resulting in phosphorylation by different kinases. Depending on the type of kinase the receptor can be rapidly endocytosed, resensitized and recycled to the cell surface, preventing the development of tolerance.

The formation of an opioid ligand-receptor complex results in structural changes at the extracellular and transmembrane domains, which are propagated to the intracellular

Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 49

result in down-regulation of the -opioid receptor. Mutation of Ser356 and Ser363 simultaneously did not alter receptor phosphorylation, but the mutations prevented downregulation of the receptor suggesting that the absence of down-regulation was not due to the removal of phosphorylation sites. Down-regulation may be occurring through a phosphorylation-independent mechanism or these two sites are not phosphorylated. This is contrary to later studies that demonstrated that Ser363 is phosphorylated.(El Kouhen, Burd et al. 2001) The T394A mutant is more rapidly internalized and resensitized relative to the wildtype -opioid receptor. These mutation studies show that multiple phosphorylation motifs may be needed for internalization and that not every phosphorylation site is phosphorylated.

Extensive structural and pharmacological studies have been performed to understand the mechanisms of action of opioids as well as for the design of new and more efficient opioidbased painkillers. The opioid agonists propagate their analgesic effects by interacting with opioid receptors. They are both endogenously expressed peptides and exogenous opiates. The term opiate is reserved for foreign substances introduced into the body to target opioid receptors. The endogenous peptides enkephalins, dynorphins, β-endorphins and nociceptins are excised from their precursors pro-enkephalin, pro-dynorphin, proopiomelanocortin and pro-nociceptin/orphanin FQ, respectively. The majority of these peptides comprise a conserved N-terminal YGGF motif,(Gentilucci, Squassabia et al. 2007) except the uncharacteristically short peptides endomorphin-1 (YPWF-NH2) and endomorphin-2 (YPFF-NH2) that are considered analogues of the YGGF motif. A list of endogenous peptides, their precursors and receptor selectivity is presented in Table 1.

Peptide Sequences Precursor Selectivity

NAIIKNAYKKGE Pro-opiomelanocortin μ=<sup>δ</sup>

Pro-dynorphin κ

Endomorphin-1 YPWF-NH2 NDa <sup>μ</sup> Endomorphin-2 YPFF-NH2

[Met5]enkephalin **YGGF**M Pro-enkephalin δ

Deltorphin I YaFDVVG-NH2 NDa δ

Nociceptin FGGFTGARKSARKLANQ Pro-nociceptin / Orphanin FQ ORL-1b

**3. Discovery and development of opioid receptor ligands** 

**3.1 Endogenous opioid ligands** 

<sup>β</sup>-endorphin **YGGF**MTSEKSQTPLVTLFK

Dynorphin A **YGGF**LRRIRPKLKWDNQ

a Not yet determined. The conserved YGGF sequence is shown in bold

Table 1. Endogenous opioid peptides, the precursor and receptor selectivity.

[Leu5]enkephalin **YGGF**L

Metorphinamide **YGGF**MRRV-NH2 Deltorphin A YmFHLMD-NH2

Deltorphin II YaFEVVG-NH2

Dynorphin B **YGGF**LRRQFKVVT α-neoendorphin **YGGF**LRKYPK β-neoendorphin **YGGF**LRKYP

Dynorphin A(1-8) **YGGF**LRRI

b Orphan opioid receptor-like 1

domain followed by the dissociation of G-proteins; phosphorylation by G-protein coupled receptor kinases (GRK), protein kinase A (PKA) and C (PKC); and binding by other proteins such as β-arrestins.(Eguchi 2004) The phosphorylated receptor is endocytosed, whereby it is re-sensitized and recycled to the cell surface or it is marked for degradation. Unlike PKA and PKC, specific GRK-phosphorylation triggers the recruitment of β-arrestins, receptor internalization, resensitization and recycling to the cell surface. This dynamic recycling process has been suggested as crucial to circumvent development to drug tolerance. Tolerance-causing agonists impede receptor endocytosis and/or resensitization, while nontolerance-inducing drugs promote rapid receptor desensitization-internalizationresensitization and recycling.(Martini and Whistler 2007)

The exact cause of development of tolerance is still a subject of debate. Nonetheless, it is generally accepted that chronic administration of opiates for analgesia gives rise to tolerance. The cellular mechanism of tolerance may involve downstream compensatory changes in neuronal circuits.(Eguchi 2004) The continual and sustained inhibition of adenylate cyclase activity triggers a positive feedback to compensate for the low intracellular levels of cAMP, resulting in the reversible superactivation of adenylate cyclase. This up-regulation of enzyme activity restores the cellular concentration of cAMP, resulting in cells being tolerant to the opiate and also dependent on it given that withdrawing the drug or introducing an antagonist gives rise to abnormally high levels of cAMP and also a restoration of the normal activity level of adenylate cyclase.(Sharma, Klee et al. 1975) The change is delayed but relatively stable and is known to be responsible for opiate tolerance and dependence.(Sharma, Klee et al. 1975) The combined inhibition and up-regulation of adenylyl cyclase provide a means of activating and deactivating neuronal circuits and may play a role in a memory process. It was later shown that the adenylate cyclase V and Gβγ played a role in this activation.(AvidorReiss, Nevo et al. 1996)

#### **2.4 Point-mutation studies to identify key residue targets for phosphorylation**

Mutation studies have been successful in identifying key cytosolic domains and residues of ligand-activated -opioid receptors, which are liable to phosphorylation, and potentially directly involved in agonist-dependent receptor internalization. (Celver, Lowe et al. 2001; El Kouhen, Burd et al. 2001; Celver, Xu et al. 2004) Truncation of the -opioid receptor at Ser363 produced a mutant that was not phosphorylated, and was endocytosed and recycled more slowly than the wild-type,(Qiu, Law et al. 2003) suggesting that phosphorylating residues in this segment may be important for internalization. Cleaving off the entire C-terminal resulted in increased agonist-independent internalization and recycling,(Waldhoer, Bartlett et al. 2004) indicating a greater exposure of some residues critical for the dynamic recycling machinery. Utilizing a single agonist, [D-Ala2,MePhe4,Gly5-*ol*]enkephalin (DAMGO), the mutation of Thr180 to alanine in the second intracellular loop prevented receptor desensitization, while alanine scanning of serine or threonine in the third cytoplasmic loop did not inhibit receptor desensitization.(Celver, Lowe et al. 2001) In a DAMGO-induced receptor activation study, mutations of C-terminal serine/threonine residues identified three phosphorylation sites: Ser363, Thr370 and Ser375. The S375A mutant decreased the rate of receptor internalization, while the S363A and T370A double mutant accelerated the rate of internalization,(El Kouhen, Burd et al. 2001) which may suggest that the combined phosphorylation of Ser363 and Thr370 attenuates receptor internalization. Other studies employing etorphine and multiple mutations have also identified Ser356 and Ser363,(Burd, El-Kouhen et al. 1998) and Thr394 (using DAMGO)(Pak, Odowd et al. 1997; Wolf, Koch et al. 1999) as sites for phosphorylation that result in down-regulation of the -opioid receptor. Mutation of Ser356 and Ser363 simultaneously did not alter receptor phosphorylation, but the mutations prevented downregulation of the receptor suggesting that the absence of down-regulation was not due to the removal of phosphorylation sites. Down-regulation may be occurring through a phosphorylation-independent mechanism or these two sites are not phosphorylated. This is contrary to later studies that demonstrated that Ser363 is phosphorylated.(El Kouhen, Burd et al. 2001) The T394A mutant is more rapidly internalized and resensitized relative to the wildtype -opioid receptor. These mutation studies show that multiple phosphorylation motifs may be needed for internalization and that not every phosphorylation site is phosphorylated.

#### **3. Discovery and development of opioid receptor ligands**

#### **3.1 Endogenous opioid ligands**

48 Pain Management – Current Issues and Opinions

domain followed by the dissociation of G-proteins; phosphorylation by G-protein coupled receptor kinases (GRK), protein kinase A (PKA) and C (PKC); and binding by other proteins such as β-arrestins.(Eguchi 2004) The phosphorylated receptor is endocytosed, whereby it is re-sensitized and recycled to the cell surface or it is marked for degradation. Unlike PKA and PKC, specific GRK-phosphorylation triggers the recruitment of β-arrestins, receptor internalization, resensitization and recycling to the cell surface. This dynamic recycling process has been suggested as crucial to circumvent development to drug tolerance. Tolerance-causing agonists impede receptor endocytosis and/or resensitization, while nontolerance-inducing drugs promote rapid receptor desensitization-internalization-

The exact cause of development of tolerance is still a subject of debate. Nonetheless, it is generally accepted that chronic administration of opiates for analgesia gives rise to tolerance. The cellular mechanism of tolerance may involve downstream compensatory changes in neuronal circuits.(Eguchi 2004) The continual and sustained inhibition of adenylate cyclase activity triggers a positive feedback to compensate for the low intracellular levels of cAMP, resulting in the reversible superactivation of adenylate cyclase. This up-regulation of enzyme activity restores the cellular concentration of cAMP, resulting in cells being tolerant to the opiate and also dependent on it given that withdrawing the drug or introducing an antagonist gives rise to abnormally high levels of cAMP and also a restoration of the normal activity level of adenylate cyclase.(Sharma, Klee et al. 1975) The change is delayed but relatively stable and is known to be responsible for opiate tolerance and dependence.(Sharma, Klee et al. 1975) The combined inhibition and up-regulation of adenylyl cyclase provide a means of activating and deactivating neuronal circuits and may play a role in a memory process. It was later shown that the adenylate cyclase V and Gβγ

resensitization and recycling.(Martini and Whistler 2007)

played a role in this activation.(AvidorReiss, Nevo et al. 1996)

**2.4 Point-mutation studies to identify key residue targets for phosphorylation** 

Mutation studies have been successful in identifying key cytosolic domains and residues of ligand-activated -opioid receptors, which are liable to phosphorylation, and potentially directly involved in agonist-dependent receptor internalization. (Celver, Lowe et al. 2001; El Kouhen, Burd et al. 2001; Celver, Xu et al. 2004) Truncation of the -opioid receptor at Ser363 produced a mutant that was not phosphorylated, and was endocytosed and recycled more slowly than the wild-type,(Qiu, Law et al. 2003) suggesting that phosphorylating residues in this segment may be important for internalization. Cleaving off the entire C-terminal resulted in increased agonist-independent internalization and recycling,(Waldhoer, Bartlett et al. 2004) indicating a greater exposure of some residues critical for the dynamic recycling machinery. Utilizing a single agonist, [D-Ala2,MePhe4,Gly5-*ol*]enkephalin (DAMGO), the mutation of Thr180 to alanine in the second intracellular loop prevented receptor desensitization, while alanine scanning of serine or threonine in the third cytoplasmic loop did not inhibit receptor desensitization.(Celver, Lowe et al. 2001) In a DAMGO-induced receptor activation study, mutations of C-terminal serine/threonine residues identified three phosphorylation sites: Ser363, Thr370 and Ser375. The S375A mutant decreased the rate of receptor internalization, while the S363A and T370A double mutant accelerated the rate of internalization,(El Kouhen, Burd et al. 2001) which may suggest that the combined phosphorylation of Ser363 and Thr370 attenuates receptor internalization. Other studies employing etorphine and multiple mutations have also identified Ser356 and Ser363,(Burd, El-Kouhen et al. 1998) and Thr394 (using DAMGO)(Pak, Odowd et al. 1997; Wolf, Koch et al. 1999) as sites for phosphorylation that Extensive structural and pharmacological studies have been performed to understand the mechanisms of action of opioids as well as for the design of new and more efficient opioidbased painkillers. The opioid agonists propagate their analgesic effects by interacting with opioid receptors. They are both endogenously expressed peptides and exogenous opiates. The term opiate is reserved for foreign substances introduced into the body to target opioid receptors. The endogenous peptides enkephalins, dynorphins, β-endorphins and nociceptins are excised from their precursors pro-enkephalin, pro-dynorphin, proopiomelanocortin and pro-nociceptin/orphanin FQ, respectively. The majority of these peptides comprise a conserved N-terminal YGGF motif,(Gentilucci, Squassabia et al. 2007) except the uncharacteristically short peptides endomorphin-1 (YPWF-NH2) and endomorphin-2 (YPFF-NH2) that are considered analogues of the YGGF motif. A list of endogenous peptides, their precursors and receptor selectivity is presented in Table 1.


a Not yet determined. The conserved YGGF sequence is shown in bold

b Orphan opioid receptor-like 1

Table 1. Endogenous opioid peptides, the precursor and receptor selectivity.

Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 51

Fig. 2. The chemical structures of some exogenous opiates. The classical "message" tyramine

The classification of some opioid compounds is given in Table 2. The chemical structures of selected compounds are shown in Figure 2. Several factors affect the potency of an analgesic, including route of administration, whether they act as full or partial agonists, ability to cross the blood-brain barrier (physico-chemical properties) and their effects on other major physiological systems.(Volpe, Tobin et al. 2011) Some potency comparisons with morphine worth mentioning include the following: fentanyl when administered intramuscularly is about 100 fold more potent; hydromorphone is 6-8 fold more potent;(Inturrisi 2002) and oral oxycodone is about 1.8 times more potent.(Curtis, Johnson et al. 1999) Though a partial agonist buprenorphine is reported to be 25-40 times more potent than morphine.(Blakeney,

Numerous structure-activity relations (SAR) studies have been carried out on opioid receptor ligands to determine features that drive affinity or efficacy with the goal of generating more effective therapeutic compounds,(Eguchi 2004; Metcalf and Coop 2005; Prisinzano and Rothman 2008; Yongye, Appel et al. 2009, amongst others). SAR studies employing site-directed substitutions and constraints of endogenous peptides, as well as modifications of morphine have provided valuable insights about the pharmacophoric features, ligand selectivity and biological roles of opioid receptors.(Blakeney, Reid et al. 2007) For example it has been determined that a positively charged amine group, an aromatic moiety and a hydrophobic group result in tight binding of morphine. A saltbridge is formed between the protonated amine and an aspartate residue in TM3, π-π

moiety is colored in red in the structure of morphine.

**3.3 Pharmacophoric features of opioid ligands** 

Reid et al. 2007)

Endomorphin-1 and endomorphin-2 are highly potent, selective μ-opioid receptor endogenous peptides isolated from mammals, and elicit responses similar to that of morphine.(Zadina, Hackler et al. 1997; Horvath 2000) The endogenous peptides are advantageous in that they do not display any of the side effects of opiates (see below); however, they are not effective in clinical settings because of *in vivo* degradation by peptidases.(Witt, Gillespie et al. 2001) Notwithstanding their degradation, these peptides and their analogues have been utilized extensively as tools to probe receptor categorization and structure-activity relationships.(Hruby and Agnes 1999; Gentilucci, Squassabia et al. 2007) The exogenous opiates on the other hand are more effective in pain management, but present numerous undesirable side effects, some of which are highlighted below. As such, several efforts are being undertaken to identify beneficial analgesics with minimal to no side effects.

#### **3.2 Potent opioid-based analgesics**

Interests in identifying more effective analgesics have led to the reporting of a large number potent opioid peptide and non-peptide compounds that are generally classified as agonists or antagonists.(Pan 1998; Stevens, Jones et al. 2000; Eguchi 2004; Waldhoer, Bartlett et al. 2004; Gentilucci, Squassabia et al. 2007; Prisinzano and Rothman 2008; Volpe, Tobin et al. 2011) In spite of the multitude of known opioid compounds, only a relatively small number has been approved for clinical use. The majority of these prescribed analgesics are relatively selective for the μ-opioid receptor,(Volpe, Tobin et al. 2011) though at sufficiently higher doses interactions with the other opioid receptors will occur. While some of these compounds are selective for either the μ (morphine), κ (salvinorin A), or δ (naltrindole) opioid receptors, some are non-selective and display mixed agonist/antagonist responses, for example buprenorphine, pentazocine and butorphanol. Buprenorphine is a partial μagonist and partial κ-antagonist that is administered clinically for opioid detoxification and maintenance.(Blakeney, Reid et al. 2007)


A = agonist; AN = antagonist

\*Currently in clinical use. a GNTI: guanidino-naltrindole

Table 2. Opioid receptor ligands.

Endomorphin-1 and endomorphin-2 are highly potent, selective μ-opioid receptor endogenous peptides isolated from mammals, and elicit responses similar to that of morphine.(Zadina, Hackler et al. 1997; Horvath 2000) The endogenous peptides are advantageous in that they do not display any of the side effects of opiates (see below); however, they are not effective in clinical settings because of *in vivo* degradation by peptidases.(Witt, Gillespie et al. 2001) Notwithstanding their degradation, these peptides and their analogues have been utilized extensively as tools to probe receptor categorization and structure-activity relationships.(Hruby and Agnes 1999; Gentilucci, Squassabia et al. 2007) The exogenous opiates on the other hand are more effective in pain management, but present numerous undesirable side effects, some of which are highlighted below. As such, several efforts are being undertaken to identify beneficial analgesics with minimal to no side

Interests in identifying more effective analgesics have led to the reporting of a large number potent opioid peptide and non-peptide compounds that are generally classified as agonists or antagonists.(Pan 1998; Stevens, Jones et al. 2000; Eguchi 2004; Waldhoer, Bartlett et al. 2004; Gentilucci, Squassabia et al. 2007; Prisinzano and Rothman 2008; Volpe, Tobin et al. 2011) In spite of the multitude of known opioid compounds, only a relatively small number has been approved for clinical use. The majority of these prescribed analgesics are relatively selective for the μ-opioid receptor,(Volpe, Tobin et al. 2011) though at sufficiently higher doses interactions with the other opioid receptors will occur. While some of these compounds are selective for either the μ (morphine), κ (salvinorin A), or δ (naltrindole) opioid receptors, some are non-selective and display mixed agonist/antagonist responses, for example buprenorphine, pentazocine and butorphanol. Buprenorphine is a partial μagonist and partial κ-antagonist that is administered clinically for opioid detoxification and

Compound Receptor Functiona Compound Receptor Functiona Morphine\* Μ A Cyclazocine\* μ/κ A/AN Fentanyl\* " " Pentazocine\* " " Hydrocodone\* " " Nalbuphine\* " " Levorphanol\* " " SIOM Δ A Meperidine\* " " SCN-80 " " Sufentanyl\* " " TAN-67 " " Methadone\* " " Ketocyclazocine Κ A Oxycodone\* " " Ethyl Ketocyclazocine " " Oxymorphone\* " " U-50,488 " " Codeine\* " " Salvinorin A " " Naloxone\* " AN 6'-GNTIa " " Buprenorphine\* μ/κ A/AN 5'-GNTIa " AN Butorphanol\* " " Bremazocine μ/δ/κ A/AN

effects.

**3.2 Potent opioid-based analgesics** 

maintenance.(Blakeney, Reid et al. 2007)

A = agonist; AN = antagonist

Table 2. Opioid receptor ligands.

\*Currently in clinical use. a GNTI: guanidino-naltrindole

Fig. 2. The chemical structures of some exogenous opiates. The classical "message" tyramine moiety is colored in red in the structure of morphine.

The classification of some opioid compounds is given in Table 2. The chemical structures of selected compounds are shown in Figure 2. Several factors affect the potency of an analgesic, including route of administration, whether they act as full or partial agonists, ability to cross the blood-brain barrier (physico-chemical properties) and their effects on other major physiological systems.(Volpe, Tobin et al. 2011) Some potency comparisons with morphine worth mentioning include the following: fentanyl when administered intramuscularly is about 100 fold more potent; hydromorphone is 6-8 fold more potent;(Inturrisi 2002) and oral oxycodone is about 1.8 times more potent.(Curtis, Johnson et al. 1999) Though a partial agonist buprenorphine is reported to be 25-40 times more potent than morphine.(Blakeney, Reid et al. 2007)

#### **3.3 Pharmacophoric features of opioid ligands**

Numerous structure-activity relations (SAR) studies have been carried out on opioid receptor ligands to determine features that drive affinity or efficacy with the goal of generating more effective therapeutic compounds,(Eguchi 2004; Metcalf and Coop 2005; Prisinzano and Rothman 2008; Yongye, Appel et al. 2009, amongst others). SAR studies employing site-directed substitutions and constraints of endogenous peptides, as well as modifications of morphine have provided valuable insights about the pharmacophoric features, ligand selectivity and biological roles of opioid receptors.(Blakeney, Reid et al. 2007) For example it has been determined that a positively charged amine group, an aromatic moiety and a hydrophobic group result in tight binding of morphine. A saltbridge is formed between the protonated amine and an aspartate residue in TM3, π-π

Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 53

is of crucial interest. A recent protocol that takes into account these aspects has been proposed.(Yongye, Bender et al. 2010) This protocol and other important aspects of conformational coverage in ligand-based virtual screening methods have been recently revised.(Musafia and Senderowitz 2010) On the other hand, docking-based approaches are most valuable when experimental structures of receptors are available. The absence of experimental opioid receptor structures means docking-based methods must rely on homology models. Moreover, for docking-based virtual screening, one has to contend with

The identification of enkephalins and -opioid receptors fueled interests in developing ligands that target this receptor. The observation that co-administration of -opioid receptor antagonists with -opioid receptor agonists produced analgesia without the side effect of only agonists further served as motivation to identify -selective opioids. Hence, considerable efforts have been devoted to studying the SAR of -opioid receptor ligands using both pharmacophore and quantitative structure-activity relationship modeling. See Bernard D et al.(Bernard, Coop et al. 2007) and references therein. Employing the *conformationally sampled pharmacophore* (CSP) approach Bernard D et al. were able to differentiate between -opioid receptor agonists and antagonists.(Bernard, Coop et al. 2003; Bernard, Coop et al. 2005) An advantage of the CSP method is the inclusion of high energy conformers in describing pharmacophores, the justification stemming from the fact that ligands may bind in higher energy conformers stabilized by intermolecular interactions with receptors. The CSP methodology was later applied to peptide and nonpeptide agonists to derive pharmacophore models of -opioid receptor ligands.(Bernard, Coop et al. 2007) Three pharmacophore points were considered: aromatic (A), basic nitrogen (N) and hydrophobic

Utilizing efficacy as the activity index, CSP was extended to five peptides and twenty nonpeptides comprising -opioid receptor ligands, to derive an aggregate pharmacophore. By analyzing a diverse group of agonists, partial agonists and antagonists the following conclusions were derived: interactions with the B or hydrophobic site of oripavines (etorphine, buprenorphine and diprenorphine) modulated the degree of agonism; agonists with bulky B groups adopt a pose in which interactions occur with both the basic amine and the B site; agonists with large N-substituents are oriented such that the substituents occupy the position of the traditional B site. The resultant pharmacophore is an aromatic group (A), a basic amine (N), a hydrophobic group (B) and N-substituents (S). The investigators claim that such an approach would facilitate efforts to develop compounds that possess both μagonistic and δ-antagonistic properties even though the cell lines only expressed the μopioid receptor.(Shim, Coop et al. 2011) Furthermore, depending on the structural class of the ligand, N-subsituents can enhance agonism or antagonism. For example, *N*-allyl and *N*cyclopropylmethyl substituents in etorphines give rise to better agonists compared to morphine,(Gorin and Marshall 1977) while they induce antagonism in 4,5-

The currently known -opioid receptor agonists have been classified into eight structural classes(Yamaotsu and Hirono 2011): peptides (dynorphins), benzomorphans (pentazocine), morphinans (butorphanol), arylacetamines (U-69593), diazabicyclononanones (HZ2), bicyclic guanidines (TPI-614-1), benzodiazepines (±tifluadom) and neocleorodane diterpenes (salvinorin A). A comprehensive review of these classes and the history of the development of -opioid receptor ligand pharmacophores was published recently by Yamaotsu N et al.(Yamaotsu and Hirono 2011) Evidently, the structural diversity of these

no induced fit and the possibility of different binding modes.

group (B).

epoxymorphinans.(Shim, Coop et al. 2011)

stacking interactions between the aromatic group and residues in the binding pocket and hydrophobic-hydrophobic interactions. In endogenous peptides the N-terminal tyrosine contains a protonated amine and aromatic group, akin to the aromatic ring (A) and basic nitrogen (N) in morphine, Figure 3. This moiety termed tyramine is common to a majority of opioids, though there are some notable potent and selective opiates that lack this classical pharmacophore: Salvinorin A was the first highly potent, non-nitrogen opiate agonist selective towards the -opioid receptor;(Roth, Baner et al. 2002) one of its analogues, herkinorin became the first non-nitrogenous agonist selective towards the opioid receptor.(Harding, Tidgewell et al. 2005) Furthermore, the phenylalanine side chain in endogenous peptides mimics the hydrophobic feature (B) of morphine (ring C). It should be pointed out that due to size differences between the peptides and morphine, the interactions between their respective hydrophobic features (B) and the receptor are different.

The observation of the occurrence of a common structural feature amongst opioid ligands gave rise to the "message-address" concept of ligand-receptor interactions, i.e., the same message (signal transduction) is delivered to different addresses (receptors). For the endogenous peptides the message comprises the conserved YGGF motif, with the exceptions cited in Table 1, while for the opiates the tyramine moiety represents the message. The other varied segments of the ligands make up the address and confer selectivity.

Fig. 3. Chemical structures of morphine and the truncated "message" motif of an endogenous peptide. The YGGF represents amino acids: Y, tyrosine; G, glycine and F, phenylalanine.

Generating pharmacophore models for opioid receptors have followed two traditional approaches: ligand-based or docking-based. Ligand-based methods involve identifying and superimposing common substructures of low energy conformers from which features that drive biological activity are determined. However, because of the inherent difficulties of superimposing structurally different scaffolds these efforts have typically revolved around congeneric series. See Shim J et al.(Shim, Coop et al. 2011) and references therein. In ligandbased virtual screenings via multi-conformer ensembles, the quality and coverage of the conformational ensemble are important. The production of the conformers can be computationally intensive, especially for compounds with a large number of rotatable bonds. Thus, reducing the size of multi-conformer databases and the number of query conformers, while simultaneously reproducing the bioactive conformer with good accuracy,

stacking interactions between the aromatic group and residues in the binding pocket and hydrophobic-hydrophobic interactions. In endogenous peptides the N-terminal tyrosine contains a protonated amine and aromatic group, akin to the aromatic ring (A) and basic nitrogen (N) in morphine, Figure 3. This moiety termed tyramine is common to a majority of opioids, though there are some notable potent and selective opiates that lack this classical pharmacophore: Salvinorin A was the first highly potent, non-nitrogen opiate agonist selective towards the -opioid receptor;(Roth, Baner et al. 2002) one of its analogues, herkinorin became the first non-nitrogenous agonist selective towards the opioid receptor.(Harding, Tidgewell et al. 2005) Furthermore, the phenylalanine side chain in endogenous peptides mimics the hydrophobic feature (B) of morphine (ring C). It should be pointed out that due to size differences between the peptides and morphine, the interactions between their respective hydrophobic features (B) and the receptor are

The observation of the occurrence of a common structural feature amongst opioid ligands gave rise to the "message-address" concept of ligand-receptor interactions, i.e., the same message (signal transduction) is delivered to different addresses (receptors). For the endogenous peptides the message comprises the conserved YGGF motif, with the exceptions cited in Table 1, while for the opiates the tyramine moiety represents the message. The other varied segments of the ligands make up the address and confer

Fig. 3. Chemical structures of morphine and the truncated "message" motif of an endogenous peptide. The YGGF represents amino acids: Y, tyrosine; G, glycine and F,

Generating pharmacophore models for opioid receptors have followed two traditional approaches: ligand-based or docking-based. Ligand-based methods involve identifying and superimposing common substructures of low energy conformers from which features that drive biological activity are determined. However, because of the inherent difficulties of superimposing structurally different scaffolds these efforts have typically revolved around congeneric series. See Shim J et al.(Shim, Coop et al. 2011) and references therein. In ligandbased virtual screenings via multi-conformer ensembles, the quality and coverage of the conformational ensemble are important. The production of the conformers can be computationally intensive, especially for compounds with a large number of rotatable bonds. Thus, reducing the size of multi-conformer databases and the number of query conformers, while simultaneously reproducing the bioactive conformer with good accuracy,

different.

selectivity.

phenylalanine.

is of crucial interest. A recent protocol that takes into account these aspects has been proposed.(Yongye, Bender et al. 2010) This protocol and other important aspects of conformational coverage in ligand-based virtual screening methods have been recently revised.(Musafia and Senderowitz 2010) On the other hand, docking-based approaches are most valuable when experimental structures of receptors are available. The absence of experimental opioid receptor structures means docking-based methods must rely on homology models. Moreover, for docking-based virtual screening, one has to contend with no induced fit and the possibility of different binding modes.

The identification of enkephalins and -opioid receptors fueled interests in developing ligands that target this receptor. The observation that co-administration of -opioid receptor antagonists with -opioid receptor agonists produced analgesia without the side effect of only agonists further served as motivation to identify -selective opioids. Hence, considerable efforts have been devoted to studying the SAR of -opioid receptor ligands using both pharmacophore and quantitative structure-activity relationship modeling. See Bernard D et al.(Bernard, Coop et al. 2007) and references therein. Employing the *conformationally sampled pharmacophore* (CSP) approach Bernard D et al. were able to differentiate between -opioid receptor agonists and antagonists.(Bernard, Coop et al. 2003; Bernard, Coop et al. 2005) An advantage of the CSP method is the inclusion of high energy conformers in describing pharmacophores, the justification stemming from the fact that ligands may bind in higher energy conformers stabilized by intermolecular interactions with receptors. The CSP methodology was later applied to peptide and nonpeptide agonists to derive pharmacophore models of -opioid receptor ligands.(Bernard, Coop et al. 2007) Three pharmacophore points were considered: aromatic (A), basic nitrogen (N) and hydrophobic group (B).

Utilizing efficacy as the activity index, CSP was extended to five peptides and twenty nonpeptides comprising -opioid receptor ligands, to derive an aggregate pharmacophore. By analyzing a diverse group of agonists, partial agonists and antagonists the following conclusions were derived: interactions with the B or hydrophobic site of oripavines (etorphine, buprenorphine and diprenorphine) modulated the degree of agonism; agonists with bulky B groups adopt a pose in which interactions occur with both the basic amine and the B site; agonists with large N-substituents are oriented such that the substituents occupy the position of the traditional B site. The resultant pharmacophore is an aromatic group (A), a basic amine (N), a hydrophobic group (B) and N-substituents (S). The investigators claim that such an approach would facilitate efforts to develop compounds that possess both μagonistic and δ-antagonistic properties even though the cell lines only expressed the μopioid receptor.(Shim, Coop et al. 2011) Furthermore, depending on the structural class of the ligand, N-subsituents can enhance agonism or antagonism. For example, *N*-allyl and *N*cyclopropylmethyl substituents in etorphines give rise to better agonists compared to morphine,(Gorin and Marshall 1977) while they induce antagonism in 4,5 epoxymorphinans.(Shim, Coop et al. 2011)

The currently known -opioid receptor agonists have been classified into eight structural classes(Yamaotsu and Hirono 2011): peptides (dynorphins), benzomorphans (pentazocine), morphinans (butorphanol), arylacetamines (U-69593), diazabicyclononanones (HZ2), bicyclic guanidines (TPI-614-1), benzodiazepines (±tifluadom) and neocleorodane diterpenes (salvinorin A). A comprehensive review of these classes and the history of the development of -opioid receptor ligand pharmacophores was published recently by Yamaotsu N et al.(Yamaotsu and Hirono 2011) Evidently, the structural diversity of these

Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 55

The search of opioid receptor ligands using experimental screening of combinatorial libraries has been complemented using computational methods. *In silico* methods can be incorporated at different stages of the drug discovery process, from library design to lead optimization. (Brooijmans and Kuntz 2003) Computational methods are largely applied to corporate chemical collections (Bajorath 2002) as well as combinatorial chemical libraries. (Houghten, Pinilla et al. 2008) However, limited efforts have been reported so far to explicitly integrate information from mixture-based combinatorial libraries and computational techniques (López-Vallejo, Caulfield et al. 2011; Yongye, Pinilla et al. 2011). The structural analogy contained in combinatorial libraries in general and in mixture-based libraries in particular deserves particular considerations. Virtual screening may assist in downsizing large compound libraries and the selection of a smaller set of promising hits, whereas mixture-based screening may screen out some of the false positives of virtual screening. The integration of mixture-based combinatorial library screening data and virtual screening information has been undertaken. In the particular case of opioid receptors, the predicted activity obtained from the experimental mixture-based screening of a large library of bicyclic guanidines was combined with structural similarity methods. This approach allowed categorizing the molecules as actives, activity cliffs, diverse compounds and missed

Ever since the discovery of opioid receptors as the principal mediators of analgesia and the identification of endogenous peptides as well as opiates that elicit analgesic responses, considerable efforts have been devoted to finding compounds that target these receptors with the aim of alleviating the sensation of pain. While the endogenous peptides do not display any side effects, their use in clinical settings is hampered because of *in vivo* degradation by protein-digesting enzymes. Opiates are more effective, but adverse side effects such as tolerance, dependence and addiction limit their prolonged usage; thus the continual search for more efficient analgesics. Several compounds have been reported as opioid receptor ligands, however, only a relatively few are currently prescribed in clinical settings with morphine being the prototypical -opioid agonist. A high proportion of opioid-based drugs is selective toward the μ-opioid receptor, and still retains untoward side effects prompting extensive studies about the molecular origins of these undesirable

This review focuses on structural aspects of opioid receptors and opioid receptor ligands, with special emphasis on the -opioid receptor. The information presented here can be

1. Considerable evidence now point to the existence of opioid receptors as homo- or hetero-oligomeric complexes and that their pharmacological responses may be crossmodulated. For example the co-administration of a -opioid agonist with a δ-opioid antagonist suppressed side effects such as dependence and tolerance while retaining agonist induced analgesia. The realization of this potential for cross-modulation has generated interests in the development of bivalent ligands. The ligands may be individual compounds that possess mixed agonist/antagonist properties or a separate agonist and antagonist tethered through a linker. Future directions of research in analgesia will continue to point towards agonists with acceptable side effects, designing

bivalent ligands, or ligands with mixed receptor specificities and functions.

hits.(Yongye, Pinilla et al. 2011)

**4. Conclusions** 

properties.

summarized as follows:

classes making it difficult to construct a consensus pharmacophore model. Previous SAR and pharmacophore analyses of -opioid receptor ligands are typically confined to structural analogues. Yamaotsu N et al. proposed a consensus pharmacophore encompassing all eight classes using seven compounds in both the training and test sets. Superposition was based on the physico-chemical properties of groups of atoms. The consensus pharmacophore comprised three hydrophobic groups, a hydrogen bond donor and three hydrogen bond acceptors. These pharmacophoric features were employed to describe four binding orientations of the different classes of ligands for the -opioid receptor. It remains to be determined how this consensus pharmacophore will perform in virtual screening: for example screening a database, requiring that a given number of features match, followed by biological evaluation of the top scoring compounds. Additionally, in the search of opioid receptor ligands, structure similarity(Martinez-Mayorga, Medina-Franco et al. 2008; Yongye, Appel et al. 2009) and chemoinformatic analyses(Medina-Franco, Martínez-Mayorga et al. 2009) have been employed to develop SAR and to characterize highly dense combinatorial libraries.

#### **3.4 Identification of opioid receptor ligands**

A large and growing body of literature has reported the identification of opioid receptor ligands. In particular, improvements in high-throughput chemical synthesis have made possible the rapid and efficient generation of molecules, giving rise to thousands or millions of compounds in combinatorial libraries. Advances in molecular biology have also enabled the evaluation of millions of individual compounds against a number of different biological targets via high-throughput screening (HTS). However, some high content assays, such as *in vivo* studies, are not amenable to the high-throughput miniaturization required to screen millions of individual compounds. In such cases, screening libraries using a mixture-based format(Houghten, Pinilla et al. 1999; Pinilla, Appel et al. 2003; Houghten, Dooley et al. 2006) (also known as positional scanning-synthetic combinatorial libraries or PS-SCL) enables the evaluation of thousands to millions of molecules in approximately a hundred to a few hundred samples. PS-SCL have been used to successfully identify active molecules for a variety of biological targets. (Houghten, Pinilla et al. 1999; Pinilla, Appel et al. 2003; Houghten, Pinilla et al. 2008) In the case of opioid receptors highly active peptides (Dooley, Chung et al. 1994; Houghten, Dooley et al. 2006) and peptidomimetics have been identified. (Houghten, Dooley et al. 2006) This technique has recently found new applications in the search of conotoxins (Armishaw, Singh et al.) and *in-vivo* screening (Reilley, Giulianotti et al.). A step forward in the development of peptides with therapeutic relevance corresponds to the formation of cyclic structures. Cyclic peptides are therapeutically attractive due to their high bioavailability, potential selectivity, and scaffold novelty. In addition, the presence of D-residues induces conformational preferences not followed by peptides consisting of only naturally abundant L-residues. Therefore, the development of synthetic schemes and comprehending how amino acids induce turns in peptides is significant in peptide design. For example, a successful method for the synthesis of cyclic peptides by the intramolecular aminolysis of peptide thioesters, has been recently reported,(Li, Yongye et al. 2009) and the corresponding explicit solvent molecular dynamics simulations were produced and analyzed.(Yongye, Li et al. 2009) The cyclic tetra-peptidomimetic, JOM6, (Fowler, Pogozheva et al. 2004) is an example of a conformationally constrained peptide that retains activity against the -opioid receptor. It is anticipated that research will continue in this direction.

The search of opioid receptor ligands using experimental screening of combinatorial libraries has been complemented using computational methods. *In silico* methods can be incorporated at different stages of the drug discovery process, from library design to lead optimization. (Brooijmans and Kuntz 2003) Computational methods are largely applied to corporate chemical collections (Bajorath 2002) as well as combinatorial chemical libraries. (Houghten, Pinilla et al. 2008) However, limited efforts have been reported so far to explicitly integrate information from mixture-based combinatorial libraries and computational techniques (López-Vallejo, Caulfield et al. 2011; Yongye, Pinilla et al. 2011). The structural analogy contained in combinatorial libraries in general and in mixture-based libraries in particular deserves particular considerations. Virtual screening may assist in downsizing large compound libraries and the selection of a smaller set of promising hits, whereas mixture-based screening may screen out some of the false positives of virtual screening. The integration of mixture-based combinatorial library screening data and virtual screening information has been undertaken. In the particular case of opioid receptors, the predicted activity obtained from the experimental mixture-based screening of a large library of bicyclic guanidines was combined with structural similarity methods. This approach allowed categorizing the molecules as actives, activity cliffs, diverse compounds and missed hits.(Yongye, Pinilla et al. 2011)

#### **4. Conclusions**

54 Pain Management – Current Issues and Opinions

classes making it difficult to construct a consensus pharmacophore model. Previous SAR and pharmacophore analyses of -opioid receptor ligands are typically confined to structural analogues. Yamaotsu N et al. proposed a consensus pharmacophore encompassing all eight classes using seven compounds in both the training and test sets. Superposition was based on the physico-chemical properties of groups of atoms. The consensus pharmacophore comprised three hydrophobic groups, a hydrogen bond donor and three hydrogen bond acceptors. These pharmacophoric features were employed to describe four binding orientations of the different classes of ligands for the -opioid receptor. It remains to be determined how this consensus pharmacophore will perform in virtual screening: for example screening a database, requiring that a given number of features match, followed by biological evaluation of the top scoring compounds. Additionally, in the search of opioid receptor ligands, structure similarity(Martinez-Mayorga, Medina-Franco et al. 2008; Yongye, Appel et al. 2009) and chemoinformatic analyses(Medina-Franco, Martínez-Mayorga et al. 2009) have been employed to develop

A large and growing body of literature has reported the identification of opioid receptor ligands. In particular, improvements in high-throughput chemical synthesis have made possible the rapid and efficient generation of molecules, giving rise to thousands or millions of compounds in combinatorial libraries. Advances in molecular biology have also enabled the evaluation of millions of individual compounds against a number of different biological targets via high-throughput screening (HTS). However, some high content assays, such as *in vivo* studies, are not amenable to the high-throughput miniaturization required to screen millions of individual compounds. In such cases, screening libraries using a mixture-based format(Houghten, Pinilla et al. 1999; Pinilla, Appel et al. 2003; Houghten, Dooley et al. 2006) (also known as positional scanning-synthetic combinatorial libraries or PS-SCL) enables the evaluation of thousands to millions of molecules in approximately a hundred to a few hundred samples. PS-SCL have been used to successfully identify active molecules for a variety of biological targets. (Houghten, Pinilla et al. 1999; Pinilla, Appel et al. 2003; Houghten, Pinilla et al. 2008) In the case of opioid receptors highly active peptides (Dooley, Chung et al. 1994; Houghten, Dooley et al. 2006) and peptidomimetics have been identified. (Houghten, Dooley et al. 2006) This technique has recently found new applications in the search of conotoxins (Armishaw, Singh et al.) and *in-vivo* screening (Reilley, Giulianotti et al.). A step forward in the development of peptides with therapeutic relevance corresponds to the formation of cyclic structures. Cyclic peptides are therapeutically attractive due to their high bioavailability, potential selectivity, and scaffold novelty. In addition, the presence of D-residues induces conformational preferences not followed by peptides consisting of only naturally abundant L-residues. Therefore, the development of synthetic schemes and comprehending how amino acids induce turns in peptides is significant in peptide design. For example, a successful method for the synthesis of cyclic peptides by the intramolecular aminolysis of peptide thioesters, has been recently reported,(Li, Yongye et al. 2009) and the corresponding explicit solvent molecular dynamics simulations were produced and analyzed.(Yongye, Li et al. 2009) The cyclic tetra-peptidomimetic, JOM6, (Fowler, Pogozheva et al. 2004) is an example of a conformationally constrained peptide that retains activity against the -opioid receptor. It is anticipated that research will continue in

SAR and to characterize highly dense combinatorial libraries.

**3.4 Identification of opioid receptor ligands** 

this direction.

Ever since the discovery of opioid receptors as the principal mediators of analgesia and the identification of endogenous peptides as well as opiates that elicit analgesic responses, considerable efforts have been devoted to finding compounds that target these receptors with the aim of alleviating the sensation of pain. While the endogenous peptides do not display any side effects, their use in clinical settings is hampered because of *in vivo* degradation by protein-digesting enzymes. Opiates are more effective, but adverse side effects such as tolerance, dependence and addiction limit their prolonged usage; thus the continual search for more efficient analgesics. Several compounds have been reported as opioid receptor ligands, however, only a relatively few are currently prescribed in clinical settings with morphine being the prototypical -opioid agonist. A high proportion of opioid-based drugs is selective toward the μ-opioid receptor, and still retains untoward side effects prompting extensive studies about the molecular origins of these undesirable properties.

This review focuses on structural aspects of opioid receptors and opioid receptor ligands, with special emphasis on the -opioid receptor. The information presented here can be summarized as follows:

1. Considerable evidence now point to the existence of opioid receptors as homo- or hetero-oligomeric complexes and that their pharmacological responses may be crossmodulated. For example the co-administration of a -opioid agonist with a δ-opioid antagonist suppressed side effects such as dependence and tolerance while retaining agonist induced analgesia. The realization of this potential for cross-modulation has generated interests in the development of bivalent ligands. The ligands may be individual compounds that possess mixed agonist/antagonist properties or a separate agonist and antagonist tethered through a linker. Future directions of research in analgesia will continue to point towards agonists with acceptable side effects, designing bivalent ligands, or ligands with mixed receptor specificities and functions.

Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 57

Bernard, D., A. Coop*, et al.* (2007). "Quantitative conformationally sampled pharmacophore

Botelho, A. V., N. J. Gibson*, et al.* (2002). "Conformational energetics of rhodopsin modulated by nonlamellar-forming lipids." Biochemistry 41(20): 6354-6368. Botelho, A. V., T. Huber*, et al.* (2006). "Curvature and hydrophobic forces drive

Brooijmans, N. and I. D. Kuntz (2003). "Molecular recognition and docking algorithms."

Burd, A. L., R. El-Kouhen*, et al.* (1998). "Identification of serine 356 and serine 363 as the

Celver, J., M. Xu*, et al.* (2004). "Distinct domains of the mu-opioid receptor control uncoupling and internalization." Molecular Pharmacology 65(3): 528-537. Celver, J. P., J. Lowe*, et al.* (2001). "Threonine 180 is required for G-protein-coupled receptor

Cowan, A., X. Z. Zhu*, et al.* (1988). "Direct dependence studies in rats with agents selective

Curtis, G. B., G. H. Johnson*, et al.* (1999). "Relative potency of controlled-release oxycodone

Decaillot, F. M., K. Befort*, et al.* (2003). "Opioid receptor random mutagenesis reveals a

Dietis, N., R. Guerrini*, et al.* (2009). "Simultaneous targeting of multiple opioid receptors: a

Dooley, C. T., N. N. Chung*, et al.* (1994). "An all D-amino-acid opioid peptide with central analgesic activity from a combinatorial library." Science 266(5193): 2019-2022. Eguchi, M. (2004). "Recent advances in selective opioid receptor agonists and antagonists."

El Kouhen, R., A. L. Burd*, et al.* (2001). "Phosphorylation of Ser363, Thr370 and Ser375

Fanelli, F. and P. G. De Benedetti (2006). "Inactive and active states and supramolecular

internalization." Journal of Biological Chemistry 276(16): 12774-12780. Elling, C. E., K. Thirstrup*, et al.* (1999). "Conversion of agonist site to metal-ion chelator site

Sciences of the United States of America 96(22): 12322-12327.

Computer-Aided Molecular Design 20(7-8): 449-461.

Xenopus oocytes." Journal of Biological Chemistry 276(7): 4894-4900. Choe, H. W., Y. J. Kim*, et al.* (2011). "Crystal structure of metarhodopsin II." Nature

receptor." Journal of Biological Chemistry 273(51): 34488-34495.

biological activity." Journal of Medicinal Chemistry 50(8): 1799-1809. Blakeney, J. S., R. C. Reid*, et al.* (2007). "Nonpeptidic ligands for peptide-activated G protein-

coupled receptors." Chemical Reviews 107(7): 2960-3041.

Annu Rev Biophys Biomol Struct. 32: 335-373.

journal 91(12): 4464-4477.

471(7340): 651-U137.

10(8): 629-636.

Therapeutics 246(3): 950-955.

of Clinical Pharmacology 55(6): 425-429.

Medicinal Research Reviews 24(2): 182-212.

for delta opioid ligands: Reevaluation of hydrophobic moieties essential for

oligomerization and modulate activity of rhodopsin in membranes." Biophysical

amino acids involved in etorphine-induced down-regulation of the mu-opioid

kinase 3- and beta-arrestin 2-mediated desensitization of the mu-opioid receptor in

for different types of opioid receptor." Journal of Pharmacology and Experimental

and controlled-release morphine in a postoperative pain model." European Journal

mechanism for G protein-coupled receptor activation." Nature Structural Biology

strategy to improve side-effect profile." British Journal of Anaesthesia 103(1): 38-49.

residues within the carboxyl tail differentially regulates μ-opioid receptor

in the beta(2)-adrenergic receptor." Proceedings of the National Academy of

organization of GPCRs: insights from computational modeling." Journal of


#### **5. Acknowledgement**

This work was supported by the State of Florida, Executive Officer of the Governor's Office of Tourism, Trade and Economic Development.

#### **6. References**


2. While the exact mechanisms of development of tolerance are still under debate, the current models suggest a combination of ligand-induced conformational changes and receptor desensitization, as well as down-stream compensatory changes of secondary

3. Promising computational methods such as consensus pharmacophore models using different structural scaffold might serve a role in identifying ligands with mixed secondary functional profiles. Understanding the cross-talk between the different

4. Production and analysis of a large number of compounds with potential affinity to opioid receptors are possible. However, considerably more work will need to be done to understand and design compounds with high analgesic effect and lower side effects. To that end, a more detailed understanding of the signaling process upon opioid

This work was supported by the State of Florida, Executive Officer of the Governor's Office

Abdelhamid, E. E., M. Sultana*, et al.* (1991). "Selective blockage of delta-opioid receptors

Altenbach, C., K. W. Cai*, et al.* (2001). "Structure and function in rhodopsin: Mapping light-

Ananthan, S. (2006). "Opioid ligands with mixed m/d opioid receptor interactions: An

Armishaw, C. J., N. Singh*, et al.* (2010). "A synthetic combinatorial strategy for developing

AvidorReiss, T., I. Nevo*, et al.* (1996). "Chronic opioid treatment induces adenylyl cyclase V

Bajorath, J. (2002). "Integration of virtual and high-throughput screening." Nat. Rev. Drug

Balboni, G., S. Salvadori*, et al.* (2011). "Opioid bifunctional ligands from morphine and the

Bernard, D., A. Coop*, et al.* (2003). "2D conformationally sampled pharmacophore: A ligand-

Bernard, D., A. Coop*, et al.* (2005). "Conformationally sampled pharmacophore for peptidic delta opioid ligands." Journal of Medicinal Chemistry 48(24): 7773-7780.

Journal of the American Chemical Society 125(10): 3101-3107.

of Pharmacology and Experimental Therapeutics 258(1): 299-303.

emerging approach to novel analgesics." AAPS J. 8(1): E118-E125.

prevents the development of morphine-tolerance and dependence in mice." Journal

dependent changes in distance between residue 65 in helix TM1 and residues in the sequence 306-319 at the cytoplasmic end of helix TM7 and in helix H8."

alpha-conotoxin analogs as potent alpha7 nicotinic acetylcholine receptor

superactivation - Involvement of G beta gamma." Journal of Biological Chemistry

opioid pharmacophore Dmt-Tic." European Journal of Medicinal Chemistry 46(2):

based pharmacophore to differentiate delta opioid agonists from antagonists."

signaling pathways of the opioid receptors will also be significant.

effectors.

receptor activation is needed.

of Tourism, Trade and Economic Development.

Biochemistry 40(51): 15483-15492.

271(35): 21309-21315.

Discov. 1(11): 882.

799-803.

antagonists." J. Biol. Chem. 285: 1809-1821.

**5. Acknowledgement** 

**6. References** 


Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 59

Karnik, S. S., C. Gogonea*, et al.* (2003). "Activation of G-protein-coupled receptors: a

Knapp, R. J., E. Malatynska*, et al.* (1995). "Molecular-biology and pharmacology of cloned

Kobilka, B. and G. F. X. Schertler (2008). "New G-protein-coupled receptor crystal structures: insights and limitations." Trends in Pharmacological Sciences 29(2): 79-83. Kolinski, M. and S. Filipek (2008). "Molecular dynamics of μ opioid receptor complexes with agonists and antagonists." The Open Structural Biology Journal 2: 8-20. Lahti, R. A., M. M. Mickelson*, et al.* (1985). "[3H]U-69593 a highly selective ligand for the opioid κ-receptor." European Journal of Pharmacology 109(2): 281-284. Lau, P. W., A. Grossfield*, et al.* (2007). "Dynamic structure of retinylidene ligand of

Leighton, G. E., M. A. Johnson*, et al.* (1987). "Pharmacological profile of PD-117302, a selective kappa-opioid agonist." British Journal of Pharmacology 92(4): 915-922. Li, J. H., S. J. Han*, et al.* (2007). "Distinct structural changes in a g protein-coupled receptor

Li, Y., A. Yongye*, et al.* (2009). "Synthesis of Cyclic Peptides through Direct Aminolysis of

Lin, S. W. and T. P. Sakmar (1996). "Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state." Biochemistry 35(34): 11149-11159. López-Vallejo, F., T. Caulfield*, et al.* (2011). "Integrating virtual screening and combinatorial

Maldonado, R., S. Negus*, et al.* (1992). "Precipitation of morphine-withdrawal syndrome in

Mansour, A., H. Khachaturian*, et al.* (1988). "Anatomy of CNS opioid receptors." Trends in

Martinez-Mayorga, K., J. L. Medina-Franco*, et al.* (2008). "Conformation-opioid activity

Martinez-Mayorga, K., M. C. Pitman*, et al.* (2006). "Retinal counterion switch mechanism in vision evaluated by molecular simulations." J. Am. Chem. Soc. 128: 16502-16503. Martini, L. and J. L. Whistler (2007). "The role of mu opioid receptor desensitization and

Medina-Franco, J. L., K. Martínez-Mayorga*, et al.* (2009). "Characterization of Activity

Meng, E. C. and H. R. Bourne (2001). "Receptor activation: what does the rhodopsin structure tell us?" Trends in Pharmacological Sciences 22(11): 587-593.

opioid receptors." Faseb Journal 9(7): 516-525.

of Combinatorial Chemistry 11(6): 1066-1072.

antagonists." Neuropharmacology 31(12): 1231-1241.

431-437.

906-917.

282(36): 26284-26293.

Screening 14(6): 475-487.

Neuroscience 11: 308.

Chem. 16(11): 5932-5938.

Neurobiology 17(5): 556-564.

Chem. Inf. Model. 49(2): 477-491.

common molecular mechanism." Trends in Endocrinology and Metabolism 14(9):

rhodopsin probed by molecular simulations." Journal of Molecular Biology 372(4):

caused by different classes of agonist ligands." Journal of Biological Chemistry

Peptide Thioesters Catalyzed by Imidazole in Aqueous Organic Solutions." Journal

chemistry for accelerated drug discovery." Comb. Chem. High Throughput

rats by administration of mu-selective, delta-selective and kappa-selective opioid

relationships of bicyclic guanidines from 3D similarity analysis." Bioorg. & Med.

endocytosis in morphine tolerance and dependence." Current Opinion in

Landscapes Using 2D and 3D Similarity Methods: Consensus Activity Cliffs." J.


Finn, A. K. and J. L. Whistler (2001). "Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal." Neuron 32(5): 829-839. Fowler, C. B., I. D. Pogozheva*, et al.* (2004). "Refinement of a homology model of the μ-

Fowler, C. B., I. D. Pogozheva*, et al.* (2004). "Complex of an active μ-opioid receptor with a

Gentilucci, L., F. Squassabia*, et al.* (2007). "Re-discussion of the importance of ionic

Gether, U. (2000). "Uncovering molecular mechanisms involved in activation of G protein-

Gether, U., S. Lin*, et al.* (1997). "Agonists induce conformational changes in transmembrane domains III and VI of the beta(2) adrenoceptor." Embo Journal 16(22): 6737-6747. Gorin, F. A. and G. R. Marshall (1977). "Proposal for biologically-active conformation of

Grossfield, A., M. C. Pitman*, et al.* (2008). "Internal Hydration Increases during Activation of the G-Protein-Coupled Receptor Rhodopsin." J. Mol. Biol. 381(2): 478-486. Harding, W. W., K. Tidgewell*, et al.* (2005). "Neoclerodane diterpenes as a novel scaffold for mu opioid receptor ligands." Journal of Medicinal Chemistry 48(15): 4765-4771. Holst, B., C. E. Elling*, et al.* (2000). "Partial agonism through a zinc-ion switch constructed

Horvath, G. (2000). "Endomorphin-1 and endomorphin-2: pharmacology of the selective

Houghten, R. A., C. T. Dooley*, et al.* (2006). "In vitro and direct in vivo testing of mixture-

Houghten, R. A., C. Pinilla*, et al.* (1999). "Mixture-based synthetic combinatorial libraries." J.

Houghten, R. A., C. Pinilla*, et al.* (2008). "Strategies for the use of mixture-based synthetic

Inturrisi, C. (2002). "Clinical pharmacology of opioids for pain." Clinical Journal of Pain 18:

Jaakola, V. P., M. T. Griffith*, et al.* (2008). "The 2.6 Angstrom Crystal Structure of a Human

deconvolution by computational methods." J. Comb. Chem. 10(1): 3-19. Hruby, V. and R. S. Agnes (1999). "Conformation-activity relationships of opioid peptides with selective activities at opioid receptors." Biopolymers 51: 391-410. Hubbell, W. L., C. Altenbach*, et al.* (2003). "Rhodopsin structure, dynamics, and activation: A

and disulfide cross-linking." Membrane Proteins 63: 243-290.

binding sites." Biochemistry 43: 8700-8710.

transduction." Current Drug Targets 8(1): 185-196.

United States of America 74(11): 5179-5183.

Molecular Pharmacology 58(2): 263-270.

opiate ligands." Aaps Journal 8(2): E371-E382.

Med. Chem. 42(19): 3743-3778.

coupled receptors." Endocrine Reviews 21(1): 90-113.

15796-15810.

437-463.

S3-S13.

opioid receptor using distance constraints from intrinsic and engineered zinc-

cyclic peptide agonist modeled from experimental constraints." Biochemistry 43:

interactions in stabilizing ligand-opioid receptor complex and in activating signal

opiates and enkephalin." Proceedings of the National Academy of Sciences of the

between transmembrane domains III and VII in the tachykinin NK1 receptor."

endogenous mu-opioid receptor agonists." Pharmacology & Therapeutics 88(3):

based combinatorial libraries for the identification of highly active and specific

combinatorial libraries: Scaffold ranking, direct testing, in vivo, and enhanced

perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity,

A(2A) Adenosine Receptor Bound to an Antagonist." Science 322(5905): 1211-1217.


Molecular Aspects of Opioid Receptors and Opioid Receptor Painkillers 61

Rutherford, J. M., J. Wang*, et al.* (2008). "Evidence for a mu-delta opioid receptor complex in

Schmauss, C. and T. L. Yaksh (1984). "*In vivo* studies on spinal opiate receptor systems

Sharma, S. K., W. A. Klee*, et al.* (1975). "Dual regulation of adenylate cyclase accounts for

Shim, J., A. Coop*, et al.* (2011). "Consensus 3D Model of mu-Opioid Receptor Ligand Efficacy

Stevens, W. C., R. M. Jones*, et al.* (2000). "Potent and Selective Indolomorphinan Antagonists

Struts, A. V., G. F. J. Salgado*, et al.* (2011). "Retinal dynamics underlie its switch from inverse

Swaminath, G., T. W. Lee*, et al.* (2003). "Identification of an allosteric binding site for ZN(2+)

Swaminath, G., Y. Xiang*, et al.* (2004). "Sequential binding of agonists to the beta(2)

Volpe, D. A., G. A. M. Tobin*, et al.* (2011). "Uniform assessment and ranking of opioid Mu

von Voigtlander, P. F., R. A. Lahti*, et al.* (1983). "U-50,488: a selective and structurally novel

Waldhoer, M., S. E. Bartlett*, et al.* (2004). "Opioid receptors." Annual Review of Biochemistry

Waldhoer, M., J. Fong*, et al.* (2005). "A heterodimer-selective agonist shows in vivo relevance

Whistler, J. L., H. H. Chuang*, et al.* (1999). "Functional dissociation of mu opioid receptor

Witt, K. A., T. J. Gillespie*, et al.* (2001). "Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability." Peptides 22(12): 2329-2343. Wolf, R., T. Koch*, et al.* (1999). "Replacement of threonine 394 by alanine facilitates

Wollemann, M., S. Benyhe*, et al.* (1993). "The kappa-opioid receptor: evidence for the

Sciences of the United States of America 102(25): 9050-9055.

of the Kappa-Opioid Receptor." J. Med. Chem. 43(14): 2759-2769.

Sciences of the United States of America 72(8): 3092-3096.

1424-1431.

Experimental Therapeutics 228(1): 1-12.

Physical Chemistry B 115(22): 7487-7496.

Biological Chemistry 279(1): 686-691.

Pharmacology 59(3): 385-390.

addiction." Neuron 23(4): 737-746.

Pharmacology 55(2): 263-268.

different subtypes." Life Sciences 52(7): 599-611.

Therapeutics 224(1): 7-12.

73: 953-990.

Biology 18(3): 392-394.

CHO cells co-expressing mu and delta opioid peptide receptors." Peptides 29(8):

mediating antinociception. 2. Pharmacological profiles suggesting a differential association of mu-receptor, delta-receptor and kappa-receptor with visceral chemical and cutaneous thermal stimuli in the rat." Journal of Pharmacology and

narcotic dependence and tolerance." Proceedings of the National Academy of

Based on a Quantitative Conformationally Sampled Pharmacophore." Journal of

agonist to agonist during rhodopsin activation." Nature Structural & Molecular

on the beta(2) adrenergic receptor." Journal of Biological Chemistry 278(1): 352-356.

adrenoceptor - Kinetic evidence for intermediate conformational states." Journal of

receptor binding constants for selected opioid drugs." Regulatory Toxicology and

non-*mu*-(*kappa*)-opioid agonist." Journal of Pharmacology and Experimental

of G protein-coupled receptor dimers." Proceedings of the National Academy of

signaling and endocytosis: Implications for the biology of opiate tolerance and

internalization and resensitization of the rat mu opioid receptor." Molecular


Metcalf, M. D. and A. Coop (2005). "Kappa opioid antagonists: Past successes and future

Musafia, B. and H. Senderowitz (2010). "Biasing conformational ensembles towards

Pak, Y., B. F. Odowd*, et al.* (1997). "Agonist-induced desensitization of the mu opioid

COOH-terminal tail." Journal of Biological Chemistry 272(40): 24961-24965. Palczewski, K., T. Kumasaka*, et al.* (2000). "Crystal structure of rhodopsin: A G protein-

Pan, Z. Z. (1998). "mu-opposing actions of the kappa-opioid receptor." Trends in

Parnot, C., S. Miserey-Lenkei*, et al.* (2002). "Lessons from constitutively active mutants of G

Pasternak, G. W. (1993). "Pharmacological mechanisms of opioid analgesics." Clinical

Pert, C. B., G. Pasternak*, et al.* (1973). "Opiate agonists and antagonist discriminated by

Pinilla, C., J. R. Appel*, et al.* (2003). "Advances in the use of synthetic combinatorial

Pogozheva, I. D., A. L. Lomize*, et al.* (1998). "Opioid receptor three-dimensional structures

Pogozheva, I. D., M. J. Przydzial*, et al.* (2005). "Homology modeling of opioid receptor-

Prisinzano, T. E. and R. B. Rothman (2008). "Salvinorin A analogs as probes in opiold

Qiu, Y., P. Y. Law*, et al.* (2003). "mu-opioid receptor desensitization - Role of receptor

Reilley, K., M. A. Giulianotti*, et al.* (2010). "Identification of Two Novel, Potent, Low-

Reisine, T. and G. Pasternak (1996). Opioid analgesics and antagonists, In: *Goodman and* 

Ridge, K. D. and K. Palczewski (2007). "Visual rhodopsin sees the light: Structure and

Roth, B. L., K. Baner*, et al.* (2002). "Salvinorin A: A potent naturally occurring

Academy of Sciences of the United States of America 99(18): 11934-11939.

Mixture-Based Combinatorial Library." AAAPS J. 12: 318-329.

from distance geometry calculations with hydrogen bonding constraints."

ligand complexes using experimental constraints." The AAAPS Journal 7(2): E434-

phosphorylation, internalization, and resensitization." Journal of Biological

Liability Antinociceptive Compounds from the Direct In Vivo Screening of a Large

*Gilman's The Pharmacological Basis of Therapeutics*. Hardman J.G., Limbird L.E., pp

mechanism of G protein signaling." Journal of Biological Chemistry 282(13): 9297-

nonnitrogenous kappa opioid selective agonist." Proceedings of the National

receptor binding in brain." Science 182(4119): 1359-1361.

pharmacology." Chemical Reviews 108(5): 1732-1743.

chemistry: mixture-based libraries." Nat. Med. 9(1): 118-122.

bioactive-like conformers for ligand-based drug design." Expert Opinion on Drug

receptor is determined by threonine 394 preceded by acidic amino acids in the

protein-coupled receptors." Trends in Endocrinology and Metabolism 13(8): 336-

prospects." Aaps Journal 7(3): E704-E722.

coupled receptor." Science 289(5480): 739-745.

Pharmacological Sciences 19(3): 94-98.

Neuropharmacology 16(1): 1-18.

Biophysical Journal 75: 612-634.

Chemistry 278(38): 36733-36739.

521-555, McGraw-Hill, New York.

Discovery 5(10): 943-959.

343.

E448.

9301.


**4** 

Igor Ukrainets

*Ukraine* 

*National University of Pharmacy* 

**Creation of New Local Anesthetics Based on** 

**Quinoline Derivatives and Related Heterocycles** 

Pain is a widely spread symptom and one of the most common causes making people seek medical attention. Though at present, different methods of pain control such as general narcosis, acupuncture, hypnosis, electroanaesthesia, homeopathy, etc., are known, nothing is better for safety and reliability than local anaesthesia. More often it is an effective alternative to general narcosis and promotes decreasing and even eliminating the use of narcotic analgesics in surgery. Dentists, dermatologists and other medical professionals apply it in their work. Unfortunately, an "ideal" local anesthetic has not been created yet, and all current medicines of the given pharmacological group have some drawbacks. The most serious disadvantages are high neuro- and cardiotoxicity, as well as tendency to cause allergy. Thus, the search for new, more effective and safe local anesthetics is ongoing and

Quinolines are the interesting compounds for research in this area. Numerous derivatives of this azaheterocycle are widely distributed in nature. Some of them are well-known to man and used for curative purposes from ancient times. For example, alkaloids cinchonine (**1a**, R = Н) and quinine (**1b**, R = ОМе, Figure 1) with antimalarial properties are isolated from *Cinchona* L. The utility of the majority of other natural quinolines prospects are to be determined. However, recently there has been a noticeable progress toward a solution of this problem. Natural compounds themselves more often attract the attention of scientists working in different fields of science and engineering. The stimulating motive for their research is the widely spread conviction that the living nature does nothing without purpose and everything it synthesizes is important at all events for life and, therefore, for man (Bochkov & Smith, 1987). This conviction finds the experimental confirmation constantly, as a result, at present the spectrum of biological

Fig. 1. Natural antimalarial drugs and the first synthetic local anesthetic of the quinoline group

N OBu

**2**

N H

N Et

Et

**.** HCl

O

scientists all over the world continue to work on this problem.

properties of natural quinolines has expanded significantly (Kartsev, 2007).

N

N

**1a,b**

HO

R

**1. Introduction** 


### **Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles**

Igor Ukrainets *National University of Pharmacy Ukraine* 

#### **1. Introduction**

62 Pain Management – Current Issues and Opinions

Xu, W., A. Sanz*, et al.* (2008). "Activation of the mu opioid receptor involves conformational

Yamaotsu, N. and S. Hirono (2011). "3D-pharmacophore identification for κ-opioid agonists

Yongye, A. B., J. R. Appel*, et al.* (2009). "Identification, structure-activity relationships and

Yongye, A. B., A. Bender*, et al.* (2010). "Dynamic clustering threshold reduces conformer

Yongye, A. B., Y. M. Li*, et al.* (2009). "Modeling of peptides containing D-amino acids: implications on cyclization." J. Comp.-Aid. Mol. Des. 23(9): 677-689. Yongye, A. B., C. Pinilla*, et al.* (2011). "Integrating computational and mixture-based

Zadina, J. E., L. Hackler*, et al.* (1997). "A potent and selective endogenous agonist for the mu-

Zhu, Y. X., M. A. King*, et al.* (1999). "Retention of supraspinal delta-like analgesia and loss of

10586.

307.

Med. Chem. 17(15): 5583-5597.

Aided. Mol. Des. 24: 675-686.

opiate receptor." Nature 386(6624): 499-502.

rearrangements of multiple transmembrane domains." Biochemistry 47(40): 10576-

using ligand-based drug-design techniques." Topics in Current Chemistry 299: 277-

molecular modeling of potent triamine and piperazine opioid ligands." Bioorg. &

ensemble size while maintaining a biologically relevant ensemble." J. Comput.

screening of combinatorial libraries." Journal of Molecular Modeling 17: 1473-1482.

morphine tolerance in delta opioid receptor knockout mice." Neuron 24(1): 243-252.

Pain is a widely spread symptom and one of the most common causes making people seek medical attention. Though at present, different methods of pain control such as general narcosis, acupuncture, hypnosis, electroanaesthesia, homeopathy, etc., are known, nothing is better for safety and reliability than local anaesthesia. More often it is an effective alternative to general narcosis and promotes decreasing and even eliminating the use of narcotic analgesics in surgery. Dentists, dermatologists and other medical professionals apply it in their work. Unfortunately, an "ideal" local anesthetic has not been created yet, and all current medicines of the given pharmacological group have some drawbacks. The most serious disadvantages are high neuro- and cardiotoxicity, as well as tendency to cause allergy. Thus, the search for new, more effective and safe local anesthetics is ongoing and scientists all over the world continue to work on this problem.

Quinolines are the interesting compounds for research in this area. Numerous derivatives of this azaheterocycle are widely distributed in nature. Some of them are well-known to man and used for curative purposes from ancient times. For example, alkaloids cinchonine (**1a**, R = Н) and quinine (**1b**, R = ОМе, Figure 1) with antimalarial properties are isolated from *Cinchona* L. The utility of the majority of other natural quinolines prospects are to be determined. However, recently there has been a noticeable progress toward a solution of this problem. Natural compounds themselves more often attract the attention of scientists working in different fields of science and engineering. The stimulating motive for their research is the widely spread conviction that the living nature does nothing without purpose and everything it synthesizes is important at all events for life and, therefore, for man (Bochkov & Smith, 1987). This conviction finds the experimental confirmation constantly, as a result, at present the spectrum of biological properties of natural quinolines has expanded significantly (Kartsev, 2007).

Fig. 1. Natural antimalarial drugs and the first synthetic local anesthetic of the quinoline group

Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 65

And recently (Davidenko, 2011) a new pharmacological property – the ability to block opioid receptors – has been revealed in 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides. It has also been found that substances closely related in structure can reveal quite opposite biological effects. Hydrochloride of 1-allyl-4-hydroxy-6,7-dimethoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (3-morpholin-4-ylpropyl)amide (**5**) in the dose of 1 mg/kg completely eliminates the analgesic effect of Tramadol and its homologue – (2-morpholin-4 yl-ethyl)amide **6** – prolongs the analgesic effect significantly. This fact requires further research and is doubtless of interest for researchers engaged in searching not only new

**3. Chemical modification of Сhinoxicaine by its transformation into pro-drugs**  All compounds that passed the stage of primary pharmacological screening were subjected to more profound and thorough analysis in pre-clinical trials. To evaluate the local anaesthetic properties a greater number of parameters were taken into account; these parameters characterized the main specific manifestations of the biological effect: potency of local anaesthesia, the rate of its onset and duration. Additionally at this stage some experimental models, such as repeated infiltration and additional conduction anaesthesia, epidural and surface anaesthesia, were involved. The local irritant properties of the

From the experiments, only one compound emerged – hydrochloride of 4-hydroxy-2-oxo-1 propyl-1,2-dihydroquinoline-3-carboxylic acid (2-diethylaminoethyl)amide (**3b**), which further was studied as a privileged structure under the name of Chinoxicaine and was transferred to the next level of investigations. This amide causes a rapid, deep and long local anaesthesia on all models studied and has a low toxicity. It has been found that prolonged introduction of Chinoxicaine to the experimental animals does not produce any statistically significant changes in the activity of central nervous and cardio-vascular systems and does not cause negative reactions of the liver and gastrointestinal tract. The medicine does not produce nephrotoxic action and, thus, it can be used safely by the patients with renal pathology. While using Chinoxicaine, there were no cases of blood pressure decrease, which is its beneficial advantage over many known anesthetics. Additional advantages of Chinoxicaine are that together with the high specific activity it shows clear antiarrhythmic,

Simultaneously with the pharmacological studies, diversified synthetic research to find the most available method for obtaining Chinoxicaine substance was carried out. As the result, principally different synthetic schemes providing a high quality of the final product have been suggested (Ukrainets et al., 1998; Ukrayinecz & Bezuhliy, 2002; Romanov & Ukrainets,

Unfortunately, the "Chinoxicaine" project faced some problems. For example, possessing a unique set of pharmacological properties Chinoxicaine appeared to be surprisingly poorly soluble in water. Its solubility is only 13.85 g in 100 ml of water at 20°С, and this caused great difficulty when preparing a stable medicinal form for injections. We solved this

At the later stages of introduction of a new local anesthetic into medical practice, namely at the stage of clinical trials, one more serious drawback was revealed. In some patients, Chinoxicaine solution in the site of injection caused a transient feeling of burning. Though this undesirable effect lasted less than one minute, further work with the medicine

problem rather rapidly, though water had to be replaced by the combined solvent.

opioid receptors antagonists, but highly effective pain-killers as well.

compounds, as well as their acute and chronic toxicity were studied.

antimicrobial, antioxidant, and fungicidal effects.

2006).

Hence the increased interest in quinolines by synthetic chemists becomes clear. Their belonging to natural metabolites, as well as practically unlimited possibilities for chemical transformations make this molecular system, especially its hydroxyanalogues, rather convenient matrices for fixing various structural elements-pharmacophores on them. It allows making systematic changes into the structure of the finished products and thus to purposefully change their physical and chemical, as well as biological properties. Finally one can succeed in obtaining new substances corresponding to high requirements for medicines. So, in particular, the first local anesthetic of the quinoline group – Cinchocaine (**2**, Figure 1) was synthesized; though it was created 85 ago (Kleemann & Engel, 2001), it has been applied successfully in medical practice nowadays (Tomoda et al., 2009; Kang & Shin, 2010; Douglas et al., 2011).

#### **2. 1-R-4-Hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides as a source of new privileged structures with the local anesthetics activity**

When systematically studying the biological properties of 1-R-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides we repeatedly noted the opportunity of creating new potential medicines with various effects on a living organism on their basis, including local anesthetics (Ukrainets, 1992; Ukrainets et al., 1994). After the experimental study of anaesthetic properties of a large group of compounds of this chemical range, our attention was paid to the most active of them. Hydrochlorides of (2-diethylaminoethyl)amides of 1 ethyl- (**3a**, R = Et) and, especially, 1-propyl- (**3b**, R = Pr) substituted 4-hydroxy-2-oxo-1,2 dihydroquinoline-3-carboxylic acids (Figure 2), were superior the known local anesthetic Lidocaine by the specific activity possessing at the same time the lower toxicity.

Later (Gorokhova, 1993) in the same range one more compound – hydrochloride of 1-ethyl-4 hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (2-morpholin-4-ylethyl)amide (**4**) was found. By the level of infiltration anaesthesia this amide had some more activity than its 1-Nethyl analogue **3a**, but it was noticeably inferior to 1-N-propyl derivative **3b**. However, after the primary screening it was also included into the list of candidates for profound research as it possessed another important for future medicine property – a relatively low toxicity. By this parameter amide **4** prevailed over its acyclic analogues **3a,b** by a factor of almost 2.

Fig. 2. Biologically active 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides

Hence the increased interest in quinolines by synthetic chemists becomes clear. Their belonging to natural metabolites, as well as practically unlimited possibilities for chemical transformations make this molecular system, especially its hydroxyanalogues, rather convenient matrices for fixing various structural elements-pharmacophores on them. It allows making systematic changes into the structure of the finished products and thus to purposefully change their physical and chemical, as well as biological properties. Finally one can succeed in obtaining new substances corresponding to high requirements for medicines. So, in particular, the first local anesthetic of the quinoline group – Cinchocaine (**2**, Figure 1) was synthesized; though it was created 85 ago (Kleemann & Engel, 2001), it has been applied successfully in medical practice nowadays (Tomoda et al., 2009; Kang & Shin, 2010; Douglas et al., 2011).

**2. 1-R-4-Hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides as a source of** 

When systematically studying the biological properties of 1-R-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides we repeatedly noted the opportunity of creating new potential medicines with various effects on a living organism on their basis, including local anesthetics (Ukrainets, 1992; Ukrainets et al., 1994). After the experimental study of anaesthetic properties of a large group of compounds of this chemical range, our attention was paid to the most active of them. Hydrochlorides of (2-diethylaminoethyl)amides of 1 ethyl- (**3a**, R = Et) and, especially, 1-propyl- (**3b**, R = Pr) substituted 4-hydroxy-2-oxo-1,2 dihydroquinoline-3-carboxylic acids (Figure 2), were superior the known local anesthetic

Later (Gorokhova, 1993) in the same range one more compound – hydrochloride of 1-ethyl-4 hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (2-morpholin-4-ylethyl)amide (**4**) was found. By the level of infiltration anaesthesia this amide had some more activity than its 1-Nethyl analogue **3a**, but it was noticeably inferior to 1-N-propyl derivative **3b**. However, after the primary screening it was also included into the list of candidates for profound research as it possessed another important for future medicine property – a relatively low toxicity. By this

MeO

MeO

MeO

MeO

Fig. 2. Biologically active 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides

N

O

**6**

O

**5**

O

O

N H

N H N

O

**.** HCl

N

O

**.** HCl

OH

N

OH

**new privileged structures with the local anesthetics activity** 

Lidocaine by the specific activity possessing at the same time the lower toxicity.

parameter amide **4** prevailed over its acyclic analogues **3a,b** by a factor of almost 2.

N

R

N

Et

OH

O

**3a,b**

O

**4**

O

N H

O

N H N Et

N

O

**.** HCl

Et

**.** HCl

OH

And recently (Davidenko, 2011) a new pharmacological property – the ability to block opioid receptors – has been revealed in 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides. It has also been found that substances closely related in structure can reveal quite opposite biological effects. Hydrochloride of 1-allyl-4-hydroxy-6,7-dimethoxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (3-morpholin-4-ylpropyl)amide (**5**) in the dose of 1 mg/kg completely eliminates the analgesic effect of Tramadol and its homologue – (2-morpholin-4 yl-ethyl)amide **6** – prolongs the analgesic effect significantly. This fact requires further research and is doubtless of interest for researchers engaged in searching not only new opioid receptors antagonists, but highly effective pain-killers as well.

#### **3. Chemical modification of Сhinoxicaine by its transformation into pro-drugs**

All compounds that passed the stage of primary pharmacological screening were subjected to more profound and thorough analysis in pre-clinical trials. To evaluate the local anaesthetic properties a greater number of parameters were taken into account; these parameters characterized the main specific manifestations of the biological effect: potency of local anaesthesia, the rate of its onset and duration. Additionally at this stage some experimental models, such as repeated infiltration and additional conduction anaesthesia, epidural and surface anaesthesia, were involved. The local irritant properties of the compounds, as well as their acute and chronic toxicity were studied.

From the experiments, only one compound emerged – hydrochloride of 4-hydroxy-2-oxo-1 propyl-1,2-dihydroquinoline-3-carboxylic acid (2-diethylaminoethyl)amide (**3b**), which further was studied as a privileged structure under the name of Chinoxicaine and was transferred to the next level of investigations. This amide causes a rapid, deep and long local anaesthesia on all models studied and has a low toxicity. It has been found that prolonged introduction of Chinoxicaine to the experimental animals does not produce any statistically significant changes in the activity of central nervous and cardio-vascular systems and does not cause negative reactions of the liver and gastrointestinal tract. The medicine does not produce nephrotoxic action and, thus, it can be used safely by the patients with renal pathology. While using Chinoxicaine, there were no cases of blood pressure decrease, which is its beneficial advantage over many known anesthetics. Additional advantages of Chinoxicaine are that together with the high specific activity it shows clear antiarrhythmic, antimicrobial, antioxidant, and fungicidal effects.

Simultaneously with the pharmacological studies, diversified synthetic research to find the most available method for obtaining Chinoxicaine substance was carried out. As the result, principally different synthetic schemes providing a high quality of the final product have been suggested (Ukrainets et al., 1998; Ukrayinecz & Bezuhliy, 2002; Romanov & Ukrainets, 2006).

Unfortunately, the "Chinoxicaine" project faced some problems. For example, possessing a unique set of pharmacological properties Chinoxicaine appeared to be surprisingly poorly soluble in water. Its solubility is only 13.85 g in 100 ml of water at 20°С, and this caused great difficulty when preparing a stable medicinal form for injections. We solved this problem rather rapidly, though water had to be replaced by the combined solvent.

At the later stages of introduction of a new local anesthetic into medical practice, namely at the stage of clinical trials, one more serious drawback was revealed. In some patients, Chinoxicaine solution in the site of injection caused a transient feeling of burning. Though this undesirable effect lasted less than one minute, further work with the medicine

Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 67

form of 2% aqueous solution which causes only insignificant hyperemia of conjunctiva of the rabbit's eye. At the same time in spite of expectations, dissolution in water decreased significantly (up to 8.86 g per 100 ml), but usually it increases sharply in pro-drugs of this type in 1-2 thresholds comparing to hydrohalides (Vinogradova et al., 1980). Significantly there is almost a threefold shortening of the duration of the surface anesthesia by the bromoacetoxymethylate **9** and this is evidently due to the low rate of liberation of the

Me

N

Pr

N

Pr

O

O

N

Pr

OH

N H

O

Me SO3H

O

HBr or

N H

MeCOOCH2CH2Br

N Et

Et

**.**

MeSO3H

OH

O

O

N H N Et

N Et

Et

Et

**.** HBr

OH

O

O

**9 10**

starting tertiary amine.

N

Pr

N

Pr

N

Pr

O

O

N H

Fig. 3. Modification of Chinoxicaine into pro-drugs

OH

O

O

N H

OH

O

O

N H N+ Et

N Et

N

**.** HCl

O

**7 11**

**3b 8**

HCl

The attempt to optimize the value by substitution of 2-bromomethyl acetate with 2-bromoethyl failed. Under the action of amide **8** the reagent is dehydrobrominated, as a result, instead of bromoacetoxyethylate hydrobromide **10** was isolated, it could be also obtained by neutralization of the tertiary amino group of amide **8** by hydrobromic acid. Though salt formation is not accompanied with the change of number, character and location of covalent bonds, it is widely used as an individual type of chemical modification of medicinal

NaOH

Et

**.** HCl

MeCOOCH2Br

Br

Et

OH

practically lost any progress without its removal. Theoretically a rather simple and effective solution of the problem has been found. The irritant action of Chinoxicaine is completely eliminated by addition such substances as adrenaline in insignificant concentrations in its solution.

However, we tried to solve the problem by structure modification well known in the art to modern researchers (Kubinyi, 2006).

For example, on the basis of structural biological regularities previously revealed a quite new analogue of Chinoxicaine with the improved properties can be synthesized. But is should be taken into account that in such case all complex of biological and pharmaceutical trials have to be carried out in a full volume. Besides to achieve the aim is quite unreal as a result of synthesis of only one new substance. Most likely, to solve the task successfully is possible only after the study of the series of new compounds.

Taking this into account we began to improve pharmaceutical properties of Chinoxicaine from the most rational variant – creation of pro-drugs on its basis. Biologically active source in this approach remains the same, that is why both the terms of development and costs for its implementation are greatly reduced.

However, the practical realization of the method is linked with certain difficulties. In particular, to increase the water solubility, as a rule, it is necessary to introduce additional ionizing groups into the structure of the modified compounds, while to eliminate the irritant action the same ionizing groups in the molecule should be masked (Kuznetsov et al., 1991). In other words, theoretically possible methods of elimination of the revealed drawbacks of Chinoxicaine mutually exclude each other.

Most likely the irritant action of Chinoxicaine is related to the presence of 4-ОН-group in its structure, which accounts for the marked acid properties in 1-R-4-hydroxy-2-oxo-1,2 dihydroquinoline-3-carboxamides (Ukrainets, 1988). However, it has been noted (Gorokhova, 1993) that the potency of the given side effect to a great extent depends on the structure of the amide fragment as well. For example, hydrochloride of 4-hydroxy-2-oxo-1 propyl-1,2-dihydroquinoline-3-carboxylic acid (2-morpholin-4-ylethyl)amide (**7**, Figure 3) and its 1-N-ethyl analogue **4** mentioned above do not yield to Chinoxicaine in the specific activity, but they do not practically render the irritant action. This fact was the foundation for performing bioreversible chemical modification of Chinoxicaine by the tertiary amino group (Ukrainets et al., 2009).

One of the obvious solutions of the target trasformation of the Chinoxicaine molecule is trasformation into quaternary ammonium salts, which is simple in its performance. It should be noted here that common alkyl halides are not suitable for such transformation since they form with the medicine – tertiary amine – the stable compounds, which are almost not subjected to metabolism and are excreted from the organism unchanged (Kuznetsov et al., 1991). Carboxylic acid haloalkyl esters are more interesting. They allow to transform tertiary amines in quaternary ammonium salts with labile grouping N+−C−O, which is capable of relatively easily to be splitted by hydrolysis and release the initial medicine in the form of the corresponding hydrohalide (Kuznetsov et al., 1991; Vinogradova et al., 1980). One of this reagents is commercially available bromomethylacetate, by its interaction with (2-diethylaminoethyl)amide of 4-hydroxy-2-oxo-1-propyl-1,2-dihydroquinoline-3-carboxylic acid (**8**) in the anhydrous acetonitrile medium the target bromoacetoxymethylate **9** was obtained (Figure 3).

The biological screening has demonstrated that quaternization conducted eliminated the irritant action of Chinoxicaine almost completely, unlike it bromoacetoxy-methylate **9** in the

practically lost any progress without its removal. Theoretically a rather simple and effective solution of the problem has been found. The irritant action of Chinoxicaine is completely eliminated by addition such substances as adrenaline in insignificant concentrations in its

However, we tried to solve the problem by structure modification well known in the art to

For example, on the basis of structural biological regularities previously revealed a quite new analogue of Chinoxicaine with the improved properties can be synthesized. But is should be taken into account that in such case all complex of biological and pharmaceutical trials have to be carried out in a full volume. Besides to achieve the aim is quite unreal as a result of synthesis of only one new substance. Most likely, to solve the task successfully is

Taking this into account we began to improve pharmaceutical properties of Chinoxicaine from the most rational variant – creation of pro-drugs on its basis. Biologically active source in this approach remains the same, that is why both the terms of development and costs for

However, the practical realization of the method is linked with certain difficulties. In particular, to increase the water solubility, as a rule, it is necessary to introduce additional ionizing groups into the structure of the modified compounds, while to eliminate the irritant action the same ionizing groups in the molecule should be masked (Kuznetsov et al., 1991). In other words, theoretically possible methods of elimination of the revealed drawbacks of

Most likely the irritant action of Chinoxicaine is related to the presence of 4-ОН-group in its structure, which accounts for the marked acid properties in 1-R-4-hydroxy-2-oxo-1,2 dihydroquinoline-3-carboxamides (Ukrainets, 1988). However, it has been noted (Gorokhova, 1993) that the potency of the given side effect to a great extent depends on the structure of the amide fragment as well. For example, hydrochloride of 4-hydroxy-2-oxo-1 propyl-1,2-dihydroquinoline-3-carboxylic acid (2-morpholin-4-ylethyl)amide (**7**, Figure 3) and its 1-N-ethyl analogue **4** mentioned above do not yield to Chinoxicaine in the specific activity, but they do not practically render the irritant action. This fact was the foundation for performing bioreversible chemical modification of Chinoxicaine by the tertiary amino

One of the obvious solutions of the target trasformation of the Chinoxicaine molecule is trasformation into quaternary ammonium salts, which is simple in its performance. It should be noted here that common alkyl halides are not suitable for such transformation since they form with the medicine – tertiary amine – the stable compounds, which are almost not subjected to metabolism and are excreted from the organism unchanged (Kuznetsov et al., 1991). Carboxylic acid haloalkyl esters are more interesting. They allow to transform tertiary amines in quaternary ammonium salts with labile grouping N+−C−O, which is capable of relatively easily to be splitted by hydrolysis and release the initial medicine in the form of the corresponding hydrohalide (Kuznetsov et al., 1991; Vinogradova et al., 1980). One of this reagents is commercially available bromomethylacetate, by its interaction with (2-diethylaminoethyl)amide of 4-hydroxy-2-oxo-1-propyl-1,2-dihydroquinoline-3-carboxylic acid (**8**) in the anhydrous acetonitrile medium the target bromo-

The biological screening has demonstrated that quaternization conducted eliminated the irritant action of Chinoxicaine almost completely, unlike it bromoacetoxy-methylate **9** in the

solution.

modern researchers (Kubinyi, 2006).

its implementation are greatly reduced.

Chinoxicaine mutually exclude each other.

acetoxymethylate **9** was obtained (Figure 3).

group (Ukrainets et al., 2009).

possible only after the study of the series of new compounds.

form of 2% aqueous solution which causes only insignificant hyperemia of conjunctiva of the rabbit's eye. At the same time in spite of expectations, dissolution in water decreased significantly (up to 8.86 g per 100 ml), but usually it increases sharply in pro-drugs of this type in 1-2 thresholds comparing to hydrohalides (Vinogradova et al., 1980). Significantly there is almost a threefold shortening of the duration of the surface anesthesia by the bromoacetoxymethylate **9** and this is evidently due to the low rate of liberation of the starting tertiary amine.

Fig. 3. Modification of Chinoxicaine into pro-drugs

The attempt to optimize the value by substitution of 2-bromomethyl acetate with 2-bromoethyl failed. Under the action of amide **8** the reagent is dehydrobrominated, as a result, instead of bromoacetoxyethylate hydrobromide **10** was isolated, it could be also obtained by neutralization of the tertiary amino group of amide **8** by hydrobromic acid. Though salt formation is not accompanied with the change of number, character and location of covalent bonds, it is widely used as an individual type of chemical modification of medicinal

Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 69

N(19) 176.2 and 176.3º respectively). The plane of the carbon atoms of the propyl group on the N(1) atom is virtually perpendicular to the mean-square plane of the dihydropyridine ring,

In the crystal the molecules of the methanesulfonate **11** form dimers *via* stacking interactions between the dihydroquinolone fragments, the benzene rings being situated over the dihydropyridines. The distance between the ring centroids is 3.54 Å and the mean-square

The cation and anion are mutually bonded by an intermolecular hydrogen bond N(19)–

The biological screening has shown that methanesulfonate **11** demonstrates a significant improvement of all pharmaceutical properties comparing to the initial hydrochloride **3b** (Chinoxicaine). In particular, the local irritant action was successfully decreased to the level of bromoacetoxymethylate **9**. Dissolution in water increased in more than six times – up to 85.72 g per 100 ml, and it has eliminated the problem of choosing a solvent for preparation of a stable medicinal form for injections. Finally, there are also some positive aspects of revealing the specific activity: the rate of anaesthesia onset remains the same, but the total

planes of the dihydropyridine and benzene fragments form a dihedral angle of 2.2º.

duration of the surface anaesthesia and the deep anaesthetization phase increased.

**improve pharmaceutical properties of Сhinoxicaine** 

**4. Synthesis of conformation stable forms of quinolones as an attempt to** 

In modern medical chemistry several standard methods are successfully applied for improving the privileged structures chosen according to the results of preliminary pharmacological trials. Recently with accumulation of information about the spatial structure of active binding sites for many types of receptors a greater attention has been paid to methodology of conformation restrictions (Chen et al., 2010; Watanabe et al., 2010; Nirogia et al., 2011). In general, this method of the structural transformation of a molecule suggests the preservation of all functional groups contacting with a biological target in their original form and at the same time it is directed to fixing of some of them in "active"

One of the most wide-spread ways of practical realization of the method is cyclization, which allows transformation of the open side chains of the initial molecule in endo- or exocyclic fragments, making possible the change of the pharmaceutical and (or) pharmacokinetic properties. Taking into account the given data it is quite logical to study N-R-amides of 1-hydroxy-3-oxo-5,6-dihydro-3H-pyrrolo- (**12a-m**) and 1-hydroxy-3-oxo-6,7 dihydro-3H,5H-pyrido- (**13a-m**) [3,2,1-*ij*]quinoline-2-carboxylic acids as potential local

The interest of these compounds is caused by the fact that they are very similar to Chinoxicaine (**3b**) and its 1-N-ethyl analogue **3a** by their structure. At the same time amides **12-13a-m** have a principally important structural difference: though their 1-Nalkyl substituents contain the same two-three carbon atoms, they are situated not in the open alkyl chains, but are included in the composition of pyrrole or tetrahydropyridine cycles annelation with the quinolone nucleus. Such modification is known to lead to the essential spatial trasformation of the molecule. In particular, in 1-propylsubstituted 4 hydroxy-quinolones-2 the ethyl fragment is placed perpendicular the plane of the quinolone nucleus, as a result the terminal methyl group is far from the bicycle more than 3 Å (Ukrainets et al., 2009). And on the contrary, the tricyclic pyrido[3,2,1-*ij*]quinoline

the angle between them being 89.1º.

H(19)O(11) (HO 1.88 Å, N–HO 176º).

conformation.

anesthetics (Figure 5).

substances in medical chemistry. Hence, hydrobromide **10** can be considered as an original pro-drug of Chinoxicaine. However, there was no positive results due to transfer of hydrochloride to hydrobromide. Absolutely all the parameters worsened: solubility decreased to 3.40 g in 100 ml of water, the irritant action increased considerably, and the local anaesthetic activity decreased.

The substitution of hydrogen chloride as a salt-forming reagent of methanesulfonic acid, which forms methanesulfonate **11** practically with the quantitative yield reacting with amide **8** in the anhydrous diethyl ester medium, was more successful.

According to the X-ray structural data, in the symmetrically independent part of the unit cell of the methanesulfonate **11** there is a molecule of the 4-hydroxy-2-oxo-1-propyl-1,2 dihydroquinoline-3-carboxylic acid (2-diethylaminoethyl)amide protonated at atom N(19) and the methanesulfonic acid anion (see Figure 4).

The dihydroquinolone fragment is planar within 0.02 Å. The deviations of atoms C(11) and C(15) from the mean square plane of the dihydropyridine ring are 0.067 and 0.022 Å respectively. A marked deviation of atom C(11) from the ring plane is explained by the presence of a shortened intramolecular contact H(9)H(11B) of 1.986 Å. The amide fragment is virtually coplanar with the dihydroquinolone (torsional angle C(4)–C(3)–C(15)–O(15) = 4º). Such an orientation is stabilized by two intramolecular hydrogen bonds: O(4)–H(4)O(15) (HO 1.74 Å, O–HO 155º) and N(16)–H(16)O(2) (HN 1.91 Å, NH–O 140º).

The O(4)–C(4) 1.319(3), N(16)–C(15) 1.313(3), and C(2)–C(3) 1.451(3) Å bonds in the compound studied are shortened (mean values 1.331, 1.334, and 1.464 Å respectively) but the O(15)–C(15) 1.264(3) and C(3)–C(4) 1.379(3) Å bonds are lengthened (mean values 1.231 and 1.363 Å respectively).

Fig. 4. The structure of the methanesulfonate **11** molecule with atomic numbering. The dotted lines indicate the intra- and intermolecular hydrogen bonds

Atom N(1) has a planar trigonal configuration. The substituents at atoms N(1) and N(16) have an *anti*-periplanar conformation (torsional angles N(1)–C(11)–C(12)–C(13) and N(16)–C(17)–C(18)–

substances in medical chemistry. Hence, hydrobromide **10** can be considered as an original pro-drug of Chinoxicaine. However, there was no positive results due to transfer of hydrochloride to hydrobromide. Absolutely all the parameters worsened: solubility decreased to 3.40 g in 100 ml of water, the irritant action increased considerably, and the

The substitution of hydrogen chloride as a salt-forming reagent of methanesulfonic acid, which forms methanesulfonate **11** practically with the quantitative yield reacting with

According to the X-ray structural data, in the symmetrically independent part of the unit cell of the methanesulfonate **11** there is a molecule of the 4-hydroxy-2-oxo-1-propyl-1,2 dihydroquinoline-3-carboxylic acid (2-diethylaminoethyl)amide protonated at atom N(19)

The dihydroquinolone fragment is planar within 0.02 Å. The deviations of atoms C(11) and C(15) from the mean square plane of the dihydropyridine ring are 0.067 and 0.022 Å respectively. A marked deviation of atom C(11) from the ring plane is explained by the presence of a shortened intramolecular contact H(9)H(11B) of 1.986 Å. The amide fragment is virtually coplanar with the dihydroquinolone (torsional angle C(4)–C(3)–C(15)–O(15) = 4º). Such an orientation is stabilized by two intramolecular hydrogen bonds: O(4)–H(4)O(15)

The O(4)–C(4) 1.319(3), N(16)–C(15) 1.313(3), and C(2)–C(3) 1.451(3) Å bonds in the compound studied are shortened (mean values 1.331, 1.334, and 1.464 Å respectively) but the O(15)–C(15) 1.264(3) and C(3)–C(4) 1.379(3) Å bonds are lengthened (mean values 1.231 and 1.363 Å

Fig. 4. The structure of the methanesulfonate **11** molecule with atomic numbering. The

Atom N(1) has a planar trigonal configuration. The substituents at atoms N(1) and N(16) have an *anti*-periplanar conformation (torsional angles N(1)–C(11)–C(12)–C(13) and N(16)–C(17)–C(18)–

dotted lines indicate the intra- and intermolecular hydrogen bonds

(HO 1.74 Å, O–HO 155º) and N(16)–H(16)O(2) (HN 1.91 Å, NH–O 140º).

amide **8** in the anhydrous diethyl ester medium, was more successful.

local anaesthetic activity decreased.

respectively).

and the methanesulfonic acid anion (see Figure 4).

N(19) 176.2 and 176.3º respectively). The plane of the carbon atoms of the propyl group on the N(1) atom is virtually perpendicular to the mean-square plane of the dihydropyridine ring, the angle between them being 89.1º.

In the crystal the molecules of the methanesulfonate **11** form dimers *via* stacking interactions between the dihydroquinolone fragments, the benzene rings being situated over the dihydropyridines. The distance between the ring centroids is 3.54 Å and the mean-square planes of the dihydropyridine and benzene fragments form a dihedral angle of 2.2º.

The cation and anion are mutually bonded by an intermolecular hydrogen bond N(19)– H(19)O(11) (HO 1.88 Å, N–HO 176º).

The biological screening has shown that methanesulfonate **11** demonstrates a significant improvement of all pharmaceutical properties comparing to the initial hydrochloride **3b** (Chinoxicaine). In particular, the local irritant action was successfully decreased to the level of bromoacetoxymethylate **9**. Dissolution in water increased in more than six times – up to 85.72 g per 100 ml, and it has eliminated the problem of choosing a solvent for preparation of a stable medicinal form for injections. Finally, there are also some positive aspects of revealing the specific activity: the rate of anaesthesia onset remains the same, but the total duration of the surface anaesthesia and the deep anaesthetization phase increased.

#### **4. Synthesis of conformation stable forms of quinolones as an attempt to improve pharmaceutical properties of Сhinoxicaine**

In modern medical chemistry several standard methods are successfully applied for improving the privileged structures chosen according to the results of preliminary pharmacological trials. Recently with accumulation of information about the spatial structure of active binding sites for many types of receptors a greater attention has been paid to methodology of conformation restrictions (Chen et al., 2010; Watanabe et al., 2010; Nirogia et al., 2011). In general, this method of the structural transformation of a molecule suggests the preservation of all functional groups contacting with a biological target in their original form and at the same time it is directed to fixing of some of them in "active" conformation.

One of the most wide-spread ways of practical realization of the method is cyclization, which allows transformation of the open side chains of the initial molecule in endo- or exocyclic fragments, making possible the change of the pharmaceutical and (or) pharmacokinetic properties. Taking into account the given data it is quite logical to study N-R-amides of 1-hydroxy-3-oxo-5,6-dihydro-3H-pyrrolo- (**12a-m**) and 1-hydroxy-3-oxo-6,7 dihydro-3H,5H-pyrido- (**13a-m**) [3,2,1-*ij*]quinoline-2-carboxylic acids as potential local anesthetics (Figure 5).

The interest of these compounds is caused by the fact that they are very similar to Chinoxicaine (**3b**) and its 1-N-ethyl analogue **3a** by their structure. At the same time amides **12-13a-m** have a principally important structural difference: though their 1-Nalkyl substituents contain the same two-three carbon atoms, they are situated not in the open alkyl chains, but are included in the composition of pyrrole or tetrahydropyridine cycles annelation with the quinolone nucleus. Such modification is known to lead to the essential spatial trasformation of the molecule. In particular, in 1-propylsubstituted 4 hydroxy-quinolones-2 the ethyl fragment is placed perpendicular the plane of the quinolone nucleus, as a result the terminal methyl group is far from the bicycle more than 3 Å (Ukrainets et al., 2009). And on the contrary, the tricyclic pyrido[3,2,1-*ij*]quinoline

Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 71

In general, based on the biological trials conducted, it can be stated that the structural transformation of the molecule, which accompanies the transfer from 1-alkylsubstitued 4 hydroxyquinolin-2-ones to conformation limited tricyclic pyrrolo- or tetrahydropyridoquinolones, allows to decrease the irritant action of compounds of this class, but at the same time it has a strong negative effect on the local anesthetic properties and that is why it can

Infiltration anaesthesia

**12а** 4.32 ± 0.28 1.1 Undetermined 0 **12b** 3.81 ± 0.21 2.7 Undetermined 0 **12c** 4.60 ± 0.33 2.0 Undetermined 0 **12d** 4.93 ± 0.39 1.2 Undetermined 0 **12e** 3.27 ± 0.30 5.1 Undetermined 0 **12f** 3.66 ± 0.27 3.5 Undetermined 0 **12g** 2.82 ± 0.31 12.2 10.64 ± 1.20 2 **12h** 2.05 ± 0.17 36.0 39.82 ± 2.37 1 **12i** 3.09 ± 0.28 9.8 9.27 ± 1.33 2 **12j** 2.91 ± 0.32 14.2 15.92 ± 1.24 1 **12k** 3.87 ± 0.45 6.4 5.30 ± 1.45 1 **12l** 2.24 ± 0.26 27.0 25.56 ± 1.62 0 **12m** 3.04 ± 0.34 16.2 14.75 ± 1.18 0 **13а** 5.26 ± 0.44 2.8 Undetermined 0 **13b** 4.32 ± 0.35 3.9 Undetermined 0 **13c** 4.63 ± 0.41 4.2 Undetermined 0 **13d** 5.65 ± 0.48 2.3 Undetermined 0 **13e** 3.72 ± 0.33 7.6 Undetermined 0 **13f** 4.08 ± 0.29 5.4 Undetermined 0 **13g** 3.26 ± 0.30 16.7 13.51 ± 1.81 1 **13h** 2.23 ± 0.23 36.0 40.24 ± 3.06 1 **13i** 3.92 ± 0.22 14.1 8.83 ± 1.26 1 **13j** 3.10 ± 0.25 17.3 11.48 ± 2.55 1 **13k** 4.05 ± 0.36 9.7 6.63 ± 1.14 0 **13l** 2.64 ± 0.23 21.4 28.96 ± 3.73 0 **13m** 3.22 ± 0.31 15.8 15.37 ± 2.02 0 **3a** 1.84 ± 0.10 36.0 58.92 ± 4.11 2 **Сhinoxicaine** 1.61 ± 0.13 36.0 74.79 ± 4.71 2

Index Duration of

total anaesthesia, min

Irritative effect, points

be considered as unperspective.

The start of anaesthesia, min

Table 1. Biological properties of tricyclic compounds **12-13** 

**of the Сhinoxicaine molecule** 

**5. Application of the bioisosteric replacements methodology for optimization** 

The term "isosters" was introduced by Irwing Langmuir at the beginning of the 20th century. By his definition, isosters are molecules or ions containing the same number of

Compound

**13a-m**

system is much compact – in spite of the *sofa* conformation of the tetrahydropyridine ring, С(6) atom deviates from its relative plane only in 0.56 Å (Ukrainets et al., 2008). Unlike 1- N-ethylsubstituted 4-hydroxyquinolones-2, in which the methyl group of ethyl substituent is never located in the quinolone cycle plane (Baumer et al., 2004; Ukrainets et al., 2007), tricyclic pyrrolo[3,2,1-*ij*]- quinoline system is practically flat (Ukrainets et al., 2006a). It is clear that transfer from 1-N-ethyl- and 1-N-propylsubtituted **3a,b** to conformation limited pyrrolo- and pyridoquinolines **12-13** should be obligatory reflected to the biological properties. The answer to the question about this influence has been found in one of the recent investigations (Kravtsova, 2011).

**12-13**: **a** R = 2-aminoethyl; **b** R = 3-aminopropyl; **c** R = 4-aminobutyl; **d** R = 6-aminohexyl; **e** R = 2-ethylaminoethyl; **f** R = 2-(2-hydroxyethylamino)ethyl; **g** R = 2-dimetylaminoethyl; **h** R = 2-diethylaminoethyl; **i** R = 3-dimethylaminopropyl; **j** R = 3-diethylaminopropyl;  **k** R = 2-piperazin-1-ylethyl; **l** R = 2-morpholin-4-ylethyl; **m** R = 3-morpholin-4-ylpropyl

#### Fig. 5. Tricyclic analogues of Chinoxicaine

**12a-m**

Testing of the samples synthesized has been carried out on the infiltration anaesthesia model by Buelbring-Yueid method (Table 1).

Analysis of the experimental data obtained demonstrates that compounds with the primary amino groups, i.e. amides **12-13а-d**, do not practically show the anesthetic properties. The weak activity (the anaesthesia lasts for not more 5 min, and the phase of complete sensitivity loss has not come) has appeared in monoalkylaminoalkylamides **12-13е,f**. And only when the second alkyl residue is introduced into the terminal amino group (amides **12-13g-m**), the local anaesthetic action increases noticeably, but though in this case its duration remains rather short. For example, for the most active diethylaminoethyl derivative **13h** this index is approximately 40 min, though the infiltration anaesthesia index reaches the maximum possible value. As compared, Chinoxicaine in the similar conditions causes total anaesthesia lasting for approximately 75 min (with the general duration of anaesthesia of 4 hours), and its 1-N-ethyl analogue **3a** – approximately 60 min (with the general duration of anaesthesia of 2 hours).

It is interesting to note that the irritant action of 2% aqueous solutions of tricyclic amides **12-13,** determined by the rabbit's eye cornea according to the simplified modification of Setnikar method, is absent in most examples at all or decreases significantly comparing to its bicyclic prototypes **3a,b**. And it is in spite of the fact that acidity of enolic ОН-groups in 1-hydroxy-3-oxo-5,6-dihydro-3H-pyrrolo[3,2,1-*ij*]quinoline-2-carboxylic acid and its 1-N-ethyl analogue is practically the same, and in 1-hydroxy-3-oxo-6,7-dihydro-3H,5Hpyrido[3,2,1-*ij*]-quinoline-2-carboxylic acid is similar with its 1-N-propyl analogue (p*K*a*OH* = 13,44-13,48).

system is much compact – in spite of the *sofa* conformation of the tetrahydropyridine ring, С(6) atom deviates from its relative plane only in 0.56 Å (Ukrainets et al., 2008). Unlike 1- N-ethylsubstituted 4-hydroxyquinolones-2, in which the methyl group of ethyl substituent is never located in the quinolone cycle plane (Baumer et al., 2004; Ukrainets et al., 2007), tricyclic pyrrolo[3,2,1-*ij*]- quinoline system is practically flat (Ukrainets et al., 2006a). It is clear that transfer from 1-N-ethyl- and 1-N-propylsubtituted **3a,b** to conformation limited pyrrolo- and pyridoquinolines **12-13** should be obligatory reflected to the biological properties. The answer to the question about this influence has been

O N

**12-13**: **a** R = 2-aminoethyl; **b** R = 3-aminopropyl; **c** R = 4-aminobutyl; **d** R = 6-aminohexyl; **e** R = 2-ethylaminoethyl; **f** R = 2-(2-hydroxyethylamino)ethyl; **g** R = 2-dimetylaminoethyl; **h** R = 2-diethylaminoethyl; **i** R = 3-dimethylaminopropyl; **j** R = 3-diethylaminopropyl;  **k** R = 2-piperazin-1-ylethyl; **l** R = 2-morpholin-4-ylethyl; **m** R = 3-morpholin-4-ylpropyl

Testing of the samples synthesized has been carried out on the infiltration anaesthesia

Analysis of the experimental data obtained demonstrates that compounds with the primary amino groups, i.e. amides **12-13а-d**, do not practically show the anesthetic properties. The weak activity (the anaesthesia lasts for not more 5 min, and the phase of complete sensitivity loss has not come) has appeared in monoalkylaminoalkylamides **12-13е,f**. And only when the second alkyl residue is introduced into the terminal amino group (amides **12-13g-m**), the local anaesthetic action increases noticeably, but though in this case its duration remains rather short. For example, for the most active diethylaminoethyl derivative **13h** this index is approximately 40 min, though the infiltration anaesthesia index reaches the maximum possible value. As compared, Chinoxicaine in the similar conditions causes total anaesthesia lasting for approximately 75 min (with the general duration of anaesthesia of 4 hours), and its 1-N-ethyl analogue **3a** – approximately 60 min (with the general duration of anaesthesia

It is interesting to note that the irritant action of 2% aqueous solutions of tricyclic amides **12-13,** determined by the rabbit's eye cornea according to the simplified modification of Setnikar method, is absent in most examples at all or decreases significantly comparing to its bicyclic prototypes **3a,b**. And it is in spite of the fact that acidity of enolic ОН-groups in 1-hydroxy-3-oxo-5,6-dihydro-3H-pyrrolo[3,2,1-*ij*]quinoline-2-carboxylic acid and its 1-N-ethyl analogue is practically the same, and in 1-hydroxy-3-oxo-6,7-dihydro-3H,5Hpyrido[3,2,1-*ij*]-quinoline-2-carboxylic acid is similar with its 1-N-propyl analogue

O

NH-R

HCl **.**

OH O

**13a-m**

found in one of the recent investigations (Kravtsova, 2011).

HCl **.**

NH-R

OH O

N

**12a-m**

Fig. 5. Tricyclic analogues of Chinoxicaine

model by Buelbring-Yueid method (Table 1).

of 2 hours).

(p*K*a*OH* = 13,44-13,48).

In general, based on the biological trials conducted, it can be stated that the structural transformation of the molecule, which accompanies the transfer from 1-alkylsubstitued 4 hydroxyquinolin-2-ones to conformation limited tricyclic pyrrolo- or tetrahydropyridoquinolones, allows to decrease the irritant action of compounds of this class, but at the same time it has a strong negative effect on the local anesthetic properties and that is why it can be considered as unperspective.


Table 1. Biological properties of tricyclic compounds **12-13** 

#### **5. Application of the bioisosteric replacements methodology for optimization of the Сhinoxicaine molecule**

The term "isosters" was introduced by Irwing Langmuir at the beginning of the 20th century. By his definition, isosters are molecules or ions containing the same number of

Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 73

had been stipulated mainly by the presence of 4-ОН-group. Thus, the next step of potentially bioisosteric transformation of Chinoxicaine was the synthesis of compounds known to be without groupings with acid properties. One of the examples of such substances were 1-R-3-(2-diethylaminoethyl)-1H-quinazoline-2,4-diones hydrochlorides **15a-f** (Figure 7). We considered various variants of obtaining compounds of this class allowing to choose the most suitable of them depending on the structure of the target product

N

R

O

N

**15: a** R = H; **b** R = Me; **c** R = Et; **d** R = Pr; **e** R = Bu; **f** R = *i*-Bu

The study of the local irritant action of 1-R-3-(2-diethylaminoethyl)-1H-quinazoline-2,4 diones hydrochlorides **15** conducted in rabbits by the method of Lebo and Camage, has shown that the substances under research in the form of aqueous solutions with 2% concentration do not cause any reactive changes on the surface of the skin of the experimental animals. It should be worth mentioning that in similar conditions Chinoxicaine also does not reveal the irritating effect. That is why other, more sensitive,

The ability of 2% aqueous solutions of the compounds synthesized to cause infiltration anaesthesia of the skin and subcutaneous cellulose has been studied in guinea pigs (Buelbring-Yueid method). Simultaneously several parameters characterizing the basic specific manifestations of the pharmacological effect such as the rate of anaesthesia onset, its depth (potency) and duration were taken into account. The data given in Table 2 shows that all 1-R-3-(2-diethylaminoethyl)-1H-quinazoline-2,4-diones hydrochlorides **15**, without exception, possess the local anaesthetic properties in some degree. In most cases anaesthesia occurs rather quickly and in some minutes after injection, the phase of deep anaesthesia begins. However, in spite of high values of the infiltration anaesthesia index, sometimes reaching the duration of the total anaesthesia caused by quinazolones **15** remains comparatively short and they yield to Chinoxicaine and Lidocaine greatly by this parameter. However, unlike the reference drugs the most active of the compounds synthesized – hydrochlorides **15a,e,f** – reveal a number of new properties, which can be considered as useful in the complex of the short, but powerful local anaesthetic action. They are sedation as well as movement disorder or motor block on the site of introduction of the substance examined. The motor block was estimated on the "peak" of the local anesthesia by 5 point scale: 0 points - the tail root tone preserved, movements preserved in full; 1 point weakening of the tail root tone; 2 points - the weak tail root tone, sluggish movement, the animal sitting more; 3 points - lowering of the tail root tone and possible slight movement of the animal during stimulation of the skin section not occurring in the anesthetized zone, slight inhibition of the animal; 4 points - general atonia of the tail root, appearance of some inhibition of movement in response to stimulation, overall inhibition of the animal; 5 points

O

N

Et Et **.** HCl

(Ukrainets et al., 2010)

Fig. 7. Derivatives of 1H-quinazoline-2,4-dione

models should be involved in further research.

atoms, as well as the same number and arrangement of electrons. Therefore, "isosteric replacements" in the created drugs are replacement of an atom or the group to the similar one by size or valency. If the physiological activity remains at the same time, then such replacement is called "bioisosteric". After a while the term "bioisoster" has been referred to compounds obtained by replacement of quite "unsimilar" groupings, but with preserving their biological properties (King, 2002). As a result, the concept of bioisosteric replacements at present has become one of the most powerful means for creating effective and safe medicines (Devereux & Popelier, 2010; Wassermann & Bajorath, 2011; Large et al., 2011). Its application allows not only to optimise the known biologically active substances, but to reveal new structures with the similar or related properties and, thus, to increase the patent protection of a future medicine.

#### **5.1 Hydrochlorides of 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-carboxylic acids N-R-amides**

The first attempt to optimize the Chinoxicaine molecule by the method of bioisosteric replacements was replacement of its 1,2-dihydroquinoline nucleus by 1,2,5,6,7,8**-**hexahydroquinoline. We did it expecting that such transformation may appear to be bioisosteric. With this aim a large group of hydrochlorides of 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-carboxylic acids N-R-amides **14a-y** has been synthesized by the method developed earlier (Kolisnyk, 2009) (Figure 6).


Fig. 6. Hydrogenated analogues of Chinoxicaine

The biological screening conducted allow to state that reduction of the benzene part of the quinolone ring, unfortunately, leads to practically complete loss of local anaesthetic properties and that is why such modification should be considered unsuccessful. In other words, there is no reason to declare 4-hydroxy-2-oxo-1,2-dihydroquinoline and 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydroquinoline molecular systems to be bioisosteric (at least, in relation to local anaesthesia).

#### **5.2 1-R-3-(2-Diethylaminoethyl)-1H-quinazoline-2,4-diones hydrochlorides**

All ways of modification of Chinoxicaine molecule considered by us previously could not remove the local irritant action completely, therefore, it can be assumed that this drawback had been stipulated mainly by the presence of 4-ОН-group. Thus, the next step of potentially bioisosteric transformation of Chinoxicaine was the synthesis of compounds known to be without groupings with acid properties. One of the examples of such substances were 1-R-3-(2-diethylaminoethyl)-1H-quinazoline-2,4-diones hydrochlorides **15a-f** (Figure 7). We considered various variants of obtaining compounds of this class allowing to choose the most suitable of them depending on the structure of the target product (Ukrainets et al., 2010)

**15: a** R = H; **b** R = Me; **c** R = Et; **d** R = Pr; **e** R = Bu; **f** R = *i*-Bu

Fig. 7. Derivatives of 1H-quinazoline-2,4-dione

72 Pain Management – Current Issues and Opinions

atoms, as well as the same number and arrangement of electrons. Therefore, "isosteric replacements" in the created drugs are replacement of an atom or the group to the similar one by size or valency. If the physiological activity remains at the same time, then such replacement is called "bioisosteric". After a while the term "bioisoster" has been referred to compounds obtained by replacement of quite "unsimilar" groupings, but with preserving their biological properties (King, 2002). As a result, the concept of bioisosteric replacements at present has become one of the most powerful means for creating effective and safe medicines (Devereux & Popelier, 2010; Wassermann & Bajorath, 2011; Large et al., 2011). Its application allows not only to optimise the known biologically active substances, but to reveal new structures with the similar or related properties and, thus, to increase the patent

**5.1 Hydrochlorides of 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-carboxylic** 

The first attempt to optimize the Chinoxicaine molecule by the method of bioisosteric replacements was replacement of its 1,2-dihydroquinoline nucleus by 1,2,5,6,7,8**-**hexahydroquinoline. We did it expecting that such transformation may appear to be bioisosteric. With this aim a large group of hydrochlorides of 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-carboxylic acids N-R-amides **14a-y** has been synthesized by the method

protection of a future medicine.

developed earlier (Kolisnyk, 2009) (Figure 6).

 **q** R = 4-diethylamino-1-methylbutyl

Fig. 6. Hydrogenated analogues of Chinoxicaine

relation to local anaesthesia).

N

R'

**14a-y**

R' = Pr: **r** R = 2-diethylaminoethyl; **s** R = 3-diethylaminopropyl

O

O

N H R

HCl **.**

**14**: R' = H: **a** R = 2-aminoethyl; **b** R = 3-aminopropyl; **c** R = 4-aminobutyl; **d** R = 6-aminohexyl; **e** R = 2-ethylaminoethyl; **f** R = 2-(2-hydroxyethylamino)ethyl; **g** R = 2-dimetylaminoethyl; **h** R = 2-diethylaminoethyl; **i** R = 3-dimethylaminopropyl; **j** R = 3-diethylaminopropyl;  **k** R = 1-ethylpyrrolidin-2-ylmethyl; **l** R = 2-morpholin-4-ylethyl; **m** R = 3-morpholin-4 ylpropyl; **o** R = 3-piperidin-1-ylpropyl; **p** R = 3-(4-methylpiperazin-1-yl)propyl;

 R' = *cyclo*-Pr: **t** R = 2-ethylaminoethyl; **u** R = 2-(2-hydroxyethylamino)ethyl; **v** R = 2-dimetylaminoethyl; **w** R = 2-diethylaminoethyl; **x** R = 3-dimethylaminopropyl; **y** R = 3-diethylaminopropyl

The biological screening conducted allow to state that reduction of the benzene part of the quinolone ring, unfortunately, leads to practically complete loss of local anaesthetic properties and that is why such modification should be considered unsuccessful. In other words, there is no reason to declare 4-hydroxy-2-oxo-1,2-dihydroquinoline and 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydroquinoline molecular systems to be bioisosteric (at least, in

All ways of modification of Chinoxicaine molecule considered by us previously could not remove the local irritant action completely, therefore, it can be assumed that this drawback

**5.2 1-R-3-(2-Diethylaminoethyl)-1H-quinazoline-2,4-diones hydrochlorides** 

OH

**acids N-R-amides** 

The study of the local irritant action of 1-R-3-(2-diethylaminoethyl)-1H-quinazoline-2,4 diones hydrochlorides **15** conducted in rabbits by the method of Lebo and Camage, has shown that the substances under research in the form of aqueous solutions with 2% concentration do not cause any reactive changes on the surface of the skin of the experimental animals. It should be worth mentioning that in similar conditions Chinoxicaine also does not reveal the irritating effect. That is why other, more sensitive, models should be involved in further research.

The ability of 2% aqueous solutions of the compounds synthesized to cause infiltration anaesthesia of the skin and subcutaneous cellulose has been studied in guinea pigs (Buelbring-Yueid method). Simultaneously several parameters characterizing the basic specific manifestations of the pharmacological effect such as the rate of anaesthesia onset, its depth (potency) and duration were taken into account. The data given in Table 2 shows that all 1-R-3-(2-diethylaminoethyl)-1H-quinazoline-2,4-diones hydrochlorides **15**, without exception, possess the local anaesthetic properties in some degree. In most cases anaesthesia occurs rather quickly and in some minutes after injection, the phase of deep anaesthesia begins. However, in spite of high values of the infiltration anaesthesia index, sometimes reaching the duration of the total anaesthesia caused by quinazolones **15** remains comparatively short and they yield to Chinoxicaine and Lidocaine greatly by this parameter. However, unlike the reference drugs the most active of the compounds synthesized – hydrochlorides **15a,e,f** – reveal a number of new properties, which can be considered as useful in the complex of the short, but powerful local anaesthetic action. They are sedation as well as movement disorder or motor block on the site of introduction of the substance examined. The motor block was estimated on the "peak" of the local anesthesia by 5 point scale: 0 points - the tail root tone preserved, movements preserved in full; 1 point weakening of the tail root tone; 2 points - the weak tail root tone, sluggish movement, the animal sitting more; 3 points - lowering of the tail root tone and possible slight movement of the animal during stimulation of the skin section not occurring in the anesthetized zone, slight inhibition of the animal; 4 points - general atonia of the tail root, appearance of some inhibition of movement in response to stimulation, overall inhibition of the animal; 5 points

Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 75

The first example of such transformation was 4-chloro-2-oxo-1-propyl-1,2-dihydroquinoline-

A high reactivity of the chlorine atom in 1-R-4-chloro-3-ethoxycarbonyl-2-oxo-1,2-dihydroquinolines in relation to nucleophilic reagents allows to transform them easily into 4 methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acids, one being the basis for synthesis of

N-R-Amides of 2-oxo-1,2-dihydroquinoline-3-carboxylic acid with a primary amino group in position 4 of the quinolone ring exist in the 2-hydroxy-4-imino form rapidly hydrolyzed by mineral acids to 4-hydroxy-2-quinolones (Ukrainets et al., 2006b). Proceeding from it as the next object for pharmacological screening we deliberately obtained hydrochloride of 4 diethylamino-2-oxo-1-propyl-1,2-dihydroquinoline-3-carboxylic acid (2-diethylaminoethyl) amide **18** as chemically more stable product. Amides **19a-d** containing no substituents at position 4 are of particular interest, in spite of the fact that due to the absence of these

The study of local irritant action of the compounds synthesized, the ability to cause infiltration anaesthesia of the skin and subcutaneous cellulose, as well as the evaluation of the motor block and the sedative effect were carried out by standard methods previously described in detail by us (Ukrainets et al., 2010). It has been determined that all substances tested in the form of aqueous solutions with 2% concentration do not cause any reactive

From the data presented in Table 3 it follows that bioisosteric replacement of 4-ОН-group to the chlorine atom – amide **16** – leads to significant decrease of all pharmacological indexes

> N H

OBu-*i*

More interesting was the replacement of the hydroxyl group to the methyl one. From all substances of the last series 4-methyl-substituted amide **17** possesses the most rapid development of the biological effect (less than 2 min after injection). The infiltration anaesthesia index reaches the maximum possible value, and the total anaesthesia or the time of absence of pain and all types of sensitivity (tactile, temperature, etc.), during which the surgical intervention can be made (the section of tissues, wound suture, etc.), last

N Et

Et

**.** HCl

O

N O R

N H

**19: a** R = Et; **b** R = Pr; **c** R = Bu; **d** R = *i*-Bu

N Et

Et

**.** HCl

O

3-carboxylic acid (2-diethylaminoethyl)amide hydrochloride **16** (Figure 8).

one more bioisoster of Сhinoxicaine – 4-methyl substituted analogue **17**.

substituents they cannot be considered to be classical bioisosters of Сhinoxicaine.

changes on the skin surface of the experimental animals.

N Et

N

**20**

Et

**.** HCl

and, therefore, it is unsuccessful.

R

N

Pr

**16** R = Cl **17** R = Me **18** R = N(Et)2

O

O

N H

Fig. 8. Modification of 4-ОН-group of Сhinoxicaine



The sedative effect was estimated in the following way: 0 points - absent, the animal moving independently in cage; 1 point - the animal calm, sitting more, moving around the cage only when disturbed by the researcher; 2 points - the animal slowed down, sitting in the corner of the cage, anxiety with the researcher significantly set aside and again sitting, often closing eyelids, sleep onset; 3 points - the animal sleeping, lying on side, not responsive to stimulation by the researcher or to needle stick.

In general, the combination of analgesic, sedative and immobile extremities effects rendering by hydrochlorides **15a,e,f** can be used in creating medicines on their basis that are available for practical application in tiny surgical interventions, for example, in veterinary medicine. Thus, it can be stated confidently that 1-R-quinazoline-2,4-dionic cycle is bioisoster of 4-hydroxy-2-oxo-1,2-dihydroquinoline nucleus.

#### **5.3 The irreversible chemical modification of Сhinoxicaine at position 4 of the quinolone nucleus**

The complex research described by us above has shown convincingly that 4-ОН-group is the main cause of the local irritant properties of Сhinoxicaine. Therefore, after its blocking one can expect the elimination of the undesired side effect. Meanwhile, we have not even considered alkylation or acylation of 4-ОН-group as the most obvious variant of another bioreversible modification of Сhinoxicaine. The reason is quite simple. Within a rather limited choice of pharmacologically available protective groups, neither 4-О-alkyl, nor 4-Оacyl derivatives of 4-hydroxyquinolin-2-ones have a high chemical stability. It is the tendency to hydrolysis that is a serious obstacle when synthesizing such compounds, as well as when further preparing sterile solutions for injections on their basis.

Taking it into account we tried to modify 4-ОН-group of Сhinoxicaine not by means of forming pro-drugs, but by using the same method of bioisosteric replacements, i.e. by its irreversible replacement with the groupings similar not by sizes or volume, but having the same physical and chemical properties and that is why inducing the similar pharmacological effect (King, 2002).

Table 2. Biological properties of the quinazoline-2,4-diones hydrochlorides **15** 


**15a** 1.14 ± 0.16 36.0 32.35 ± 1.38 4 1 **15b** 1.53 ± 0.19 35.8 30.19 ± 0.75 0 0 **15c** 4.46 ± 0.29 18.5 15.74 ± 1.05 0 1 **15d** 2.97 ± 0.32 35.7 29.82 ± 0.59 0 0 **15e** 2.82 ± 0.43 36.0 36.46 ± 2.53 5 1 **15f** 1.59 ± 0.25 34.2 29.20 ± 1.43 5 3 **Chinoxicaine** 1.50 ± 0.04 36.0 75.61 ± 4.54 0 0 **Lidocaine** 2.12 ± 0.19 36.0 52.80 ± 3.76 0 0

The sedative effect was estimated in the following way: 0 points - absent, the animal moving independently in cage; 1 point - the animal calm, sitting more, moving around the cage only when disturbed by the researcher; 2 points - the animal slowed down, sitting in the corner of the cage, anxiety with the researcher significantly set aside and again sitting, often closing eyelids, sleep onset; 3 points - the animal sleeping, lying on side, not responsive to

In general, the combination of analgesic, sedative and immobile extremities effects rendering by hydrochlorides **15a,e,f** can be used in creating medicines on their basis that are available for practical application in tiny surgical interventions, for example, in veterinary medicine. Thus, it can be stated confidently that 1-R-quinazoline-2,4-dionic cycle is

The complex research described by us above has shown convincingly that 4-ОН-group is the main cause of the local irritant properties of Сhinoxicaine. Therefore, after its blocking one can expect the elimination of the undesired side effect. Meanwhile, we have not even considered alkylation or acylation of 4-ОН-group as the most obvious variant of another bioreversible modification of Сhinoxicaine. The reason is quite simple. Within a rather limited choice of pharmacologically available protective groups, neither 4-О-alkyl, nor 4-Оacyl derivatives of 4-hydroxyquinolin-2-ones have a high chemical stability. It is the tendency to hydrolysis that is a serious obstacle when synthesizing such compounds, as

Taking it into account we tried to modify 4-ОН-group of Сhinoxicaine not by means of forming pro-drugs, but by using the same method of bioisosteric replacements, i.e. by its irreversible replacement with the groupings similar not by sizes or volume, but having the same physical and chemical properties and that is why inducing the similar

**5.3 The irreversible chemical modification of Сhinoxicaine at position 4 of the** 

well as when further preparing sterile solutions for injections on their basis.

Duration of total anaesthesia, min

Motor block, points

Sedative effect, points

stimulation of the skin outside the area of anesthesia, the animal lying on side.

Infiltration anaesthesia

Table 2. Biological properties of the quinazoline-2,4-diones hydrochlorides **15** 

Index

Compound

**quinolone nucleus** 

pharmacological effect (King, 2002).

The start of anaesthesia, min

stimulation by the researcher or to needle stick.

bioisoster of 4-hydroxy-2-oxo-1,2-dihydroquinoline nucleus.

The first example of such transformation was 4-chloro-2-oxo-1-propyl-1,2-dihydroquinoline-3-carboxylic acid (2-diethylaminoethyl)amide hydrochloride **16** (Figure 8).

A high reactivity of the chlorine atom in 1-R-4-chloro-3-ethoxycarbonyl-2-oxo-1,2-dihydroquinolines in relation to nucleophilic reagents allows to transform them easily into 4 methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acids, one being the basis for synthesis of one more bioisoster of Сhinoxicaine – 4-methyl substituted analogue **17**.

N-R-Amides of 2-oxo-1,2-dihydroquinoline-3-carboxylic acid with a primary amino group in position 4 of the quinolone ring exist in the 2-hydroxy-4-imino form rapidly hydrolyzed by mineral acids to 4-hydroxy-2-quinolones (Ukrainets et al., 2006b). Proceeding from it as the next object for pharmacological screening we deliberately obtained hydrochloride of 4 diethylamino-2-oxo-1-propyl-1,2-dihydroquinoline-3-carboxylic acid (2-diethylaminoethyl) amide **18** as chemically more stable product. Amides **19a-d** containing no substituents at position 4 are of particular interest, in spite of the fact that due to the absence of these substituents they cannot be considered to be classical bioisosters of Сhinoxicaine.

The study of local irritant action of the compounds synthesized, the ability to cause infiltration anaesthesia of the skin and subcutaneous cellulose, as well as the evaluation of the motor block and the sedative effect were carried out by standard methods previously described in detail by us (Ukrainets et al., 2010). It has been determined that all substances tested in the form of aqueous solutions with 2% concentration do not cause any reactive changes on the skin surface of the experimental animals.

From the data presented in Table 3 it follows that bioisosteric replacement of 4-ОН-group to the chlorine atom – amide **16** – leads to significant decrease of all pharmacological indexes and, therefore, it is unsuccessful.

Fig. 8. Modification of 4-ОН-group of Сhinoxicaine

More interesting was the replacement of the hydroxyl group to the methyl one. From all substances of the last series 4-methyl-substituted amide **17** possesses the most rapid development of the biological effect (less than 2 min after injection). The infiltration anaesthesia index reaches the maximum possible value, and the total anaesthesia or the time of absence of pain and all types of sensitivity (tactile, temperature, etc.), during which the surgical intervention can be made (the section of tissues, wound suture, etc.), last

Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 77

index and reduction of duration of the deep anaesthesia phase. At the same time the general duration of anaesthesia increases a little, as well as duration of the sedative effect. Unfortunately, transfer of the isobutyl substituent from the nitrogen atom to the oxygen atom is accompanied by appearance of undesirable properties – unlike amide **19d** its

The research carried out by us gives reason to suppose that 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acids are of great interest as a base in creating new effective medicines to eliminate pain. Such medicines can be not only local anesthetics possessing the unique complex of pharmacological properties, but, as it has been found quite recently, nonnarcotic analgesics with high activity and low toxicity as well. The rich arsenal of structural and biological regularities accumulated, as well as practically unlimited synthetic potential of 4-hydroxyquinolin-2-ones allow to change the character of impact of such compounds on a living organism easily and in the required direction, and thus, to provide their direct

We appreciate the assistance of professor V.I. Mamchur (Dnepropetrovsk State Medical Academy, Ukraine) in studying biological properties of the compounds synthesized and

Bochkov, A.F. & Smit, V.A. (1987). *Organic Synthesis. Purposes, Methods, Tactics, Strategy* [in

Kartsev, V.G. (Ed.). (2007). *Selected Methods for Synthesis and Modification of Heterocycles. Vol. 6.* 

Kleemann, A. & Engel J. (2001). *Pharmaceutical Substances: Syntheses, Patents, Applications,* 

Tomoda, K.; Asahiyama, M.; Ohtsuki, E.; Nakajima, T.; Terada, H.; Kanebako, M.; Inagi, T. &

Kang, C. & Shin, S.C. (2010). Preparation and Evaluation of Bioadhesive Dibucaine Gels for

Douglas, H.A.; Callaway, J.K.; Sword, J.; Kirov, S.A. & Andrew, R.D. (2011). Potent

*Journal of Neurophysiology,* Vol.105, No.4, pp. 1482-1494, ISSN 0022-3077 Ukrainets, I.V. (1992). Synthesis, Chemical Transformation and Biological Properties of

Thieme Medical Publishers, ISBN 1588900312, Stuttgart, Germany

*Quinolines: Chemistry and Biological Activity* [in Russian]*,* International charitable foundation "Scientific Partnership Foundation" (ICSPF), ISBN 978-5-903078-10-3,

Makino, K. (2009). Preparation and Properties of Carrageenan Microspheres Containing Allopurinol and Local Anesthetic Agents for the Treatment of Oral Mucositis. *Colloids and Surfaces. B, Biointerfaces,* Vol.71, No.1, pp. 27-35, ISSN 0927-

Enhanced Local Anesthetic Action. *Archives of Pharmacal Research,* Vol.33, No.8, pp.

Inhibition of Anoxic Depolarization by the Sodium Channel Blocker Dibucaine.

Alkyl(aryl)amides of Malonic Acid Derivatives [in Russian]. *Thesis for Doctor Degree* 

aromatic isomer **20** has been found to have the irritant action, though a transient one.

**6. Conclusion** 

practical value and a great perspective.

Moscow, Russia

1277-1283, ISSN 0253-6269

7765

useful comments while discussing the results obtained.

Russian]*,* Nauka, Moscow, Russia

**7. Acknowledgment** 

**8. References** 

approximately 55 min. These data prove the sufficient high activity of amide **17**, which are comparable to the reference drugs - Chinoxicaine and Lidocaine. However, amide **17** yields them significantly in the total duration of anaesthesia, i.e. time when the sensitivity increases gradually and then restores completely.


Table 3. Biological properties of 4-ОН-modified derivatives of Chinoxicaine

A special attention should be paid to 4-diethylamine derivative **18**, not only for its high anaesthetic properties, but for the perspective to perform further modifications of such type easily and practically in unlimited quantity as well and to reach the result required.

From the series of non-substituted amides **19** at position 4 it is worth mentioning only compounds with butyl and *iso-*butyl substituents at the cyclic nitrogen atom (amides **19c** and **19d** respectively). Both are characterized by a rather rapid onset of action and high values of infiltration anaesthesia indexes. The distinctive feature of the first one is the signs of drowsiness, inertia in animals in 10-15 min after the injection and complete sleepiness can occur at 15-20 min. The motor block with the strength of 5 points lasts for approximately 20 min on the site of introduction of the substance examined. In the case of amide **19d** already by 7-10 min after injection the animals had the state of deep sleep: they slept on their side without the reaction to the active stimulation by the needle (tactile, pain and temperature sensitivity is absent). In 15-20 min the animals awoke, but they were drowsy and motionless for approximately 20 min and then began to move their paws. Therefore, one can speak about the deep and prolonged motor block and the marked sedative effect, which can be very useful properties of local anesthetic while conducting a number of short-termed surgical interventions, especially when rendering aid to patients with the increased excitability and possible fear before any surgical manipulations.

The study of hydrochloride of 2-isobutoxyquinoline-3-carboxylic acid (2-diethylaminoethyl)amide **20** is of particular interest. This compound has been specially synthesized by us as an aromatic analogue of the most active of 1,2-dihydro derivatives, i.e. amide **19d**. A comparative analysis of biological properties of these isomers demonstrates that with transfer to the aromatic structure some parameters decrease, and others, vice versa, intensify. For example, amide **20** differs with the later start of anaesthesia, decrease of the index and reduction of duration of the deep anaesthesia phase. At the same time the general duration of anaesthesia increases a little, as well as duration of the sedative effect. Unfortunately, transfer of the isobutyl substituent from the nitrogen atom to the oxygen atom is accompanied by appearance of undesirable properties – unlike amide **19d** its aromatic isomer **20** has been found to have the irritant action, though a transient one.

#### **6. Conclusion**

76 Pain Management – Current Issues and Opinions

approximately 55 min. These data prove the sufficient high activity of amide **17**, which are comparable to the reference drugs - Chinoxicaine and Lidocaine. However, amide **17** yields them significantly in the total duration of anaesthesia, i.e. time when the sensitivity

> The total anaesthesia, min

**16** 3.96 ± 0.42 26.3 14.25 ± 1.11 24.72 ± 2.18 0 0 **17** 1.94 ± 0.21 36.0 55.33 ± 2.74 68.38 ± 2.68 0 0 **18** 2.28 ± 0.31 36.0 37.51 ± 2.83 67.85 ± 2.37 0 0 **19a** 4.52 ± 0.32 19.3 13.20 ± 1.00 21.01 ± 1.67 0 0 **19b** 4.50 ± 0.36 35.5 27.89 ± 1.89 32.34 ± 2.92 0 0 **19c** 3.03 ± 0.28 36.0 39.04 ± 2.12 58.26 ± 2.81 5 2 **19d** 2.71 ± 0.37 36.0 53.77 ± 1.93 83.28 ± 2.05 5 3 **20** 2.82 ± 0.44 35.6 47.56 ± 1.74 85.48 ± 2.33 5 3 **Chinoxicaine** 1.62 ± 0.13 36.0 74.74 ± 4.71 236.89 ± 9.34 0 0 **Lidocaine** 2.34 ± 0.20 36.0 51.26 ± 3.45 140.27 ± 6.20 0 0

A special attention should be paid to 4-diethylamine derivative **18**, not only for its high anaesthetic properties, but for the perspective to perform further modifications of such type

From the series of non-substituted amides **19** at position 4 it is worth mentioning only compounds with butyl and *iso-*butyl substituents at the cyclic nitrogen atom (amides **19c** and **19d** respectively). Both are characterized by a rather rapid onset of action and high values of infiltration anaesthesia indexes. The distinctive feature of the first one is the signs of drowsiness, inertia in animals in 10-15 min after the injection and complete sleepiness can occur at 15-20 min. The motor block with the strength of 5 points lasts for approximately 20 min on the site of introduction of the substance examined. In the case of amide **19d** already by 7-10 min after injection the animals had the state of deep sleep: they slept on their side without the reaction to the active stimulation by the needle (tactile, pain and temperature sensitivity is absent). In 15-20 min the animals awoke, but they were drowsy and motionless for approximately 20 min and then began to move their paws. Therefore, one can speak about the deep and prolonged motor block and the marked sedative effect, which can be very useful properties of local anesthetic while conducting a number of short-termed surgical interventions, especially when rendering aid to patients with the increased

The study of hydrochloride of 2-isobutoxyquinoline-3-carboxylic acid (2-diethylaminoethyl)amide **20** is of particular interest. This compound has been specially synthesized by us as an aromatic analogue of the most active of 1,2-dihydro derivatives, i.e. amide **19d**. A comparative analysis of biological properties of these isomers demonstrates that with transfer to the aromatic structure some parameters decrease, and others, vice versa, intensify. For example, amide **20** differs with the later start of anaesthesia, decrease of the

easily and practically in unlimited quantity as well and to reach the result required.

Motor block, points

The general duration of anaesthesia, min

Sedative effect, points

Infiltration anaesthesia

Index

Table 3. Biological properties of 4-ОН-modified derivatives of Chinoxicaine

excitability and possible fear before any surgical manipulations.

increases gradually and then restores completely.

The start of anaesthesia, min

Compound

The research carried out by us gives reason to suppose that 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylic acids are of great interest as a base in creating new effective medicines to eliminate pain. Such medicines can be not only local anesthetics possessing the unique complex of pharmacological properties, but, as it has been found quite recently, nonnarcotic analgesics with high activity and low toxicity as well. The rich arsenal of structural and biological regularities accumulated, as well as practically unlimited synthetic potential of 4-hydroxyquinolin-2-ones allow to change the character of impact of such compounds on a living organism easily and in the required direction, and thus, to provide their direct practical value and a great perspective.

#### **7. Acknowledgment**

We appreciate the assistance of professor V.I. Mamchur (Dnepropetrovsk State Medical Academy, Ukraine) in studying biological properties of the compounds synthesized and useful comments while discussing the results obtained.

#### **8. References**


Creation of New Local Anesthetics Based on Quinoline Derivatives and Related Heterocycles 79

Chen, H.; Gong, Y.; Gries, R.M. & Plettner, E. (2010). Synthesis and Biological Activity of

Watanabe, M.; Hirokawa, T.; Kobayashi, T.; Yoshida, A.; Ito, Y.; Yamada, S.; Orimoto, N.;

Nirogia, R.V.; Kambhampati, R.; Daulatabad, A.V.; Gudla, P.; Shaikh, M.; Achanta, P.K.;

Ukrainets, I.V.; Tkach, A.A. & Grinevich, L.A. (2008). 4-Hydroxy-2-Quinolones. 148. Synthe-

Baumer, V.N.; Shishkin, O.V.; Ukrainets, I.V.; Sidorenko, L.V. & El Kayal, S.A. (2004). 1-

Ukrainets, I.V.; Gorokhova, O.V.; Sidorenko, L.V. & Bereznyakova, N.L. (2007). 4-Hydroxy-

Ukrainets, I.V.; Sidorenko, L.V.; Gorokhova, O.V.; Mospanova, E.V. & Shishkin, O.V.

Kravtsova, V.V. (2011). The Search of New Local Anesthetics in the Range of Amide Derivati-

King, F.D. (Ed.). (2002). *Medicinal Chemistry: Principles and Practice,* Royal Society of

Devereux, M. & Popelier, P.L. (2010). In Silico Techniques for the Identification of Bioisoste-

Wassermann, A.M. & Bajorath, J. (2011). Large-scale Exploration of Bioisosteric Replace-

Large, J.M.; Torr, J.E.; Raynaud, F.I.; Clarke, P.A.; Hayes, A.; Stefano, F.; Urban, F.; Shuttle-

and Medicinal Chemistry*,* Vol.19, No.2, pp. 836-851, ISSN 0968-0896

Medicinal Chemistry, Vol.18, No.8, pp. 2920-2929, ISSN 0968-0896

*Chemistry,* Vol.26, No.3, pp. 341-349, ISSN 1475-6366

*Compounds,* Vol.44, No.8, pp. 956-966, ISSN 0009-3122

pp. 3585-93, ISSN 0022-2623

pp. o2356-o2358, ISSN 1600-5368

No.1, pp. 58-62, ISSN 0009-3122

Manuscript, Kharkov, Ukraine

No.6, pp. 657-668, ISSN 1568-0266

No.4, pp. 425-36, ISSN 1756-8919

Chemistry, ISBN 0854046313, Cambridge, UK

Conformationally Restricted Gypsy Moth Pheromone Mimics. Bioorganic and

Yamasaki, Y.; Arisawa, M. & Shuto, S. (2010). Investigation of the Bioactive Conformation of Histamine H3 Receptor Antagonists by the Cyclopropylic Strain-based Conformational Restriction Strategy. Journal of Medicinal Chemistry, Vol.53, No.9,

Shinde, A.K. & Dubey, P.K. (2011). Design, Synthesis and Pharmacological Evaluation of Conformationally Restricted N-Arylsulfonyl-3-Aminoalkoxy Indoles as a Potential 5-HT(6) Receptor Ligands. *Journal of Enzyme Inhibition and Medicinal* 

sis and Anti-tubercular Activity of 1-Hydroxy-3-Oxo-6,7-Dihydro-3H,5H-Pyrido- [3,2,1-*ij*]quinoline-2-Carboxylic Acid N-R-Amides. *Chemistry of Heterocyclic* 

Ethyl-4-Hydroxyquinolin-2(1H)-one. *Acta Crystallographica Section E,* Vol.60, No.12,

2-Quinolones. 111. Simple Synthesis of 1-Substituted 4-Methyl-2-Oxo-1,2- Dihydroquino-line-3-Carboxylic Acids. *Chemistry of Heterocyclic Compounds,* Vol.43,

(2006a). 4-Hydroxy-2-Quinolones. 94. Improved Synthesis and Structure of 1- Hydroxy-3-Oxo-5,6-Dihydro-3H-Pyrrolo[3,2,1-*i,j*]quinoline-2-Carboxylic Acid Ethyl Ester. *Chemistry of Heterocyclic Compounds,* Vol.42, No.5, pp. 631-635, ISSN 0009-3122

ves of Oxoquinoline-3-Carboxylic Acids [in Ukrainian]. *Thesis for Candidate Degree in Pharmacy in speciality 15.00.02 – Pharmaceutical Chemistry and Pharmacognosy,* 

ric Replacements for Drug Design. *Current Topics in Medicinal Chemistry,* Vol.10,

ments on the Basis of Matched Molecular Pairs. *Future Medicinal Chemistry*, Vol.3,

worth, S.J.; Saghir, N.; Sheldrake, P.; Workman, P. & McDonald, E. (2011). Preparation and Evaluation of Trisubstituted Pyrimidines as Phosphatidylinositol 3-Kinase Inhibitors. 3-Hydroxyphenol Analogues and Bioisosteric Replacements. Bioorganic

*in Chemistry in speciality 15.00.02 – Pharmaceutical Chemistry and Pharmacognosy,*  Manuscript, Kharkov, Ukraine


Ukrainets, I.V.; Gorokhova, O.V.; Taran, S.G.; Bezugly, P.A.; Filimonova, N.I. & Turov, A.V.

Gorokhova, O.V. (1993). Synthesis, Chemical and Biological Properties of Alkyl- and

Davidenko, O.O. (2011). Synthesis, Physical, Chemical Properties and Biological Activity of

Ukrainets, I.V.; Bezugly, P.A.; Gorokhova, O.V.; Taran, S.G. & Treskach, V.I. (1998). Method

Ukrayinecz, I.V. & Bezuhliy, P.A. (2002). Injectable Anesthetic. *Patent USA 6340692*,

http://worldwide.espacenet.com/searchResults?NUM=US6340692&DB=EPODOC

Romanov, I.V. & Ukrainets, I.V. (2006). Method for Preparing 1-Propyl-2-Oxo-4-Hydroxyquinoline-3-Carboxylic Acid Diethylaminoethylamide Hydrochloride ( Chinoxy-

http://worldwide.espacenet.com/searchResults?NUM=RU2285692&DB=EPODO

Kubinyi, H. (2006). In Looking ups of the New Compounds-leaders for Creation of Drugs

Ukrainets, I.V. (1988). Synthesis and Research of New Biological Active Derivatives of

Ukrainets, I.V.; Kravtzova, V.V.; Tkach, A.A. & Rybakov, V.B. (2009). 4-Hydroxy-2-Quino-

Vinogradova, N.D.; Kuznetsov, S.G. & Chigareva, S.M. (1980). Quaternary Ammonium Salts

*Heterocyclic Compounds,* Vol.45, No.6, pp. 698-704, ISSN 0009-3122

[in Russian]. *Russian Chemical Journal*, Vol.L, No.2, pp. 5-17, ISSN 0373-0247, Available from http://www.chem.msu.su/rus/journals/jvho/2006-2/5.pdf Kuznetsov, S.G.; Chigareva, S.M. & Ramsh, S.M. (1991). Pro-drugs. Chemical Aspect.

*Summaries in Science and Technology. Organic Chemistry* [in Russian], VINITI, ISSN

Malonic Acid 2-Carboxyphenylamide [in Russian]. *Thesis for Candidate Degree in Pharmacy in speciality 15.00.02 – Pharmaceutical Chemistry and Pharmacognosy,* 

lones. 155. Bioreversible Chemical Modification of Chinoxycaine at the Tertiary Amino Group as a Method of Improving its Pharmaceutical Activity. *Chemistry of* 

with Labile N+-C Bonds as Drug Precursors. *Pharmaceutical Chemistry Journal*,

Manuscript, Kharkov, Ukraine

Manuscript, Kharkov, Ukraine

http://base.ukrpatent.org/searchINV/

&locale=en\_EP&ST=number&compact=false

caine). *Patent Russia 2285692*, Available from

C&locale=en\_EP&ST=number&compact=false

0137-0251, Moscow, Russia

Manuscript, Kharkov, Ukraine

Vol.14, No.9, pp. 604-609, ISSN 0091-150X

ISSN 0009-3122

Ukraine

Available from

*in Chemistry in speciality 15.00.02 – Pharmaceutical Chemistry and Pharmacognosy,* 

(1994). 4-Hydroxy-2-Quinolones. 24. Improved Synthesis and Biological Properties of 1-Alkyl-4-Hydroxy-2-Quinoline-3-Carboxylic Acid β-Dialkylaminoalkylamide Hydrochlorides. *Chemistry of Heterocyclic Compounds,* Vol.30, No.10, pp. 1214-1219,

Arylamides of Malonic Acid Derivatives [in Russian]. *Thesis for Candidate Degree in Chemistry in speciality 15.00.02 – Pharmaceutical Chemistry and Pharmacognosy,* 

Substituted 4-Hydroxy-2-Oxo-1,2-Dihydroquinoline-3-Carboxylic Acids and Their Derivatives [in Ukrainian]. *Thesis for Candidate Degree in Pharmacy in speciality 15.00.02 – Pharmaceutical Chemistry and Pharmacognosy,* Manuscript, Kharkov,

for Preparing 1-Propyl-2-Oxo-4-Hydroxyquinoline-3-Carboxylic Acid Diethylaminoethylamide Hydrochloride (Chinoxycaine). *Patent Ukraine 24967*, Available from


**5** 

*Iran* 

**Neuroprotection and Pain Management** 

Pain, as a sub modality of somatic sensation, has been defined as a complex constellation of unpleasant sensory, emotional and cognitive experiences provoked by real or perceived tissue damage and manifested by certain autonomic, psychological, and behavioral reactions. The benefit of these unpleasant sensations, however, is underscored by extreme cases: patients lacking the ability to perceive pain due to hereditary neuropathies often maintain unrealized infections; self mutilate, and have curtailed life spans. Normally, nociception and the perception of pain are evoked only at pressures and temperatures extreme enough to potentially injured tissues and by toxic molecules and inflammatory mediators. As opposed to the relatively more objective nature of other senses, pain is highly individual and subjective and the translation of nociception into pain perception can be

Chronic pain is estimated to affect millions of people worldwide and is one of the most common reasons for physician visits (Scascighini et al. 2008). Inflammation may cause direct painful stimuli as well as sensitize nociceptors to stimulation (McMahon et al. 2005). Thus, there are multiple points along the pain pathway that represent opportunities for therapeutic intervention. Despite this, there are only a limited number of mechanisms through which current pain medications work. Major classes of analgesics include opioids, non-steroidal anti-inflammatory drugs, antidepressants, and anticonvulsants. Although these treatments provide relief, the effects are often incomplete and complicated by serious side effects and/or tolerance. Thus, therapeutics with novel mechanisms of actions are

What exactly, from a neurobiological perspective, is pain? Pain is actually three quite different things, although it is difficult to make the distinction; nociceptive pain, inflammatory pain and neuropathic pain. Nociceptive pain is not a clinical problem, except in the specific context of surgery and other clinical procedures that necessarily involve noxious stimuli, where it must be suppressed by local and general anesthetics or high-dose

Nociception involves multiple steps from the peripheral receptor, the afferent nerve transmitting the impulse to the spinal cord, the signal processing in the dorsal horn, with inhibitory and facilitatory elements and finally transmission to higher cerebral centers where the peripheral nociceptive stimulus is perceived as pain (Arendt-Nielsen and

The second kind of pain is also adaptive and protective. By heightening sensory sensitivity after unavoidable tissue damage, this pain assists in the healing of the injured body part by

curtailed by stress or exacerbated by anticipation (Woolf).

desperately needed (Finnerup et al. 2005).

opioids (Woolf).

Sumikura 2002).

**1. Introduction** 

Kambiz Hassanzadeh and Esmael Izadpanah *Kurdistan University of Medical Sciences, Sanandaj* 


### **Neuroprotection and Pain Management**

Kambiz Hassanzadeh and Esmael Izadpanah

*Kurdistan University of Medical Sciences, Sanandaj Iran* 

#### **1. Introduction**

80 Pain Management – Current Issues and Opinions

Kolisnyk, O.V. (2009). Synthesis, Physical, Chemical and Biological Properties of 4-Hydroxy-

*Pharmaceutical Chemistry and Pharmacognosy,* Manuscript, Kharkov, Ukraine Ukrainets, I.V., Kravtsova, V.V., Tkach, A.A., Mamchur, V.I. & Kovalenko, E.Yu. (2010). 4-

Ukrainets, I.V.; Sidorenko, L.V.; Gorokhova, O.V. & Jaradat, N.A. (2006b). 4-Hydroxy-2-

*Compounds,* Vol.46, No.1, pp. 96-105, ISSN 0009-3122

*Compounds,* Vol.42, No.4, pp. 475-487, ISSN 0009-3122

2-Oxo-1,2,5,6,7,8-Hexahydroquinoline-3-Carboxylic Acids Amidation Derivatives. [in Ukrainian]. *Thesis for Candidate Degree in Pharmacy in speciality 15.00.02 –* 

Hydroxy-2-Quinolones. 173. 1-R-3-(2-Diethylaminoethyl)-1H-Quinazoline-2,4-Dione Hydrochlorides as Potential Local Anesthetic Agents. *Chemistry of Heterocyclic* 

Quinolones. 93. Synthesis and Biological Properties of 2-Hydroxy-4-Imino-1,4- Dihydroquinoline-3-Carboxylic Acid N-R-Amides. *Chemistry of Heterocyclic* 

> Pain, as a sub modality of somatic sensation, has been defined as a complex constellation of unpleasant sensory, emotional and cognitive experiences provoked by real or perceived tissue damage and manifested by certain autonomic, psychological, and behavioral reactions. The benefit of these unpleasant sensations, however, is underscored by extreme cases: patients lacking the ability to perceive pain due to hereditary neuropathies often maintain unrealized infections; self mutilate, and have curtailed life spans. Normally, nociception and the perception of pain are evoked only at pressures and temperatures extreme enough to potentially injured tissues and by toxic molecules and inflammatory mediators. As opposed to the relatively more objective nature of other senses, pain is highly individual and subjective and the translation of nociception into pain perception can be curtailed by stress or exacerbated by anticipation (Woolf).

> Chronic pain is estimated to affect millions of people worldwide and is one of the most common reasons for physician visits (Scascighini et al. 2008). Inflammation may cause direct painful stimuli as well as sensitize nociceptors to stimulation (McMahon et al. 2005). Thus, there are multiple points along the pain pathway that represent opportunities for therapeutic intervention. Despite this, there are only a limited number of mechanisms through which current pain medications work. Major classes of analgesics include opioids, non-steroidal anti-inflammatory drugs, antidepressants, and anticonvulsants. Although these treatments provide relief, the effects are often incomplete and complicated by serious side effects and/or tolerance. Thus, therapeutics with novel mechanisms of actions are desperately needed (Finnerup et al. 2005).

> What exactly, from a neurobiological perspective, is pain? Pain is actually three quite different things, although it is difficult to make the distinction; nociceptive pain, inflammatory pain and neuropathic pain. Nociceptive pain is not a clinical problem, except in the specific context of surgery and other clinical procedures that necessarily involve noxious stimuli, where it must be suppressed by local and general anesthetics or high-dose opioids (Woolf).

> Nociception involves multiple steps from the peripheral receptor, the afferent nerve transmitting the impulse to the spinal cord, the signal processing in the dorsal horn, with inhibitory and facilitatory elements and finally transmission to higher cerebral centers where the peripheral nociceptive stimulus is perceived as pain (Arendt-Nielsen and Sumikura 2002).

> The second kind of pain is also adaptive and protective. By heightening sensory sensitivity after unavoidable tissue damage, this pain assists in the healing of the injured body part by

Neuroprotection and Pain Management 83

Glutamatergic receptors include both iontropic and metabotropic receptor subtypes. The iontropic receptors include N-Methyl-D-Aspartat (NMDA), α-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA), and kainate receptors. Binding of glutamate to these receptors causes Ca2+ and Na+ entry into neurons, resulting in excitatory postsynaptic potentials and membrane depolarization. In addition, increased intracellular Ca2+ levels activate a number of signaling cascades (Berridge 1998). The NMDA receptor forms a channel allowing for ion influx, whereas the AMPA and kainate receptors open voltagesensitive ion channels on the cell membrane. The NMDA receptor is voltage-gated and is blocked by magnesium and modulated by two coagonists, glycine and d-serine, as well as by several intracellular and extracellular mediators (Millan 2005)). It has been proposed that NMDA receptor hypofunction may lead to excessive stimulation of other iontropic receptors, causing a cascade of excitotoxic events including oxidative stress and apoptosis (Deutsch et al. 2001). Dysregulation of glutamateric functioning has been observed across

The mechanism of glutamate-induced neuronal death has been extensively studied: glutamate induces neuronal death *via* stimulation of NMDA receptor through which Ca2+ enters the cell and activates Ca2+-dependent nitric oxide (NO) synthase, resulting in excessive nitric oxide formation, production of radicals, mitochondrial dysfunction and cell death (Kaneko et al. 1997). It has been shown that glutamate induces neuronal death associated with necrosis and apoptosis. Necrosis is caused by catastrophic cell damage and is characterized by cell swelling, injury to cytoplasmic organelles and rapid collapse of internal homeostasis, leading to the lysis of membranes and the release of cellular contents, resulting in inflammation. On the other hand, apoptosis is a process characterized by cell shrinkage, membrane blebbing, nuclear pyknosis, chromatin condensation and genomic fragmentation (Kerr et al. 1972; Schulte-Hermann et al. 1992;

In rodents, blocking of NMDA receptors is associated with increased release of glutamate within the cerebral cortex (Moghaddam et al. 1997), (Adams and Moghaddam 1998) and nucleus accumbens (Razoux et al. 2007). However, elevations in glutamate within the prefrontal cortex of rodents occurs during short-term administration of NMDA antagonists, whereas long-term administration over 7 consecutive days actually results in a trend for lower basal levels and lower dialysate levels of glutamate upon challenge (Zuo et al. 2006). Thus, excitotoxic events associated with NMDA antagonists may be reflected by initial increases in glutamatergic neurotransmission that are followed subsequently and

As noted before glutamate can induce apoptosis via NMDA receptor activation. Apoptosis or programmed cell death is a process normally associated with the elimination of redundant neurons during neurodevelopment (Johnson et al. 1995). Apoptosis involves the regulation of a complex molecular cascade controlling the activation of a family of cysteine proteases known as caspase proteins (Glantz et al. 2006). Caspases are responsible for breaking down important structural and functional proteins, leading to cellular degradation and eventually death. Apoptosis results from a cascade of gene activation and involves genes that both promote (i.e., Bax) (Schlesinger et al. 1997), (Gross et al. 1998) and oppose

**4. Apoptosis and** *N***-Methyl-***D***-Aspartate antagonist-induced** 

many components of the glutamate neurotransmission system.

Takada-Takatori et al. 2009).

chronically by lower levels.

**neurodegeneration**

creating a situation that discourages physical contact and movement. Pain hypersensitivity, or tenderness, reduces further risk of damage and promotes recovery, as after a surgical wound or in an inflamed joint, where normally innocuous stimuli now elicit pain. This pain is caused by activation of the immune system by tissue injury or infection, and is therefore called inflammatory pain.

Finally, there is the pain that is not protective, but maladaptive, resulting from abnormal functioning of the nervous system. This pathological pain, which is not a symptom of some disorder but rather a disease state of the nervous system, can occur after damage to the nervous system (neuropathic pain), but also in conditions in which there is no such damage or inflammation (dysfunctional pain) (Woolf).

The incidence of pain rises as people get older and women are more likely to be in pain than men. Pain management strategies include pain relieving medications, physical or occupational therapy and complementary therapies (such as acupuncture and massage).

Pharmacologic therapies are the foundation of chronic pain management. These therapies include nonopioids, opioids, and adjuvant analgesics, physical techniques physical measures, such as physical activity, physical and occupational therapy, orthotics, and assistive devices can serve as adjuncts to analgesics in the management of chronic pain (Paice and Ferrell).

On the other hand in recent years, we and others have focused on the relationship between neuroprotection and pain mechanism and management. Thus in this chapter we will review recent progress related to neuronal mechanism for using neuroprotective agents alone or in combination with antinociceptive drugs to reduce the pain. In addition we will focus on the effect of neuroprotective agents on prevention of tolerance to the analgesic effect of opiates.

#### **2. Neuroprotection**

Neuroprotection is the mechanism and strategies used to protect against neural injury or degeneration in the central nervous system (CNS). There is a wide range of neuroprotective products available or under investigation. Some products with neuroprotective effects are grouped into the following categories:


To better understand, we first discuss the mechanism by which neurotoxins induce toxicity.

#### **3. Glutamate**

Glutamate is a neurotransmitter with roles such as long-term potentiation and synaptic plasticity of the brain (Harris et al. 1984) and is also a exitotoxin whose neurotoxicity has been associated with numerous neurodegenerative diseases, such as Alzheimer disease (AD), (Kihara et al. 2002) vascular dementia, (Martinez et al. 1993) Parkinson disease (Greenamyre 2001) and amyotrophic lateral sclerosis (Cid et al. 2003).

Glutamatergic synapses are the key excitatory synapses within the brain, and mechanisms of both hyperglutamatergic and hypoglutamatergic functioning have been implicated in the pathophysiology of CNS disorders (Olney et al. 1999).

creating a situation that discourages physical contact and movement. Pain hypersensitivity, or tenderness, reduces further risk of damage and promotes recovery, as after a surgical wound or in an inflamed joint, where normally innocuous stimuli now elicit pain. This pain is caused by activation of the immune system by tissue injury or infection, and is therefore

Finally, there is the pain that is not protective, but maladaptive, resulting from abnormal functioning of the nervous system. This pathological pain, which is not a symptom of some disorder but rather a disease state of the nervous system, can occur after damage to the nervous system (neuropathic pain), but also in conditions in which there is no such damage

The incidence of pain rises as people get older and women are more likely to be in pain than men. Pain management strategies include pain relieving medications, physical or occupational therapy and complementary therapies (such as acupuncture and massage). Pharmacologic therapies are the foundation of chronic pain management. These therapies include nonopioids, opioids, and adjuvant analgesics, physical techniques physical measures, such as physical activity, physical and occupational therapy, orthotics, and assistive devices can serve as adjuncts to analgesics in the management of chronic pain

On the other hand in recent years, we and others have focused on the relationship between neuroprotection and pain mechanism and management. Thus in this chapter we will review recent progress related to neuronal mechanism for using neuroprotective agents alone or in combination with antinociceptive drugs to reduce the pain. In addition we will focus on the effect of neuroprotective agents on prevention of tolerance to the analgesic effect of opiates.

Neuroprotection is the mechanism and strategies used to protect against neural injury or degeneration in the central nervous system (CNS). There is a wide range of neuroprotective products available or under investigation. Some products with neuroprotective effects are

To better understand, we first discuss the mechanism by which neurotoxins induce toxicity.

Glutamate is a neurotransmitter with roles such as long-term potentiation and synaptic plasticity of the brain (Harris et al. 1984) and is also a exitotoxin whose neurotoxicity has been associated with numerous neurodegenerative diseases, such as Alzheimer disease (AD), (Kihara et al. 2002) vascular dementia, (Martinez et al. 1993) Parkinson disease

Glutamatergic synapses are the key excitatory synapses within the brain, and mechanisms of both hyperglutamatergic and hypoglutamatergic functioning have been implicated in the

(Greenamyre 2001) and amyotrophic lateral sclerosis (Cid et al. 2003).

pathophysiology of CNS disorders (Olney et al. 1999).

called inflammatory pain.

(Paice and Ferrell).

**2. Neuroprotection** 

grouped into the following categories:

 Free radical scavengers Anti excitotoxic agents Anti apoptotic agents Anti inflammatory agents Neurotrophic factors

**3. Glutamate** 

or inflammation (dysfunctional pain) (Woolf).

Glutamatergic receptors include both iontropic and metabotropic receptor subtypes. The iontropic receptors include N-Methyl-D-Aspartat (NMDA), α-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA), and kainate receptors. Binding of glutamate to these receptors causes Ca2+ and Na+ entry into neurons, resulting in excitatory postsynaptic potentials and membrane depolarization. In addition, increased intracellular Ca2+ levels activate a number of signaling cascades (Berridge 1998). The NMDA receptor forms a channel allowing for ion influx, whereas the AMPA and kainate receptors open voltagesensitive ion channels on the cell membrane. The NMDA receptor is voltage-gated and is blocked by magnesium and modulated by two coagonists, glycine and d-serine, as well as by several intracellular and extracellular mediators (Millan 2005)). It has been proposed that NMDA receptor hypofunction may lead to excessive stimulation of other iontropic receptors, causing a cascade of excitotoxic events including oxidative stress and apoptosis (Deutsch et al. 2001). Dysregulation of glutamateric functioning has been observed across many components of the glutamate neurotransmission system.

The mechanism of glutamate-induced neuronal death has been extensively studied: glutamate induces neuronal death *via* stimulation of NMDA receptor through which Ca2+ enters the cell and activates Ca2+-dependent nitric oxide (NO) synthase, resulting in excessive nitric oxide formation, production of radicals, mitochondrial dysfunction and cell death (Kaneko et al. 1997). It has been shown that glutamate induces neuronal death associated with necrosis and apoptosis. Necrosis is caused by catastrophic cell damage and is characterized by cell swelling, injury to cytoplasmic organelles and rapid collapse of internal homeostasis, leading to the lysis of membranes and the release of cellular contents, resulting in inflammation. On the other hand, apoptosis is a process characterized by cell shrinkage, membrane blebbing, nuclear pyknosis, chromatin condensation and genomic fragmentation (Kerr et al. 1972; Schulte-Hermann et al. 1992; Takada-Takatori et al. 2009).

In rodents, blocking of NMDA receptors is associated with increased release of glutamate within the cerebral cortex (Moghaddam et al. 1997), (Adams and Moghaddam 1998) and nucleus accumbens (Razoux et al. 2007). However, elevations in glutamate within the prefrontal cortex of rodents occurs during short-term administration of NMDA antagonists, whereas long-term administration over 7 consecutive days actually results in a trend for lower basal levels and lower dialysate levels of glutamate upon challenge (Zuo et al. 2006). Thus, excitotoxic events associated with NMDA antagonists may be reflected by initial increases in glutamatergic neurotransmission that are followed subsequently and chronically by lower levels.

#### **4. Apoptosis and** *N***-Methyl-***D***-Aspartate antagonist-induced neurodegeneration**

As noted before glutamate can induce apoptosis via NMDA receptor activation. Apoptosis or programmed cell death is a process normally associated with the elimination of redundant neurons during neurodevelopment (Johnson et al. 1995). Apoptosis involves the regulation of a complex molecular cascade controlling the activation of a family of cysteine proteases known as caspase proteins (Glantz et al. 2006). Caspases are responsible for breaking down important structural and functional proteins, leading to cellular degradation and eventually death. Apoptosis results from a cascade of gene activation and involves genes that both promote (i.e., Bax) (Schlesinger et al. 1997), (Gross et al. 1998) and oppose

Neuroprotection and Pain Management 85

shown that acetylcholinesterase inhibitors protect neurons from glutamate-induced

The long-standing belief was that acetylcholinesterase inhibitors are symptomatic agents that ameliorate cholinergic deficits by slowing the hydrolysis of acetylcholinesterase at synaptic nerve termini; however, recent studies have shown that acetylcholinesterase inhibitors have other pharmacological properties, for example, neuroprotection against toxic insults, such as glutamate and up-regulation of nicotinic receptors (Akaike 2006), (Takada-

Several reports have indicated that activation of cholinergic neurons in the central nervous system produces antinociception and analgesia in a variety of animals, including humans (Harte et al. 2004) provide evidence supporting the involvement of the intralaminar thalamus in muscarinic induced antinociception. Pharmacological experiments have shown that the microinjection of acetylcholine or carbachol into specific brainstem nuclei can produce antinociception and can be reversed by muscarinic receptor antagonists (Brodie and Proudfit 1984), (Yaksh et al. 1985). Meanwhile some other types of receptors or drugs produce analgesia by mediation of ACh. Sumatriptan (5- HT1agonist) is able to induce antinociception by increasing cholinergic neurotransmission (Ghelardini et al. 1997). D2 antagonist prochlorperazine exerts an antinocicptive effect mediated by a central cholinergic mechanism (Ghelardini et al. 2004), (Yang et al. 2008). In addition, more recently we showed that an acethylcolinesterase inhibitor, donepezil, could prevent tolerance to the analgesic

Pain severely impairs quality of life. Currently available treatments, generally opioids and anti-inflammatory drugs, are not always effective for certain painful conditions. The discovery of the cannabinoid receptors in the 1990s led to the characterization of the endogenous cannabinoid system in terms of its components and numerous basic physiologic functions. Cannabinoid1 (CB1) receptors are present in nervous system areas involved in modulating nociception and evidence supports a role of the endocannabinoids in pain modulation. Cannabinoids have antinociceptive mechanisms different from that of other drugs currently in use, which thus opens a new line of promising treatment to mitigate pain that fails to respond to the pharmacologic treatments available, especially for

Cannabis extracts and synthetic cannabinoids are still widely considered illegal substances. The Cannabis sativa plant has been exploited for medicinal, agricultural and spiritual purposes in diverse cultures over thousands of years. Cannabis has been used recreationally for its psychotropic properties, while effects such as stimulation of appetite, analgesia and anti-emesis have lead to the medicinal application of cannabis. Indeed, reports of medicinal efficacy of cannabis can been traced back as far as 2700 BC, and even at that time reports

Preclinical and clinical studies have suggested that they may result useful to treat diverse diseases, including those related with acute or chronic pain. The discovery of cannabinoid receptors, their endogenous ligands, and the machinery for the synthesis, transport, and degradation of these retrograde messengers, has equipped us with neurochemical tools for novel drug design. Agonist-activated cannabinoid receptors, modulate nociceptive thresholds, inhibit release of pro-inflammatory molecules, and display synergistic effects

neurotoxicity in the primary culture of rat cortical neurons.

Takatori et al. 2009).

effect of morphine (unpublished data).

**7. Cannabinoids, pain and neuroprotection** 

neuropathic and inflammatory pains (Manzanares et al. 2006).

also suggested a neuroprotective effect of the cultivar (Scotter et al.).

the process (i.e., Bcl-2) (Craig 1995), (Schlesinger et al. 1997), (Adams and Cory 1998). In a study we showed that there is a relation between glutamate increase and apoptosis promotion and increase in proapoptotic agent activity in both cerebral cortex and lumbar spinal cord of rat (Hassanzadeh et al.).

A vast array of stimuli can activate apoptosis in neurons (Sastry and Rao 2000). Many of these stimuli have been implicated in the pathophysiology of opioid–induced tolerance including glutamate excitotoxicity, increased calcium flux and mitochondria dysfunction and these mechanisms are discussed in detail later in this chapter.

#### **5. Neuroactive steroids are neuroprotective**

Neuroactive steroids are endogenous neuromodulators synthesized either within the brain (neurosteroids) or in the periphery by the adrenal glands and gonads. In addition to the classic effect of steroids on gene transcription via binding to intracellular steroid receptors, neuroactive steroids can alter neuronal excitability via nongenomic effects by acting at inhibitory Gama Amino Butiric Acid A (GABAA) receptors and/or excitatory NMDA receptors, among others (Shulman and Tibbo 2005), (Marx et al. 2006). There is also evidence for a potential role of these neurosteroids in controlling GABA and glutamate release. Neuroactive steroids have also been implicated in neuroprotection, myelination, and modulation of the stress response. A number of neuroactive steroids are present in human postmortem brain at physiologically relevant nanomolar concentrations and serve as allosteric modulators of the GABAA receptor (Marx et al. 2006). Neuroactive steroids that are effective modulators of GABAA and/or T-type Ca2+ channels are promising tools for studying the role of these channels in peripheral pain perception. They appear to be very effective in alleviating peripheral Nociception in rat models of acute and chronic pain (Jevtovic-Todorovic et al. 2009).

#### **6. Acetyl Choline Receptors (AChRs) and neuroprotection**

Agonists and antagonist selective for AChR subtypes have been used in experimental and clinical research. Some of those compounds are potential candidates for the treatment of neurodegenerative disease such as Alzheimer's disease, Parkinson's disease and others. A growing list of *in vivo* and *in vitro* research suggest that AChRs modulators are gaining importance as clinically relevant neuroprotective drugs (Mudo et al. 2007).

The inhibition of α7 AChRs decreases the GABAergic tone causing increased ACh release into the synaptic cleft (Giorgetti et al. 2000), which then activates the α4β2 AChRs located post-synaptically. The selective α7 inhibitor methyllycaconitine (Ivy Carroll et al. 2007) mimics, at least in part, the neuroprotective effect of 4R (Ferchmin et al. 2003). Other i*n vivo*  and *in vitro* studies confirm that α7 inhibition can be neuroprotective (de Fiebre and de Fiebre 2005), (Laudenbach et al. 2002), (Martin et al. 2004).

Protection of neurons from neuronal damage and cell death in neurodegenerative disease is a major challenge in neuroscience research. Donepezil, galantamine and tacrine are acetylcholinesterase inhibitors used for the treatment of Alzheimer's disease, and were believed to be symptomatic drugs whose therapeutic effects are achieved by slowing the hydrolysis of acetylcholine at synaptic termini. However, recent accumulated evidence strongly suggests that these acetylcholinesterase inhibitors also possess neuroprotective properties whose mechanism is independent of acetylcholinesterase inhibition. It has been

the process (i.e., Bcl-2) (Craig 1995), (Schlesinger et al. 1997), (Adams and Cory 1998). In a study we showed that there is a relation between glutamate increase and apoptosis promotion and increase in proapoptotic agent activity in both cerebral cortex and lumbar

A vast array of stimuli can activate apoptosis in neurons (Sastry and Rao 2000). Many of these stimuli have been implicated in the pathophysiology of opioid–induced tolerance including glutamate excitotoxicity, increased calcium flux and mitochondria dysfunction

Neuroactive steroids are endogenous neuromodulators synthesized either within the brain (neurosteroids) or in the periphery by the adrenal glands and gonads. In addition to the classic effect of steroids on gene transcription via binding to intracellular steroid receptors, neuroactive steroids can alter neuronal excitability via nongenomic effects by acting at inhibitory Gama Amino Butiric Acid A (GABAA) receptors and/or excitatory NMDA receptors, among others (Shulman and Tibbo 2005), (Marx et al. 2006). There is also evidence for a potential role of these neurosteroids in controlling GABA and glutamate release. Neuroactive steroids have also been implicated in neuroprotection, myelination, and modulation of the stress response. A number of neuroactive steroids are present in human postmortem brain at physiologically relevant nanomolar concentrations and serve as allosteric modulators of the GABAA receptor (Marx et al. 2006). Neuroactive steroids that are effective modulators of GABAA and/or T-type Ca2+ channels are promising tools for studying the role of these channels in peripheral pain perception. They appear to be very effective in alleviating peripheral Nociception in rat models of acute and chronic pain

Agonists and antagonist selective for AChR subtypes have been used in experimental and clinical research. Some of those compounds are potential candidates for the treatment of neurodegenerative disease such as Alzheimer's disease, Parkinson's disease and others. A growing list of *in vivo* and *in vitro* research suggest that AChRs modulators are gaining

The inhibition of α7 AChRs decreases the GABAergic tone causing increased ACh release into the synaptic cleft (Giorgetti et al. 2000), which then activates the α4β2 AChRs located post-synaptically. The selective α7 inhibitor methyllycaconitine (Ivy Carroll et al. 2007) mimics, at least in part, the neuroprotective effect of 4R (Ferchmin et al. 2003). Other i*n vivo*  and *in vitro* studies confirm that α7 inhibition can be neuroprotective (de Fiebre and de

Protection of neurons from neuronal damage and cell death in neurodegenerative disease is a major challenge in neuroscience research. Donepezil, galantamine and tacrine are acetylcholinesterase inhibitors used for the treatment of Alzheimer's disease, and were believed to be symptomatic drugs whose therapeutic effects are achieved by slowing the hydrolysis of acetylcholine at synaptic termini. However, recent accumulated evidence strongly suggests that these acetylcholinesterase inhibitors also possess neuroprotective properties whose mechanism is independent of acetylcholinesterase inhibition. It has been

and these mechanisms are discussed in detail later in this chapter.

**6. Acetyl Choline Receptors (AChRs) and neuroprotection** 

importance as clinically relevant neuroprotective drugs (Mudo et al. 2007).

Fiebre 2005), (Laudenbach et al. 2002), (Martin et al. 2004).

**5. Neuroactive steroids are neuroprotective** 

spinal cord of rat (Hassanzadeh et al.).

(Jevtovic-Todorovic et al. 2009).

shown that acetylcholinesterase inhibitors protect neurons from glutamate-induced neurotoxicity in the primary culture of rat cortical neurons.

The long-standing belief was that acetylcholinesterase inhibitors are symptomatic agents that ameliorate cholinergic deficits by slowing the hydrolysis of acetylcholinesterase at synaptic nerve termini; however, recent studies have shown that acetylcholinesterase inhibitors have other pharmacological properties, for example, neuroprotection against toxic insults, such as glutamate and up-regulation of nicotinic receptors (Akaike 2006), (Takada-Takatori et al. 2009).

Several reports have indicated that activation of cholinergic neurons in the central nervous system produces antinociception and analgesia in a variety of animals, including humans (Harte et al. 2004) provide evidence supporting the involvement of the intralaminar thalamus in muscarinic induced antinociception. Pharmacological experiments have shown that the microinjection of acetylcholine or carbachol into specific brainstem nuclei can produce antinociception and can be reversed by muscarinic receptor antagonists (Brodie and Proudfit 1984), (Yaksh et al. 1985). Meanwhile some other types of receptors or drugs produce analgesia by mediation of ACh. Sumatriptan (5- HT1agonist) is able to induce antinociception by increasing cholinergic neurotransmission (Ghelardini et al. 1997). D2 antagonist prochlorperazine exerts an antinocicptive effect mediated by a central cholinergic mechanism (Ghelardini et al. 2004), (Yang et al. 2008). In addition, more recently we showed that an acethylcolinesterase inhibitor, donepezil, could prevent tolerance to the analgesic effect of morphine (unpublished data).

#### **7. Cannabinoids, pain and neuroprotection**

Pain severely impairs quality of life. Currently available treatments, generally opioids and anti-inflammatory drugs, are not always effective for certain painful conditions. The discovery of the cannabinoid receptors in the 1990s led to the characterization of the endogenous cannabinoid system in terms of its components and numerous basic physiologic functions. Cannabinoid1 (CB1) receptors are present in nervous system areas involved in modulating nociception and evidence supports a role of the endocannabinoids in pain modulation. Cannabinoids have antinociceptive mechanisms different from that of other drugs currently in use, which thus opens a new line of promising treatment to mitigate pain that fails to respond to the pharmacologic treatments available, especially for neuropathic and inflammatory pains (Manzanares et al. 2006).

Cannabis extracts and synthetic cannabinoids are still widely considered illegal substances. The Cannabis sativa plant has been exploited for medicinal, agricultural and spiritual purposes in diverse cultures over thousands of years. Cannabis has been used recreationally for its psychotropic properties, while effects such as stimulation of appetite, analgesia and anti-emesis have lead to the medicinal application of cannabis. Indeed, reports of medicinal efficacy of cannabis can been traced back as far as 2700 BC, and even at that time reports also suggested a neuroprotective effect of the cultivar (Scotter et al.).

Preclinical and clinical studies have suggested that they may result useful to treat diverse diseases, including those related with acute or chronic pain. The discovery of cannabinoid receptors, their endogenous ligands, and the machinery for the synthesis, transport, and degradation of these retrograde messengers, has equipped us with neurochemical tools for novel drug design. Agonist-activated cannabinoid receptors, modulate nociceptive thresholds, inhibit release of pro-inflammatory molecules, and display synergistic effects

Neuroprotection and Pain Management 87

cell under the influence of a drug tries to compensate for its acute effects by promoting changes in the opposite direction, transiently restoring its homeostasis. However, when the acute action of the drug is finished, the cell is imbalanced by its own reactive response(Sharma SK et al. 1975). As a consequence, the phenomenon of tolerance develops, that is, the need for an increased dose of the drug to produce the same effect (McQuay 1999). After tolerance is established, the withdrawal of the drug may produce physical or psychological symptoms opposed to the acute pharmacological actions of the drug itself. Opioid drugs are used clinically as unsurpassed analgesic agents but are also illegally abused on the street to induce a sense of well-being and euphoria. Tolerance to opioids, defined as a loss of effect following repeated treatments such that a higher dose is required for equivalent effect, limits the analgesic efficacy of these drugs and contributes to the social

In order to safely use morphine in clinic, we need to know how morphine tolerance and dependence are developed and what kinds of medicines could inhibit or prevent such mechanisms. In line with this, various approaches have been attempted to clarify the mechanisms underlying morphine tolerance and dependence. Here we summarize various

Prolonged and repeated exposures to opioid agonists reduce the responsiveness of G protein coupled opioid receptors. This reduction in receptor function is hypothesized to contribute to opioid tolerance, dependence, and addiction in humans (Nestler 1992). Substantial experimental evidence has divided this reduced function into separate but correlated receptor traffickings, 1) desensitization, 2) internalization, 3) sequestration/recycling, 4) down regulation (Law et al. 2000). The molecular events underlying opioid tolerance are currently discussed in relation to all these receptor trafficking mechanisms. According to current understanding, opioid receptors are desensitized on the cell surface through a phosphorylation process in the C-terminal (Afify et al. 1998) and/or third intracellular loop. On the other hand, receptor internalization or receptor disappearance from the cell surface, is now believed to contribute to resensitization through dephosphorylation during endosomal stages (Krueger et al. 1997; Zhang et al. 1997). Down-regulation is a loss of receptor protein in cells through increased degradation or decreased synthesis of the receptor. Little is known, however, regarding the regulation of this mechanism and involvement in opioid tolerance. Thus, much research has been done on the molecular basis of events in receptor phosphorylation in the membranes and internalization. Recent studies revealed that cAMP-dependent protein kinase A (PKA) (Harada et al. 1990), protein kinase C (PKC) (Ueda et al. 1995), Ca2 +/calmodulin-dependent protein kinases (Koch et al. 1997), G protein-coupled receptor kinases (GRKs) (Zhang et al. 1998), and mitogen-activated protein kinase (Polakiewicz et al. 1998) have roles in opioid receptor phosphorylation. PKC and GRK mechanisms are likely candidates for opioid

problems surrounding recreational opioid abuse.

**8.2 Mechanisms for acute morphine tolerance** 

proposed hypotheses and introduce our new approaches in this area.

desensitization and internalization (Ueda et al. 1995; Zhang et al. 1998).

A number of reports have demonstrated that PKC is involved in the opioid tolerance or desensitization. Most of recent reports have demonstrated that PKC activators or inhibitors modulate opioid signaling in cells expressing opioid receptors. A series of reports have demonstrated the involvement of PKC in opioid tolerance by correlating both in vitro and in

**8.3 PKC hypothesis**

vivo studies.

with other systems that influence analgesia, especially the endogenous opioid system. Cannabinoid receptor agonists have shown therapeutic value against inflammatory and neuropathic pains, conditions that are often refractory to therapy. Although the psychoactive effects of these substances have limited clinical progress to study cannabinoid actions in pain mechanisms, preclinical research is progressing rapidly.

There has been anecdotal and preliminary scientific evidence of cannabis affording symptomatic relief in diverse neurodegenerative disorders. These include multiple sclerosis, Huntington's, Parkinson's and Alzheimer's diseases, and amyotrophic lateral sclerosis. This evidence implied that hypofunction or dysregulation of the endocannabinoid system may be responsible for some of the symptomatology of these diseases.

In Huntington's disease, Alzheimer's disease, as well as in ALS, pathologic changes in endocannabinoid levels and CB2 expression are induced by the inflammatory environment.

CB1 activation has been shown to be effective in limiting cell death following excitotoxic lesions, while CB2 is involved in dampening inflammatory immune cell response to disease. These two targets may therefore work together to provide both neuroprotection to acute injury and immune suppression during more chronic responses (Scotter et al.).

During the last two decades, a large number of research papers have demonstrated the efficacy of cannabinoids and modulators of the endocannabinoid system in suppressing neuropathic pain in animal models. Cannabinoids suppress hyperalgesia and allodynia (i.e. mechanical allodynia, mechanical hyperalgesia, thermal hyperalgesia and, where evaluated, cold allodynia), induced by diverse neuropathic pain states through CB1 and CB2-specific mechanisms (Rahn and Hohmann 2009).

On the other hand, responses to cannabinoid (CB) receptor activation include opening of potassium channels, inhibition of calcium currents, and stimulation of various protein kinases (Deadwyler et al. 1995; Gomez del Pulgar et al. 2000; Galve-Roperh et al. 2002; Karanian et al. 2005b; Molina-Holgado et al. 2005; Karanian et al. 2007). Some of the many such signaling elements activated by endocannabinoids play important roles in neuronal maintenance (Bahr et al. 2006; Galve-Roperh et al. 2008). CB receptor transmission elicits modulatory effects on calcium channels, resulting in reduced neurotransmitter (e.g., GABA, glutamate) release (Hajos et al. 2000; Kreitzer and Regehr 2001; Wilson et al. 2001). One particular mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), is involved in cannabinergic signaling, as are focal adhesion kinase (FAK) and phosphatidylinositol 3′-kinase (PI3K). These signaling elements appear to play key roles in the neuroprotective nature of the endocannabinoid system, and the associated signaling pathways are disrupted by blocking CB receptor activation (Hwang et al.; Wallace et al. 2003; Khaspekov et al. 2004; Karanian et al. 2005a; Karanian et al. 2005b).

Together, these studies indicate that the neuroprotectant cannabinoids have antinociceptive properties.

#### **8. Neuroprotection and tolerance to the analgesic effect**

#### **8.1 Opioid tolerance**

Many types of neuronal cells and brain nuclei have the property of changing, acutely or chronically, their regular behavior by the action of pharmacological agents, such as psychoactive drugs. Acute changes, those that cease in a short time, would not be important to the chronic altered behavior if the cell recovered its original drug-free state, but it is observed that some adaptation occurs that impairs such a recovery. In fact, the disturbed

with other systems that influence analgesia, especially the endogenous opioid system. Cannabinoid receptor agonists have shown therapeutic value against inflammatory and neuropathic pains, conditions that are often refractory to therapy. Although the psychoactive effects of these substances have limited clinical progress to study cannabinoid

There has been anecdotal and preliminary scientific evidence of cannabis affording symptomatic relief in diverse neurodegenerative disorders. These include multiple sclerosis, Huntington's, Parkinson's and Alzheimer's diseases, and amyotrophic lateral sclerosis. This evidence implied that hypofunction or dysregulation of the endocannabinoid system may

In Huntington's disease, Alzheimer's disease, as well as in ALS, pathologic changes in endocannabinoid levels and CB2 expression are induced by the inflammatory environment. CB1 activation has been shown to be effective in limiting cell death following excitotoxic lesions, while CB2 is involved in dampening inflammatory immune cell response to disease. These two targets may therefore work together to provide both neuroprotection to acute

During the last two decades, a large number of research papers have demonstrated the efficacy of cannabinoids and modulators of the endocannabinoid system in suppressing neuropathic pain in animal models. Cannabinoids suppress hyperalgesia and allodynia (i.e. mechanical allodynia, mechanical hyperalgesia, thermal hyperalgesia and, where evaluated, cold allodynia), induced by diverse neuropathic pain states through CB1 and CB2-specific

On the other hand, responses to cannabinoid (CB) receptor activation include opening of potassium channels, inhibition of calcium currents, and stimulation of various protein kinases (Deadwyler et al. 1995; Gomez del Pulgar et al. 2000; Galve-Roperh et al. 2002; Karanian et al. 2005b; Molina-Holgado et al. 2005; Karanian et al. 2007). Some of the many such signaling elements activated by endocannabinoids play important roles in neuronal maintenance (Bahr et al. 2006; Galve-Roperh et al. 2008). CB receptor transmission elicits modulatory effects on calcium channels, resulting in reduced neurotransmitter (e.g., GABA, glutamate) release (Hajos et al. 2000; Kreitzer and Regehr 2001; Wilson et al. 2001). One particular mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), is involved in cannabinergic signaling, as are focal adhesion kinase (FAK) and phosphatidylinositol 3′-kinase (PI3K). These signaling elements appear to play key roles in the neuroprotective nature of the endocannabinoid system, and the associated signaling pathways are disrupted by blocking CB receptor activation (Hwang et al.; Wallace et al.

Together, these studies indicate that the neuroprotectant cannabinoids have antinociceptive

Many types of neuronal cells and brain nuclei have the property of changing, acutely or chronically, their regular behavior by the action of pharmacological agents, such as psychoactive drugs. Acute changes, those that cease in a short time, would not be important to the chronic altered behavior if the cell recovered its original drug-free state, but it is observed that some adaptation occurs that impairs such a recovery. In fact, the disturbed

injury and immune suppression during more chronic responses (Scotter et al.).

2003; Khaspekov et al. 2004; Karanian et al. 2005a; Karanian et al. 2005b).

**8. Neuroprotection and tolerance to the analgesic effect** 

actions in pain mechanisms, preclinical research is progressing rapidly.

be responsible for some of the symptomatology of these diseases.

mechanisms (Rahn and Hohmann 2009).

properties.

**8.1 Opioid tolerance** 

cell under the influence of a drug tries to compensate for its acute effects by promoting changes in the opposite direction, transiently restoring its homeostasis. However, when the acute action of the drug is finished, the cell is imbalanced by its own reactive response(Sharma SK et al. 1975). As a consequence, the phenomenon of tolerance develops, that is, the need for an increased dose of the drug to produce the same effect (McQuay 1999). After tolerance is established, the withdrawal of the drug may produce physical or psychological symptoms opposed to the acute pharmacological actions of the drug itself. Opioid drugs are used clinically as unsurpassed analgesic agents but are also illegally abused on the street to induce a sense of well-being and euphoria. Tolerance to opioids, defined as a loss of effect following repeated treatments such that a higher dose is required for equivalent effect, limits the analgesic efficacy of these drugs and contributes to the social problems surrounding recreational opioid abuse.

In order to safely use morphine in clinic, we need to know how morphine tolerance and dependence are developed and what kinds of medicines could inhibit or prevent such mechanisms. In line with this, various approaches have been attempted to clarify the mechanisms underlying morphine tolerance and dependence. Here we summarize various proposed hypotheses and introduce our new approaches in this area.

#### **8.2 Mechanisms for acute morphine tolerance**

Prolonged and repeated exposures to opioid agonists reduce the responsiveness of G protein coupled opioid receptors. This reduction in receptor function is hypothesized to contribute to opioid tolerance, dependence, and addiction in humans (Nestler 1992). Substantial experimental evidence has divided this reduced function into separate but correlated receptor traffickings, 1) desensitization, 2) internalization, 3) sequestration/recycling, 4) down regulation (Law et al. 2000). The molecular events underlying opioid tolerance are currently discussed in relation to all these receptor trafficking mechanisms. According to current understanding, opioid receptors are desensitized on the cell surface through a phosphorylation process in the C-terminal (Afify et al. 1998) and/or third intracellular loop. On the other hand, receptor internalization or receptor disappearance from the cell surface, is now believed to contribute to resensitization through dephosphorylation during endosomal stages (Krueger et al. 1997; Zhang et al. 1997). Down-regulation is a loss of receptor protein in cells through increased degradation or decreased synthesis of the receptor. Little is known, however, regarding the regulation of this mechanism and involvement in opioid tolerance. Thus, much research has been done on the molecular basis of events in receptor phosphorylation in the membranes and internalization. Recent studies revealed that cAMP-dependent protein kinase A (PKA) (Harada et al. 1990), protein kinase C (PKC) (Ueda et al. 1995), Ca2 +/calmodulin-dependent protein kinases (Koch et al. 1997), G protein-coupled receptor kinases (GRKs) (Zhang et al. 1998), and mitogen-activated protein kinase (Polakiewicz et al. 1998) have roles in opioid receptor phosphorylation. PKC and GRK mechanisms are likely candidates for opioid desensitization and internalization (Ueda et al. 1995; Zhang et al. 1998).

#### **8.3 PKC hypothesis**

A number of reports have demonstrated that PKC is involved in the opioid tolerance or desensitization. Most of recent reports have demonstrated that PKC activators or inhibitors modulate opioid signaling in cells expressing opioid receptors. A series of reports have demonstrated the involvement of PKC in opioid tolerance by correlating both in vitro and in vivo studies.

Neuroprotection and Pain Management 89

The induction of apoptosis in neurons has been demonstrated to share the same basic mechanisms with all other cell types (Sastry and Rao 2000). In vitro studies also indicate that exposure to μ- and/or κ-opioid receptor agonists of neuronal cultures from embryonic chick brain (Goswami et al. 1998) and specific cell lines (Dawson et al. 1997; Singhal et al. 1998; Singhal et al. 1999) increases their vulnerability to death by apoptotic mechanisms. The molecular mechanisms of apoptosis (i.e., the detailed cascade of events from the cell surface to final changes in the nucleus) have not been established yet, but various key proteins are involved in the regulation of programmed cell death (Sastry and Rao 2000). Some members of the Bcl-2 family of proteins, such as Bcl-2 and Bcl-xL, suppresses apoptosis, while the expression of other, such as the homologues Bax and Bak, are pro-apoptotic (Adams and Cory 1998). Specifically, the Bcl-2 oncoprotein, localized mainly to the mitochondrial membranes, has been shown to play an important role in protecting neurons from apoptotic cell death (Hockenbery et al. 1990), probably by preventing the release of cytochrome c (induced by Bax) and the subsequent activation of specific proteases termed caspases, the proteolytic enzymes which are crucial for the execution of nuclear fragmentation and apoptosis (Adams and Cory 1998; Sastry and Rao 2000). In fact, Bax mRNA and Bax protein are increased in the substantia nigra of MPTP-treated mice (degeneration of dopamine neurons by apoptosis) (Hassouna et al. 1996), and the release of cytochrome c from the mitochondria and the subsequent activation of caspases-3/9 was shown to play a key role in cocaine-induced apoptosis in foetal rat myocardial cells (Xiao et al. 2000). The results of our studies demonstrated that chronic morphine administration in rat, induced apoptosis; decrease in Bcl-2 and increase in caspase3 activity in both cerebral cortex and lumbar spinal cord in rat (Hassanzadeh et al.). Another key element involved in the regulation of apoptosis is the Fas glycoprotein (also known as CD95 or Apo1), a cell surface receptor that belongs to the tumor necrosis factor receptor family (death receptors) and that is expressed abundantly in various tissues (Nagata 1999). In contrast to Bcl-2 mitochondrial protein, the Fas receptor triggers cell apoptosis when it binds to its ligand, Fas, and Fas-mediated death bypasses the usual long sequence of signaling enzymes and immediately activates a pre-existing caspase cascade (Nagata 1999). In the context of the induction of aberrant apoptosis in opioid addiction, it was of great interest the in vitro study demonstrating the ability of morphine to increase, through a naloxone-sensitive mechanism, the expression (mRNA) of the pro-apoptotic receptor Fas in mouse splenocytes and in human blood lymphocytes (Yin et al. 1999). A relevant consequence of the morphine-induced potentiation of apoptosis in lymphocytes (Singhal et al. 1999; Yin et al. 1999) is the reduction of the immune response (and the increase in recurrent infections) observed in heroin addicts

On the other hand, over a decade, the NMDA receptor (NMDAR), a subgroup of glutamate receptors, has been implicated in the development of opioid tolerance (Trujillo and Akil 1991; Mao et al. 1994). Activation of NMDARs can lead to neurotoxicity under many circumstances (Rothman and Olney 1986; Moncada et al. 1992; Catania et al. 1993) For instance, peripheral nerve injury has been shown to activate spinal cord NMDARs, which results in not only intractable neuropathic pain but also neuronal cell death by means of apoptosis (Mao et al. 1997; Whiteside and Munglani 2001). Furthermore, cross talk between the cellular mechanisms of opioid tolerance and neuropathic pain has been proposed, suggesting that a common cellular mechanism may be involved in both neuropathic pain and opioid tolerance (Mayer et al. 1999). Thus, it is possible that the cellular process leading to the development of opioid tolerance may also cause neurotoxic changes in response to prolonged opioid administration. More recently, we examined the hypothesis that neurotoxicity in the form of apoptotic cell

(Govitrapong et al. 1998).

#### **8.4 Mechanisms for chronic morphine tolerance and dependence**

Clear difference between acute morphine tolerance and chronic one has not been demonstrated for a long time. In algogenic-induced nociceptive flexion (ANF) test in mice the peripheral morphine analgesia developed the acute tolerance by 4 h pretreatment with morphine (Ueda et al. 2001). However, the peripheral analgesia had no change in mice that were given morphine for 5 days, a treatment which caused a marked chronic tolerance to systemic morphine analgesia (Ueda and Inoue 1999). Thus, it is evident that acute morphine tolerance mediates distinct mechanisms from the chronic one, and chronic tolerance is likely mediated through a complicated neuronal network present in the central nervous system.

#### **8.5 cAMP hypothesis**

Since the report by Sharma et al. (1975), it has been accepted that cAMP may play a key role in the morphine tolerance and dependence. According to this so-called cAMP hypothesis, a morphine-induced decrease in cAMP production is getting disappeared during long-period exposure to morphine (Sharma SK et al. 1975). As the naloxone application causes an abrupt increase in cAMP production, some unidentified mechanisms are supposed to mediate an increase in cAMP production through specific gene expressions during chronic morphine treatment. A candidate could be a cAMP-responsive element binding protein (CREB), which is involved in the gene expression of adenylyl cyclase. In vivo study using knockout mice demonstrates that CREB plays roles in the development of morphine dependence (Maldonado et al. 1996). Although several compounds possessing the antagonistic activity are reported to inhibit morphine tolerance and dependence, they have serious side effects at the same time (Trujillo and Akil 1991; Mao et al. 1992; Trujillo 1995; Mao 1999; Habibi-Asl and Hassanzadeh 2004; Habibi-Asl 2005; Asl et al. 2008).

#### **8.6 Anti-opioid hypothesis**

In addition to mechanisms at the single cellular level, the plasticity through neuronal networks would be involved in the development of morphine tolerance and dependence, as above-mentioned. One of approaches to cut in the mechanisms is based on the view that enhanced anti-opioid neuronal activity during chronic morphine treatments might suppress the acute morphine actions. The candidates include cholecystokinin (Mitchell et al. 2000 ), neuropeptide FF (Lake et al. 1992), nociceptin (Ueda et al. 2000) and glutamate, as an NMDA receptor ligand (Ueda et al. 2000; Mao and Mayer 2001). Among them the nociceptin (N/OFQ) system has been extensively characterized to be involved in the development of morphine tolerance and dependence. NMDA receptor has been long supposed to play important roles in the development of morphine tolerance and dependence (Trujillo and Akil 1991). Although several compounds possessing the antagonistic activity are reported to inhibit morphine tolerance and dependence, they have serious side effects at the same time (Trujillo and Akil 1991; Mao et al. 1992; Trujillo 1995; Mao 1999; Habibi-Asl and Hassanzadeh 2004; Habibi-Asl 2005; Asl et al. 2008).

#### **8.7 Apoptosis hypothesis**

Apoptosis, or programmed cell death, is an active process of normal cell death during development and also occurs as a consequence of the cytotoxic effect of various neurotoxins (e.g., MPTP/MPP+, MDMA, ethanol and cocaine) (Sastry and Rao 2000). Among the drugs of abuse, cocaine has been shown to cause a direct cytotoxic effect on the foetal rat heart, and to induce apoptosis in foetal rat myocardial cells in a dose-dependent manner (Xiao et al. 2000).

Clear difference between acute morphine tolerance and chronic one has not been demonstrated for a long time. In algogenic-induced nociceptive flexion (ANF) test in mice the peripheral morphine analgesia developed the acute tolerance by 4 h pretreatment with morphine (Ueda et al. 2001). However, the peripheral analgesia had no change in mice that were given morphine for 5 days, a treatment which caused a marked chronic tolerance to systemic morphine analgesia (Ueda and Inoue 1999). Thus, it is evident that acute morphine tolerance mediates distinct mechanisms from the chronic one, and chronic tolerance is likely mediated through a complicated neuronal network present in the central nervous system.

Since the report by Sharma et al. (1975), it has been accepted that cAMP may play a key role in the morphine tolerance and dependence. According to this so-called cAMP hypothesis, a morphine-induced decrease in cAMP production is getting disappeared during long-period exposure to morphine (Sharma SK et al. 1975). As the naloxone application causes an abrupt increase in cAMP production, some unidentified mechanisms are supposed to mediate an increase in cAMP production through specific gene expressions during chronic morphine treatment. A candidate could be a cAMP-responsive element binding protein (CREB), which is involved in the gene expression of adenylyl cyclase. In vivo study using knockout mice demonstrates that CREB plays roles in the development of morphine dependence (Maldonado et al. 1996). Although several compounds possessing the antagonistic activity are reported to inhibit morphine tolerance and dependence, they have serious side effects at the same time (Trujillo and Akil 1991; Mao et al. 1992; Trujillo 1995; Mao 1999; Habibi-Asl

In addition to mechanisms at the single cellular level, the plasticity through neuronal networks would be involved in the development of morphine tolerance and dependence, as above-mentioned. One of approaches to cut in the mechanisms is based on the view that enhanced anti-opioid neuronal activity during chronic morphine treatments might suppress the acute morphine actions. The candidates include cholecystokinin (Mitchell et al. 2000 ), neuropeptide FF (Lake et al. 1992), nociceptin (Ueda et al. 2000) and glutamate, as an NMDA receptor ligand (Ueda et al. 2000; Mao and Mayer 2001). Among them the nociceptin (N/OFQ) system has been extensively characterized to be involved in the development of morphine tolerance and dependence. NMDA receptor has been long supposed to play important roles in the development of morphine tolerance and dependence (Trujillo and Akil 1991). Although several compounds possessing the antagonistic activity are reported to inhibit morphine tolerance and dependence, they have serious side effects at the same time (Trujillo and Akil 1991; Mao et al. 1992; Trujillo 1995; Mao 1999; Habibi-Asl and

Apoptosis, or programmed cell death, is an active process of normal cell death during development and also occurs as a consequence of the cytotoxic effect of various neurotoxins (e.g., MPTP/MPP+, MDMA, ethanol and cocaine) (Sastry and Rao 2000). Among the drugs of abuse, cocaine has been shown to cause a direct cytotoxic effect on the foetal rat heart, and to induce apoptosis in foetal rat myocardial cells in a dose-dependent manner (Xiao et al. 2000).

**8.4 Mechanisms for chronic morphine tolerance and dependence**

and Hassanzadeh 2004; Habibi-Asl 2005; Asl et al. 2008).

Hassanzadeh 2004; Habibi-Asl 2005; Asl et al. 2008).

**8.5 cAMP hypothesis**

**8.6 Anti-opioid hypothesis**

**8.7 Apoptosis hypothesis** 

The induction of apoptosis in neurons has been demonstrated to share the same basic mechanisms with all other cell types (Sastry and Rao 2000). In vitro studies also indicate that exposure to μ- and/or κ-opioid receptor agonists of neuronal cultures from embryonic chick brain (Goswami et al. 1998) and specific cell lines (Dawson et al. 1997; Singhal et al. 1998; Singhal et al. 1999) increases their vulnerability to death by apoptotic mechanisms. The molecular mechanisms of apoptosis (i.e., the detailed cascade of events from the cell surface to final changes in the nucleus) have not been established yet, but various key proteins are involved in the regulation of programmed cell death (Sastry and Rao 2000). Some members of the Bcl-2 family of proteins, such as Bcl-2 and Bcl-xL, suppresses apoptosis, while the expression of other, such as the homologues Bax and Bak, are pro-apoptotic (Adams and Cory 1998). Specifically, the Bcl-2 oncoprotein, localized mainly to the mitochondrial membranes, has been shown to play an important role in protecting neurons from apoptotic cell death (Hockenbery et al. 1990), probably by preventing the release of cytochrome c (induced by Bax) and the subsequent activation of specific proteases termed caspases, the proteolytic enzymes which are crucial for the execution of nuclear fragmentation and apoptosis (Adams and Cory 1998; Sastry and Rao 2000). In fact, Bax mRNA and Bax protein are increased in the substantia nigra of MPTP-treated mice (degeneration of dopamine neurons by apoptosis) (Hassouna et al. 1996), and the release of cytochrome c from the mitochondria and the subsequent activation of caspases-3/9 was shown to play a key role in cocaine-induced apoptosis in foetal rat myocardial cells (Xiao et al. 2000). The results of our studies demonstrated that chronic morphine administration in rat, induced apoptosis; decrease in Bcl-2 and increase in caspase3 activity in both cerebral cortex and lumbar spinal cord in rat (Hassanzadeh et al.). Another key element involved in the regulation of apoptosis is the Fas glycoprotein (also known as CD95 or Apo1), a cell surface receptor that belongs to the tumor necrosis factor receptor family (death receptors) and that is expressed abundantly in various tissues (Nagata 1999). In contrast to Bcl-2 mitochondrial protein, the Fas receptor triggers cell apoptosis when it binds to its ligand, Fas, and Fas-mediated death bypasses the usual long sequence of signaling enzymes and immediately activates a pre-existing caspase cascade (Nagata 1999). In the context of the induction of aberrant apoptosis in opioid addiction, it was of great interest the in vitro study demonstrating the ability of morphine to increase, through a naloxone-sensitive mechanism, the expression (mRNA) of the pro-apoptotic receptor Fas in mouse splenocytes and in human blood lymphocytes (Yin et al. 1999). A relevant consequence of the morphine-induced potentiation of apoptosis in lymphocytes (Singhal et al. 1999; Yin et al. 1999) is the reduction of the immune response (and the increase in recurrent infections) observed in heroin addicts (Govitrapong et al. 1998).

On the other hand, over a decade, the NMDA receptor (NMDAR), a subgroup of glutamate receptors, has been implicated in the development of opioid tolerance (Trujillo and Akil 1991; Mao et al. 1994). Activation of NMDARs can lead to neurotoxicity under many circumstances (Rothman and Olney 1986; Moncada et al. 1992; Catania et al. 1993) For instance, peripheral nerve injury has been shown to activate spinal cord NMDARs, which results in not only intractable neuropathic pain but also neuronal cell death by means of apoptosis (Mao et al. 1997; Whiteside and Munglani 2001). Furthermore, cross talk between the cellular mechanisms of opioid tolerance and neuropathic pain has been proposed, suggesting that a common cellular mechanism may be involved in both neuropathic pain and opioid tolerance (Mayer et al. 1999). Thus, it is possible that the cellular process leading to the development of opioid tolerance may also cause neurotoxic changes in response to prolonged opioid administration. More recently, we examined the hypothesis that neurotoxicity in the form of apoptotic cell

Neuroprotection and Pain Management 91

Opioid tolerance manifests as a loss of agonist potency and as a shift of the dose-response curve to the right. During the past decades, many studies have focused on excitatory amino acid receptors to investigate the role which they play in the development of tolerance to the antinociceptive action of opiates. This idea was suggested by Trujillo and Akil who reported that the NMDA receptor antagonist, MK801 (dizocilpine), inhibited the development of tolerance to the antinociceptive effect of morphine and morphine physical dependence

Using behavioral studies, we and others have shown that a variety of NMDA receptor antagonists have the ability to inhibit the development of opiate tolerance and dependence (Trujillo and Akil 1991; Trujillo 1995; Habibi-Asl and Hassanzadeh 2004; Asl et al. 2008; Habibi-Asl et al. 2009b). There are also several lines of evidence which suggest that activation of NMDARs leads to removing the magnesium blockade (Begon et al. 2001) in the calcium channel and toxic calcium influx, which activates numerous enzymes, including neuronal nitric oxide (NO) synthase (NOS). In our unpublished data we observed that nitric oxide donors such as nitroglycerin or nicorandil increased the tolerance to the analgesic effect of morphine. On the other hand the nitric oxide synthase inhibitor, N-Nitro-L-Arginine Methyl Ester (LNAME) could prevent the tolerance. It has been demonstrated that Magnesium (Mg)-deficient rats develop a mechanical hyperalgesia which is reversed by a N-Methyl-D-Aspartate (NMDA) receptor antagonist (Begon et al. 2001). Our study in agreement with those studies showed that systemic administration of magnesium sulfate could attenuate morphine tolerance to the analgesic effect (Habibi-Asl 2005; Habibi-Asl et al. 2009b). Also we showed that selenium with similar mechanism appeared to have a weaker

Our recently published finding, indicated that riluzole (2- amino-6-[trifluoromethoxy] benzothiazole), an antiglutamatergic agent, decreases the development of tolerance, shifting the first day of established tolerance from the 8th day in the control group to the 13th day (Habibi-Asl et al. 2009a). Riluzole interferes with responses mediated by excitatory amino acids, even though it does not interact with any known binding sites on the NMDA, kainate or AMPA glutamate receptors (Debono et al. 1993). The neuroprotective effect of riluzole, which has been shown both in vivo and in vitro, is believed to be beneficial in various neurodegenerative diseases and amelioration of trauma and stroke (Doble 1999; Albo et al.

The results indicated that there was a significant shift to the right in the dose-response curve as well as an increase in the antinociceptive 50% effective dose (ED50) of morphine for animals who received morphine also compared with those that received morphine and riluzole. On the other hand, co-administration of riluzole delayed the onset of morphineinduced apoptosis and significantly decreased the average number of TUNEL-positive cells (p < 0.01). This finding is in line with our recent results concerning the lumbar region of the spinal cord (Hassanzadeh et al.). In addition, we found that the group that received morphine and riluzole for 13 days had developed tolerance; they showed an increase in the number of apoptotic cells, as under control conditions. This result indicates that after the completion of tolerance in both the control and the treated groups, apoptosis had already developed. Previous studies have indicated that certain addictive drugs, such as morphine, could induce apoptosis in cultured neuronal cell lines as well as human cells (Singhal et al. 1998; Singhal et al. 1999). More recently, it has been shown that in vivo neuronal apoptosis occurs in the rat's spinal cord dorsal horn after chronic morphine treatment that was associated with the expression of activated caspase- 3 and the involvement of mitogen-

(Trujillo and Akil 1991).

2004).

effect than magnesium (Charkhpour M et al. 2009).

death would be induced in association with the development of morphine tolerance. In confirmation of Mao et al. findings, we demonstrated that chronic opioid injection leads to apoptosis in the CNS which was in association with the development of tolerance to the analgesic effect (Habibi-Asl et al. 2009a). Figure1 shows the possible mechanisms of opioidinduced neuronal apoptosis and its association with opioid tolerance.

Fig. 1. Schematic diagram illustrating the possible mechanisms of opioid-induced neuronal apoptosis and tolerance. The results of before studies suggest that chronic opioid administration may induce NMDAR, microglia, FAAD/P53,… activation resulting in intracellular positive apoptosis regulators induction. The resultant apoptosis contributes to the cellular mechanism of opioid tolerance. NMDA: N-Methyl-D-Aspartate, NO: Nitric Oxide, AIF: Apoptosis-Inducing Factor, FADD: Fas-Associated Death Domain,

death would be induced in association with the development of morphine tolerance. In confirmation of Mao et al. findings, we demonstrated that chronic opioid injection leads to apoptosis in the CNS which was in association with the development of tolerance to the analgesic effect (Habibi-Asl et al. 2009a). Figure1 shows the possible mechanisms of opioid-

Fig. 1. Schematic diagram illustrating the possible mechanisms of opioid-induced neuronal

apoptosis and tolerance. The results of before studies suggest that chronic opioid administration may induce NMDAR, microglia, FAAD/P53,… activation resulting in intracellular positive apoptosis regulators induction. The resultant apoptosis contributes to the cellular mechanism of opioid tolerance. NMDA: N-Methyl-D-Aspartate, NO: Nitric

Oxide, AIF: Apoptosis-Inducing Factor, FADD: Fas-Associated Death Domain,

induced neuronal apoptosis and its association with opioid tolerance.

Opioid tolerance manifests as a loss of agonist potency and as a shift of the dose-response curve to the right. During the past decades, many studies have focused on excitatory amino acid receptors to investigate the role which they play in the development of tolerance to the antinociceptive action of opiates. This idea was suggested by Trujillo and Akil who reported that the NMDA receptor antagonist, MK801 (dizocilpine), inhibited the development of tolerance to the antinociceptive effect of morphine and morphine physical dependence (Trujillo and Akil 1991).

Using behavioral studies, we and others have shown that a variety of NMDA receptor antagonists have the ability to inhibit the development of opiate tolerance and dependence (Trujillo and Akil 1991; Trujillo 1995; Habibi-Asl and Hassanzadeh 2004; Asl et al. 2008; Habibi-Asl et al. 2009b). There are also several lines of evidence which suggest that activation of NMDARs leads to removing the magnesium blockade (Begon et al. 2001) in the calcium channel and toxic calcium influx, which activates numerous enzymes, including neuronal nitric oxide (NO) synthase (NOS). In our unpublished data we observed that nitric oxide donors such as nitroglycerin or nicorandil increased the tolerance to the analgesic effect of morphine. On the other hand the nitric oxide synthase inhibitor, N-Nitro-L-Arginine Methyl Ester (LNAME) could prevent the tolerance. It has been demonstrated that Magnesium (Mg)-deficient rats develop a mechanical hyperalgesia which is reversed by a N-Methyl-D-Aspartate (NMDA) receptor antagonist (Begon et al. 2001). Our study in agreement with those studies showed that systemic administration of magnesium sulfate could attenuate morphine tolerance to the analgesic effect (Habibi-Asl 2005; Habibi-Asl et al. 2009b). Also we showed that selenium with similar mechanism appeared to have a weaker effect than magnesium (Charkhpour M et al. 2009).

Our recently published finding, indicated that riluzole (2- amino-6-[trifluoromethoxy] benzothiazole), an antiglutamatergic agent, decreases the development of tolerance, shifting the first day of established tolerance from the 8th day in the control group to the 13th day (Habibi-Asl et al. 2009a). Riluzole interferes with responses mediated by excitatory amino acids, even though it does not interact with any known binding sites on the NMDA, kainate or AMPA glutamate receptors (Debono et al. 1993). The neuroprotective effect of riluzole, which has been shown both in vivo and in vitro, is believed to be beneficial in various neurodegenerative diseases and amelioration of trauma and stroke (Doble 1999; Albo et al. 2004).

The results indicated that there was a significant shift to the right in the dose-response curve as well as an increase in the antinociceptive 50% effective dose (ED50) of morphine for animals who received morphine also compared with those that received morphine and riluzole. On the other hand, co-administration of riluzole delayed the onset of morphineinduced apoptosis and significantly decreased the average number of TUNEL-positive cells (p < 0.01). This finding is in line with our recent results concerning the lumbar region of the spinal cord (Hassanzadeh et al.). In addition, we found that the group that received morphine and riluzole for 13 days had developed tolerance; they showed an increase in the number of apoptotic cells, as under control conditions. This result indicates that after the completion of tolerance in both the control and the treated groups, apoptosis had already developed. Previous studies have indicated that certain addictive drugs, such as morphine, could induce apoptosis in cultured neuronal cell lines as well as human cells (Singhal et al. 1998; Singhal et al. 1999). More recently, it has been shown that in vivo neuronal apoptosis occurs in the rat's spinal cord dorsal horn after chronic morphine treatment that was associated with the expression of activated caspase- 3 and the involvement of mitogen-

Neuroprotection and Pain Management 93

triggering alterations in their morphology, metabolism, and function (Watkins et al. 2005). Mika et al. concluded that the effect of minocycline on morphine tolerance is related to microglia. Their results provide evidence that systemic administration of minocycline in mice influences morphine's effectiveness and delays the development of morphine tolerance

In summary, we believe that adding the neuroprotective agents to analgesic drugs specially opioids, increase the analgesic effect and prevents the hyperalgesia and tolerance to their

Adams B, Moghaddam B. Corticolimbic dopamine neurotransmission is temporally

Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science

Adayev T, Ray I, Sondhi R, Sobocki T, Banerjee P. The G protein-coupled 5-HT1A receptor

Afify EA, Law PY, Riedl M, Elde R, Loh HH. Role of carboxyl terminus of mu-and delta-

Ahlemeyer B, Glaser A, Schaper C, Semkova I, Krieglstein J. The 5-HT1A receptor agonist

Ahlemeyer B, Krieglstein J. Stimulation of 5-HT1A receptor inhibits apoptosis induced by

Akaike A. Preclinical evidence of neuroprotection by cholinesterase inhibitors. Alzheimer

Albo F, Pieri M, Zona C. Modulation of AMPA receptors in spinal motor neurons by the

Alessandri B, Tsuchida E, Bullock RM. The neuroprotective effect of a new serotonin

Arendt-Nielsen L, Sumikura H. From pain research to pain treatment: role of human pain

Asl BH, Hassanzadeh K, Khezri E, Mohammadi S. Evaluation the effects of

Bahr BA, Karanian DA, Makanji SS, Makriyannis A. Targeting the endocannabinoid system in treating brain disorders. Expert Opin Investig Drugs 2006;15(4):351-365. Begon S, Pickering G, Eschalier A, Mazur A, Rayssiguier Y, Dubray C. Role of spinal NMDA

by magnesium deficiency in rats. Br J Pharmacol 2001;134(6):1227-1236.

Brodie MS, Proudfit HK. Hypoalgesia induced by the local injection of carbachol into the

neuroprotective agent riluzole. J Neurosci Res 2004;78(2):200-207.

subdural hematoma in the rat. Brain Res 1999;845(2):232-235.

dependence in mice. Pak J Biol Sci 2008;11(13):1690-1695.

Berridge MJ. Neuronal calcium signaling. Neuron 1998;21(1):13-26.

nucleus raphe magnus. Brain Res 1984;291(2):337-342.

dissociated from the cognitive and locomotor effects of phencyclidine. J Neurosci

causes suppression of caspase-3 through MAPK and protein kinase Calpha.

opioid receptor in agonist-induced down-regulation. Brain Res Mol Brain Res

Bay x 3702 inhibits apoptosis induced by serum deprivation in cultured neurons.

serum deprivation in cultured neurons from chick embryo. Brain Res 1997;777(1-

receptor agonist, BAY X3702, upon focal ischemic brain damage caused by acute

dextromethorphan and midazolam on morphine induced tolerance and

receptors, protein kinase C and nitric oxide synthase in the hyperalgesia induced

by attenuating microglial activation and its markers (Mika et al. 2009).

analgesic effects.

**9. References** 

1998;18(14):5545-5554.

1998;54(1):24-34.

2):179-186.

1998;281(5381):1322-1326.

Biochim Biophys Acta 2003;1640(1):85-96.

Eur J Pharmacol 1999;370(2):211-216.

Dis Assoc Disord 2006;20(2 Suppl 1):S8-11.

models. J Nihon Med Sch 2002;69(6):514-524.

activated protein kinase (MAPK) (Mao et al. 2002), suggesting that chronic morphine may lead to changes within the central nervous system.

Our more recent studies demonstrated that prolonged morphine administration induces upregulation of proapoptotic elements such as caspase-3 and down regulation of the antiapoptotic factors Bcl-2 and HSP70 in the rat cerebral cortex and spinal cord (Hassanzadeh et al.; Hassanzadeh et al.; Tikka and Koistinaho 2001; Gabra et al. 2005; Hassanzadeh K et al. 2011). Importantly, up-regulation of caspase-3 and Bax was inhibited when morphine was co-administered with the noncompetitive NMDAR antagonist MK-801, thereby supporting a link between NMDAR activation and intracellular changes in caspase-3 and Bax in response to prolonged morphine administration (Jordan et al. 2007).

Interestingly, our results demonstrated that neuroprotective agents such as serotonin1A receptor agonist, minocycline (Habibi-Asl 2009; Habibi-Asl et al. 2009a), selegiline,… could prevent morphine induced tolerance and apoptosis. The stimulation of serotonin1A (5HT1A) receptors induces a variable level of neuroprotection in different animal models of central nervous system injury such as ischemia, (Prehn et al. 1993; Semkova et al. 1998; Schaper et al. 2000; Kukley et al. 2001; Torup et al. 2000) N-methyl-D-aspartate (NMDA) excitotoxicity, (Oosterink et al. 1998; Oosterink et al. 2003) acute subdural hematoma, (Alessandri et al. 1999) and traumatic brain injury (Kline et al. 2001). Furthermore, in vitro evidence indicates that 5HT1A agonists are able to protect neurons from apoptosis induced by staurosporine (Suchanek et al. 1998), glutamate (Semkova et al. 1998), or serum deprivation (Ahlemeyer and Krieglstein 1997; Ahlemeyer et al. 1999). There are different hypotheses on the mechanisms involved in 5HT1A-mediated neuroprotection, including neuronal membrane hyperpolarization that reduces excitability,(Ahlemeyer and Krieglstein 1997; Krüger et al. 1999), reduced glutamate release, (Mauler et al. 2001) and blockade of voltage-sensitive Na channels (Melena et al. 2000).

Other neuroprotective mechanisms have also been proposed for 5HT1A agonists such as stimulation of the anti-apoptotic proto-oncogene B-cell lymphoma protein 2 (BCL-2) expression through the mitogen-activated protein kinase (MAPK/ERK) signaling pathway (Kukley et al. 2001) and suppression of the proapoptotic protein caspase-3 in a MAPK- and protein kinase C alfa-dependent manner (Adayev et al. 2003).

More recently we examined the effect of 8-OH-DPAT, a specific 5-HT1A receptor agonist, on morphine induced tolerance to an analgesic effect in rat. We found that Intra-dorsal raphe nucleus (DRN) administration of the 5-HT1A receptor agonist, 8-OH-DPAT, prevented morphine-induced apoptosis after tolerance to the analgesic effect. On the other hand, the total analgesic effect of morphine significantly increased in animals treated with morphine and 8-OH-DPAT in comparison with the control group. In addition, the results indicated that administration of both 5HT1 agonist (8-OH-DPAT) and antagonist (NAN-190) together with morphine prevent the antiapoptotic activity of the 5HT1A agonist. This means that after antagonizing the 5HT1A receptor, the apoptosis process has already developed. Another mechanism contributes to the morphine tolerance is microglial activation. Studies showed that NMDA-induced neuronal death involved proliferation and activation of microglial cells and that neuroprotective agents such as minocycline completely prevented NMDA toxicity and the preceding activation and proliferation of microglial cells. These results support the notion that microglial activation contributes to excitotoxic neuronal death, which can be inhibited by anti- inflammatory compounds, such as minocycline (Tikka and Koistinaho 2001). The mechanism underlying the role of glial cells in the effects of morphine on naive mice is unclear. It is possible that morphine acts directly on microglia, triggering alterations in their morphology, metabolism, and function (Watkins et al. 2005). Mika et al. concluded that the effect of minocycline on morphine tolerance is related to microglia. Their results provide evidence that systemic administration of minocycline in mice influences morphine's effectiveness and delays the development of morphine tolerance by attenuating microglial activation and its markers (Mika et al. 2009).

In summary, we believe that adding the neuroprotective agents to analgesic drugs specially opioids, increase the analgesic effect and prevents the hyperalgesia and tolerance to their analgesic effects.

#### **9. References**

92 Pain Management – Current Issues and Opinions

activated protein kinase (MAPK) (Mao et al. 2002), suggesting that chronic morphine may

Our more recent studies demonstrated that prolonged morphine administration induces upregulation of proapoptotic elements such as caspase-3 and down regulation of the antiapoptotic factors Bcl-2 and HSP70 in the rat cerebral cortex and spinal cord (Hassanzadeh et al.; Hassanzadeh et al.; Tikka and Koistinaho 2001; Gabra et al. 2005; Hassanzadeh K et al. 2011). Importantly, up-regulation of caspase-3 and Bax was inhibited when morphine was co-administered with the noncompetitive NMDAR antagonist MK-801, thereby supporting a link between NMDAR activation and intracellular changes in caspase-3 and Bax in

Interestingly, our results demonstrated that neuroprotective agents such as serotonin1A receptor agonist, minocycline (Habibi-Asl 2009; Habibi-Asl et al. 2009a), selegiline,… could prevent morphine induced tolerance and apoptosis. The stimulation of serotonin1A (5HT1A) receptors induces a variable level of neuroprotection in different animal models of central nervous system injury such as ischemia, (Prehn et al. 1993; Semkova et al. 1998; Schaper et al. 2000; Kukley et al. 2001; Torup et al. 2000) N-methyl-D-aspartate (NMDA) excitotoxicity, (Oosterink et al. 1998; Oosterink et al. 2003) acute subdural hematoma, (Alessandri et al. 1999) and traumatic brain injury (Kline et al. 2001). Furthermore, in vitro evidence indicates that 5HT1A agonists are able to protect neurons from apoptosis induced by staurosporine (Suchanek et al. 1998), glutamate (Semkova et al. 1998), or serum deprivation (Ahlemeyer and Krieglstein 1997; Ahlemeyer et al. 1999). There are different hypotheses on the mechanisms involved in 5HT1A-mediated neuroprotection, including neuronal membrane hyperpolarization that reduces excitability,(Ahlemeyer and Krieglstein 1997; Krüger et al. 1999), reduced glutamate release, (Mauler et al. 2001) and blockade of voltage-sensitive Na

Other neuroprotective mechanisms have also been proposed for 5HT1A agonists such as stimulation of the anti-apoptotic proto-oncogene B-cell lymphoma protein 2 (BCL-2) expression through the mitogen-activated protein kinase (MAPK/ERK) signaling pathway (Kukley et al. 2001) and suppression of the proapoptotic protein caspase-3 in a MAPK- and

More recently we examined the effect of 8-OH-DPAT, a specific 5-HT1A receptor agonist, on morphine induced tolerance to an analgesic effect in rat. We found that Intra-dorsal raphe nucleus (DRN) administration of the 5-HT1A receptor agonist, 8-OH-DPAT, prevented morphine-induced apoptosis after tolerance to the analgesic effect. On the other hand, the total analgesic effect of morphine significantly increased in animals treated with morphine and 8-OH-DPAT in comparison with the control group. In addition, the results indicated that administration of both 5HT1 agonist (8-OH-DPAT) and antagonist (NAN-190) together with morphine prevent the antiapoptotic activity of the 5HT1A agonist. This means that after antagonizing the 5HT1A receptor, the apoptosis process has already developed. Another mechanism contributes to the morphine tolerance is microglial activation. Studies showed that NMDA-induced neuronal death involved proliferation and activation of microglial cells and that neuroprotective agents such as minocycline completely prevented NMDA toxicity and the preceding activation and proliferation of microglial cells. These results support the notion that microglial activation contributes to excitotoxic neuronal death, which can be inhibited by anti- inflammatory compounds, such as minocycline (Tikka and Koistinaho 2001). The mechanism underlying the role of glial cells in the effects of morphine on naive mice is unclear. It is possible that morphine acts directly on microglia,

lead to changes within the central nervous system.

channels (Melena et al. 2000).

response to prolonged morphine administration (Jordan et al. 2007).

protein kinase C alfa-dependent manner (Adayev et al. 2003).


Neuroprotection and Pain Management 95

Giorgetti M, Bacciottini L, Giovannini MG, Colivicchi MA, Goldfarb J, Blandina P. Local

Glantz LA, Gilmore JH, Lieberman JA, Jarskog LF. Apoptotic mechanisms and the synaptic

Gomez del Pulgar T, Velasco G, Guzman M. The CB1 cannabinoid receptor is coupled to the activation of protein kinase B/Akt. Biochem J 2000;347(Pt 2):369-373. Goswami R, Dawson SA, Dawson G. Cyclic AMP protects against staurosporine and

Greenamyre JT. Glutamatergic influences on the basal ganglia. Clin Neuropharmacol

Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. Embo J 1998;17(14):3878-3885. Habibi-Asl B, Alimohammadi, B., Charkhpour, M., Hassanzadeh, K. Evaluation the Effects

Habibi-Asl B, Hassanzadeh K. Effects of ketamine and midazolam on morphine induced

Habibi-Asl B, Hassanzadeh K, Charkhpour M. Central administration of minocycline and

Habibi-Asl B, Hassanzadeh K, Vafai H, Mohammadi S. Development of morphine induced

Habibi-Asl B, Hassanzadeh, K., Moosazadeh, S. Effects of ketamine and magnesium on morphine induced tolerance and dependence in mice. DARU 2005;13:110-115. Hajos N, Katona I, Naiem SS, MacKie K, Ledent C, Mody I, Freund TF. Cannabinoids inhibit

Harada H, Ueda H, Katada T, Ui M, Satoh M. Phosphorylated mu-opioid receptor purified

Harris EW, Ganong AH, Cotman CW. Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors. Brain Res 1984;323(1):132-137. Harte SE, Hoot MR, Borszcz GS. Involvement of the intralaminar parafascicular nucleus in muscarinic-induced antinociception in rats. Brain Res 2004;1019(1-2):152-161. Hassanzadeh K, L R, Habibi-asl B, Farajnia S, Izadpanah E, Nemati M, Arasteh M,

Hassanzadeh K, Habibi-asl B, Farajnia S, Roshangar L. Minocycline prevents morphine-

mechanism for attenuating morphine tolerance. Neurotox Res;19(4):649-659.

pathology of schizophrenia. Schizophr Res 2006;81(1):47-63.

Tabriz University of Medical Sciences) 2009;15:205-212.

sulfate in mice. Pak J Biol Sci 2009b;12(10):798-803.

cortex. Pharamacol Rep 2011;63:697-707.

dependence and tolerance in mice. DARU 2004;12:101-105.

reconstituted lipid vesicles. Neurosci Lett 1990;113(1):47-49.

rats. Eur J Neurosci 2000;12(6):1941-1948.

1998;286(2):883-889.

2009a;109(3):936-942.

2000;12(9):3239-3249.

2001;24(2):65-70.

GABAergic modulation of acetylcholine release from the cortex of freely moving

wortmannin-induced apoptosis and opioid-enhanced apoptosis in both embryonic and immortalized (F-11kappa7) neurons. J Neurochem 1998;70(4):1376-1382. Govitrapong P, Suttitum T, Kotchabhakdi N, Uneklabh T. Alterations of immune functions

in heroin addicts and heroin withdrawal subjects. J Pharmacol Exp Ther

of Systemic Administration of Minocycline and Riluzole on Tolerance to Morphine Analgesic effect in rat. . Pharmaceutical Sciences (Journal of Faculty of Pharmacy,

riluzole prevents morphine-induced tolerance in rats. Anesth Analg

tolerance and withdrawal symptoms is attenuated by lamotrigine and magnesium

hippocampal GABAergic transmission and network oscillations. Eur J Neurosci

from rat brains lacks functional coupling with Gi1, a GTP-binding protein in

Mohammadi S. Riluzole prevents morphine-induced apoptosis in rat cerebral

induced apoptosis in rat cerebral cortex and lumbar spinal cord: a possible


Catania MV, Hollingsworth Z, Penney JB, Young AB. Phospholipase A2 modulates different

Charkhpour M, Habibi Asl B, Yagobifard S, Hassanzadeh K. Evaluation the effect of co-

Cid C, Alvarez-Cermeno JC, Regidor I, Salinas M, Alcazar A. Low concentrations of

Dawson G, Dawson SA, Goswami R. Chronic exposure to kappa-opioids enhances the

de Fiebre NC, de Fiebre CM. alpha7 Nicotinic acetylcholine receptor knockout selectively

Deadwyler SA, Hampson RE, Mu J, Whyte A, Childers S. Cannabinoids modulate voltage

Debono MW, Le GJ, Canton T, Doble A, Pradier L. Inhibition by riluzole of

Ferchmin PA, Perez D, Eterovic VA, de Vellis J. Nicotinic receptors differentially regulate N-

Finnerup NB, Otto M, McQuay HJ, Jensen TS, Sindrup SH. Algorithm for neuropathic pain

Gabra BH, Afify EA, Daabees TT, Abou Zeit-Har MS. The role of the NO/NMDA pathways

Galve-Roperh I, Aguado T, Palazuelos J, Guzman M. Mechanisms of control of neuron survival by the endocannabinoid system. Curr Pharm Des 2008;14(23):2279-2288. Galve-Roperh I, Rueda D, Gomez del Pulgar T, Velasco G, Guzman M. Mechanism of

Ghelardini C, Galeotti N, Nicolodi M, Donaldson S, Sicuteri F, Bartolini A. Involvement of

Ghelardini C, Galeotti N, Uslenghi C, Grazioli I, Bartolini A. Prochlorperazine induces

treatment: an evidence based proposal. Pain 2005;118(3):289-305.

expressed in Xenopus oocytes. Eur J Pharmacol 1993;235(2-3):283-289. Deutsch SI, Rosse RB, Schwartz BL, Mastropaolo J. A revised excitotoxic hypothesis of schizophrenia: therapeutic implications. Clin Neuropharmacol 2001;24(1):43-49. Doble A. The role of excitotoxicity in neurodegenerative disease: implications for therapy.

Neurochem 1993;60(1):236-245.

lateral sclerosis. J Neurol Sci 2003;206(1):91-95. Craig RW. The bcl-2 gene family. Semin Cancer Biol 1995;6(1):35-43.

by a mechanism that may involve ceramide.

process. J Pharmacol Exp Ther 1995;273(2):734-743.

Pharmacol Ther 1999;81(3):163-221.

Pharmacol Res 2005;51(4):319-327.

Mol Pharmacol 2002;62(6):1385-1392.

J Clin Pharmacol Res 1997;17(2-3):105-109.

2003;305(3):1071-1078.

2004;50(3):351-358.

2009;14(4):209-217.

J Neurochem 1997;68(6):2363-2370.

2005;373(1):42-47.

subtypes of excitatory amino acid receptors: autoradiographic evidence. J

administration of gabapentin and sodium selenite on the development of tolerance to morphine analgesia and dependence in mice. Pharmaceutical Sciences

glutamate induce apoptosis in cultured neurons: implications for amyotrophic

susceptibility of immortalized neurons (F-11kappa 7) to apoptosis-inducing drugs

enhances ethanol-, but not beta-amyloid-induced neurotoxicity. Neurosci Lett

sensitive potassium A-current in hippocampal neurons via a cAMP-dependent

electrophysiological responses mediated by rat kainate and NMDA receptors

methyl-D-aspartate damage in acute hippocampal slices. J Pharmacol Exp Ther

in the development of morphine withdrawal induced by naloxone in vitro.

extracellular signal-regulated kinase activation by the CB(1) cannabinoid receptor.

central cholinergic system in antinociception induced by sumatriptan in mouse. Int

central antinociception mediated by the muscarinic system. Pharmacol Res


Neuroprotection and Pain Management 97

Koch T, Kroslak T, Mayer P, Raulf E, Höllt V. Site mutation in the rat mu-opioid receptor

Kreitzer AC, Regehr WG. Retrograde inhibition of presynaptic calcium influx by

Krueger KM, Daaka Y, Pitcher JA, Lefkowitz RJ. The role of sequestration in G protein-

dephosphorylation by vesicular acidification. J Biol Chem 1997;272(1):5-8. Krüger H, Heinemann U, Luhmann HJ. Effects of ionotropic glutamate receptor blockade

Kukley M, Schaper C, Becker A, Rose K, Krieglstein J. Effect of 5-hydroxytryptamine 1A receptor

Lake JR, Hebert KM, Payza K, Deshotel KD, Hausam DD, Witherspoon WE, Arcangeli KA,

Laudenbach V, Medja F, Zoli M, Rossi FM, Evrard P, Changeux JP, Gressens P. Selective

Maldonado R, Blendy JA, Tzavara E, Gass P, Roques BP, Hanoune J, Schütz G. Reduction of

Manzanares J, Julian M, Carrascosa A. Role of the cannabinoid system in pain control and

Mao J. NMDA and opioid receptors: their interactions in antinociception, tolerance and

Mao J, Mayer DJ. Spinal cord neuroplasticity following repeated opioid exposure and its

Mao J, Mayer DJ, Hayes RL, Lu J, Price DD. Differential roles of NMDA and non-NMDA

Mao J, Price DD, Zhu J, Lu J, Mayer DJ. The inhibition of nitric oxide-activated poly(ADP-

Mao J, Sung B, Ji RR, Lim G. Neuronal apoptosis associated with morphine tolerance: evidence for an opioid-induced neurotoxic mechanism. J Neurosci 2002;22(17):7650-7661. Martin SE, de Fiebre NE, de Fiebre CM. The alpha7 nicotinic acetylcholine receptor-selective

in primary neuron-enriched cultures. Brain Res 2004;1022(1-2):254-256.

with painful peripheral mononeuropathy Brain Res 1992;598:271–278. Mao J, Price DD, Mayer DJ. Thermal hyperalgesia in association with the development of

effects on neonatal excitotoxic brain injuries. Faseb J 2002;16(3):423-425. Law PY, Wong YH, Loh HH. Molecular mechanisms and regulation of opioid receptor

signaling. Annu Rev Pharmacol Toxicol 2000;40:389-430.

neuroplasticity. Brain Res Brain Res Rev 1999;30(3):289-304.

relation to pathological pain. Ann N Y Acad Sci 2001;933(175-84).

neurons and neuropathic pain in the rat. Pain 1997;72(3):355-366.

rats after transient focal ischaemia. Neuroscience 2001;107(3):405-413.

in agonist-mediated desensitization.

Neuroreport 1999;10(12):2651-2656.

Lett 1992;146(2):203-206.

1996;273(5275):657-659.

Curr Neuropharmacol 2006;4(3):239-257.

kinase C. J Neurosci 1994;14(4):2301-2312.

J Neurochem 1997;69(4):1767-1770.

2001;29(3):717-727.

demonstrates the involvement of calcium/calmodulin-dependent protein kinase II

endogenous cannabinoids at excitatory synapses onto Purkinje cells. Neuron

coupled receptor resensitization. Regulation of beta2-adrenergic receptor

and 5-HT1A receptor activation on spreading depression in rat neocortical slices.

agonist BAY X 3702 on BCL-2 and BAX proteins level in the ipsilateral cerebral cortex of

Malin DH. Analog of neuropeptide FF attenuates morphine tolerance. Neurosci

activation of central subtypes of the nicotinic acetylcholine receptor has opposite

morphine abstinence in mice with a mutation in the gene encoding CREB. Science

therapeutic implications for the management of acute and chronic pain episodes.

receptor activation in induction and maintenance of thermal hyperalgesia in rats

morphine tolerance in rats: roles of excitatory amino acid receptors and protein

ribose) synthetase attenuates transsynaptic alteration of spinal cord dorsal horn

antagonist, methyllycaconitine, partially protects against beta-amyloid1-42 toxicity


Hassanzadeh K, Habibi-asl B, Roshangar L, Nemati M, Ansarin M, Farajnia S.

Hockenbery D, Nuñez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner

Hwang J, Adamson C, Butler D, Janero DR, Makriyannis A, Bahr BA. Enhancement of

Ivy Carroll F, Ma W, Navarro HA, Abraham P, Wolckenhauer SA, Damaj MI, Martin BR.

Johnson EM, Jr., Greenlund LJ, Akins PT, Hsu CY. Neuronal apoptosis: current

Jordan J, Fernandez-Gomez FJ, Ramos M, Ikuta I, Aguirre N, Galindo MF. Minocycline and

Kaneko S, Maeda T, Kume T, Kochiyama H, Akaike A, Shimohama S, Kimura J. Nicotine

neuronal receptors and neuronal CNS receptors. Brain Res 1997;765(1):135-140. Karanian DA, Brown QB, Makriyannis A, Bahr BA. Blocking cannabinoid activation of FAK

Karanian DA, Brown QB, Makriyannis A, Kosten TA, Bahr BA. Dual modulation of

Karanian DA, Karim SL, Wood JT, Williams JS, Lin S, Makriyannis A, Bahr BA.

associated brain damage. J Pharmacol Exp Ther 2007;322(3):1059-1066. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br J Cancer 1972;26(4):239-257. Khaspekov LG, Brenz Verca MS, Frumkina LE, Hermann H, Marsicano G, Lutz B.

phosphatidylinositol 3 kinase cascade. J Neurosci Res 2002;70(3):274-282. Kline AE, Yu J, Horváth E, Marion DW, Dixon CE. The selective 5-HT(1A) receptor agonist

traumatic brain injury in rats. Neuroscience 2001;106(3):547-555.

neuroprotective therapeutic modality. Life Sci;86(15-16):615-623.

treatment of mice. Neurosci Lett 1996;204(1-2):85-88.

Psychoneuroendocrinology 2009;34 Suppl 1:S178-185.

injury. J Neurotrauma 1995;12(5):843-852.

excitotoxicity. J Neurosci 2005b;25(34):7813-7820.

1990;348(6299):334-336.

2007;4(3):225-231.

2005a;508(1-3):47-56.

Intracerebroventricular administration of riluzole prevents morphine-induced apoptosis in the lumbar region of the rat spinal cord. Pharmacol Rep;62(4):664-673. Hassouna I, Wickert H, Zimmermann M, Gillardon F. Increase in bax expression in

substantia nigra following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

mitochondrial membrane protein that blocks programmed cell death. Nature

endocannabinoid signaling by fatty acid amide hydrolase inhibition: a

Synthesis, nicotinic acetylcholine receptor binding, antinociceptive and seizure properties of methyllycaconitine analogs. Bioorg Med Chem 2007;15(2):678-685. Jevtovic-Todorovic V, Covey DF, Todorovic SM. Are neuroactive steroids promising

therapeutic agents in the management of acute and chronic pain?

understanding of molecular mechanisms and potential role in ischemic brain

cytoprotection: shedding new light on a shadowy controversy. Curr Drug Deliv

protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha7-

and ERK1/2 compromises synaptic integrity in hippocampus. Eur J Pharmacol

endocannabinoid transport and fatty acid amide hydrolase protects against

Endocannabinoid enhancement protects against kainic acid-induced seizures and

Involvement of brain-derived neurotrophic factor in cannabinoid receptordependent protection against excitotoxicity. Eur J Neurosci 2004;19(7):1691-1698. Kihara T, Shimohama S, Sawada H, Honda K, Nakamizo T, Kanki R, Yamashita H, Akaike

A. Protective effect of dopamine D2 agonists in cortical neurons via the

repinotan HCl attenuates histopathology and spatial learning deficits following


Neuroprotection and Pain Management 99

Polakiewicz RD, Schieferl SM, Dorner LF, Kansra V, Comb MJ. A mitogen-activated protein

Prehn JH, Welsch M, Backhauss C, Nuglisch J, Ausmeier F, Karkoutly C, Krieglstein J.

Razoux F, Garcia R, Lena I. Ketamine, at a dose that disrupts motor behavior and latent

Scascighini L, Toma V, Dober-Spielmann S, Sprott H. Multidisciplinary treatment for

Schaper C, Zhu Y, Kouklei M, Culmsee C, Krieglstein J. Stimulation of 5-HT(1A) receptors

Schlesinger PH, Gross A, Yin XM, Yamamoto K, Saito M, Waksman G, Korsmeyer SJ.

Scotter EL, Abood ME, Glass M. The endocannabinoid system as a target for the treatment

Semkova I, Wolz P, Krieglstein J. Neuroprotective effect of 5-HT1A receptor agonist, Bay X 3702, demonstrated in vitro and in vivo. Eur J Pharmacol 1998;359(2-3):251-260. Sharma SK, Klee WA, Nirenberg M. Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc Natl Acad Sci U S A 1975;72(8):3092-3096. Shulman Y, Tibbo PG. Neuroactive steroids in schizophrenia. Can J Psychiatry

Singhal PC, Kapasi AA, Reddy K, Franki N, Gibbons N, Ding G. Morphine promotes

Singhal PC, Sharma P, Kapasi AA, Reddy K, Franki N, Gibbons N. Morphine enhances

Suchanek B, Struppeck H, Fahrig T. The 5-HT1A receptor agonist BAY x 3702 prevents staurosporine-induced apoptosis. Eur J Pharmacol 1998;355(1):95-101. Takada-Takatori Y, Kume T, Izumi Y, Ohgi Y, Niidome T, Fujii T, Sugimoto H, Akaike A.

Tikka TM, Koistinaho JE. Minocycline provides neuroprotection against N-methyl-D-

Roles of nicotinic receptors in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation. Biol Pharm Bull

aspartate neurotoxicity by inhibiting microglia. J Immunol 2001;166(12):7527-7533.

of neurodegenerative disease. Br J Pharmacol;160(3):480-498.

apoptosis in Jurkat cells. J Leukoc Biol 1999;66(4):650-658.

macrophage apoptosis. J Immunol 1998;160(4):1886-1893.

antiapoptotic BCL-2. Proc Natl Acad Sci U S A 1997;94(21):11357-11362. Schulte-Hermann R, Bursch W, Kraupp-Grasl B, Oberhammer F, Wagner A. Programmed

nucleus accumbens. Neuropsychopharmacology 2007;32(3):719-727. Rothman SM, Olney JW. Glutamate and the pathophysiology of hypoxic--ischemic brain

Sastry PS, Rao KS. Apoptosis and the nervous system. J Neurochem 2000;74(1):1-20.

kinase pathway is required for mu-opioid receptor desensitization. J Biol Chem

Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia. Brain Res 1993;630(1-2):10-20. Rahn EJ, Hohmann AG. Cannabinoids as pharmacotherapies for neuropathic pain: from the

inhibition, enhances prefrontal cortex synaptic efficacy and glutamate release in the

chronic pain: a systematic review of interventions and outcomes. Rheumatology

reduces apoptosis after transient forebrain ischemia in the rat. Brain Res

Comparison of the ion channel characteristics of proapoptotic BAX and

cell death and its protective role with particular reference to apoptosis. Toxicol Lett

Paice JA, Ferrell B. The management of cancer pain. CA Cancer J Clin;61(3):157-182.

bench to the bedside. Neurotherapeutics 2009;6(4):713-737.

damage. Ann Neurol 1986;19(2):105-111.

(Oxford) 2008;47(5):670-678.

1992;64-65 Spec No:569-574.

2005;50(11):695-702.

2009;32(3):318-324.

2000;883(1):41-50.

1998;273(20):12402-12406.


Martinez M, Frank A, Diez-Tejedor E, Hernanz A. Amino acid concentrations in

Marx CE, Stevens RD, Shampine LJ, Uzunova V, Trost WT, Butterfield MI, Massing MW,

Mauler F, Fahrig T, Horváth E, Jork R. Inhibition of evoked glutamate release by the

Mayer DJ, Mao J, Holt J, Price DD. Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions. Proc Natl Acad Sci U S A 1999;96(14):7731-7736. McMahon SB, Cafferty WB, Marchand F. Immune and glial cell factors as pain mediators

Melena J, Chidlow G, Osborne NN. Blockade of voltage-sensitive Na(+) channels by the 5-

Mika J, Wawrzczak-Bargiela A, Osikowicz M, Makuch W, Przewlocka B. Attenuation of

Millan MJ. N-Methyl-D-aspartate receptors as a target for improved antipsychotic agents: novel insights and clinical perspectives. Psychopharmacology (Berl) 2005;179(1):30-53. Mitchell JM, Basbaum AI, Fields HL. A locus and mechanism of action for associative

Moghaddam B, Adams B, Verma A, Daly D. Activation of glutamatergic neurotransmission

Molina-Holgado F, Pinteaux E, Heenan L, Moore JD, Rothwell NJ, Gibson RM.

Moncada C, Lekieffre D, Arvin B, Meldrum B. Effect of NO synthase inhibition on NMDAand ischaemia-induced hippocampal lesions. Neuroreport 1992;3(6):530-532. Mudo G, Belluardo N, Fuxe K. Nicotinic receptor agonists as neuroprotective/neurotrophic drugs. Progress in molecular mechanisms. J Neural Transm 2007;114(1):135-147.

Nestler EJ. Molecular mechanisms of drug addiction. Journal of Neuroscience

Olney JW, Newcomer JW, Farber NB. NMDA receptor hypofunction model of

Oosterink BJ, Harkany T, Luiten PG. Post-lesion administration of 5-HT1A receptor agonist

Oosterink BJ, Korte SM, Nyakas C, Korf J, Luiten PGM. Neuroprotection against N-methyl-

HT1A receptor agonist 8-OH-DPAT. Eur J Pharmacol 1998;358(2):147-152.

8-OH-DPAT protects cholinergic nucleus basalis neurons against NMDA

D-aspartate-induced excitotoxicity in rat magnocellular nucleus basalis by the 5-

therapeutics. Neuropsychopharmacology 2006;31(6):1249-1263.

Neural Transm Park Dis Dement Sect 1993;6(1):1-9.

and modulators. Exp Neurol 2005;192(2):444-462. McQuay H. Opioids in pain management. Lancet 1999;353:2229-2232.

Eur J Pharmacol 2000;406(3):319-324.

Neurosci 1997;17(8):2921-2927.

Neurosci 2005;28(1):189-194.

1992;12(7):2439-2450.

mice. Brain Behav Immun 2009;23(1):75-84.

morphine tolerance. Nat Neurosci 2000 3(1):47-53.

Nagata S. Fas ligand-induced apoptosis. Annu Rev Genet 1999;33:29-55.

schizophrenia. J Psychiatr Res 1999;33(6):523-533.

excitotoxicity. Neuroreport 2003;14(1):57-60.

Res 2001;888(1):150-157.

cerebrospinal fluid and serum in Alzheimer's disease and vascular dementia. J

Hamer RM, Morrow AL, Lieberman JA. Neuroactive steroids are altered in schizophrenia and bipolar disorder: relevance to pathophysiology and

neuroprotective 5-HT(1A) receptor agonist BAY x 3702 in vitro and in vivo. Brain

HT(1A) receptor agonist 8-OH-DPAT: possible significance for neuroprotection.

morphine tolerance by minocycline and pentoxifylline in naive and neuropathic

by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex. J

Neuroprotective effects of the synthetic cannabinoid HU-210 in primary cortical neurons are mediated by phosphatidylinositol 3-kinase/AKT signaling. Mol Cell Paice JA, Ferrell B. The management of cancer pain. CA Cancer J Clin;61(3):157-182.


**6** 

*1,3,4USA 2UK* 

**Reduced Antinociceptive** 

**Effect of Repeated Treatment** 

**with a Cannabinoid Receptor Type 2** 

**Following Spinal Nerve Transection** 

Joyce A. DeLeo1,3,4 and E. Alfonso Romero-Sandoval1,3,4 *1Neuroscience Center at Dartmouth, Dartmouth Medical School,* 

*3Department of Anesthesiology, Dartmouth-Hitchcock Medical Center,* 

 **Agonist in Cannabinoid-Tolerant Rats** 

Matthew S. Alkaitis1,2, Christian Ndong1,3, Russell P. Landry III1,3,

*2Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital,* 

*4Department of Pharmacology and Toxicology, Dartmouth-Hitchcock Medical Center,* 

In both preclinical and clinical studies, agents that activate cannabinoid receptors type 1 (CB1) and 2 (CB2) have shown promise in the treatment of pain (Wade et al., 2004; Romero-Sandoval and Eisenach, 2007). Cannabinoids are licensed for the clinical treatment of cancer chemotherapy-associated nausea and vomiting (USA and Canada), immunodeficiency syndrome-associated loss of appetite and weight loss (USA and Canada), multiple sclerosisassociated spasticity (United Kingdom and Canada) and neuropathic pain (Canada). However, clinical use of cannabinoid compounds is limited both by undesirable neurological side effects and by induction of tolerance. In animal models, neurological side effects have been shown to be dependent on CB1 receptor but not CB2 receptor activation (Romero-Sandoval and Eisenach, 2007). Furthermore, sustained spinal or subcutaneous administration of the CB1 receptor agonist, WIN 55,212-2 has been shown to induce hypersensitivity and antinociceptive tolerance in naive mice and rats. In contrast, we (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a) and others (Yao et al., 2009) have shown that spinal CB2 receptor agonists (such as JWH015) relieve postoperative and neuropathic pain in rodent models without inducing neurological side effects or antinociceptive tolerance. Despite advancements in the molecular mechanisms involved in cannabinoid tolerance (Martini et al., 2010), a better understanding of the respective roles of CB1 and CB2 receptors is required to design effective therapies that do not induce tolerance. Further advances in this area may also guide clinical treatment of patients who have already developed tolerance through prior exposure to non-selective cannabinoid agonists for

**1. Introduction** 

recreational or medical purposes.


## **Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection**

Matthew S. Alkaitis1,2, Christian Ndong1,3, Russell P. Landry III1,3,

Joyce A. DeLeo1,3,4 and E. Alfonso Romero-Sandoval1,3,4 *1Neuroscience Center at Dartmouth, Dartmouth Medical School, 2Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, 3Department of Anesthesiology, Dartmouth-Hitchcock Medical Center, 4Department of Pharmacology and Toxicology, Dartmouth-Hitchcock Medical Center, 1,3,4USA 2UK* 

#### **1. Introduction**

100 Pain Management – Current Issues and Opinions

Torup L, Møller A, Sager TN, Diemer NH. Neuroprotective effect of 8-OH-DPAT in global

Trujillo KA. Effects of noncompetitive N-methyl-D-aspartate receptor antagonists on opiate

Trujillo KA, Akil H. Inhibition of morphine tolerance and dependence by the NMDA

Ueda H, Inoue M. Peripheral morphine analgesia resistant to tolerance in chronic morphine-

Ueda H, Inoue M, Matsumoto T. Protein kinase C-mediated inhibition of mu-opioid

Ueda H, Miyamae T, Hayashi C, Watanabe S, Fukushima N, Sasaki Y, Iwamura T, Misu Y.

Wallace MJ, Blair RE, Falenski KW, Martin BR, DeLorenzo RJ. The endogenous cannabinoid

Watkins LR, Hutchinson MR, Johnston IN, Maier SF. Glia: novel counter-regulators of

Whiteside GT, Munglani R. Cell death in the superficial dorsal horn in a model of

Wilson RI, Kunos G, Nicoll RA. Presynaptic specificity of endocannabinoid signaling in the

Xiao Y, He J, Gilbert RD, Zhang L. Cocaine induces apoptosis in fetal myocardial cells through a mitochondria-dependent pathway. J Pharmacol Exp Ther 2000;292(1):8-14. Yaksh TL, Dirksen R, Harty GJ. Antinociceptive effects of intrathecally injected cholinomimetic drugs in the rat and cat. Eur J Pharmacol 1985;117(1):81-88. Yang XF, Xiao Y, Xu MY. Both endogenous and exogenous ACh plays antinociceptive role

Yin D, Mufson RA, Wang R, Shi Y. Fas-mediated cell death promoted by opioids. Nature

Zhang J, Barak LS, Winkler KE, Caron MG, Ferguson SS. A central role for beta-arrestins

Zhang J, Ferguson SS, Barak LS, Bodduluri SR, Laporte SA, Law PY, Caron MG. Role for G

receptor responsiveness. Proc Natl Acad Sci U S A 1998;95(12):7157-7162. Zuo DY, Zhang YH, Cao Y, Wu CF, Tanaka M, Wu YL. Effect of acute and chronic MK-801

and clathrin-coated vesicle-mediated endocytosis in beta2-adrenergic receptor resensitization. Differential regulation of receptor resensitization in two distinct cell

protein-coupled receptor kinase in agonist-specific regulation of mu-opioid

administration on extracellular glutamate and ascorbic acid release in the prefrontal cortex of freely moving mice on line with open-field behavior. Life Sci

in the hippocampus CA1 of rats. J Neural Transm 2008;115(1):1-6.

to peripheral mu-agonist analgesia. J Neurosci 2001;21(9):2967-2973. Ueda H, Inoue M, Takeshima H, Iwasawa Y. Enhanced spinal nociceptin receptor

receptor antagonist MK-801. Science 1991;251(4989):85-87.

treated mice. Neurosci Lett 1999;266(2):105-108.

epilepsy. J Pharmacol Exp Ther 2003;307(1):129-137.

opioid analgesia. Trends Neurosci 2005;28(12):661-669.

neuropathic pain. J Neurosci Res 2001;64(2):168-173.

Woolf CJ. What is this thing called pain? J Clin Invest;120(11):3742-3744.

hippocampus. Neuron 2001;31(3):453-462.

types. J Biol Chem 1997;272(43):27005-27014.

2000;395(2):137-141.

2000;20(20):7640-7647.

1995;15(11):7485-4799.

1999;397(6716):218.

2006;78(19):2172-2178.

cerebral ischemia assessed by stereological cell counting. Eur J Pharmacol

tolerance and physical dependence. Neuropsychopharmacology 1995;13(4):301-307.

receptor internalization and its involvement in the development of acute tolerance

expression develops morphine tolerance and dependence. J Neurosci

Protein kinase C involvement in homologous desensitization of delta-opioid receptor coupled to Gi1-phospholipase C activation in Xenopus oocytes. J Neurosci

system regulates seizure frequency and duration in a model of temporal lobe

In both preclinical and clinical studies, agents that activate cannabinoid receptors type 1 (CB1) and 2 (CB2) have shown promise in the treatment of pain (Wade et al., 2004; Romero-Sandoval and Eisenach, 2007). Cannabinoids are licensed for the clinical treatment of cancer chemotherapy-associated nausea and vomiting (USA and Canada), immunodeficiency syndrome-associated loss of appetite and weight loss (USA and Canada), multiple sclerosisassociated spasticity (United Kingdom and Canada) and neuropathic pain (Canada). However, clinical use of cannabinoid compounds is limited both by undesirable neurological side effects and by induction of tolerance. In animal models, neurological side effects have been shown to be dependent on CB1 receptor but not CB2 receptor activation (Romero-Sandoval and Eisenach, 2007). Furthermore, sustained spinal or subcutaneous administration of the CB1 receptor agonist, WIN 55,212-2 has been shown to induce hypersensitivity and antinociceptive tolerance in naive mice and rats. In contrast, we (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a) and others (Yao et al., 2009) have shown that spinal CB2 receptor agonists (such as JWH015) relieve postoperative and neuropathic pain in rodent models without inducing neurological side effects or antinociceptive tolerance. Despite advancements in the molecular mechanisms involved in cannabinoid tolerance (Martini et al., 2010), a better understanding of the respective roles of CB1 and CB2 receptors is required to design effective therapies that do not induce tolerance. Further advances in this area may also guide clinical treatment of patients who have already developed tolerance through prior exposure to non-selective cannabinoid agonists for recreational or medical purposes.

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

examined per animal.

Chemicon, Billerica, Massachusetts).

(Molecular Probes, Eugene, Oregon).

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 103

pixels above a preset intensity threshold using SigmaScan Pro 5 as previously described (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008b). For both CB1 receptor and CB2 receptor expression, the staining intensity was examined in a standardized area of superficial laminae (I-II) and deep laminae (III-V) of the L5 dorsal horn in 3–4 slices

Immunofluorescence was also used for dual labeling with specific cell markers and CB1 receptor or CB2 receptor. All sections were blocked in 5% Normal Goat Serum (NGS) and 0.01% Triton-X-100 for 1 hour at 4 °C. Sections were incubated in the appropriate primary antibody or antibodies diluted in a buffer composed of 1% NGS and 1% Triton-X-100 in PBS overnight at 4 °C. To determine the cellular localization of CB1 receptor or CB2 receptor we co-labeled antibodies for CB1 receptor and CB2 receptor with the following cellular markers (antibodies): rabbit polyclonal anti-Iba-1 for microglia (1:1000, Wako Pure Chemical Industries, Richmond, VA), mouse polyclonal anti-GFAP for astrocytes (1:400, Sigma, Saint Louis, Missouri), mouse polyclonal antibody anti- ED2/CD163 for perivascular cells (1:150, Serotec, Raleigh, NC), mouse polyclonal anti-Neuronal Nuclei, NeuN for neurons (1:10,000,

The following secondary antibodies were used as indicated in table 1: Alexa-Fluor™ 488 Goat anti-Rabbit IgG1 (Molecular Probes, Eugene, Oregon), Alexa-Fluor™ 488 Goat anti-Mouse IgG1 (Molecular Probes, Eugene, Oregon), Alexa-Fluor™ 555 Goat anti-Mouse IgG (Molecular Probes, Eugene, Oregon) and Alexa-Fluor™ 555 Donkey anti-Goat IgG

To avoid cross-reactivity between the secondary antibodies in the CB2 receptor colocalization experiments, sections were first incubated in Alexa-Fluor™ 555 Donkey anti-Goat IgG (Molecular Probes, Eugene, Oregon) as described above, washed 2 times in PBS and then incubated in the appropriate Alexa-Fluor™ 488 secondary antibody as described above. This protocol modification prevented binding of the Alexa-Fluor™ 555 Donkey anti-Goat IgG to the goat-derived Alexa-Fluor™ 488. The specificity of each antibody was tested by omitting the primary antibody on 1-3 additional sections. To avoid crossreactivity when co-staining with primary antibodies against Iba-1 and CB1 receptors that are both rabbit-derived, a TSA Signal Amplification Kit was used following the manufacturer instructions (PerkinElmer LifeSciences Inc, Boston, MA). On the first day, normal immunofluorescence protocol was followed except that sections were incubated only in anti-CB1 receptor antibody at a concentration of 1:10,000. On the second day sections were washed 2 times for 5 minutes in PBS then incubated in a biotinylated Goat α Rabbit secondary antibody for 1 hour at 4 °C. Sections were then subjected to another wash, incubated in SA-HRP (1:100) for 1 hour at 4 °C, washed again and incubated in the TSA fluorophore (1:250) for 10 minutes at 4 °C. Sections were then washed again and incubated overnight in the Iba-1 primary antibody (1:1000). The next day sections were subjected to normal day 2 immunofluorescence protocol to visualize Iba-1 (described above). One control was included with only the anti-CB1 receptor primary antibody (1:10,000) and the Alexa 555 Goat α Rabbit secondary antibody to control for any crossreactivity that might cause CB1 receptor expression to appear in red. A second control included only the anti-CB1 receptor primary antibody and the TSA kit in order to visualize the staining achieved in the absence of the co-stain. Finally, a third control included the TSA kit, Iba-1 primary and the Alexa 555 Goat α Rabbit secondary antibody but excluded the anti-CB1 receptor primary antibody. This third control provided

Using the L5 nerve transection (L5NT) rodent model of chronic neuropathic pain, this study was designed to test: 1) whether a non-selective cannabinoid agonist (CP55940) induces tolerance following repeated intrathecal (i.t.) administration in a model of neuropathic pain; 2) whether this antinociceptive tolerance could be reversed by the cessation of drug exposure; and 3) whether sustained spinal administration of the nonselective cannabinoid CP55940 affects antinociception induced by a CB2 receptor agonist (JWH015). To determine the site of action of these agonists we additionally examined expression levels and cellular localization of CB1 and CB2 receptors in the spinal cord of rats receiving either L5NT or sham surgery.

#### **2. Materials and methods**

#### **2.1 Animals and surgical procedures**

These studies were performed in accordance with the Guidelines for Animal Experimentation of the International Association for the Study of Pain (IASP) and after approval by the Institutional Animal Care and Use Committee at Dartmouth College (Dartmouth Medical School, Hanover, New Hampshire). Male Sprague-Dawley rats weighing 200–250 g (Harlan, Indianapolis, IN) at the start of surgery underwent L5NT surgery as previous described (Tanga et al., 2005). Briefly, rats were anesthetized with 2% isoflurane in oxygen and a small incision to the skin overlying L5–S1 was made followed by retraction of the paravertebral musculature from the vertebral transverse processes. The L6 transverse process was then partially removed to expose the L4 and L5 spinal nerves. The L5 spinal nerve was identified, lifted slightly, and transected. The wound was irrigated with saline and sutured in two layers. Sham surgeries were performed in other group of rats following the same procedure but without manipulating or injuring the nerves. The surgeries and anesthesia exposure lasted 15 – 20 minutes. Animals were housed individually and maintained in a 12:12 hr light/dark cycle with *ad libitum* access to food and water. Efforts were made to limit animal distress and to use the minimum number of animals necessary to achieve statistical significance.

#### **2.2 Tissue preparation, immunohistochemistry, imaging and image analysis**

After being anesthetized with 2-4% isoflurane in oxygen, rats were perfused transcardially with phosphate buffered saline (0.01 M, 150 ml) followed by 4% formaldehyde (350 ml) at room temperature. The L5 spinal cord section was collected and placed in 30% sucrose for 48–72 hr at 4 °C. The tissue was then frozen in O.C.T. Compound (Sakura Finetek, Torrance, CA) and stored at -80 °C. To determine the expression of spinal CB2 receptor immunohistochemistry was performed on transverse 20-µm L5 spinal cord free-floating sections by using the Vector ELITE ABC (Vector Labs, Burlingame, CA), avidin-biotin complex technique and a goat polyclonal antibody against the C-terminus of CB2 receptor (1:150, Santa Cruz biotechnology, Santa Cruz, CA, sc10076) as we have previously described (Romero-Sandoval et al., 2008a). Immunofluorescence was performed to determine the spinal CB1 receptor expression level using a rabbit polyclonal antibody (1:200, Cayman, Ann Arbor, MI) and a Alexa-Fluor™ 488 Goat anti-Rabbit IgG1 secondary antibody (Molecular Probes, Eugene, Oregon). For CB1 receptor and CB2 receptor expression quantification, the sections were examined with an Olympus microscope, and images were captured with a Q-Fire cooled camera (Olympus, Melville, NY). We quantified the CB1 receptor or CB2 receptor expression, blinded to experimental conditions, as the number of

Using the L5 nerve transection (L5NT) rodent model of chronic neuropathic pain, this study was designed to test: 1) whether a non-selective cannabinoid agonist (CP55940) induces tolerance following repeated intrathecal (i.t.) administration in a model of neuropathic pain; 2) whether this antinociceptive tolerance could be reversed by the cessation of drug exposure; and 3) whether sustained spinal administration of the nonselective cannabinoid CP55940 affects antinociception induced by a CB2 receptor agonist (JWH015). To determine the site of action of these agonists we additionally examined expression levels and cellular localization of CB1 and CB2 receptors in the spinal cord of

These studies were performed in accordance with the Guidelines for Animal Experimentation of the International Association for the Study of Pain (IASP) and after approval by the Institutional Animal Care and Use Committee at Dartmouth College (Dartmouth Medical School, Hanover, New Hampshire). Male Sprague-Dawley rats weighing 200–250 g (Harlan, Indianapolis, IN) at the start of surgery underwent L5NT surgery as previous described (Tanga et al., 2005). Briefly, rats were anesthetized with 2% isoflurane in oxygen and a small incision to the skin overlying L5–S1 was made followed by retraction of the paravertebral musculature from the vertebral transverse processes. The L6 transverse process was then partially removed to expose the L4 and L5 spinal nerves. The L5 spinal nerve was identified, lifted slightly, and transected. The wound was irrigated with saline and sutured in two layers. Sham surgeries were performed in other group of rats following the same procedure but without manipulating or injuring the nerves. The surgeries and anesthesia exposure lasted 15 – 20 minutes. Animals were housed individually and maintained in a 12:12 hr light/dark cycle with *ad libitum* access to food and water. Efforts were made to limit animal distress and to use the minimum number of

**2.2 Tissue preparation, immunohistochemistry, imaging and image analysis** 

After being anesthetized with 2-4% isoflurane in oxygen, rats were perfused transcardially with phosphate buffered saline (0.01 M, 150 ml) followed by 4% formaldehyde (350 ml) at room temperature. The L5 spinal cord section was collected and placed in 30% sucrose for 48–72 hr at 4 °C. The tissue was then frozen in O.C.T. Compound (Sakura Finetek, Torrance, CA) and stored at -80 °C. To determine the expression of spinal CB2 receptor immunohistochemistry was performed on transverse 20-µm L5 spinal cord free-floating sections by using the Vector ELITE ABC (Vector Labs, Burlingame, CA), avidin-biotin complex technique and a goat polyclonal antibody against the C-terminus of CB2 receptor (1:150, Santa Cruz biotechnology, Santa Cruz, CA, sc10076) as we have previously described (Romero-Sandoval et al., 2008a). Immunofluorescence was performed to determine the spinal CB1 receptor expression level using a rabbit polyclonal antibody (1:200, Cayman, Ann Arbor, MI) and a Alexa-Fluor™ 488 Goat anti-Rabbit IgG1 secondary antibody (Molecular Probes, Eugene, Oregon). For CB1 receptor and CB2 receptor expression quantification, the sections were examined with an Olympus microscope, and images were captured with a Q-Fire cooled camera (Olympus, Melville, NY). We quantified the CB1 receptor or CB2 receptor expression, blinded to experimental conditions, as the number of

rats receiving either L5NT or sham surgery.

animals necessary to achieve statistical significance.

**2.1 Animals and surgical procedures** 

**2. Materials and methods** 

pixels above a preset intensity threshold using SigmaScan Pro 5 as previously described (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008b). For both CB1 receptor and CB2 receptor expression, the staining intensity was examined in a standardized area of superficial laminae (I-II) and deep laminae (III-V) of the L5 dorsal horn in 3–4 slices examined per animal.

Immunofluorescence was also used for dual labeling with specific cell markers and CB1 receptor or CB2 receptor. All sections were blocked in 5% Normal Goat Serum (NGS) and 0.01% Triton-X-100 for 1 hour at 4 °C. Sections were incubated in the appropriate primary antibody or antibodies diluted in a buffer composed of 1% NGS and 1% Triton-X-100 in PBS overnight at 4 °C. To determine the cellular localization of CB1 receptor or CB2 receptor we co-labeled antibodies for CB1 receptor and CB2 receptor with the following cellular markers (antibodies): rabbit polyclonal anti-Iba-1 for microglia (1:1000, Wako Pure Chemical Industries, Richmond, VA), mouse polyclonal anti-GFAP for astrocytes (1:400, Sigma, Saint Louis, Missouri), mouse polyclonal antibody anti- ED2/CD163 for perivascular cells (1:150, Serotec, Raleigh, NC), mouse polyclonal anti-Neuronal Nuclei, NeuN for neurons (1:10,000, Chemicon, Billerica, Massachusetts).

The following secondary antibodies were used as indicated in table 1: Alexa-Fluor™ 488 Goat anti-Rabbit IgG1 (Molecular Probes, Eugene, Oregon), Alexa-Fluor™ 488 Goat anti-Mouse IgG1 (Molecular Probes, Eugene, Oregon), Alexa-Fluor™ 555 Goat anti-Mouse IgG (Molecular Probes, Eugene, Oregon) and Alexa-Fluor™ 555 Donkey anti-Goat IgG (Molecular Probes, Eugene, Oregon).

To avoid cross-reactivity between the secondary antibodies in the CB2 receptor colocalization experiments, sections were first incubated in Alexa-Fluor™ 555 Donkey anti-Goat IgG (Molecular Probes, Eugene, Oregon) as described above, washed 2 times in PBS and then incubated in the appropriate Alexa-Fluor™ 488 secondary antibody as described above. This protocol modification prevented binding of the Alexa-Fluor™ 555 Donkey anti-Goat IgG to the goat-derived Alexa-Fluor™ 488. The specificity of each antibody was tested by omitting the primary antibody on 1-3 additional sections. To avoid crossreactivity when co-staining with primary antibodies against Iba-1 and CB1 receptors that are both rabbit-derived, a TSA Signal Amplification Kit was used following the manufacturer instructions (PerkinElmer LifeSciences Inc, Boston, MA). On the first day, normal immunofluorescence protocol was followed except that sections were incubated only in anti-CB1 receptor antibody at a concentration of 1:10,000. On the second day sections were washed 2 times for 5 minutes in PBS then incubated in a biotinylated Goat α Rabbit secondary antibody for 1 hour at 4 °C. Sections were then subjected to another wash, incubated in SA-HRP (1:100) for 1 hour at 4 °C, washed again and incubated in the TSA fluorophore (1:250) for 10 minutes at 4 °C. Sections were then washed again and incubated overnight in the Iba-1 primary antibody (1:1000). The next day sections were subjected to normal day 2 immunofluorescence protocol to visualize Iba-1 (described above). One control was included with only the anti-CB1 receptor primary antibody (1:10,000) and the Alexa 555 Goat α Rabbit secondary antibody to control for any crossreactivity that might cause CB1 receptor expression to appear in red. A second control included only the anti-CB1 receptor primary antibody and the TSA kit in order to visualize the staining achieved in the absence of the co-stain. Finally, a third control included the TSA kit, Iba-1 primary and the Alexa 555 Goat α Rabbit secondary antibody but excluded the anti-CB1 receptor primary antibody. This third control provided

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

from Tocris, Ellisville, MI.

**non-tolerant animals** 

paws were evaluated as described above.

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 105

gauge 5/8-inch hypodermic needle. The needle was inserted intrathecally, on the midline between the fourth and fifth lumbar vertebrae. The correct injection site was confirmed with the stimulation of nerves in the cauda equina when the lumbar needle penetrated the dura and produced a brief but obvious movement of the tail and/or the hind paws. The animals regained consciousness 2–3 min after the discontinuation of anesthesia. Drugs were diluted in dimethylsulfoxide and saline in a ratio of 1:1 and administered in a volume of 15 µl as previously described (Romero-Sandoval et al., 2008a). The drugs used were: the dual (CB1 receptor and CB2 receptor) cannabinoid receptor agonist CP55940 (5-(1,1-Dimethylheptyl)-2- [5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]phenol; Sigma Chemical Co., St. Louis, MO); the CB2 receptor agonist JWH015 ((2-Methyl-1-propyl-1*H*-indol-3-yl)-1-naphthalenylmethanone), the CB1 receptor antagonist AM281 (1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-*N*-4 morpholinyl-1*H*-pyrazole-3-carboxamide) and the CB2 receptor antagonist AM630 (6-Iodo-2 methyl-1-[2-(4-morpholinyl)ethyl]-1*H*-indol-3-yl](4-methoxyphenyl)methanone), purchased

**2.5 Repeated CP55940 administration and monitoring of behavioral effects** 

**2.6 Evaluation of response to acute CP55940 dose escalation in tolerant and** 

CP55940 was acutely administered i.t. in 30-min interval escalating doses: 0.4, 2, 10 and 50 µg in L5NT animals 24 hr before and 24 hr after the repeated (5 day) treatment with CP55940 (n=5) or vehicle (n=6). As a control, vehicle was administered i.t. using the same dose escalation paradigm in animals that had previously received L5NT followed by repeated (5 days) treatment with CP55940 (n=8). The antinociceptive effect of escalating doses of CP55940 was evaluated 15 min after every injection. The effectiveness and potency of CP55940 were calculated using these dose responses and were compared in both repeated CP55940 and repeated vehicle treatment groups. To determine whether cannabinoidmediated tolerance was reversed following the discontinuation of sustained CP55940 administration, the antinociceptive response to escalating doses of CP55940 were also measured two weeks after the last day of repeated CP55940 treatment (washout period). In summary, responses to acute CP55940 dose escalation (or vehicle) was evaluated in the following cases: 1) prior to any additional treatment, 2) 24 hours after repeated (5-day) treatment with CP55940, 3) 24 hours after repeated (5-day) treatment with vehicle, and 4) 2 weeks (washout period) after repeated (5-day) treatment with CP55940. To confirm that CP55940 induced its effects via CB1 receptor and CB2 receptor as we have previously demonstrated (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a), we

Beginning four days after surgery, CP55940 (100 µg/injection, n=18) or vehicle (n=17) was administered in single daily injections (8:00-9:00 AM) for five days. This dose and i.t. administration method were chosen based on our previous study using CP55940 in the same model of neuropathic pain (Romero-Sandoval and Eisenach, 2007), and on a previous study that demonstrated induction of antinociceptive tolerance with another non-selective cannabinoid agonist WIN 55,212-2 (Gardell et al., 2002) at a dose of 100 µg twice daily. Drugs and vehicle were administered i.t. based on previous evidence that spinal cord mechanisms drive induction of cannabinoid tolerance (Gardell et al., 2002). Two hours after each injection, mechanical withdrawal thresholds in both ipsilateral and contralateral hind-


visualization of the non-specific background staining produced by the kit alone. All controls confirmed the specificity of the co-stain.

Table 1. Details of antibody selections for all immunofluorescense experiments, CB1: Cannabinoid receptor type 1, CB2: Cannabinoid receptor type 2, ED2: Perivascular cell marker, GFAP: Glial Fibrillary Acidic Protein, Iba-1: Ionized Calcium–Binding Adapter Molecule 1, NeuN: Neuronal Nuclei.

Stained sections were examined with an Olympus fluorescence microscope, and images were captured with a Q-Fire cooled camera (Olympus, Melville, NY). Confocal microscopy was also performed using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss AG, Oberkochen, Germany; Englert Cell Analysis Laboratory, Dartmouth). Merged color images were processed using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA).

#### **2.3 Behavioral testing**

Mechanical allodynia was evaluated by measuring the 50% withdrawal threshold using an up–down statistical method (Chaplan et al., 1994) and calibrated von Frey filaments (1 – 60 g, Stoelting, Wood Dale, IL). At each time point, two measurements were made on the paw ipsilateral to surgery in 5-10 min intervals, and the average of these values was used for data analyses. As an internal control, withdrawal thresholds were also measured in the paw contralateral to surgery (uninjured side). The withdrawal threshold was determined for each animal before surgery, 4 days after surgery (immediately before any pharmacological treatment), and after drug administration (different time points for different paradigms, see below). The investigator was blinded to drug treatment in all behavioral tests.

#### **2.4 Drugs and treatments**

Drugs were administered by intrathecal (i.t.) injection by means of lumbar puncture under brief inhalational anesthesia (2-4% isoflurane in oxygen) using a Hamilton syringe and a 28-

visualization of the non-specific background staining produced by the kit alone. All

**(Co-stain) Primary Secondary Fluorophore optimal** 

(TSA Signal Amplification Kit) 488 (555)

(Goat α Rabbit) 488 (555)

(Goat α Rabbit) 488 (555)

(Donkey α Goat) 488 (555)

(Donkey α Goat) 488 (555)

(Donkey α Goat) 488 (555)

(Donkey α Goat) 488 (555)

CB1 Rabbit Goat α Rabbit 488

Table 1. Details of antibody selections for all immunofluorescense experiments,

were processed using Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA).

below). The investigator was blinded to drug treatment in all behavioral tests.

CB1: Cannabinoid receptor type 1, CB2: Cannabinoid receptor type 2, ED2: Perivascular cell marker, GFAP: Glial Fibrillary Acidic Protein, Iba-1: Ionized Calcium–Binding Adapter

Stained sections were examined with an Olympus fluorescence microscope, and images were captured with a Q-Fire cooled camera (Olympus, Melville, NY). Confocal microscopy was also performed using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss AG, Oberkochen, Germany; Englert Cell Analysis Laboratory, Dartmouth). Merged color images

Mechanical allodynia was evaluated by measuring the 50% withdrawal threshold using an up–down statistical method (Chaplan et al., 1994) and calibrated von Frey filaments (1 – 60 g, Stoelting, Wood Dale, IL). At each time point, two measurements were made on the paw ipsilateral to surgery in 5-10 min intervals, and the average of these values was used for data analyses. As an internal control, withdrawal thresholds were also measured in the paw contralateral to surgery (uninjured side). The withdrawal threshold was determined for each animal before surgery, 4 days after surgery (immediately before any pharmacological treatment), and after drug administration (different time points for different paradigms, see

Drugs were administered by intrathecal (i.t.) injection by means of lumbar puncture under brief inhalational anesthesia (2-4% isoflurane in oxygen) using a Hamilton syringe and a 28-

**excitation (nm)** 

controls confirmed the specificity of the co-stain.

Iba1 (CB1) Rabbit (Rabbit) Goat α Rabbit

GFAP (CB1) Mouse (Rabbit) Goat α Mouse

ED2 (CB1) Mouse (Rabbit) Goat α Mouse

Iba1 (CB2) Rabbit (Goat) Goat α Rabbit

GFAP (CB2) Rabbit (Goat) Goat α Rabbit

ED2 (CB2) Mouse (Goat) Goat α Mouse

NeuN (CB2) Mouse (Goat) Goat α Mouse

Molecule 1, NeuN: Neuronal Nuclei.

**2.3 Behavioral testing** 

**2.4 Drugs and treatments** 

**Antigen** 

gauge 5/8-inch hypodermic needle. The needle was inserted intrathecally, on the midline between the fourth and fifth lumbar vertebrae. The correct injection site was confirmed with the stimulation of nerves in the cauda equina when the lumbar needle penetrated the dura and produced a brief but obvious movement of the tail and/or the hind paws. The animals regained consciousness 2–3 min after the discontinuation of anesthesia. Drugs were diluted in dimethylsulfoxide and saline in a ratio of 1:1 and administered in a volume of 15 µl as previously described (Romero-Sandoval et al., 2008a). The drugs used were: the dual (CB1 receptor and CB2 receptor) cannabinoid receptor agonist CP55940 (5-(1,1-Dimethylheptyl)-2- [5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]phenol; Sigma Chemical Co., St. Louis, MO); the CB2 receptor agonist JWH015 ((2-Methyl-1-propyl-1*H*-indol-3-yl)-1-naphthalenylmethanone), the CB1 receptor antagonist AM281 (1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-*N*-4 morpholinyl-1*H*-pyrazole-3-carboxamide) and the CB2 receptor antagonist AM630 (6-Iodo-2 methyl-1-[2-(4-morpholinyl)ethyl]-1*H*-indol-3-yl](4-methoxyphenyl)methanone), purchased from Tocris, Ellisville, MI.

#### **2.5 Repeated CP55940 administration and monitoring of behavioral effects**

Beginning four days after surgery, CP55940 (100 µg/injection, n=18) or vehicle (n=17) was administered in single daily injections (8:00-9:00 AM) for five days. This dose and i.t. administration method were chosen based on our previous study using CP55940 in the same model of neuropathic pain (Romero-Sandoval and Eisenach, 2007), and on a previous study that demonstrated induction of antinociceptive tolerance with another non-selective cannabinoid agonist WIN 55,212-2 (Gardell et al., 2002) at a dose of 100 µg twice daily. Drugs and vehicle were administered i.t. based on previous evidence that spinal cord mechanisms drive induction of cannabinoid tolerance (Gardell et al., 2002). Two hours after each injection, mechanical withdrawal thresholds in both ipsilateral and contralateral hindpaws were evaluated as described above.

#### **2.6 Evaluation of response to acute CP55940 dose escalation in tolerant and non-tolerant animals**

CP55940 was acutely administered i.t. in 30-min interval escalating doses: 0.4, 2, 10 and 50 µg in L5NT animals 24 hr before and 24 hr after the repeated (5 day) treatment with CP55940 (n=5) or vehicle (n=6). As a control, vehicle was administered i.t. using the same dose escalation paradigm in animals that had previously received L5NT followed by repeated (5 days) treatment with CP55940 (n=8). The antinociceptive effect of escalating doses of CP55940 was evaluated 15 min after every injection. The effectiveness and potency of CP55940 were calculated using these dose responses and were compared in both repeated CP55940 and repeated vehicle treatment groups. To determine whether cannabinoidmediated tolerance was reversed following the discontinuation of sustained CP55940 administration, the antinociceptive response to escalating doses of CP55940 were also measured two weeks after the last day of repeated CP55940 treatment (washout period). In summary, responses to acute CP55940 dose escalation (or vehicle) was evaluated in the following cases: 1) prior to any additional treatment, 2) 24 hours after repeated (5-day) treatment with CP55940, 3) 24 hours after repeated (5-day) treatment with vehicle, and 4) 2 weeks (washout period) after repeated (5-day) treatment with CP55940. To confirm that CP55940 induced its effects via CB1 receptor and CB2 receptor as we have previously demonstrated (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a), we

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

used for analyses.

**2.10 Statistical analyses** 

were used for statistical analyses.

**3. Results** 

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 107

~1 cm of diameter and 10 cm above and parallel to a table, leaving the hind paws resting on the table. A cataleptic animal will stay in that position longer than a normal animal. The time in which the animal puts its forelimb on the table was recorded, using a cut off time of 60 s. The righting test consists of placing the animal supine and recording the ability to right itself. Righting was scored on a scale of 0-3, 0 indicating normal righting reflex (an immediate and coordinated twisting of the body to an upright position), 1 indicating mild impairment (ability to completely right, but slowly), 2 indicating moderate impairment (ability to right the forelimbs slowly followed by the hind limbs with more difficulty) and 3 indicating severe impairment (inability to right in 20 sec). Vocalization was rated on a scale of 0-3, 0 indicating absent vocalization, 1 indicating some vocalization when manipulated, 2 indicating consistent vocalization when manipulated and 3 indicating vocalization even light touch. Exploratory activity was rated on a scale of 0-3 with 0 indicating normal activity, 1 indicating only head movements without vertical and/or horizontal exploration, 2 indicating no spontaneous movements and 3 indicating splayed posture with no spontaneous movements. All behavioral measures were performed twice and the average

The effects of L5NT surgery and drug injections on bar test, placing-stepping test and withdrawal thresholds were examined using the repetitive measurements one-way analysis of variance. If significant effects were found, Tukey's multiple comparison or Dunnett's test was conducted. Differences between groups were examined using two-way analysis of variance. If differences were found, the Bonferroni post test was used. In the acute antinociceptive effect studies, acute i.t. JWH015 50% of maximum efficacy (ED50) and its 95% confidence limits were calculated and compared between repeated CP55940 and repeated vehicle groups using Student's *t* test. ED50s were calculated using the baseline and after-surgery withdrawal thresholds as maximum and minimum effect values respectively. Vocalization, righting test and exploratory activity data following treatment were compared using the Friedman Repeated Measures Analysis of Variance on Rank test. If significant effects were found, non-parametric Wilcoxon signed ranks tests were conducted comparing each time point to the baseline value (before surgery). Between group differences were compared at each time period using the Kruskal-Wallis test. Significant effects were further evaluated using the Mann-Whitney U test comparing only the novel treatment to control or agonist group. The effects of CP55940 in acute antinociception vs. CP55940 in the presence of CB1 receptor or CB2 receptor antagonist was evaluated by one-way ANOVA followed by Dunnett's post-test. The effects of JWH015 in acute antinociception in the presence of the CB2 receptor antagonist was evaluated by unpaired Student's t-test. Data are presented as mean ± SEM. In all cases a *P*  value less than 0.05 was considered significant. SigmaStat and GraphPad inStat software

**3.1 Spinal cord CB1 and CB2 receptor expression and cellular localization** 

Compared to rats receiving sham surgery, rats receiving L5NT surgery demonstrated significantly higher CB1 receptor expression in the L5 dorsal horn on postoperative days 4 and 7 (Figure 1). The changes in CB1 receptor expression were primarily apparent in

administered CP55940 at a dose of 50 µg in combination with vehicle, the CB1 receptor antagonist AM281 at a dose of 50 µg or the CB2 receptor antagonist AM630 at a dose of 50 µg in a separate group of rats. Mechanical withdrawal threshold was determined 2 hr after treatments.

#### **2.7 Evaluation of response to acute JWH015 dose escalation in tolerant and non-tolerant animals**

JWH015, a CB2 receptor agonist, was acutely administered i.t. in 30-min interval escalating doses: 0.4, 2, 10 and 50 µg in L5NT animals that had previously received repeated (5 days) treatment with CP55940 (n=8) or vehicle (n=8). Vehicle was acutely administered i.t. using the same dose escalation paradigm in animals that had previously received L5NT followed by repeated (5 days) treatment with CP55940 (n=8). The antinociceptive effect of escalating doses of JWH015 was evaluated 15 min after every injection and its efficacy and potency were quantified. The first set of experiments was performed 24 hr after the last day of repeated CP55940 administration to test whether the cannabinoid-mediated tolerance influenced the antinociceptive effects of a CB2 receptor agonist administered acutely. The second set of experiments was performed two weeks after the last day of repeated CP55940 treatment (washout period) to test whether the potency and/or efficacy of the CB2 receptor agonist, JWH015 improves following the discontinuation of sustained CP55940 treatment. In summary, responses to acute JWH015 dose escalation (or vehicle) were evaluated in the following cases: 1) 24 hours after repeated (5-day) treatment with CP55940 or vehicle and 2) 2 weeks (washout period) after repeated (5-day) treatment with CP55940 or vehicle.

To confirm that JWH015 induced its effects via CB2 receptors as we have previously demonstrated (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a), we administered JWH015 at a dose of 50 µg in combination with the CB2 receptor antagonist AM630 at a dose of 50 µg or vehicle in a separate group of animals. Mechanical withdrawal threshold was determined 2 hr after treatments.

#### **2.8 Evaluation of response to repeated JWH015 administration in tolerant and non-tolerant animals**

Following the washout period (two weeks after repeated administration of CP55940 or vehicle), JWH015 (50 µg/injection, n=9) or vehicle (n=8) was administered in single daily injections (8:00-9:00 AM) for four days. Behavioral testing were performed before and 2 hr after each injection. Antinociceptive tolerance was evaluated by testing mechanical withdrawal thresholds in the paw ipsilateral or contralateral to surgery.

#### **2.9 Assessment of neurological side effects**

Based on our previous studies (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a) righting and placing-stepping tests were used to evaluate motor reflexes; the bar test was used to evaluate catalepsy; vocalization was used as a sign of irritability or discomfort to manipulation and exploratory activity was used as a measure of awareness. These parameters were evaluated before, 20 minutes and 2.5 hr after each injection (following behavioral mechanical hypersensitivity testing). The placing-stepping reflex was tested by placing the rostral aspect of the hind paws on the edge of a table and was quantified as the seconds in which the animals put the paws up and forward into a position to walk. A cut-off of 60 s was used. The bar test consists of placing the forelimbs on a bar of

~1 cm of diameter and 10 cm above and parallel to a table, leaving the hind paws resting on the table. A cataleptic animal will stay in that position longer than a normal animal. The time in which the animal puts its forelimb on the table was recorded, using a cut off time of 60 s. The righting test consists of placing the animal supine and recording the ability to right itself. Righting was scored on a scale of 0-3, 0 indicating normal righting reflex (an immediate and coordinated twisting of the body to an upright position), 1 indicating mild impairment (ability to completely right, but slowly), 2 indicating moderate impairment (ability to right the forelimbs slowly followed by the hind limbs with more difficulty) and 3 indicating severe impairment (inability to right in 20 sec). Vocalization was rated on a scale of 0-3, 0 indicating absent vocalization, 1 indicating some vocalization when manipulated, 2 indicating consistent vocalization when manipulated and 3 indicating vocalization even light touch. Exploratory activity was rated on a scale of 0-3 with 0 indicating normal activity, 1 indicating only head movements without vertical and/or horizontal exploration, 2 indicating no spontaneous movements and 3 indicating splayed posture with no spontaneous movements. All behavioral measures were performed twice and the average used for analyses.

#### **2.10 Statistical analyses**

106 Pain Management – Current Issues and Opinions

administered CP55940 at a dose of 50 µg in combination with vehicle, the CB1 receptor antagonist AM281 at a dose of 50 µg or the CB2 receptor antagonist AM630 at a dose of 50 µg in a separate group of rats. Mechanical withdrawal threshold was determined 2 hr after

JWH015, a CB2 receptor agonist, was acutely administered i.t. in 30-min interval escalating doses: 0.4, 2, 10 and 50 µg in L5NT animals that had previously received repeated (5 days) treatment with CP55940 (n=8) or vehicle (n=8). Vehicle was acutely administered i.t. using the same dose escalation paradigm in animals that had previously received L5NT followed by repeated (5 days) treatment with CP55940 (n=8). The antinociceptive effect of escalating doses of JWH015 was evaluated 15 min after every injection and its efficacy and potency were quantified. The first set of experiments was performed 24 hr after the last day of repeated CP55940 administration to test whether the cannabinoid-mediated tolerance influenced the antinociceptive effects of a CB2 receptor agonist administered acutely. The second set of experiments was performed two weeks after the last day of repeated CP55940 treatment (washout period) to test whether the potency and/or efficacy of the CB2 receptor agonist, JWH015 improves following the discontinuation of sustained CP55940 treatment. In summary, responses to acute JWH015 dose escalation (or vehicle) were evaluated in the following cases: 1) 24 hours after repeated (5-day) treatment with CP55940 or vehicle and 2) 2 weeks (washout period) after

To confirm that JWH015 induced its effects via CB2 receptors as we have previously demonstrated (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a), we administered JWH015 at a dose of 50 µg in combination with the CB2 receptor antagonist AM630 at a dose of 50 µg or vehicle in a separate group of animals. Mechanical withdrawal

Following the washout period (two weeks after repeated administration of CP55940 or vehicle), JWH015 (50 µg/injection, n=9) or vehicle (n=8) was administered in single daily injections (8:00-9:00 AM) for four days. Behavioral testing were performed before and 2 hr after each injection. Antinociceptive tolerance was evaluated by testing mechanical

Based on our previous studies (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a) righting and placing-stepping tests were used to evaluate motor reflexes; the bar test was used to evaluate catalepsy; vocalization was used as a sign of irritability or discomfort to manipulation and exploratory activity was used as a measure of awareness. These parameters were evaluated before, 20 minutes and 2.5 hr after each injection (following behavioral mechanical hypersensitivity testing). The placing-stepping reflex was tested by placing the rostral aspect of the hind paws on the edge of a table and was quantified as the seconds in which the animals put the paws up and forward into a position to walk. A cut-off of 60 s was used. The bar test consists of placing the forelimbs on a bar of

**2.8 Evaluation of response to repeated JWH015 administration in tolerant and** 

withdrawal thresholds in the paw ipsilateral or contralateral to surgery.

**2.7 Evaluation of response to acute JWH015 dose escalation in tolerant and** 

repeated (5-day) treatment with CP55940 or vehicle.

threshold was determined 2 hr after treatments.

**2.9 Assessment of neurological side effects** 

treatments.

**non-tolerant animals** 

**non-tolerant animals** 

The effects of L5NT surgery and drug injections on bar test, placing-stepping test and withdrawal thresholds were examined using the repetitive measurements one-way analysis of variance. If significant effects were found, Tukey's multiple comparison or Dunnett's test was conducted. Differences between groups were examined using two-way analysis of variance. If differences were found, the Bonferroni post test was used. In the acute antinociceptive effect studies, acute i.t. JWH015 50% of maximum efficacy (ED50) and its 95% confidence limits were calculated and compared between repeated CP55940 and repeated vehicle groups using Student's *t* test. ED50s were calculated using the baseline and after-surgery withdrawal thresholds as maximum and minimum effect values respectively. Vocalization, righting test and exploratory activity data following treatment were compared using the Friedman Repeated Measures Analysis of Variance on Rank test. If significant effects were found, non-parametric Wilcoxon signed ranks tests were conducted comparing each time point to the baseline value (before surgery). Between group differences were compared at each time period using the Kruskal-Wallis test. Significant effects were further evaluated using the Mann-Whitney U test comparing only the novel treatment to control or agonist group. The effects of CP55940 in acute antinociception vs. CP55940 in the presence of CB1 receptor or CB2 receptor antagonist was evaluated by one-way ANOVA followed by Dunnett's post-test. The effects of JWH015 in acute antinociception in the presence of the CB2 receptor antagonist was evaluated by unpaired Student's t-test. Data are presented as mean ± SEM. In all cases a *P*  value less than 0.05 was considered significant. SigmaStat and GraphPad inStat software were used for statistical analyses.

#### **3. Results**

#### **3.1 Spinal cord CB1 and CB2 receptor expression and cellular localization**

Compared to rats receiving sham surgery, rats receiving L5NT surgery demonstrated significantly higher CB1 receptor expression in the L5 dorsal horn on postoperative days 4 and 7 (Figure 1). The changes in CB1 receptor expression were primarily apparent in

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

group.

all groups.

perivascular cells.

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 109

observed on postoperative days 1 or 7 following nerve injury compared to the sham surgery

Fig. 2. CB2 receptor expression is increased on day 4 following L5 nerve transection. Representative images (A-B) show CB2 receptor expression at postoperative day 4 (D4) in the L5 dorsal horn of rats receiving sham surgery or L5 nerve transection. Details of the superficial laminae (II-III) of the dorsal horn of these spinal cord tissues are shown next to each original image (Aa and Bb). CB2 expression was quantified in the ipsilateral whole dorsal horn (C), laminae I-II (D) and laminae III-IV (E) of rats receiving sham surgery or L5 nerve transection at postoperative days 1, 4 and 7. Receptor expression was quantified as the number of pixels above a set threshold per total pixels in the selected area and normalized to percent of each control, sham group. \*p<0.05 vs. respective sham group by t test. N=3 for

Using confocal microscopy, we observed that spinal CB1 receptors were primarily expressed on NeuN-positive neurons in the dorsal horns of animals receiving L5NT surgery (Figure 3). Occasionally, CB1 receptors expression co-localized with the astrocyte marker GFAP (Figure 3). CB1 receptor expression did not co-localize with Iba-1-positive microglia or ED2/CD163 positive perivascular cells at any observed time point following L5NT (Figure 3). However, cells expressing CB1 receptor were in close proximity to Iba-1-positive microglia and

the deeper laminae (III-V) of the dorsal horn in rats that had received L5NT surgery. CB1 receptor expression on day 1 after surgery was not significantly different between groups.

Fig. 1. CB1 receptor expression is increased on days 4 and 7 after L5 nerve transection. Representative images (A-D) show CB1 receptor expression at postoperative days 4 (D4) and 7 (D7) in the L5 dorsal horn of rats receiving sham surgery or L5 nerve transection. Details of the deep laminae (III-IV) of the dorsal horn of these spinal cord tissues are shown next to each original image (Aa-Dd). CB1 expression was quantified in the ipsilateral whole dorsal horn (E), laminae I-II (F) and laminae III-IV (G) of rats receiving sham surgery or L5 nerve transection at postoperative days 1, 4 and 7. Receptor expression was quantified as the number of pixels above a set threshold per total pixels in the selected area and normalized to percent of each control, sham group. \*p<0.05 vs. respective sham group by t test. N=3 for all groups.

Compared to the sham surgery group, rats receiving L5NT also demonstrated significantly higher spinal CB2 receptor expression on postoperative day 4 (Figure 2). This increased CB2 receptor expression was mainly observed in the superficial laminae (I-II) of the dorsal horn in animals with L5NT surgery. No significant changes in CB2 receptor expression were

the deeper laminae (III-V) of the dorsal horn in rats that had received L5NT surgery. CB1 receptor expression on day 1 after surgery was not significantly different between

Fig. 1. CB1 receptor expression is increased on days 4 and 7 after L5 nerve transection. Representative images (A-D) show CB1 receptor expression at postoperative days 4 (D4) and 7 (D7) in the L5 dorsal horn of rats receiving sham surgery or L5 nerve transection. Details of the deep laminae (III-IV) of the dorsal horn of these spinal cord tissues are shown next to each original image (Aa-Dd). CB1 expression was quantified in the ipsilateral whole dorsal horn (E), laminae I-II (F) and laminae III-IV (G) of rats receiving sham surgery or L5 nerve transection at postoperative days 1, 4 and 7. Receptor expression was quantified as the number of pixels above a set threshold per total pixels in the selected area and normalized to percent of each control, sham group. \*p<0.05 vs. respective sham group by t test. N=3 for

Compared to the sham surgery group, rats receiving L5NT also demonstrated significantly higher spinal CB2 receptor expression on postoperative day 4 (Figure 2). This increased CB2 receptor expression was mainly observed in the superficial laminae (I-II) of the dorsal horn in animals with L5NT surgery. No significant changes in CB2 receptor expression were

groups.

all groups.

observed on postoperative days 1 or 7 following nerve injury compared to the sham surgery group.

Fig. 2. CB2 receptor expression is increased on day 4 following L5 nerve transection. Representative images (A-B) show CB2 receptor expression at postoperative day 4 (D4) in the L5 dorsal horn of rats receiving sham surgery or L5 nerve transection. Details of the superficial laminae (II-III) of the dorsal horn of these spinal cord tissues are shown next to each original image (Aa and Bb). CB2 expression was quantified in the ipsilateral whole dorsal horn (C), laminae I-II (D) and laminae III-IV (E) of rats receiving sham surgery or L5 nerve transection at postoperative days 1, 4 and 7. Receptor expression was quantified as the number of pixels above a set threshold per total pixels in the selected area and normalized to percent of each control, sham group. \*p<0.05 vs. respective sham group by t test. N=3 for all groups.

Using confocal microscopy, we observed that spinal CB1 receptors were primarily expressed on NeuN-positive neurons in the dorsal horns of animals receiving L5NT surgery (Figure 3). Occasionally, CB1 receptors expression co-localized with the astrocyte marker GFAP (Figure 3). CB1 receptor expression did not co-localize with Iba-1-positive microglia or ED2/CD163 positive perivascular cells at any observed time point following L5NT (Figure 3). However, cells expressing CB1 receptor were in close proximity to Iba-1-positive microglia and perivascular cells.

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 111

expression was occasionally observed on NeuN-positive neuronal somata (Figure 4). Even though GFAP-positive spinal cord astrocytes did not demonstrate CB2 receptor expression,

these cells were in close proximity to cells that expressed CB2 receptor (Figure 4).

Fig. 4. CB2 receptors are mainly expressed in microglial cells. Representative confocal images show CB2 receptor cell localization in the ipsilateral L5 dorsal horn of rats at days 1, 4 and 7 after L5 nerve transection. CB2 receptor appears in red. NeuN (marker for neurons), Iba-1 (marker for microglia) and ED2/CD163 (ED2, marker for perivascular microglia) appear in green, and GFAP (marker for astrocytes) appears in grey. GFAP color (originally in green) was changed to grey to obtain a better visualization of this specific marker and any potential expression of CB2 receptors. The colocalization of CB2 receptors with the other

cellular markers is visualized in yellow.

Fig. 3. CB1 receptor is expressed primarily in neurons. Representative confocal images show CB1 receptor cell localization in the ipsilateral L5 dorsal horn of rats at days 1, 4 and 7 after L5 nerve transection. CB1 receptor staining appears in red. NeuN (marker for neurons), Iba-1 (marker for microglia) and ED2/CD163 (ED2, marker for perivascular microglia) appear in green, and GFAP (marker for astrocytes) appears in grey. In the images of CB1 receptors and Iba-1, Iba-1 (originally in red) was changed to green, and CB1 receptor (originally in green) was changed to red to consistently show CB1 receptors in red in all images. GFAP color (originally in green) was changed to grey to obtain a better visualization of occasional expression of CB1 receptors on GFAP-positive cells. The colocalization of CB1 receptors with NeuN appears in yellow.

Microglia (Iba-1 positive cells) and perivascular cells (ED2/CD163 positive cells) displayed localized areas of CB2 receptor expression (Figure 4). Diffuse, punctate CB2 receptor

Fig. 3. CB1 receptor is expressed primarily in neurons. Representative confocal images show CB1 receptor cell localization in the ipsilateral L5 dorsal horn of rats at days 1, 4 and 7 after L5 nerve transection. CB1 receptor staining appears in red. NeuN (marker for neurons), Iba-1 (marker for microglia) and ED2/CD163 (ED2, marker for perivascular microglia) appear in green, and GFAP (marker for astrocytes) appears in grey. In the images of CB1 receptors and Iba-1, Iba-1 (originally in red) was changed to green, and CB1 receptor (originally in green) was changed to red to consistently show CB1 receptors in red in all

Microglia (Iba-1 positive cells) and perivascular cells (ED2/CD163 positive cells) displayed localized areas of CB2 receptor expression (Figure 4). Diffuse, punctate CB2 receptor

images. GFAP color (originally in green) was changed to grey to obtain a better visualization of occasional expression of CB1 receptors on GFAP-positive cells.

The colocalization of CB1 receptors with NeuN appears in yellow.

expression was occasionally observed on NeuN-positive neuronal somata (Figure 4). Even though GFAP-positive spinal cord astrocytes did not demonstrate CB2 receptor expression, these cells were in close proximity to cells that expressed CB2 receptor (Figure 4).

Fig. 4. CB2 receptors are mainly expressed in microglial cells. Representative confocal images show CB2 receptor cell localization in the ipsilateral L5 dorsal horn of rats at days 1, 4 and 7 after L5 nerve transection. CB2 receptor appears in red. NeuN (marker for neurons), Iba-1 (marker for microglia) and ED2/CD163 (ED2, marker for perivascular microglia) appear in green, and GFAP (marker for astrocytes) appears in grey. GFAP color (originally in green) was changed to grey to obtain a better visualization of this specific marker and any potential expression of CB2 receptors. The colocalization of CB2 receptors with the other cellular markers is visualized in yellow.

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

injection on day 5 compared to day 3 (Figure 5).

activation of both CB1 and CB2 receptors.

L5NT no previous treatment

24 hr after

24 hr after

2 weeks after

50% w.t. for the 50 µg dose (efficacy in g)

CP55940 JWH015 CP55940 JWH015

32.9±1.94 14.7 (10.91-19.9)

repeated vehicle 29.7±2.85 17.0±2.7 11.9 (7.6-18.6) 26.4 (13.8-50.5)

repeated CP55940 14.9±1.12 \* 16.3±3.9 112.6 (21.2-596.6) \* 37.4 (26.7-52.5)

repeated CP55940 15.7±4.8 \* 14.2±2.5 162.6 (5.7-4567) \* 32.5 (1.2-872)

Table 2. Effect of the highest dose (50 µg) and ED50 (95% confidence limits) of acute i.t. administration of CP55940 and JWH015 in L5NT, \*P<0.05 vs. L5NT no previous treatment

and 24 hr after repeated vehicle groups. Withdrawal threshold = w.t.

ED50 (95% confidence limits)

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 113

administration of the non-selective cannabinoid agonist CP55940 (100 µg, i.t.) resulted in significantly higher withdrawal thresholds (measured 2 hours following injection) compared to vehicle-treated controls on each day observations were made (Figure 1). However, ipsilateral withdrawal thresholds in animals treated with CP55940 were significantly lower at 2 hr after injection on days 3 - 5 compared to day 1 values (Figure 1). Additionally, the anti-allodynic effect of CP55940 was significantly lower at 2 hr after

In order to test the efficacy and potency of CP55940 before and after its repeated administration, we performed an acute dose escalation with i.t. CP55940. CP55940 reduced L5NT-induced hypersensitivity in a significant and dose-dependent manner before and 24 hr after the 5-day course of daily CP55940 administration (Figure 6). Compared to acute i.t. vehicle treatment, the minimum effective dose of CP55940 was 10 µg, and its maximum effective dose (dose that induced a return to base line values) was 50 µg (the maximum dose tested) before and after its repeated administration. However, CP55940 displayed an approximately 2-fold higher efficacy (p<0.05, Table 2) and an approximately 7-fold higher potency (p<0.05, Table 2) in untreated animals (Figure 6A) than in animals previously treated with CP55940 for five days (repeated CP55940 group, Figure 6B). The higher efficacy and potency of CP55940 observed in untreated animals were similar to the ones observed in animals previously treated for five days with vehicle (repeated vehicle group, Figure 6C, Table 2). We then evaluated the effects of acute CP55940 two weeks after repeated treatment with CP55940 was discontinued (washout period). Even though acute CP55940 was still effective (at 10 and 50 µg doses vs. vehicle) following 2 weeks of washout period, its efficacy and potency were significantly lower than in animals that had not received repeated CP55940 treatment (Figure 6D, Table 2). The acute antinociception induced by CP55940 50 µg (plus vehicle, 32.9 2.1 g, n=6) in the L5NT group was blocked by either the CB1 receptor antagonist AM281 50 µg (14.5 4.3 g, n=4, P<0.05) or the CB2 receptor antagonist AM630 50 µg (15.2 4.3 g, n=4, P<0.05), confirming that the activity of this compound depends on

#### **3.2 CP55940 antinociceptive tolerance**

Mechanical withdrawal thresholds on the uninjured side (paw contralateral to L5NT) were not affected by surgery (26.7±1.4 g vs. 23.1±1.1 g, before and after surgery respectively), nor were they significantly different at any observed time point during the five subsequent days of intrathecal vehicle or CP55940 administration (Figure 5). In the paw ipsilateral to L5NT surgery, withdrawal thresholds were significantly reduced after surgery (26.6±1.3 g vs. 5.4±0.5 g, before and after surgery respectively, p<0.05).

Fig. 5. Antinociceptive effects of repeated i.t. administration of CP55940. Paw withdrawal thresholds indicate responses to von Frey stimulation ipsilateral to L5NT or contralateral to surgery (uninjured side) before surgery (base line = BL), four days after surgery (S), and 2 hr after i.t. injections of vehicle (n=17) or CP55940 (n=18) on days 1, 3 and 5. Withdrawal thresholds on day 1 (D1), day 3 (D3) and day 5 (D5) vs. after surgery data significantly differ by repeated measures one way ANOVA; \*p<0.05 vs. after surgery, +p<0.05 vs. D1 L5NT-CP55940, # p<0.05 vs. D3 L5NT-CP55940 by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups significantly differ by two way ANOVA; p<0.05 compared to vehicle and both contralateral groups by two way ANOVA followed by Bonferroni post tests.

Administration of vehicle (i.t.) on each of the subsequent 5 days did not significantly alter this L5NT-induced hypersensitivity at any time point observed (Figure 5). In contrast,

Mechanical withdrawal thresholds on the uninjured side (paw contralateral to L5NT) were not affected by surgery (26.7±1.4 g vs. 23.1±1.1 g, before and after surgery respectively), nor were they significantly different at any observed time point during the five subsequent days of intrathecal vehicle or CP55940 administration (Figure 5). In the paw ipsilateral to L5NT surgery, withdrawal thresholds were significantly reduced after surgery (26.6±1.3 g vs. 5.4±0.5 g, before and after surgery respectively, p<0.05).

Fig. 5. Antinociceptive effects of repeated i.t. administration of CP55940. Paw withdrawal thresholds indicate responses to von Frey stimulation ipsilateral to L5NT or contralateral to surgery (uninjured side) before surgery (base line = BL), four days after surgery (S), and 2 hr after i.t. injections of vehicle (n=17) or CP55940 (n=18) on days 1, 3 and 5. Withdrawal thresholds on day 1 (D1), day 3 (D3) and day 5 (D5) vs. after surgery data significantly differ

Administration of vehicle (i.t.) on each of the subsequent 5 days did not significantly alter this L5NT-induced hypersensitivity at any time point observed (Figure 5). In contrast,

by repeated measures one way ANOVA; \*p<0.05 vs. after surgery, +p<0.05 vs. D1 L5NT-CP55940, # p<0.05 vs. D3 L5NT-CP55940 by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups significantly differ by two way ANOVA; p<0.05 compared to vehicle and both contralateral groups by two way

ANOVA followed by Bonferroni post tests.

**3.2 CP55940 antinociceptive tolerance** 

administration of the non-selective cannabinoid agonist CP55940 (100 µg, i.t.) resulted in significantly higher withdrawal thresholds (measured 2 hours following injection) compared to vehicle-treated controls on each day observations were made (Figure 1). However, ipsilateral withdrawal thresholds in animals treated with CP55940 were significantly lower at 2 hr after injection on days 3 - 5 compared to day 1 values (Figure 1). Additionally, the anti-allodynic effect of CP55940 was significantly lower at 2 hr after injection on day 5 compared to day 3 (Figure 5).

In order to test the efficacy and potency of CP55940 before and after its repeated administration, we performed an acute dose escalation with i.t. CP55940. CP55940 reduced L5NT-induced hypersensitivity in a significant and dose-dependent manner before and 24 hr after the 5-day course of daily CP55940 administration (Figure 6). Compared to acute i.t. vehicle treatment, the minimum effective dose of CP55940 was 10 µg, and its maximum effective dose (dose that induced a return to base line values) was 50 µg (the maximum dose tested) before and after its repeated administration. However, CP55940 displayed an approximately 2-fold higher efficacy (p<0.05, Table 2) and an approximately 7-fold higher potency (p<0.05, Table 2) in untreated animals (Figure 6A) than in animals previously treated with CP55940 for five days (repeated CP55940 group, Figure 6B). The higher efficacy and potency of CP55940 observed in untreated animals were similar to the ones observed in animals previously treated for five days with vehicle (repeated vehicle group, Figure 6C, Table 2). We then evaluated the effects of acute CP55940 two weeks after repeated treatment with CP55940 was discontinued (washout period). Even though acute CP55940 was still effective (at 10 and 50 µg doses vs. vehicle) following 2 weeks of washout period, its efficacy and potency were significantly lower than in animals that had not received repeated CP55940 treatment (Figure 6D, Table 2). The acute antinociception induced by CP55940 50 µg (plus vehicle, 32.9 2.1 g, n=6) in the L5NT group was blocked by either the CB1 receptor antagonist AM281 50 µg (14.5 4.3 g, n=4, P<0.05) or the CB2 receptor antagonist AM630 50 µg (15.2 4.3 g, n=4, P<0.05), confirming that the activity of this compound depends on activation of both CB1 and CB2 receptors.


Table 2. Effect of the highest dose (50 µg) and ED50 (95% confidence limits) of acute i.t. administration of CP55940 and JWH015 in L5NT, \*P<0.05 vs. L5NT no previous treatment and 24 hr after repeated vehicle groups. Withdrawal threshold = w.t.

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

**3.3 CP55940 neurological side effects** 

test followed by Mann-Whitney U test.

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 115

In order to investigate the neurological side effects of CP55940 administration, we evaluated the place-stepping reflex, vocalization, exploratory activity and the bar test. Repeated vehicle injection did not significantly affect any of these behaviors at any time point observed. CP55940 significantly impaired the placing-stepping reflex (Figure 7A), induced vocalization (Figure 7B) and reduced exploratory activity (Figure 7C) on days 1, 2 and 3 compared to vehicle group, and induced catalepsy (Figure 7D) on days 1, 2, 3 and 4

Fig. 7. Neurological side effects in response to repeated treatment with CP55940. Placingstepping (A), vocalization (B), exploratory activity (C) and bar test (C) scores are shown from before the first injection (base line = BL), and 0.5, 2 and 24 hr after each i.t. injection (days 1 - 5) of vehicle (n=8) or CP55940 (n=13) during five consecutive days. Withdrawal thresholds on days 1, 3 and 5 in placing-stepping and bar test vs. base line data significantly differ by repeated measures one way ANOVA, \*p<0.05 vs. base line, #p<0.05 vs. 0.5 hr, ^p<0.05 vs. 2 hr by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups differ in placing-stepping and bar test by repetitive measurements two-way ANOVA, +p<0.05 vs. CP55940 group by two way ANOVA followed by Bonferroni post tests. Days 1, 3 and 5 values in vocalization and exploratory activity vs. base line significantly differ by Friedman test, \*p<0.05 vs. base line, #p<0.05 vs. 0.5 hr, ^p<0.05 vs. 2 hr by Friedman test followed by Wilcoxon test. Groups in vocalization and exploratory activity significantly differ by Kruskal-Wallis test; +p<0.05 vs. CP55940 by Kruskal-Wallis

Fig. 6. Antinociceptive effects of acute i.t. administration of CP55940. Withdrawal thresholds (95% confidence limits, doted lines) indicate responses to von Frey stimulation ipsilateral to L5NT surgery 15 min after escalating doses (0.4, 2, 10 and 50 µg) of i.t. CP55940 in animals receiving no additional treatment (A, n=5), 24 hr after the discontinuation of repeated treatment (5 days) with CP55940 i.t., 100 µg (B, n=6) or vehicle (C, n=6) and 2 weeks (washout period) after the discontinuation of repeated treatment (5 days) with CP55940 100 µg (D, n=5). Withdrawal thresholds in response to dose escalation of CP55940 significantly differ from after-surgery values by repeated measures one way ANOVA, \*p<0.05 vs. after surgery by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups significantly differ by two way ANOVA; p<0.05 L5NT or L5NT 24 hr after repeated vehicle groups vs. L5NT 24 hr after repeated CP55940 or L5NT 2 weeks after repeated CP55940 groups for 50 µg by two way ANOVA followed by Bonferroni post tests.

#### **3.3 CP55940 neurological side effects**

114 Pain Management – Current Issues and Opinions

Fig. 6. Antinociceptive effects of acute i.t. administration of CP55940. Withdrawal thresholds (95% confidence limits, doted lines) indicate responses to von Frey stimulation ipsilateral to L5NT surgery 15 min after escalating doses (0.4, 2, 10 and 50 µg) of i.t. CP55940 in animals receiving no additional treatment (A, n=5), 24 hr after the discontinuation of repeated treatment (5 days) with CP55940 i.t., 100 µg (B, n=6) or vehicle (C, n=6) and 2 weeks (washout period) after the discontinuation of repeated treatment (5 days) with CP55940 100 µg (D, n=5). Withdrawal thresholds in response to dose escalation of CP55940 significantly differ from after-surgery values by repeated measures one way ANOVA, \*p<0.05 vs. after surgery by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups significantly differ by two way ANOVA; p<0.05 L5NT or L5NT 24 hr after repeated vehicle groups vs. L5NT 24 hr after repeated CP55940 or L5NT 2 weeks after repeated CP55940 groups for 50 µg by two way ANOVA followed by Bonferroni post tests.

In order to investigate the neurological side effects of CP55940 administration, we evaluated the place-stepping reflex, vocalization, exploratory activity and the bar test. Repeated vehicle injection did not significantly affect any of these behaviors at any time point observed. CP55940 significantly impaired the placing-stepping reflex (Figure 7A), induced vocalization (Figure 7B) and reduced exploratory activity (Figure 7C) on days 1, 2 and 3 compared to vehicle group, and induced catalepsy (Figure 7D) on days 1, 2, 3 and 4

Fig. 7. Neurological side effects in response to repeated treatment with CP55940. Placingstepping (A), vocalization (B), exploratory activity (C) and bar test (C) scores are shown from before the first injection (base line = BL), and 0.5, 2 and 24 hr after each i.t. injection (days 1 - 5) of vehicle (n=8) or CP55940 (n=13) during five consecutive days. Withdrawal thresholds on days 1, 3 and 5 in placing-stepping and bar test vs. base line data significantly differ by repeated measures one way ANOVA, \*p<0.05 vs. base line, #p<0.05 vs. 0.5 hr, ^p<0.05 vs. 2 hr by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups differ in placing-stepping and bar test by repetitive measurements two-way ANOVA, +p<0.05 vs. CP55940 group by two way ANOVA followed by Bonferroni post tests. Days 1, 3 and 5 values in vocalization and exploratory activity vs. base line significantly differ by Friedman test, \*p<0.05 vs. base line, #p<0.05 vs. 0.5 hr, ^p<0.05 vs. 2 hr by Friedman test followed by Wilcoxon test. Groups in vocalization and exploratory activity significantly differ by Kruskal-Wallis test; +p<0.05 vs. CP55940 by Kruskal-Wallis test followed by Mann-Whitney U test.

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

last injection respectively, n=6) 24 hr after repeated treatment with vehicle.

the CB2 receptor antagonist AM630 50 µg (2.4 0.4 g, n=4, P<0.05).

**CP55940 tolerant animals studies** 

**4. Discussion** 

**3.5 Antinociceptive effect of a CB2 receptor agonist administered repeatedly in** 

the repeated CP55940 group after the two-week washout period (data not shown).

The main findings of our study are: 1) the repeated administration of a non-selective cannabinoid agonist (CP55940) induces antinociceptive tolerance and tolerance to cannabinoid-induced neurological side effects in a rat model of neuropathic pain; 2) CP55940 tolerance persists two weeks after the discontinuation of cannabinoid administration; 3) prior induction of CP55940 tolerance reduced the antinociceptive effect of

JWH015 injected i.t. for four consecutive days induced similar antinociceptive effects on all days tested in animals previously exposed to repeated i.t. vehicle treatment (for 5 days) and a washout period of two weeks. However, JWH015 injected i.t. for four consecutive days induced antinociception only on days 1 and 4 in animals previously exposed to sustained spinal CP55940 administration (for 5 days) and a washout period of two weeks. Repeated i.t. JWH015 was significantly less effective on the last three days of treatment in animals previously exposed to repeated CP55940 when compared to those previously exposed to repeated vehicle (Figure 9A). The JWH015 repeated treatment did not modify the mechanical withdrawal threshold in the contralateral paw in the repeated vehicle or CP55940 group, and the effects of repeated JWH015 did not differ between groups, except on day 3 when the withdrawal threshold was significantly higher in the repeated vehicle group than the CP55940 one (Figure 9B). Vehicle (same paradigm as repeated JWH015) did not modify the withdrawal thresholds ipsilateral (n=6) or contralateral (n=6) to surgery in

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 117

maximum effective doses of JWH015 in the repeated CP55940 group were 10 and 50 µg respectively (50 µg was the highest dose used). JWH015 was equally effective in both repeated CP55940 and vehicle groups since no significant difference in withdrawal thresholds was observed between groups in any dose tested. As a result, the ED50 value [95% confidence limits] of JWH015 was not significantly different in animals previously treated with repeated CP55940 compared to animals previously treated with vehicle (Table 1 and Figure 8A). Vehicle (same paradigm as cumulative JWH015) did not modify the withdrawal thresholds ipsilateral to surgery (3.5±0.6 vs. 5.3±1.4 g before and 15 min after the

JWH015 was also effective in reversing the L5NT-induced hypersensitivity when it was administered in a cumulative manner two weeks after the cessation of CP5940 treatment (washout period). In this case, the minimum and maximum effective dose of JWH015 were 2 and 50 µg respectively in animals previously exposed to CP55940 (repeated CP55940 group), and 10 and 50 µg respectively in animals previously treated with vehicle (repeated vehicle group). Similar efficacy and potency of JWH015 were observed in both repeated CP55940 and vehicle groups (Table 2). No significant difference in withdrawal thresholds was observed between groups in any dose tested (Figure 8B). Vehicle (same paradigm as cumulative JWH015) did not modify the withdrawal thresholds ipsilateral to surgery in the repeated vehicle group after the two-week washout period (3.5±0.6 vs. 3.6±0.7 g before and 15 min after the last injection respectively, n=6). The acute antinociception induced by JWH015 50 µg (plus vehicle, 17 2.7 g, n=8) in the L5NT group was completely blocked by

compared to vehicle group. The magnitude of these neurological side effects decreased over the 5-day course of daily CP55940 injections until they were not significantly different compared to vehicle group on days 4 and 5 (except for catalepsy, 2 hr after CP55940 injection on day 4 vs. vehicle group, p<0.05). The righting reflex was significantly impaired by CP55940 compared to base line on days 1 and 3 (30 min and 2 hr after injections, data not shown). The effects of CP55940 on placing-stepping reflex, vocalization and bar test on day 1 were significantly higher compared to its effects on days 4 and 5. The effects of CP55940 on exploratory activity on day 1 were significantly higher compared to its effects on days 3, 4 and 5. For clarity, only the data obtained on days 1, 3 and 5 of treatment are shown.

#### **3.4 Acute antinociceptive effect of JWH015 in CP55940-tolerant animals**

JWH015, a selective CB2 receptor agonist, reduced mechanical hypersensitivity ipsilateral to surgery in a dose-dependent fashion when administered i.t. in cumulative, escalating doses in animals previously exposed to CP55940 or vehicle (Figure 8A). The minimum and

Fig. 8. Antinociceptive effects of acute i.t. administration of JWH015 in CP55940-mediated tolerant animals. Withdrawal thresholds (95% confidence limits, doted lines) indicate responses to von Frey stimulation ipsilateral to L5NT surgery 15 min after escalating doses (0.4, 2, 10 and 50 µg) of i.t. JWH015 administered 24 hr (A) or two weeks (washout period, B) after the discontinuation of repeated treatment with CP55940 100 µg (Repeated CP55940) or vehicle (Repeated Vehicle). Groups did not differ by two-way ANOVA. Withdrawal thresholds after each dose vs. after surgery values significantly differ by repeated measures one way ANOVA, \*p<0.05 vs. after surgery by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Twenty-four hr after repeated treatment cessation: Repeated CP55940 n=8, Repeated Vehicle n=8, Repeated CP55940-washout period n=6 and Repeated Vehicle-washout period n=5.

maximum effective doses of JWH015 in the repeated CP55940 group were 10 and 50 µg respectively (50 µg was the highest dose used). JWH015 was equally effective in both repeated CP55940 and vehicle groups since no significant difference in withdrawal thresholds was observed between groups in any dose tested. As a result, the ED50 value [95% confidence limits] of JWH015 was not significantly different in animals previously treated with repeated CP55940 compared to animals previously treated with vehicle (Table 1 and Figure 8A). Vehicle (same paradigm as cumulative JWH015) did not modify the withdrawal thresholds ipsilateral to surgery (3.5±0.6 vs. 5.3±1.4 g before and 15 min after the last injection respectively, n=6) 24 hr after repeated treatment with vehicle.

JWH015 was also effective in reversing the L5NT-induced hypersensitivity when it was administered in a cumulative manner two weeks after the cessation of CP5940 treatment (washout period). In this case, the minimum and maximum effective dose of JWH015 were 2 and 50 µg respectively in animals previously exposed to CP55940 (repeated CP55940 group), and 10 and 50 µg respectively in animals previously treated with vehicle (repeated vehicle group). Similar efficacy and potency of JWH015 were observed in both repeated CP55940 and vehicle groups (Table 2). No significant difference in withdrawal thresholds was observed between groups in any dose tested (Figure 8B). Vehicle (same paradigm as cumulative JWH015) did not modify the withdrawal thresholds ipsilateral to surgery in the repeated vehicle group after the two-week washout period (3.5±0.6 vs. 3.6±0.7 g before and 15 min after the last injection respectively, n=6). The acute antinociception induced by JWH015 50 µg (plus vehicle, 17 2.7 g, n=8) in the L5NT group was completely blocked by the CB2 receptor antagonist AM630 50 µg (2.4 0.4 g, n=4, P<0.05).

#### **3.5 Antinociceptive effect of a CB2 receptor agonist administered repeatedly in CP55940 tolerant animals studies**

JWH015 injected i.t. for four consecutive days induced similar antinociceptive effects on all days tested in animals previously exposed to repeated i.t. vehicle treatment (for 5 days) and a washout period of two weeks. However, JWH015 injected i.t. for four consecutive days induced antinociception only on days 1 and 4 in animals previously exposed to sustained spinal CP55940 administration (for 5 days) and a washout period of two weeks. Repeated i.t. JWH015 was significantly less effective on the last three days of treatment in animals previously exposed to repeated CP55940 when compared to those previously exposed to repeated vehicle (Figure 9A). The JWH015 repeated treatment did not modify the mechanical withdrawal threshold in the contralateral paw in the repeated vehicle or CP55940 group, and the effects of repeated JWH015 did not differ between groups, except on day 3 when the withdrawal threshold was significantly higher in the repeated vehicle group than the CP55940 one (Figure 9B). Vehicle (same paradigm as repeated JWH015) did not modify the withdrawal thresholds ipsilateral (n=6) or contralateral (n=6) to surgery in the repeated CP55940 group after the two-week washout period (data not shown).

#### **4. Discussion**

116 Pain Management – Current Issues and Opinions

compared to vehicle group. The magnitude of these neurological side effects decreased over the 5-day course of daily CP55940 injections until they were not significantly different compared to vehicle group on days 4 and 5 (except for catalepsy, 2 hr after CP55940 injection on day 4 vs. vehicle group, p<0.05). The righting reflex was significantly impaired by CP55940 compared to base line on days 1 and 3 (30 min and 2 hr after injections, data not shown). The effects of CP55940 on placing-stepping reflex, vocalization and bar test on day 1 were significantly higher compared to its effects on days 4 and 5. The effects of CP55940 on exploratory activity on day 1 were significantly higher compared to its effects on days 3, 4 and 5. For clarity, only the data obtained on

JWH015, a selective CB2 receptor agonist, reduced mechanical hypersensitivity ipsilateral to surgery in a dose-dependent fashion when administered i.t. in cumulative, escalating doses in animals previously exposed to CP55940 or vehicle (Figure 8A). The minimum and

Fig. 8. Antinociceptive effects of acute i.t. administration of JWH015 in CP55940-mediated tolerant animals. Withdrawal thresholds (95% confidence limits, doted lines) indicate responses to von Frey stimulation ipsilateral to L5NT surgery 15 min after escalating doses (0.4, 2, 10 and 50 µg) of i.t. JWH015 administered 24 hr (A) or two weeks (washout period, B) after the discontinuation of repeated treatment with CP55940 100 µg (Repeated CP55940) or vehicle (Repeated Vehicle). Groups did not differ by two-way ANOVA. Withdrawal thresholds after each dose vs. after surgery values significantly differ by repeated measures one way ANOVA, \*p<0.05 vs. after surgery by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Twenty-four hr after repeated treatment cessation: Repeated CP55940 n=8, Repeated Vehicle n=8, Repeated CP55940-washout period

**3.4 Acute antinociceptive effect of JWH015 in CP55940-tolerant animals** 

days 1, 3 and 5 of treatment are shown.

n=6 and Repeated Vehicle-washout period n=5.

The main findings of our study are: 1) the repeated administration of a non-selective cannabinoid agonist (CP55940) induces antinociceptive tolerance and tolerance to cannabinoid-induced neurological side effects in a rat model of neuropathic pain; 2) CP55940 tolerance persists two weeks after the discontinuation of cannabinoid administration; 3) prior induction of CP55940 tolerance reduced the antinociceptive effect of

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

induced tolerance on JWH015's antinociceptive effectiveness.

expression and sensitivity.

**5. Conclusion** 

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 119

tolerance observed in response to CP55940 administration in the current study. Repeated administration of CP55940 also induced tolerance to a range of neurological side effects. We have previously observed that CP55940-induced neurological side effects are dependent on CB1 receptor activation, but not on CB2 receptor activation in rat postoperative and neuropathic pain models (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a). While these findings support the potential role of CB1 receptors in cannabinoid induced tolerance in our neuropathic pain model, CB1 receptor agonism does not induce antinociceptive tolerance in a spinal cord injury model (Hama and Sagen, 2009). Therefore, agonism of both CB1 and CB2 receptors may be required to induce antinociceptive tolerance to cannabinoid therapies in animals or patients with peripheral or central nerve injury. CB1 receptor-dependent cross-tolerance among cannabinoids has recently been described between delta-tetrahydrocannabinol (the active ingredient of cannabis) and anandamide (one of the major endocannabinoids) (Falenski et al., 2010), and between 2 arachidonylglycerol (another major endocannabinoid) and the CB1 receptor agonist WIN55,212-2 (Schlosburg et al., 2010). This cross-tolerance is thought to be CB1-dependent (Falenski et al., 2010). However, we demonstrate in our current study that repeated administration of JWH015 exhibited reduced efficacy in rats with peripheral nerve injury that have been previously exposed to a non-selective cannabinoid agonist. This finding directly contrasts with our previous observation that repeated JWH015 reduces L5NTinduced hypersensitivity without signs of tolerance in the same rat model of neuropathic pain (Romero-Sandoval et al., 2008a). Taken together, these findings indicate that cannabinoid antinociceptive tolerance to non-selective cannabinoid agonists affects subsequent responsiveness of both CB1 and CB2 receptors. We also observed that CB1 receptors are predominantly expressed in neurons and that CB2 receptors are predominantly expressed in microglia in the spinal cord of both sham surgery and L5NT groups. Therefore, neuronal and glial interactions may contribute to the effects of CP55940-

Cannabinoid tolerance depends on CB receptor availability (Tappe-Theodor et al., 2007; Martini et al., 2010) and/or sensitivity (Jin et al., 1999; Selley et al., 2004). These receptor properties may change following peripheral insults such as paw incision (Alkaitis et al., 2010), peripheral nerve injury (Lim et al., 2003) or sustained activation by endogenous (Falenski et al., 2010; Schlosburg et al., 2010) or exogenous cannabinoids (Gardell et al., 2002; Hama and Sagen, 2009). In accordance with our findings, others have shown that a single intracerebroventricular dose of CB1 receptor agonists (WIN55,212-2 or ACEA) induces antinociceptive tolerance that lasts for more than 14 days through actions on the pertussis toxin-insensitive G proteins, Gz (Garzon et al., 2009). The mechanisms involved in long lasting CB1 receptor-mediated tolerance may also include the persistent cellular internalization or degradation of CB1 receptor (Sim-Selley et al., 2006). These data suggest that cannabinoid responsiveness and tolerance are shaped by a number of factors including type of pain or injury, exposure to endogenous or exogenous cannabinoids and receptor

We demonstrate that a non-selective cannabinoid drug induces tolerance under neuropathic pain conditions, that this tolerance persists several weeks after the suspension of the treatment and that this tolerance affects the antinociceptive effects of repeated

repeated administration of a CB2 receptor agonist (JWH015), but did not alter the antinociceptive response to acute JWH015 dose escalation.

Fig. 9. Antinociceptive effects of repeated i.t. administration of JWH015 in CP55940 mediated tolerant animals. Paw withdrawal thresholds indicate responses to von Frey stimulation ipsilateral to L5NT (A) or contralateral to surgery (uninjured side, B) two weeks after the cessation of repeated CP55940 or vehicle administration (After washout period), and 2 hr after each i.t. injection of JWH015 during four consecutive days. Withdrawal thresholds on days 1-4 vs. after washout period data significantly differ by repeated measures one way ANOVA, \*p<0.05 after washout period by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups significantly differ by two way ANOVA; +p<0.05 compared to vehicle group by two way ANOVA followed by Bonferroni post tests.

We demonstrate that a non-selective cannabinoid agonist administered repeatedly at a concentration that induces neurological side effects (such as the effects that regular cannabis users seek for recreational purposes) is sufficient to produce a long lasting antinociceptive tolerance that persists weeks after the cessation of drug exposure. In agreement with these findings, diminished psychotropic effects (D'Souza et al., 2008) and analgesic tolerance to delta-9-tetrahydrocannabinol (Clark et al., 1981) have been demonstrated in frequent users of cannabis. This hypothesis has been further supported by a double-blind, placebocontrolled study demonstrating evidence of dronabinol tolerance in regular marijuana users (Bedi et al., 2010). It has also been shown that repeated administration of CB1 receptor agonists results in antinociceptive tolerance in naïve mice and rats (Gardell et al., 2002; Hama and Sagen, 2009), and that this tolerance is dependent on spinal cord mechanisms (Gardell et al., 2002). In contrast, we have previously shown that i.t. administration of the CB2 receptor agonist JWH015 effectively reverses L5 nerve transection-induced behavioral hypersensitivity without antinociceptive tolerance through at least five days of treatment (Romero-Sandoval and Eisenach, 2007). Similar findings have been described with another CB2 receptor agonist, A-836339 (Yao et al., 2009). Taken together, these previous findings suggest that CB1 rather than CB2 receptor agonism is responsible for the antinociceptive tolerance observed in response to CP55940 administration in the current study. Repeated administration of CP55940 also induced tolerance to a range of neurological side effects. We have previously observed that CP55940-induced neurological side effects are dependent on CB1 receptor activation, but not on CB2 receptor activation in rat postoperative and neuropathic pain models (Romero-Sandoval and Eisenach, 2007; Romero-Sandoval et al., 2008a). While these findings support the potential role of CB1 receptors in cannabinoid induced tolerance in our neuropathic pain model, CB1 receptor agonism does not induce antinociceptive tolerance in a spinal cord injury model (Hama and Sagen, 2009). Therefore, agonism of both CB1 and CB2 receptors may be required to induce antinociceptive tolerance to cannabinoid therapies in animals or patients with peripheral or central nerve injury.

CB1 receptor-dependent cross-tolerance among cannabinoids has recently been described between delta-tetrahydrocannabinol (the active ingredient of cannabis) and anandamide (one of the major endocannabinoids) (Falenski et al., 2010), and between 2 arachidonylglycerol (another major endocannabinoid) and the CB1 receptor agonist WIN55,212-2 (Schlosburg et al., 2010). This cross-tolerance is thought to be CB1-dependent (Falenski et al., 2010). However, we demonstrate in our current study that repeated administration of JWH015 exhibited reduced efficacy in rats with peripheral nerve injury that have been previously exposed to a non-selective cannabinoid agonist. This finding directly contrasts with our previous observation that repeated JWH015 reduces L5NTinduced hypersensitivity without signs of tolerance in the same rat model of neuropathic pain (Romero-Sandoval et al., 2008a). Taken together, these findings indicate that cannabinoid antinociceptive tolerance to non-selective cannabinoid agonists affects subsequent responsiveness of both CB1 and CB2 receptors. We also observed that CB1 receptors are predominantly expressed in neurons and that CB2 receptors are predominantly expressed in microglia in the spinal cord of both sham surgery and L5NT groups. Therefore, neuronal and glial interactions may contribute to the effects of CP55940 induced tolerance on JWH015's antinociceptive effectiveness.

Cannabinoid tolerance depends on CB receptor availability (Tappe-Theodor et al., 2007; Martini et al., 2010) and/or sensitivity (Jin et al., 1999; Selley et al., 2004). These receptor properties may change following peripheral insults such as paw incision (Alkaitis et al., 2010), peripheral nerve injury (Lim et al., 2003) or sustained activation by endogenous (Falenski et al., 2010; Schlosburg et al., 2010) or exogenous cannabinoids (Gardell et al., 2002; Hama and Sagen, 2009). In accordance with our findings, others have shown that a single intracerebroventricular dose of CB1 receptor agonists (WIN55,212-2 or ACEA) induces antinociceptive tolerance that lasts for more than 14 days through actions on the pertussis toxin-insensitive G proteins, Gz (Garzon et al., 2009). The mechanisms involved in long lasting CB1 receptor-mediated tolerance may also include the persistent cellular internalization or degradation of CB1 receptor (Sim-Selley et al., 2006). These data suggest that cannabinoid responsiveness and tolerance are shaped by a number of factors including type of pain or injury, exposure to endogenous or exogenous cannabinoids and receptor expression and sensitivity.

#### **5. Conclusion**

118 Pain Management – Current Issues and Opinions

repeated administration of a CB2 receptor agonist (JWH015), but did not alter the

Fig. 9. Antinociceptive effects of repeated i.t. administration of JWH015 in CP55940 mediated tolerant animals. Paw withdrawal thresholds indicate responses to von Frey stimulation ipsilateral to L5NT (A) or contralateral to surgery (uninjured side, B) two weeks after the cessation of repeated CP55940 or vehicle administration (After washout period), and 2 hr after each i.t. injection of JWH015 during four consecutive days. Withdrawal thresholds on days 1-4 vs. after washout period data significantly differ by repeated measures one way ANOVA, \*p<0.05 after washout period by repeated measures one way ANOVA followed by Tukey's multiple comparison test. Groups significantly differ by two way ANOVA; +p<0.05 compared to vehicle group by two way ANOVA followed by

We demonstrate that a non-selective cannabinoid agonist administered repeatedly at a concentration that induces neurological side effects (such as the effects that regular cannabis users seek for recreational purposes) is sufficient to produce a long lasting antinociceptive tolerance that persists weeks after the cessation of drug exposure. In agreement with these findings, diminished psychotropic effects (D'Souza et al., 2008) and analgesic tolerance to delta-9-tetrahydrocannabinol (Clark et al., 1981) have been demonstrated in frequent users of cannabis. This hypothesis has been further supported by a double-blind, placebocontrolled study demonstrating evidence of dronabinol tolerance in regular marijuana users (Bedi et al., 2010). It has also been shown that repeated administration of CB1 receptor agonists results in antinociceptive tolerance in naïve mice and rats (Gardell et al., 2002; Hama and Sagen, 2009), and that this tolerance is dependent on spinal cord mechanisms (Gardell et al., 2002). In contrast, we have previously shown that i.t. administration of the CB2 receptor agonist JWH015 effectively reverses L5 nerve transection-induced behavioral hypersensitivity without antinociceptive tolerance through at least five days of treatment (Romero-Sandoval and Eisenach, 2007). Similar findings have been described with another CB2 receptor agonist, A-836339 (Yao et al., 2009). Taken together, these previous findings suggest that CB1 rather than CB2 receptor agonism is responsible for the antinociceptive

antinociceptive response to acute JWH015 dose escalation.

Bonferroni post tests.

We demonstrate that a non-selective cannabinoid drug induces tolerance under neuropathic pain conditions, that this tolerance persists several weeks after the suspension of the treatment and that this tolerance affects the antinociceptive effects of repeated

Reduced Antinociceptive Effect of Repeated Treatment with a Cannabinoid

antinociceptive tolerance. *Pain*, Vol. 98, pp. 79-88

*J Rehabil Res Dev*, Vol. 46, No. 1, pp. 135-143

behaviors in rats. *Pain*, Vol. 105, pp. 275-283

*Anesthesiology*, Vol. 106, No. 4, pp. 787-794

*Brain Research*, Vol. 1219, pp. 116-26

*Neurosci*, Vol. 13, No. 9, pp. 1113-1119

No. 6, pp. 1363-1373

108, No. 4, pp. 722-734

66, No. 5, pp. 1275-1284

102, No. 16, pp. 5856-5861

pp. 986-996

11

Receptor Type 2 Agonist in Cannabinoid-Tolerant Rats Following Spinal Nerve Transection 121

Gardell, LR., Burgess, SE., Dogrul, A., Ossipov, MH., Malan, TP., Lai, J., & Porreca, F. (2002).

Garzon, J., de la Torre-Madrid, E., Rodriguez-Munoz, M., Vicente-Sanchez, A., & Sanchez-

Hama, A., & Sagen, J. (2009). Sustained antinociceptive effect of cannabinoid receptor

Jin, W., Brown, S., Roche, JP., Hsieh, C., Celver, JP., Kovoor, A., Chavkin, C., & Mackie, K.

Lim, G., Sung, B., Ji, RR., & Mao, J. (2003). Upregulation of spinal cannabinoid-1-receptors

Martini, L., Thompson, D., Kharazia, V., & Whistler, JL. (2010). Differential regulation of

Romero-Sandoval, A., & Eisenach, JC. (2007). Spinal cannabinoid receptor type 2 activation

Romero-Sandoval, A., Nutile-McMenemy, N., & DeLeo, JA. (2008a). Spinal microglial and

Romero-Sandoval, A., Chai, N., Nutile-McMenemy, N., & DeLeo, JA. (2008b). A comparison

Schlosburg, JE., Blankman, JL., Long, JZ., Nomura, DK., Pan, B., Kinsey, SG., Nguyen, PT.,

Selley, DE., Cassidy, MP., Martin, BR., & Sim-Selley, LJ. (2004). Long-term administration of

Sim-Selley, LJ., Schechter, NS., Rorrer, WK., Dalton, GD., Hernandez, J., Martin, BR., &

Tanga, FY., Nutile-McMenemy, N., & DeLeo, JA. (2005). The CNS role of Toll-like receptor 4

and internalization. *J Neurosci*, Vol. 19, No. 10, pp. 3773-3780

Pronociceptive effects of spinal dynorphin promote cannabinoid-induced pain and

Blazquez, P. (2009). Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine. *Mol Pain*, Vol. 5, No.

agonist WIN 55,212-2 over time in rat model of neuropathic spinal cord injury pain.

(1999). Distinct domains of the CB1 cannabinoid receptor mediate desensitization

following nerve injury enhances the effects of Win 55,212-2 on neuropathic pain

behavioral tolerance to WIN55,212-2 by GASP1. *Neuropsychopharmacology*, Vol. 35,

reduces hypersensitivity and spinal cord glial activation after paw incision.

perivascular cell cannabinoid receptor type 2 activation reduces behavioral hypersensitivity without tolerance after peripheral nerve injury. *Anesthesiology*, Vol.

of spinal Iba1 and GFAP expression in rodent models of acute and chronic pain.

Ramesh, D., Booker, L., Burston, JJ., Thomas, EA., Selley, DE., Sim-Selley, LJ., Liu, QS., Lichtman, AH., & Cravatt, BF. (2010). Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system. *Nat* 

Delta9-tetrahydrocannabinol desensitizes CB1-, adenosine A1-, and GABABmediated inhibition of adenylyl cyclase in mouse cerebellum. *Mol Pharmacol*, Vol.

Selley, DE. (2006). Prolonged recovery rate of CB1 receptor adaptation after cessation of long-term cannabinoid administration. *Mol Pharmacol*, Vol. 70, No. 3,

in innate neuroimmunity and painful neuropathy. *Proc Natl Acad Sci U S A*, Vol.

administration of a CB2 receptor agonist. These findings suggest that potential future analgesic drugs based on selective actions on CB2 receptor may not be a good alternative for long-term treatment in patients previously exposed to chronic cannabinoids. These results build on previous published data demonstrating that central CB1 receptor-mediated tolerance enhances tolerance to opioids (Trang et al., 2007; Garzon et al., 2009) and nonsteroidal anti-inflammatory agents (Anikwue et al., 2002). Further research is needed to determine the mechanisms for this broad cross-tolerance among distinct drug classes. Additional studies are also warranted to determine whether patients with histories of cannabis or cannabinoid-based drug use for recreational or medical purposes demonstrate tolerance to common analgesic therapies.

#### **6. Acknowledgements**

Supported in part by grants DA025211 (AR-S) and DA11276 (JAD) from the National Institutes of Health (Bethesda, MD), and the American Pain Society Future Leaders in Pain Research grant. The authors declare that there are no conflicts of interest.

#### **7. References**


administration of a CB2 receptor agonist. These findings suggest that potential future analgesic drugs based on selective actions on CB2 receptor may not be a good alternative for long-term treatment in patients previously exposed to chronic cannabinoids. These results build on previous published data demonstrating that central CB1 receptor-mediated tolerance enhances tolerance to opioids (Trang et al., 2007; Garzon et al., 2009) and nonsteroidal anti-inflammatory agents (Anikwue et al., 2002). Further research is needed to determine the mechanisms for this broad cross-tolerance among distinct drug classes. Additional studies are also warranted to determine whether patients with histories of cannabis or cannabinoid-based drug use for recreational or medical purposes demonstrate

Supported in part by grants DA025211 (AR-S) and DA11276 (JAD) from the National Institutes of Health (Bethesda, MD), and the American Pain Society Future Leaders in Pain

Alkaitis, MS., Solorzano, C., Landry, RP., Piomelli, D., DeLeo, JA., & Romero-Sandoval, EA.

Bedi, G., Foltin, RW., Gunderson, EW., Rabkin, J., Hart, CL., Comer, SD., Vosburg, SK., &

Chaplan, SR., Bach, FW., Pogrel, JW., Chung, JM., & Yaksh, TL. (1994). Quantitative

Clark, WC., Janal, MN., Zeidenberg, P., & Nahas, GG. (1981). Effects of moderate and high

D'Souza, DC., Ranganathan, M., Braley, G., Gueorguieva, R., Zimolo, Z., Cooper, T., Perry,

Falenski, KW., Thorpe, AJ., Schlosburg, JE., Cravatt, BF., Abdullah, RA., Smith, TH., Selley,

(2010). Evidence for a role of endocannabinoids, astrocytes and p38 phosphorylation in the resolution of postoperative pain. *PLoS One*, Vol. 5:e10891 Anikwue, R., Huffman, JW., Martin, ZL., & Welch, SP. (2002). Decrease in efficacy and

potency of nonsteroidal anti-inflammatory drugs by chronic delta(9) tetrahydrocannabinol administration. *J Pharmacol Exp Ther*, Vol. 303, No. 1, pp. 340-

Haney, M. (2010). Efficacy and tolerability of high-dose dronabinol maintenance in HIV-positive marijuana smokers: a controlled laboratory study. *Psychopharmacology* 

assessment of tactile allodynia in the rat paw. *J Neurosci Methods*, Vol. 43, No. 1, pp.

doses of marihuana on thermal pain: a sensory decision theory analysis. *J Clin* 

E., & Krystal, J. (2008). Blunted Psychotomimetic and Amnestic Effects of Delta-9- Tetrahydrocannabinol in Frequent Users of Cannabis. *Neuropsychopharmacology*, Vol

DE., Lichtman, AH., & Sim-Selley, LJ. (2010). FAAH-/- mice display differential tolerance, dependence, and cannabinoid receptor adaptation after delta 9 tetrahydrocannabinol and anandamide administration. *Neuropsychopharmacology*,

Research grant. The authors declare that there are no conflicts of interest.

tolerance to common analgesic therapies.

*(Berl)*, Vol. 212, No. 4, pp. 675-86

*Pharmacol*, Vol 21, pp. 299S-310S

33, No. 10, pp. 2505-16

Vol 35, No. 8, pp. 1775-1787

**6. Acknowledgements** 

**7. References** 

46

55-63


**7** 

*USA* 

Kevin L. Wininger1,2

*1Orthopaedic & Spine Center, Columbus, Ohio 2Otterbein University, Westerville, Ohio* 

**Applied Radiologic Science in the Treatment** 

Accompanied by the work from innovative physician researchers and biomedical engineers who introduced new techniques and devices to expand armamentariums in interventional pain medicine in the 1990s and 2000s, the first decade of the 21st century resulted in a significant rise in the number of interventional procedures performed for pain management. For example, data from the Centers for Medicare and Medicaid Services shows a 518% increase from 1997 through 2006 in the Medicare population receiving spinal cord stimulation therapy (Manchikanti et al., 2009). Other examples include the efforts to design radiofrequency probes to target the sacroiliac joint and subsequently denervate this relatively complex but biomechanically unique structure with as little local tissue trauma as possible (Wininger, 2010); or the application of novel neuromodulation techniques to treat

The use of ionizing radiation for image construction (x-ray imaging) continues to be the standard in image guidance at many interventional pain medicine centers. Hence, the competent use of x-radiation not only benefits patients as well as physicians and ancillary staff in close proximity to the patient at the time of treatment—but from a health physics point of view also yields benefits for the general population given that recent evidence points to an overall increase in the use of radiation in medicine (Fazel et al., 2009; U.S. National Academy of Sciences, 2006). As of 2007, for example, medical sources of radiation represented the primary source of radiation exposure in the United States. Comparatively speaking, natural sources accounted for 3.0 mSv of the total dose, whereas medical sources accounted for 3.2 mSv (which was 5.9 times higher when compared to benchmark figures from 1980). The increase was primarily due to increased use of computed tomography (CT) and nuclear medicine studies. Note that medical sources were delineated as follows: 1.5 mSv from CT, 0.7 mSv from nuclear medicine, 0.6 mSv from radiography, and 0.4 mSv from

This chapter is intended to serve as a reference to help guide interventional pain physicians in their decision-making process concerning radiation risk management. In this context, the subject matter goes beyond the traditional emphasis placed solely on the cardinal rules of radiation safety (i.e., time, distance, and shielding) to render a systematic review of the different interventional imaging modalities used in the treatment of pain, namely fluoroscopy, CT, and ultrasound. Notably, we will center our discussion on the so-called

challenging cases of headache (Deshpande & Wininger, 2011).

interventional radiology (Johnston et al., 2011).

**1. Introduction** 

**of Pain: Interventional Pain Medicine** 


### **Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine**

Kevin L. Wininger1,2

*1Orthopaedic & Spine Center, Columbus, Ohio 2Otterbein University, Westerville, Ohio USA* 

#### **1. Introduction**

122 Pain Management – Current Issues and Opinions

Tappe-Theodor, A., Agarwal, N., Katona, I., Rubino, T., Martini, L., Swiercz, J., Mackie, K.,

Trang, T., Sutak, M., & Jhamandas, K. (2007). Involvement of cannabinoid (CB1)-receptors in

Wade, DT., Makela, P., Robson, P., House, H., & Bateman, C. (2004). Do cannabis-based

Yao, BB., Hsieh, G., Daza, AV., Fan, Y., Grayson, GK., Garrison, TR., El Kouhen, O., Hooker,

resonance imaging. *J Pharmacol Exp Ther*, Vol. 328, No. 1, pp. 141-151

4177

pp. 1275-1288

*Mult Scler*, Vol. 10, No. 4, pp. 434-441

Monyer, H., Parolaro, D., Whistler, J., Kuner, T., & Kuner, R. (2007). A molecular basis of analgesic tolerance to cannabinoids. *J Neurosci*, Vol. 27, No. 15, pp. 4165-

the development and maintenance of opioid tolerance. *Neuroscience*, Vol. 146, No. 3,

medicinal extracts have general or specific effects on symptoms in multiple sclerosis? A double-blind, randomized, placebo-controlled study on 160 patients.

BA., Pai, M., Wensink, EJ., Salyers, AK., Chandran, P., Zhu, CZ., Zhong, C., Ryther, K., Gallagher, ME., Chin, CL., Tovcimak, AE., Hradil, VP., Fox, GB., Dart, MJ., Honore, P., & Meyer MD. (2009). Characterization of a cannabinoid CB2 receptorselective agonist, A-836339 [2,2,3,3-tetramethyl-cyclopropanecarboxylic acid [3-(2 methoxy-ethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]-amide], using in vitro pharmacological assays, in vivo pain models, and pharmacological magnetic

Accompanied by the work from innovative physician researchers and biomedical engineers who introduced new techniques and devices to expand armamentariums in interventional pain medicine in the 1990s and 2000s, the first decade of the 21st century resulted in a significant rise in the number of interventional procedures performed for pain management. For example, data from the Centers for Medicare and Medicaid Services shows a 518% increase from 1997 through 2006 in the Medicare population receiving spinal cord stimulation therapy (Manchikanti et al., 2009). Other examples include the efforts to design radiofrequency probes to target the sacroiliac joint and subsequently denervate this relatively complex but biomechanically unique structure with as little local tissue trauma as possible (Wininger, 2010); or the application of novel neuromodulation techniques to treat challenging cases of headache (Deshpande & Wininger, 2011).

The use of ionizing radiation for image construction (x-ray imaging) continues to be the standard in image guidance at many interventional pain medicine centers. Hence, the competent use of x-radiation not only benefits patients as well as physicians and ancillary staff in close proximity to the patient at the time of treatment—but from a health physics point of view also yields benefits for the general population given that recent evidence points to an overall increase in the use of radiation in medicine (Fazel et al., 2009; U.S. National Academy of Sciences, 2006). As of 2007, for example, medical sources of radiation represented the primary source of radiation exposure in the United States. Comparatively speaking, natural sources accounted for 3.0 mSv of the total dose, whereas medical sources accounted for 3.2 mSv (which was 5.9 times higher when compared to benchmark figures from 1980). The increase was primarily due to increased use of computed tomography (CT) and nuclear medicine studies. Note that medical sources were delineated as follows: 1.5 mSv from CT, 0.7 mSv from nuclear medicine, 0.6 mSv from radiography, and 0.4 mSv from interventional radiology (Johnston et al., 2011).

This chapter is intended to serve as a reference to help guide interventional pain physicians in their decision-making process concerning radiation risk management. In this context, the subject matter goes beyond the traditional emphasis placed solely on the cardinal rules of radiation safety (i.e., time, distance, and shielding) to render a systematic review of the different interventional imaging modalities used in the treatment of pain, namely fluoroscopy, CT, and ultrasound. Notably, we will center our discussion on the so-called

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 125

automated "negative feedback" commonly set by most operators to ensure a proper amount of x-rays in order to image patients with thin to average body types. Because of its real-time imaging capability, extended exposure times are possible when operating fluoroscopy systems, and thus, the amount of tube current is substantially less compared to that used in static film-based radiography, 1 to 5 mA versus 100 to 500 mA, respectively (Carlton & Adler, 2006; Bushong, 2004). However, the physician has likely encountered degradation of recorded detail while using fluoroscopy due to a blotchy or grainy appearance that is directly related to an insufficient amount of radiation to create a uniform image (a phenomenon common to all electromagnetic imaging modalities) (Carlton & Adler, 2006). This is referred to as quantum noise or quantum mottle, as "quantum" means counted or

With fluoroscopy, the time factor is controlled by the length of time the eye can integrate, or accumulate, light photons from the fluoro imaging chain. Because this period is 0.2 seconds, fluoroscopy must provide sufficient photons, through mA, to avoid mottle. Quantum mottle is also a large part of video noise and is a special problem during fluoroscopy because the units operate with the minimum number of photons possible to activate the fluoro screen. The factors that influence mottle are those that affect the total number of photons arriving at the retina of the eye. This includes radiation output, beam attenuation by the subject, the conversion efficiency of the input screen, minification gain, flux gain, total brightness gain, viewing system, and the distance of the eye from the viewing system. Increasing the efficiency of any of these factors can assist in reducing

quantum mottle, but the most common solution is to increase the fluoro tube mA.

Fig. 1. Inside the image intensifier tube, x-rays photons are converted to light photons at the

intensifier tube the electron stream is repelled by the negatively charged electrostatic lenses and is attracted to the positively charged anode. Electrons are converted back to light at the output screen in order to proceed to the image viewing system. The output quantity of light

Image degradation from quantum mottle not only presents patient safety concerns due to challenges surrounding needle placement, particularly in patients with hypersthenic body

input screen and then to electrons at the photocathode. Flowing through the image

photons is significantly greater than the input quantity of x-ray photons due to total

brightness gain.

measured. According to Carlton and Adler (2006):

"imaging (or 'viewing') chain" of each modality, and thus the issues surrounding image quality and signal processing relative to radiation exposure (or the lack thereof in the case of ultrasound imaging). Moreover, to develop a fundamental understanding of signal processing, key physical and mathematical concepts will be explored.

While this chapter is well-motivated allowing each section to stand alone, the subject matter is presented in such a way to promote continuity from one section to the next. We begin by focusing on fluoroscopic guidance; here analysis has been included to help establish benchmark radiation exposure for spinal cord stimulation procedures as first reported by Wininger et al. (2010). We then look closely at interventional approaches in pain medicine that utilize CT. Throughout these sections, ways to mitigate radiation dose will be considered, including recent steps to improve the shielding afforded by radiation protection apparel. Next, we provide an overview on ultrasound-guided pain medicine; here the tradeoff between spatial resolution and achievable depth of imaging is highlighted. Finally, future directions in image guidance for pain management will be surveyed, including nonionizing radiation emitting modalities such as ultrasound imaging beyond regional anesthesia and interventional magnetic resonance imaging.

#### **2. Fluoroscopy and interventional pain medicine**

#### **2.1 The fluoroscopic imaging chain**

Overall radiographic quality is based on two principal properties, photographic quality (i.e., visibility of detail) and geometric quality (i.e., sharpness of detail) (Carlton & Adler, 2006; Bushong, 2004). Photographic quality is determined by density and contrast, whereas geometric quality is governed by recorded detail (i.e., resolution) and image distortion. In fluoroscopic image acquisition, the term "density" (a term derived from static film-based radiography) is replaced by the term "brightness" to be congruent with the language used to describe the visibility of images on a display monitor. The notion of an imaging chain makes reference to highly integrated instrumentation (together with the patient), regardless of the modality of interest. The fluoroscopic imaging chain denotes the x-ray generator, x-ray tube, collimator and filtration, table and patient, grid, image intensifier, optical coupler, and the image viewing system (Schueler, 2000). To this end, while each link in the chain is of equal importance, an understanding of fluoroscopic image quality relative to the image intensifier will be emphasized. The image intensifier functions as a "pass-through" device by converting x-rays to light (fluorescence) and then to electrons by way of its input phosphor-screen with adjoined photocathode backing (see Figure 1). This design effectively and efficiently reduces overall radiation exposure (Wang & Blackburn, 2000), and at the same time, allows physicians to dynamically view anatomy with a relatively high degree of resolution due to the total brightness gain. It is the ability to view dynamically with excellent image resolution—that underpins the role of fluoroscopy in many of the modern disciplines of medicine.

The potential for x-rays to penetrate an object (i.e., soft tissue or bone) and create an image is related to the quality of the x-ray beam as a result of the operating kilovoltage (kV) at the xray tube, and may simply be referred to as the "x-ray tube intensity," "tube intensity," or "tube potential." The amount of x-rays produced is related to tube current (in milliamperage (mA)) and time (in seconds (s)). Whereas the operator presets these factors in static filmbased radiography, producing kilovoltage peak (kVp) and milliamperage seconds (mAs) upon exposure, this is not commonplace in fluoroscopic imaging due to automatic brightness control and real-time intended-use. Automatic brightness control is a type of

"imaging (or 'viewing') chain" of each modality, and thus the issues surrounding image quality and signal processing relative to radiation exposure (or the lack thereof in the case of ultrasound imaging). Moreover, to develop a fundamental understanding of signal

While this chapter is well-motivated allowing each section to stand alone, the subject matter is presented in such a way to promote continuity from one section to the next. We begin by focusing on fluoroscopic guidance; here analysis has been included to help establish benchmark radiation exposure for spinal cord stimulation procedures as first reported by Wininger et al. (2010). We then look closely at interventional approaches in pain medicine that utilize CT. Throughout these sections, ways to mitigate radiation dose will be considered, including recent steps to improve the shielding afforded by radiation protection apparel. Next, we provide an overview on ultrasound-guided pain medicine; here the tradeoff between spatial resolution and achievable depth of imaging is highlighted. Finally, future directions in image guidance for pain management will be surveyed, including nonionizing radiation emitting modalities such as ultrasound imaging beyond regional

Overall radiographic quality is based on two principal properties, photographic quality (i.e., visibility of detail) and geometric quality (i.e., sharpness of detail) (Carlton & Adler, 2006; Bushong, 2004). Photographic quality is determined by density and contrast, whereas geometric quality is governed by recorded detail (i.e., resolution) and image distortion. In fluoroscopic image acquisition, the term "density" (a term derived from static film-based radiography) is replaced by the term "brightness" to be congruent with the language used to describe the visibility of images on a display monitor. The notion of an imaging chain makes reference to highly integrated instrumentation (together with the patient), regardless of the modality of interest. The fluoroscopic imaging chain denotes the x-ray generator, x-ray tube, collimator and filtration, table and patient, grid, image intensifier, optical coupler, and the image viewing system (Schueler, 2000). To this end, while each link in the chain is of equal importance, an understanding of fluoroscopic image quality relative to the image intensifier will be emphasized. The image intensifier functions as a "pass-through" device by converting x-rays to light (fluorescence) and then to electrons by way of its input phosphor-screen with adjoined photocathode backing (see Figure 1). This design effectively and efficiently reduces overall radiation exposure (Wang & Blackburn, 2000), and at the same time, allows physicians to dynamically view anatomy with a relatively high degree of resolution due to the total brightness gain. It is the ability to view dynamically with excellent image resolution—that

underpins the role of fluoroscopy in many of the modern disciplines of medicine.

The potential for x-rays to penetrate an object (i.e., soft tissue or bone) and create an image is related to the quality of the x-ray beam as a result of the operating kilovoltage (kV) at the xray tube, and may simply be referred to as the "x-ray tube intensity," "tube intensity," or "tube potential." The amount of x-rays produced is related to tube current (in milliamperage (mA)) and time (in seconds (s)). Whereas the operator presets these factors in static filmbased radiography, producing kilovoltage peak (kVp) and milliamperage seconds (mAs) upon exposure, this is not commonplace in fluoroscopic imaging due to automatic brightness control and real-time intended-use. Automatic brightness control is a type of

processing, key physical and mathematical concepts will be explored.

anesthesia and interventional magnetic resonance imaging.

**2. Fluoroscopy and interventional pain medicine** 

**2.1 The fluoroscopic imaging chain** 

automated "negative feedback" commonly set by most operators to ensure a proper amount of x-rays in order to image patients with thin to average body types. Because of its real-time imaging capability, extended exposure times are possible when operating fluoroscopy systems, and thus, the amount of tube current is substantially less compared to that used in static film-based radiography, 1 to 5 mA versus 100 to 500 mA, respectively (Carlton & Adler, 2006; Bushong, 2004). However, the physician has likely encountered degradation of recorded detail while using fluoroscopy due to a blotchy or grainy appearance that is directly related to an insufficient amount of radiation to create a uniform image (a phenomenon common to all electromagnetic imaging modalities) (Carlton & Adler, 2006). This is referred to as quantum noise or quantum mottle, as "quantum" means counted or measured. According to Carlton and Adler (2006):

With fluoroscopy, the time factor is controlled by the length of time the eye can integrate, or accumulate, light photons from the fluoro imaging chain. Because this period is 0.2 seconds, fluoroscopy must provide sufficient photons, through mA, to avoid mottle. Quantum mottle is also a large part of video noise and is a special problem during fluoroscopy because the units operate with the minimum number of photons possible to activate the fluoro screen. The factors that influence mottle are those that affect the total number of photons arriving at the retina of the eye. This includes radiation output, beam attenuation by the subject, the conversion efficiency of the input screen, minification gain, flux gain, total brightness gain, viewing system, and the distance of the eye from the viewing system. Increasing the efficiency of any of these factors can assist in reducing quantum mottle, but the most common solution is to increase the fluoro tube mA.

Fig. 1. Inside the image intensifier tube, x-rays photons are converted to light photons at the input screen and then to electrons at the photocathode. Flowing through the image intensifier tube the electron stream is repelled by the negatively charged electrostatic lenses and is attracted to the positively charged anode. Electrons are converted back to light at the output screen in order to proceed to the image viewing system. The output quantity of light photons is significantly greater than the input quantity of x-ray photons due to total brightness gain.

Image degradation from quantum mottle not only presents patient safety concerns due to challenges surrounding needle placement, particularly in patients with hypersthenic body

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 127

cloud) on the cathode. The number of electrons boiled off is directly related to the tube current. Occurring nearly simultaneously with tube activation, the electrons in the electron cloud are forcefully attracted to the target anode due to the potential difference between the cathode and anode. The rate of speed and the efficiency of attraction are dependent on the potential difference across the tube. When high-speed (incident) electrons strike the target, the change in kinetic energy produces only less than 1% of x-rays, with most of the change occurring in the form of heat production (99% or greater) (Dowd & Tilson, 1999). More specifically, x-rays are generated by two processes. The first process involves the interaction of electrons with the nucleus of an atom of tungsten in which the incident electron slows down to change direction (called bremsstrahlung, or "braking radiation"). Bremsstrahlung radiation is emitted from zero to the maximum energy (operating kV). The second process is a collision of the incident electron and an outer shell electron of the tungsten atom. The collision knocks the outer shell electron out of orbit (producing characteristic radiation). Characteristic radiation is the term used to reference the fact that the x-ray energy produced is related to the binding energy between the outer shell electron and the nucleus of the target atom, and is always the same for a specific target atom (again, tungsten in the case of

Fig. 2. A closer look inside the x-ray tube. When **tube current** is applied the filament heats up to boil off electrons into a cloud. X-rays are produced as the **tube potential** forces the

When x-ray photons in the primary beam pass through matter, they either pass unaltered (transmission) or undergo attenuation. Moreover, attenuated x-ray photons are either absorbed (all energy lost and the photon "dies") or scattered (some energy lost and the photon changes direction). The x-ray photons which are absorbed are primarily responsible for patient radiation exposure (via the photoelectric effect) and those photons which scatter are responsible for occupational radiation exposure (via Compton scattering). Note: when tube potential increases the photoelectric effect decreases *greatly* and the percentage of Compton interactions decreases *slightly*. (Dowd & Tilson, 1999). We will further elucidate the significance of the patient as the point source when discussing radiation risk management in fluoroscopy (or otherwise briefly stated, the occupational exposure due to secondary radiation emanating from the patient due to the interactions between the primary beam and the patient).

incident (free) electrons to strike the target on the anode at a high-speed.

**2.2.2 Secondary radiation—the patient as the point source** 

x-ray production in fluoroscopy) (Dowd & Tilson, 1999).

habitus, but can also be a concern to the interventional pain physician and staff members inside the fluoroscopy suite, especially when team members are standing near the patient. We turn to x-ray attenuation physics to help us better understand this (McKetty, 1998). We see that in most fluoroscopically-guided pain procedures, the primary beam is directed at bony structures (i.e., material with a large content of calcium atoms, atomic number-20, which efficiently attenuates the beam) as opposed to soft tissues (i.e., material containing more atoms of carbon, oxygen, and hydrogen, producing an effective atomic number-7.4 and thus allowing more of the beam to transmit to the image intensifier). Moreover, Table 1 lists differences in the atomic numbers and densities of matter found in the makeup of the human body. It follows that in order to compensate for the attenuated beam within the field-of-view for bony imaging compared to soft tissue imaging, radiation output ramps up either as a result of adjustments to technique factors via automatic brightness control or by means of manual technique adjustments or activation of high-fluoro/boost mode by the operator. It is also important to note that most manufacturers incorporate an increase in mA during pulsed fluoroscopy to maintain equivalent image perception (Mahesh, 2001). With this in mind, a study on perceptual comparison between pulsed and continuous fluoroscopy concluded that the average absolute differences in the equivalent-perception dose is approximately 3% (Aufrichtig et al., 1994), where the equivalent-perception dose is defined as the dose of radiation in pulsed mode needed to give the visual equivalence in continuous mode. Thus, we find, importantly, an average radiation dose savings of 22%, 38%, and 49% for pulsed-15 frames per second, pulsed-10 frames per second, and pulsed-7.5 frames per second, respectively (Aufrichtig et al., 1994).


Adapted from Johns & Cunningham, 1983, and Dowd & Tilson, 1999.

\*Note: x-ray output relative to the density of water serves as a baseline measure of x-ray output in the original design and calibration of x-ray producing systems as well as many radiation dose models, and may still be used to check system standards during annual physics acceptance testing. To this point, CT systems assign the number zero to water when calculating voxel/pixel brightness values (see Figure 6, CT Numbers and Hounsfield Units).

Table 1. Differences between matter in the makeup of the human body.

#### **2.2 The physics of fluoroscopy**

#### **2.2.1 Primary radiation**

When the x-ray tube is activated, electrons are "boiled off" from the wire element (i.e., a thin filament of tungsten) to form an electron cloud (see Figure 2). The wire element is strategically located opposite from the spinning target anode as part of a built-in concavity (of which the rim is slightly more negatively charged to concentrate the electrons in the

habitus, but can also be a concern to the interventional pain physician and staff members inside the fluoroscopy suite, especially when team members are standing near the patient. We turn to x-ray attenuation physics to help us better understand this (McKetty, 1998). We see that in most fluoroscopically-guided pain procedures, the primary beam is directed at bony structures (i.e., material with a large content of calcium atoms, atomic number-20, which efficiently attenuates the beam) as opposed to soft tissues (i.e., material containing more atoms of carbon, oxygen, and hydrogen, producing an effective atomic number-7.4 and thus allowing more of the beam to transmit to the image intensifier). Moreover, Table 1 lists differences in the atomic numbers and densities of matter found in the makeup of the human body. It follows that in order to compensate for the attenuated beam within the field-of-view for bony imaging compared to soft tissue imaging, radiation output ramps up either as a result of adjustments to technique factors via automatic brightness control or by means of manual technique adjustments or activation of high-fluoro/boost mode by the operator. It is also important to note that most manufacturers incorporate an increase in mA during pulsed fluoroscopy to maintain equivalent image perception (Mahesh, 2001). With this in mind, a study on perceptual comparison between pulsed and continuous fluoroscopy concluded that the average absolute differences in the equivalent-perception dose is approximately 3% (Aufrichtig et al., 1994), where the equivalent-perception dose is defined as the dose of radiation in pulsed mode needed to give the visual equivalence in continuous mode. Thus, we find, importantly, an average radiation dose savings of 22%, 38%, and 49% for pulsed-15 frames per second, pulsed-10 frames per second, and pulsed-7.5 frames per

Atomic Number

Air 7.78 1.29 Fat 6.46 916. Soft Tissue 7.40 n/a \*Water 7.51 1000. Muscle 7.64 1040. Spongy Bone 12.31 1650. Compact Bone 13.80 1850. Calcium 20.00 n/a

\*Note: x-ray output relative to the density of water serves as a baseline measure of x-ray output in the original design and calibration of x-ray producing systems as well as many radiation dose models, and may still be used to check system standards during annual physics acceptance testing. To this point, CT systems assign the number zero to water when calculating voxel/pixel brightness values (see Figure 6,

When the x-ray tube is activated, electrons are "boiled off" from the wire element (i.e., a thin filament of tungsten) to form an electron cloud (see Figure 2). The wire element is strategically located opposite from the spinning target anode as part of a built-in concavity (of which the rim is slightly more negatively charged to concentrate the electrons in the

Density (kg/m3)

second, respectively (Aufrichtig et al., 1994).

CT Numbers and Hounsfield Units).

**2.2 The physics of fluoroscopy** 

**2.2.1 Primary radiation** 

Matter Effective

Adapted from Johns & Cunningham, 1983, and Dowd & Tilson, 1999.

Table 1. Differences between matter in the makeup of the human body.

cloud) on the cathode. The number of electrons boiled off is directly related to the tube current. Occurring nearly simultaneously with tube activation, the electrons in the electron cloud are forcefully attracted to the target anode due to the potential difference between the cathode and anode. The rate of speed and the efficiency of attraction are dependent on the potential difference across the tube. When high-speed (incident) electrons strike the target, the change in kinetic energy produces only less than 1% of x-rays, with most of the change occurring in the form of heat production (99% or greater) (Dowd & Tilson, 1999). More specifically, x-rays are generated by two processes. The first process involves the interaction of electrons with the nucleus of an atom of tungsten in which the incident electron slows down to change direction (called bremsstrahlung, or "braking radiation"). Bremsstrahlung radiation is emitted from zero to the maximum energy (operating kV). The second process is a collision of the incident electron and an outer shell electron of the tungsten atom. The collision knocks the outer shell electron out of orbit (producing characteristic radiation). Characteristic radiation is the term used to reference the fact that the x-ray energy produced is related to the binding energy between the outer shell electron and the nucleus of the target atom, and is always the same for a specific target atom (again, tungsten in the case of x-ray production in fluoroscopy) (Dowd & Tilson, 1999).

Fig. 2. A closer look inside the x-ray tube. When **tube current** is applied the filament heats up to boil off electrons into a cloud. X-rays are produced as the **tube potential** forces the incident (free) electrons to strike the target on the anode at a high-speed.

#### **2.2.2 Secondary radiation—the patient as the point source**

When x-ray photons in the primary beam pass through matter, they either pass unaltered (transmission) or undergo attenuation. Moreover, attenuated x-ray photons are either absorbed (all energy lost and the photon "dies") or scattered (some energy lost and the photon changes direction). The x-ray photons which are absorbed are primarily responsible for patient radiation exposure (via the photoelectric effect) and those photons which scatter are responsible for occupational radiation exposure (via Compton scattering). Note: when tube potential increases the photoelectric effect decreases *greatly* and the percentage of Compton interactions decreases *slightly*. (Dowd & Tilson, 1999). We will further elucidate the significance of the patient as the point source when discussing radiation risk management in fluoroscopy (or otherwise briefly stated, the occupational exposure due to secondary radiation emanating from the patient due to the interactions between the primary beam and the patient).

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 129

522 216 609 81.3 175 Total Fluoroscopy Time

\*\*\*Izadpanah

— — —

1245 — —

> — —

**<sup>2009</sup>** Thoracic Spine

Total Patient *ESE ESE* – AP Imaging *ESE* – Lateral Imaging

Total DAP DAP – AP Imaging DAP – Lateral Imaging

Effective Dose/Minute Physician Hand

\*\*\*Villavicencio **2005**

> — — —

> — — —

— —

Fluoroscopy time (in seconds); patient entrance skin exposure (*ESE*) and dose area product (DAP, a calculation of stochastic risk for the patient [Vano et al., 2001]) (in centiGray); physician total and hand

Table 3. Radiation exposure associated with biplanar systems or multi-directional systems. Inter-procedural variance compared to fluoroscopy times observed in Tables 2 and 3 may be attributed to differences in procedural techniques, level of experience, and/or physician preferences in imaging assistance, as well as attenuation physics relative to image quality. To illustrate this last point, we consider spinal imaging. During spinal imaging, two common challenges associated with image quality exist: 1) highly radiolucent vertebral bodies, particularly against the imaged lung field, creating excess image brightness in the region of interest, and 2) large body habitus with resultant poor image quality. In the former, tight collimation with the paired leaves shutters drawn close to the spine and continuous-mode imaging may help compensate for poor contrast resolution due to the vertebral bodies lacking enough cortical bone density to effectively attenuate the beam (i.e., low beam attenuation) (Johns & Cunningham, 1983). Subsequently, overriding automatic brightness control by manually ramping down tube current (mA) during tightly collimated bony imaging can help improve image resolution, especially for extremely radiolucent vertebral bodies. Alternatively, a manual adjustment to monitor/display window contrast may effectively improve image quality. To compensate for poor image quality secondary to large body habitus (i.e., a highly attenuated beam with resultant image granularity), it too may be necessary to operate the fluoroscope in continuous mode to increase the overall radiation at the image intensifier rather than disengaging the low dose feature. This strategy may improve image contrast while limiting patient exposure if a "manual beam on/off" operator technique is used, e.g., while panning or moving the C-arm between

anteroposterior/oblique positioning to keep region of interest in the field of view.

In recent years the assessment of radiation dose has received increased scrutiny; notably, the evaluation of deterministic effects, for which the severity of effects will vary according to the dose received and for which dose thresholds usually exist (e.g., radiation induced skin injuries) (Balter, 2006 & 2008). Moreover, dose assessment has seemingly evolved from an academic enterprise to a clinical endeavour. Direct influence on clinical practice is appreciated by The Joint Commission's recent decision to add unexpectedly prolonged fluoroscopic exposure to its list of reviewable sentinel events, as well as their suggestion to

\*\*without or \*\*\*with 3D navigation in vertebroplasty or kyphoplasty procedures.

Kallmes **2003**

> — — —

> — — —

— 236 Boszczyk **2006**

> 100 32 68

> > — — —

> > — —

exposure (in microSieverts/minute).

**2.4 Radiation risk management/safety** 

**\*\***Perisinakis **2004**

> 203 — —

3598 2294 1304

> — —

#### **2.3 Fluoroscopically-guided pain medicine procedures**

Nearly all interventional pain treatments and techniques evolved using fluoroscopic guidance. This was, in part, due to the capability of fluoroscopy systems to render high resolution images of bony anatomy (and adjacent tissues) to target pain generators. Today, with the versatility afforded by mobile C-arms (Tuohy et al., 1997), together with continued fidelity of the images rendered, the fluoroscope remains the principal modality for pain medicine image guidance. As a resulting consequence of this history, the literature not only contains several articles on the pros and cons of imaging techniques (for example, see Kapural and Goyle, 2007), but also contains multiple and diverse reports on radiation exposure associated with interventional pain procedures.

Notably, data collected on fluoroscopy time (the traditional metric used for clinical radiation management) serves to benchmark performance (Balter, 2006). While this parameter (fluoroscopy time) plays an essential role in the development of suitable registries to catalog radiation exposure levels according to the different pain procedures being performed, it may also be said that it is simply an awareness of this parameter by the physician that is inherent to optimization strategies in health physics (Shahabi, 1999). Table 2 presents the fluoroscopy times reported for the more common interventional pain medicine procedures using mobile multi-directional fluoroscopy systems (i.e., the conventional mobile C-arm). Other data noteworthy to collect includes: dose settings employed (operator chosen) and patient body mass. It is this additional information along with fluoroscopy time which may be used to calculate patient radiation dose received (i.e., entrance skin exposure). Table 3 shows fluoroscopy time as well as radiation dose using mobile multi-directional systems or biplanar systems for vertebral augmentation procedures.


\*Wininger et al. also reported calculations on entrance skin exposure.

Table 2. Fluoroscopy time (in seconds) using mobile multi-directional systems.

Nearly all interventional pain treatments and techniques evolved using fluoroscopic guidance. This was, in part, due to the capability of fluoroscopy systems to render high resolution images of bony anatomy (and adjacent tissues) to target pain generators. Today, with the versatility afforded by mobile C-arms (Tuohy et al., 1997), together with continued fidelity of the images rendered, the fluoroscope remains the principal modality for pain medicine image guidance. As a resulting consequence of this history, the literature not only contains several articles on the pros and cons of imaging techniques (for example, see Kapural and Goyle, 2007), but also contains multiple and diverse reports on radiation

Notably, data collected on fluoroscopy time (the traditional metric used for clinical radiation management) serves to benchmark performance (Balter, 2006). While this parameter (fluoroscopy time) plays an essential role in the development of suitable registries to catalog radiation exposure levels according to the different pain procedures being performed, it may also be said that it is simply an awareness of this parameter by the physician that is inherent to optimization strategies in health physics (Shahabi, 1999). Table 2 presents the fluoroscopy times reported for the more common interventional pain medicine procedures using mobile multi-directional fluoroscopy systems (i.e., the conventional mobile C-arm). Other data noteworthy to collect includes: dose settings employed (operator chosen) and patient body mass. It is this additional information along with fluoroscopy time which may be used to calculate patient radiation dose received (i.e., entrance skin exposure). Table 3 shows fluoroscopy time as well as radiation dose using mobile multi-directional systems or

> Manchikanti **2003a**

> > 4.5 — — —

— — 50.6 — — 7.5 Sacroiliac Joint Blocks

2.7 — — — —

— — — 12.7 — — Medial Branch Block 57.2 — 146.8 — — — Discography — 133.4 — — — — Spinal Cord

— — — 13.2 8.9 12.5 Per Procedure — — — 7.7 4.9 7.5 Per Patient

Manchikanti **2003b** 

> 5.8 — — —

> 3.7 — — — —

Facet Nerve Blocks Cervical Thoracic Lumbar

Epidurals Interlaminar Caudal TFESI – Cervical TFESI – Lumbar

Stimulation

**2.3 Fluoroscopically-guided pain medicine procedures** 

exposure associated with interventional pain procedures.

biplanar systems for vertebral augmentation procedures.

Manchikanti **2002** 

> — 5.9 5.5 5.7

3.75 — — 8.8 10.9

\*Wininger et al. also reported calculations on entrance skin exposure.

Table 2. Fluoroscopy time (in seconds) using mobile multi-directional systems.

Zhou **2003**

> 81.5 — — —

> 46.6 — — — —

> — — — —

— — 12.6 — 15.2 **\***Wininger **2010** 

> — — — —

> — — — — —


\*\*without or \*\*\*with 3D navigation in vertebroplasty or kyphoplasty procedures. Fluoroscopy time (in seconds); patient entrance skin exposure (*ESE*) and dose area product (DAP, a calculation of stochastic risk for the patient [Vano et al., 2001]) (in centiGray); physician total and hand exposure (in microSieverts/minute).

Table 3. Radiation exposure associated with biplanar systems or multi-directional systems.

Inter-procedural variance compared to fluoroscopy times observed in Tables 2 and 3 may be attributed to differences in procedural techniques, level of experience, and/or physician preferences in imaging assistance, as well as attenuation physics relative to image quality. To illustrate this last point, we consider spinal imaging. During spinal imaging, two common challenges associated with image quality exist: 1) highly radiolucent vertebral bodies, particularly against the imaged lung field, creating excess image brightness in the region of interest, and 2) large body habitus with resultant poor image quality. In the former, tight collimation with the paired leaves shutters drawn close to the spine and continuous-mode imaging may help compensate for poor contrast resolution due to the vertebral bodies lacking enough cortical bone density to effectively attenuate the beam (i.e., low beam attenuation) (Johns & Cunningham, 1983). Subsequently, overriding automatic brightness control by manually ramping down tube current (mA) during tightly collimated bony imaging can help improve image resolution, especially for extremely radiolucent vertebral bodies. Alternatively, a manual adjustment to monitor/display window contrast may effectively improve image quality. To compensate for poor image quality secondary to large body habitus (i.e., a highly attenuated beam with resultant image granularity), it too may be necessary to operate the fluoroscope in continuous mode to increase the overall radiation at the image intensifier rather than disengaging the low dose feature. This strategy may improve image contrast while limiting patient exposure if a "manual beam on/off" operator technique is used, e.g., while panning or moving the C-arm between anteroposterior/oblique positioning to keep region of interest in the field of view.

#### **2.4 Radiation risk management/safety**

In recent years the assessment of radiation dose has received increased scrutiny; notably, the evaluation of deterministic effects, for which the severity of effects will vary according to the dose received and for which dose thresholds usually exist (e.g., radiation induced skin injuries) (Balter, 2006 & 2008). Moreover, dose assessment has seemingly evolved from an academic enterprise to a clinical endeavour. Direct influence on clinical practice is appreciated by The Joint Commission's recent decision to add unexpectedly prolonged fluoroscopic exposure to its list of reviewable sentinel events, as well as their suggestion to

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 131

fluoroscopy was utilized differently during cases #3 and #43, the fluoroscopy time for each case was recorded as 198.9 seconds. Analysis between actual settings used and hypothetical use variances for the low dose setting and the pulsed mode feature, based on simplistic modeling, illustrated how fluoroscopy time alone may lead to inadequate skin dose assessments. In other words, analysis of incident air kerma derived from the actual settings revealed that case #43 incurred 39.4% more skin exposure than case #3. Hypothetically, if neither the low dose feature nor pulsed fluoroscopy had been utilized, the resultant incident air kerma (i.e., 38.7 mGy) would have approximated the actual estimates derived for fluoroscopy times greater than 300 seconds (i.e., approximately double) for this procedure (i.e., 25.7–43.7 mGy). However, because the earliest deterministic threshold is 2.0 Gy, the level associated with transient erythema (Geleijns & Wondergem, 2005), research indicates that induction of deterministic insults (such as skin injuries) is highly unlikely during interventional pain medicine procedures. *Rather, in interventional pain medicine, the prime objective is to safeguard to the degree possible (i.e., ALARA) against low doses of x-radiation (U.S.* 

With respect to quality assurance programs for fluoroscopy systems, it is important to address mechanical and electrical safety in addition to radiation safety and image quality. Moreover, such programs are particularly important for mobile systems due to the various uses and locations in which these units are intended to perform. Given that mobile units are often the more commonly utilized systems in pain management applications, a quality assurance protocol is essential. However, due to differences between mobile systems from the various manufacturers, as well the different regulations overseeing radiologic licensure in the various jurisdictions the reader finds him- or herself in, an outline of such program is beyond the scope of this chapter. The reader is, therefore, referred to Tuohy et al. (1997) who

Reports on occupational incurred dose from scatter radiation are typically based on radiation exposure to standardized phantoms, thus representing the symmetrical ideal "small lumbar" spine. This methodology, however, may potentially underestimate the amount of occupational radiation exposure since body habitus does not always lend itself to symmetry and body size varies from patient to patient. Hence, Whitworth (n.d.) performed scatter radiation vector analysis on the lumbar spines of five cadavers to better parallel the general experience of the interventional pain physician and those team members inside the fluoroscopy suite. The study employed an OEC 9800 mobile C-arm with automatic brightness control engaged. Radiation exposure was recorded using Geiger Mueller

**OEC 9800 mrem/hour kV mA High Level/Boost Fluoro** 204 71 3.9 **Normal Dose** 111 70 2.2 **Low Dose** 56 72 0.76 **Low Dose + Collimation** 15 74 0.82 **Pulsed-8 frames per second** 7 76 0.86

Table 4. Scatter radiation measurements: imaging the lumbar spine of a cadaver with 21.5

Meter set at 17 inches above the floor and 12 inches lateral to the image intensifier.

*National Academy of Sciences, 2006; Little et al., 2009).*

tackled these issues and made several key recommendations.

techniques (measurements are shown in Table 4).

body mass index.

follow-up qualifying events with a period of over six-months to one-year to monitor cumulative skin dose (The Joint Commission, n.d.). While fluoroscopy time alone provides inadequate skin dose estimates (Balter, 2006; Balter, 2008), the evaluation of incident air kerma (x-ray exposure to the skin, previously referred to as entrance skin exposure) is possible by simplistic modeling (Balter, 2008; American Association of Physicists in Medicine (AAPM), 2001; Bushong, 2004).

In part with its approach to minimally invasive treatments and therapeutic procedures, interventional medicine is the one branch of medicine which, in its practice, is riddled with concepts that stem from physics. It may be further stated that physicians, even interventionally-trained physicians, may feel as though they are not able to translate applicable literature into everyday practice without possessing a doctorate in the physical sciences. For example, radiation dose models can be complicated as there exists many nuances when talking about dose, and the units of measure are not intuitive. However, both radiation safety and radiation protection are fundamental considerations for the interventional pain physician, and are equally paramount responsibilities in the interventional pain practice. In the view of the author, physicians who utilize fluoroscopic guidance will find that keeping radiation exposure, and therefore radiation dose, as low as reasonably achievable (given the acronym ALARA) is a challenge that is not insurmountable. This goal is achieved by exploitation of the principles unique to image acquisition in fluoroscopy, together with radiologic physics and applied radiobiology (Dowd & Tilson, 1999), and a suitable quality assurance program. Thus, by first laying out the conversion between the basic units of measure in radiation physics,

#### ͳܴ ൎ ͳݎܽ݀ ൌ ͳͲ݉ݕܩ ൌ ͳͲ݉ܵݒ

this section will strive to provide the interventional pain physician with information on radiation risk management which may be readily acted on and implemented.

Entrance skin exposure is the radiation exposure to the skin measured in Roentgen (R) or milliRoentgen (mR) at the point of skin entrance for the nominal patient (i.e., 30 cm from the image intensifier). The measurement is made without the contributions from scatter radiation. In compliance with physics acceptance testing, the fluoroscopic tube potentials (kVp) under automatic brightness control should operate at/or between 70 and 90 kVp with 3.8 cm of aluminum (~15 cm of water or acrylic plastic) attenuation material. This produces measured fluoroscopic exposure rates in the range of 1.0 to 4.0 R/minute for all magnification modes (fields of view) for continuous mode in the normal dose setting (AAPM, 2001; Bushong, 2004). The lower portion of the exposure range accounts for the largest field of view (least magnification), and the upper portion of the exposure range accounts for the smallest field of view (most magnification). The name of the quantity which corresponds to entrance skin exposure and which is recognized by the International Commission on Radiation Units and Measurements is incident air kerma (Balter, 2008), and the unit of measurement is milligray (mGy). (Note: 1 R = 1 Roentgen = 2.58 × 10-4 coulombs/kg-m of air at standard temperature and pressure, and 1 R = 8.76 mGy [milligray].)

The dedicated use of the low dose setting (which provides 40% or more dose reduction compared to the normal dose setting, Smiddy et al., 1996; Davies et al., 2006), when paired with pulsed fluoroscopy (which provides 50% dose reduction at 7.5 frames per second, Aufrichtig et al., 1994) promotes optimal radiation risk management. This impact is best observed by a closer inspection of the work by Wininger et al. (2010) on radiation exposure in spinal cord stimulation [trialing] procedures. The authors point out that although pulsed

follow-up qualifying events with a period of over six-months to one-year to monitor cumulative skin dose (The Joint Commission, n.d.). While fluoroscopy time alone provides inadequate skin dose estimates (Balter, 2006; Balter, 2008), the evaluation of incident air kerma (x-ray exposure to the skin, previously referred to as entrance skin exposure) is possible by simplistic modeling (Balter, 2008; American Association of Physicists in

In part with its approach to minimally invasive treatments and therapeutic procedures, interventional medicine is the one branch of medicine which, in its practice, is riddled with concepts that stem from physics. It may be further stated that physicians, even interventionally-trained physicians, may feel as though they are not able to translate applicable literature into everyday practice without possessing a doctorate in the physical sciences. For example, radiation dose models can be complicated as there exists many nuances when talking about dose, and the units of measure are not intuitive. However, both radiation safety and radiation protection are fundamental considerations for the interventional pain physician, and are equally paramount responsibilities in the interventional pain practice. In the view of the author, physicians who utilize fluoroscopic guidance will find that keeping radiation exposure, and therefore radiation dose, as low as reasonably achievable (given the acronym ALARA) is a challenge that is not insurmountable. This goal is achieved by exploitation of the principles unique to image acquisition in fluoroscopy, together with radiologic physics and applied radiobiology (Dowd & Tilson, 1999), and a suitable quality assurance program. Thus, by first laying out

ͳܴ ൎ ͳݎܽ݀ ൌ ͳͲ݉ݕܩ ൌ ͳͲ݉ܵݒ this section will strive to provide the interventional pain physician with information on

Entrance skin exposure is the radiation exposure to the skin measured in Roentgen (R) or milliRoentgen (mR) at the point of skin entrance for the nominal patient (i.e., 30 cm from the image intensifier). The measurement is made without the contributions from scatter radiation. In compliance with physics acceptance testing, the fluoroscopic tube potentials (kVp) under automatic brightness control should operate at/or between 70 and 90 kVp with 3.8 cm of aluminum (~15 cm of water or acrylic plastic) attenuation material. This produces measured fluoroscopic exposure rates in the range of 1.0 to 4.0 R/minute for all magnification modes (fields of view) for continuous mode in the normal dose setting (AAPM, 2001; Bushong, 2004). The lower portion of the exposure range accounts for the largest field of view (least magnification), and the upper portion of the exposure range accounts for the smallest field of view (most magnification). The name of the quantity which corresponds to entrance skin exposure and which is recognized by the International Commission on Radiation Units and Measurements is incident air kerma (Balter, 2008), and the unit of measurement is milligray (mGy). (Note: 1 R = 1 Roentgen = 2.58 × 10-4 coulombs/kg-m of air at standard temperature

The dedicated use of the low dose setting (which provides 40% or more dose reduction compared to the normal dose setting, Smiddy et al., 1996; Davies et al., 2006), when paired with pulsed fluoroscopy (which provides 50% dose reduction at 7.5 frames per second, Aufrichtig et al., 1994) promotes optimal radiation risk management. This impact is best observed by a closer inspection of the work by Wininger et al. (2010) on radiation exposure in spinal cord stimulation [trialing] procedures. The authors point out that although pulsed

the conversion between the basic units of measure in radiation physics,

radiation risk management which may be readily acted on and implemented.

Medicine (AAPM), 2001; Bushong, 2004).

and pressure, and 1 R = 8.76 mGy [milligray].)

fluoroscopy was utilized differently during cases #3 and #43, the fluoroscopy time for each case was recorded as 198.9 seconds. Analysis between actual settings used and hypothetical use variances for the low dose setting and the pulsed mode feature, based on simplistic modeling, illustrated how fluoroscopy time alone may lead to inadequate skin dose assessments. In other words, analysis of incident air kerma derived from the actual settings revealed that case #43 incurred 39.4% more skin exposure than case #3. Hypothetically, if neither the low dose feature nor pulsed fluoroscopy had been utilized, the resultant incident air kerma (i.e., 38.7 mGy) would have approximated the actual estimates derived for fluoroscopy times greater than 300 seconds (i.e., approximately double) for this procedure (i.e., 25.7–43.7 mGy). However, because the earliest deterministic threshold is 2.0 Gy, the level associated with transient erythema (Geleijns & Wondergem, 2005), research indicates that induction of deterministic insults (such as skin injuries) is highly unlikely during interventional pain medicine procedures. *Rather, in interventional pain medicine, the prime objective is to safeguard to the degree possible (i.e., ALARA) against low doses of x-radiation (U.S. National Academy of Sciences, 2006; Little et al., 2009).*

With respect to quality assurance programs for fluoroscopy systems, it is important to address mechanical and electrical safety in addition to radiation safety and image quality. Moreover, such programs are particularly important for mobile systems due to the various uses and locations in which these units are intended to perform. Given that mobile units are often the more commonly utilized systems in pain management applications, a quality assurance protocol is essential. However, due to differences between mobile systems from the various manufacturers, as well the different regulations overseeing radiologic licensure in the various jurisdictions the reader finds him- or herself in, an outline of such program is beyond the scope of this chapter. The reader is, therefore, referred to Tuohy et al. (1997) who tackled these issues and made several key recommendations.

Reports on occupational incurred dose from scatter radiation are typically based on radiation exposure to standardized phantoms, thus representing the symmetrical ideal "small lumbar" spine. This methodology, however, may potentially underestimate the amount of occupational radiation exposure since body habitus does not always lend itself to symmetry and body size varies from patient to patient. Hence, Whitworth (n.d.) performed scatter radiation vector analysis on the lumbar spines of five cadavers to better parallel the general experience of the interventional pain physician and those team members inside the fluoroscopy suite. The study employed an OEC 9800 mobile C-arm with automatic brightness control engaged. Radiation exposure was recorded using Geiger Mueller techniques (measurements are shown in Table 4).


Meter set at 17 inches above the floor and 12 inches lateral to the image intensifier.

Table 4. Scatter radiation measurements: imaging the lumbar spine of a cadaver with 21.5 body mass index.

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 133

It is know that the dominant hand of the interventionalist receives the highest dose of radiation. It is interesting to note that a new type of sterilizable, radiation protection glove (primarily composed of tungsten) was recently tested among surgeons during a variety of cases, including micro-discectomy (Back et al., 2004). In terms of radiation protection, results revealed that the glove was superior to all other gloves in the marketplace, attenuating 90% of x-rays (see Table 6), and radiation dose to the dominant hand was reduced to less than

equivalent Pure lead Lead equivalent "Lead-free"

Attenuation at 70 kVp

Transmission at 70 kVp

Attenuation at 100 kVp

Transmission at 100 kVp

Adapted from Back et al., 2005; Christodoulou et al., 2003; and Clasper & Pinks, 1995.

79-87% 95% 95%

Henleys Medical (3 STAR)

89-95% 99% 98-99% n/a

1% 1-2%

93-96%

5% 4-7%

Henleys Medical (2 STAR)

90% 65% 57% 32% 25%

Risks of cataract development due to radiation exposure to the eyes have been investigated in interventionalists, with no conclusive evidence to date. However, the use of lead-based glasses is advocated, especially when the risk of "rescatter" (radiation which emanates from within the interventionalist's head, or so-called tertiary exposure) is considered (Cousin et al. 1987). As pointed out in a review on exposure risks of interventional pain physicians, studies demonstrate a decrease in transmission rates of 70-90% with appropriate eyewear ("lead" glasses) ( Fish et al., 2011). Moreover, because it is the patient that is the point source of occupational radiation risk coupled with the proximity of the interventional pain physician to the patient, positioning the monitor to require the interventionalist to look 90° (from the patient) with eyewear with side shields could further help reduce eye exposure. As stated by Fish et al. (2011), "…It is extremely vital for the interventionalist to be

**Gloves – supplier or \*type, and quoted decrease at 80 kVp**

98.1-98.3%

n/a 93.2-93.9%

Henleys Medical (1 STAR)

Mean Range

Mean Range

Mean Range

Mean Range

F&L Medical Products Co.

the dose received by the non-dominant hand.

Pure lead

95% 92%

85% 83%

\*Tungsten

Lead

8% 5-11%

17% 13-31%

Table 6. Radiation protection apparel.

**Aprons (0.25 mm) Aprons (0.50 mm)**

Four important concepts were drawn from the resulting data set, as follows:


It is interesting to pair the data set obtained by Whitworth with a law in radiophysics which aptly describes the phenomenon of scatter radiation in a meaningful way: as kilovoltage increases the photoelectric effect decreases *greatly* and the percentage of Compton interactions decreases *slightly*. Thus, Whitworth actually observed the following. As tube potential (kVp) increases, fewer x-rays interact with tissue, and therefore less scatter is created. However, the scatter that is created has higher energy and is more likely to reach the image intensifier [or nearby dosimeter(s)] than to interact with the patient's body. This makes increased kVp a radiation protection tool that must be counterbalanced with image concerns (Dowd & Tilson, 1999). See Table 5.


Adapted from Dowd & Tilson, 1999.

Table 5. Effects of x-ray tube potential (kilovoltage) on secondary radiation generated.

In many interventional pain suites, "lead aprons" are the principal shield for radiation protection of personnel. In addition, it should be noted that a table skirt substantially decreases occupational radiation levels—since the majority of scatter radiation is produced under the fluoroscopy table (for fluoroscopes/C-arms with under-table x-ray tubes) in the form of backscatter. Thus, significant reduction to scatter radiation is gained from the combined pair, as each scattering incidence results in x-radiation energy levels of only 1/1000 of that prior to the episode (Dowd & Tilson, 1999). Moreover, largely as a consequence of complaints of back pain over time from the wearers of lead aprons (Christodoulou et al., 2003), lead equivalent aprons became the apron-of-choice among interventionalists, and recently, "lead free" aprons have emerged in the marketplace. Such alternative materials include tin, iodine, barium, and antimony, or any combination thereof. Such "lead free" alternatives offer significant weight reduction, compared to primarilyleaded aprons, and equivalent radiation protection (Finnerty, 2005) (see Table 6). As part of an occupation radiation safety program, it is suggested that the reader ensure annual inspections of aprons are performed and rejection criteria established. Practical rejection criteria have been offered by Stam and Pillay (2008).

Scatter radiation is exponential with increasing kilovoltage (kV) and linear with

Use of low dose reduces scatter radiation by 50-75%; use of pulsed mode reduces scatter

It is interesting to pair the data set obtained by Whitworth with a law in radiophysics which aptly describes the phenomenon of scatter radiation in a meaningful way: as kilovoltage increases the photoelectric effect decreases *greatly* and the percentage of Compton interactions decreases *slightly*. Thus, Whitworth actually observed the following. As tube potential (kVp) increases, fewer x-rays interact with tissue, and therefore less scatter is created. However, the scatter that is created has higher energy and is more likely to reach the image intensifier [or nearby dosimeter(s)] than to interact with the patient's body. This makes increased kVp a radiation protection tool that must be counterbalanced with image

> **Secondary Radiation**  (The Patient as the Point Source)

50 500 (50%) 500 (50%) 1000 90 165 (33%) 335 (67%) 500

In many interventional pain suites, "lead aprons" are the principal shield for radiation protection of personnel. In addition, it should be noted that a table skirt substantially decreases occupational radiation levels—since the majority of scatter radiation is produced under the fluoroscopy table (for fluoroscopes/C-arms with under-table x-ray tubes) in the form of backscatter. Thus, significant reduction to scatter radiation is gained from the combined pair, as each scattering incidence results in x-radiation energy levels of only 1/1000 of that prior to the episode (Dowd & Tilson, 1999). Moreover, largely as a consequence of complaints of back pain over time from the wearers of lead aprons (Christodoulou et al., 2003), lead equivalent aprons became the apron-of-choice among interventionalists, and recently, "lead free" aprons have emerged in the marketplace. Such alternative materials include tin, iodine, barium, and antimony, or any combination thereof. Such "lead free" alternatives offer significant weight reduction, compared to primarilyleaded aprons, and equivalent radiation protection (Finnerty, 2005) (see Table 6). As part of an occupation radiation safety program, it is suggested that the reader ensure annual inspections of aprons are performed and rejection criteria established. Practical rejection

Table 5. Effects of x-ray tube potential (kilovoltage) on secondary radiation generated.

Total Number of Compton Scatter/ Coherent Interactions

**Total Number of Interactions in 1mm Tissue** 

Four important concepts were drawn from the resulting data set, as follows:

Scatter radiation drops 50% every 6 inches away from the image intensifier.

Total Number of Photoelectric Effects

Collimation reduces scatter radiation by 50% or more;

increasing mA;

**Tube Potential**  Kilovoltage (kVp)

Adapted from Dowd & Tilson, 1999.

criteria have been offered by Stam and Pillay (2008).

radiation by 65-90%; and

concerns (Dowd & Tilson, 1999). See Table 5.

It is know that the dominant hand of the interventionalist receives the highest dose of radiation. It is interesting to note that a new type of sterilizable, radiation protection glove (primarily composed of tungsten) was recently tested among surgeons during a variety of cases, including micro-discectomy (Back et al., 2004). In terms of radiation protection, results revealed that the glove was superior to all other gloves in the marketplace, attenuating 90% of x-rays (see Table 6), and radiation dose to the dominant hand was reduced to less than the dose received by the non-dominant hand.


Adapted from Back et al., 2005; Christodoulou et al., 2003; and Clasper & Pinks, 1995.

Table 6. Radiation protection apparel.

Risks of cataract development due to radiation exposure to the eyes have been investigated in interventionalists, with no conclusive evidence to date. However, the use of lead-based glasses is advocated, especially when the risk of "rescatter" (radiation which emanates from within the interventionalist's head, or so-called tertiary exposure) is considered (Cousin et al. 1987). As pointed out in a review on exposure risks of interventional pain physicians, studies demonstrate a decrease in transmission rates of 70-90% with appropriate eyewear ("lead" glasses) ( Fish et al., 2011). Moreover, because it is the patient that is the point source of occupational radiation risk coupled with the proximity of the interventional pain physician to the patient, positioning the monitor to require the interventionalist to look 90° (from the patient) with eyewear with side shields could further help reduce eye exposure. As stated by Fish et al. (2011), "…It is extremely vital for the interventionalist to be

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 135

1. Figure 3 shows the descriptive statistical summary for fluoroscopy time. Seven outliers were identified in each data set. Notably, less variance around the median value

occurred with the new data (i.e., the interquartile range was reduced by 47.4%).

5. Patient radiation exposure was reduced: 1.53—32.0 mGy compared to 1.8—43.7 mGy. 6. Patient size ranged from mildly underweight to morbidly obese according to BMI (as defined by the World Health Organization, n.d.). The mean BMI, by gender, bordered

 Note: trending increase in body weight among the U.S. population paired with concern about cumulative dose trends (Yanch et al., 2009; Fazel et al., 2009) underscore the need to obtain accurate reference levels on radiation exposure, as such exposure will, in general, be higher for patients with greater body mass. 7. Estimates for incident air kerma were stratified according to various SSDs, see Figure 5.

Radiation exposure from spinal cord stimulation trialing procedures remains negligible despite the likelihood, as suggested here, for this therapy to be used in a patient population

The author thanks Siva Gopal, PhD, Otterbein University, for his constructive instruction in

with a greater risk for increased irradiation based on BMI valuations.

 Figure 4 compares the grouped subsets—based on one minute intervals. 3. Mean total fluoroscopy time was 46.3% less (71.7 seconds compared to 133.4 seconds). However, it is noted that outliers from the previous data set had not been removed in the reporting of that data, and thus mean total fluoroscopy time for the previous data

2. Accounting for outliers, total fluoroscopy time was normally distributed.

Percentage pulsed imaging: Mean: 33.4%

Source-to-skin distance: Mean: not reported Range: 43 cm to 50 cm

Body mass index: n = 54 females Mean: 31.54 kg/m2 Range: 18.46-53.32 kg/m2

 n = 52 males Mean: 29.65 kg/m2 Range: 17.03-42.45 kg/m2

Incident air kerma: Mean: 8.33 mGy

Physician dosimeter:

**Discussion:** 

Range: 1.53 mGy to 32.0 mGy

Whole body cumulative dose: 73 mrem

set was artificially inflated.

pre-obese and obese.

**Conclusions:** 

**Acknowledgements:** 

descriptive statistical methods.

4. Percentage pulsed imaging was equivalent.

Range: 1.80% to 75.2%

Mode: 55.4% (compiled % most frequent)

cognizant of his/her surroundings. This reiterates the importance of increasing the distance between the physician and the source of the radiation, it also emphasizes decreasing the amount of exposure time, which can both drastically reduce unnecessary radiation via scatter."

#### **2.5 Special report: Radiation exposure during spinal cord stimulation mapping: A new data set**

**Summary of Background Data:** The increase in exposure to low-dose radiation from the growing use of medical imaging has raised concerns about cumulative dose among the general population (Fazel et al., 2009; U.S. National Academy of Sciences, 2006; Little et al., 2009), and accordingly, dose assessment has received increased scrutiny (Balter, 2008). Conversely, unique among implantable devices, some spinal cord stimulation systems utilize integrated technology to perform "electronic fluoroscopy" to assess device orientation (i.e., the leads) without irradiation (Kosek et al., 2006). Recently, however, a first look at radiation exposure from spinal cord stimulation [trialing] procedures was published to help benchmark radiation exposure reference levels for this procedure (Wininger et al., 2010). Although estimated exposure was negligible, data on patient size was unavailable and the source-to-skin distance (SSD) was not taken into account due to simplistic modeling.

**Objective:** To address the aforementioned limitations, radiation exposure was reexamined by the author for a new patient population.

**Methods:** 106 dual parallel lead spinal cord stimulation trialing procedures [using either multiple-independent current-controlled systems or constant-voltage systems] in the nonuniversity, outpatient setting, from October 2008 to October 2009, were studied prospectively. Body mass index (BMI) measurements were retrieved. The \*fluoroscopy system automatically tabulated total fluoro-time (in seconds) per case, and partitioned the absolute time- and the percentage of time allocated to- pulsed and continuous-mode imaging. High dose fluoroscopy, or "boost" mode, was not used. A study specific ‡personal dosimeter was worn by the physician. For the dose model, radiation output was measured with a §dosimeter/ion chamber located 30 cm from the image intensifier, along the central axis of an anteroposterior projected beam, and calculated based on the following equation.

$$ESE\_{pat} = ESE\_{pha} \bullet \left[ \frac{O\_{pha}}{O\_{pat}} \bullet \left( \frac{SSD\_{pat}}{SSD\_{pha}} \right)^2 \right] \bullet \ t\_{flu}$$

Where *ESEpat* and *ESEpha* are skin exposure to the patient and †phantom; *Opha* and *Opat* are radiation output for phantom and patient exposure (in Röentgens); *SSDpat* and *SSDpha* are the distances from the x-ray source to the skin for the patient and phantom; and *tflu* is fluorotime (converted to minutes). Note: incident air kerma is measured in milligray (mGy) and is converted from *ESEpat* by applying a factor of 8.76 mGy to 1 Röentgen. Incident air kerma estimates were stratified according to SSD and low dose mode engaged/disengaged.

#### **Results**:

Total fluoroscopy time:

 Mean: 71.7 seconds (standard deviation: 34.9 seconds) Range: 19.5 seconds to 166.6 seconds


#### **Discussion:**

134 Pain Management – Current Issues and Opinions

cognizant of his/her surroundings. This reiterates the importance of increasing the distance between the physician and the source of the radiation, it also emphasizes decreasing the amount of exposure time, which can both drastically reduce unnecessary radiation via

**2.5 Special report: Radiation exposure during spinal cord stimulation mapping: A new** 

**Summary of Background Data:** The increase in exposure to low-dose radiation from the growing use of medical imaging has raised concerns about cumulative dose among the general population (Fazel et al., 2009; U.S. National Academy of Sciences, 2006; Little et al., 2009), and accordingly, dose assessment has received increased scrutiny (Balter, 2008). Conversely, unique among implantable devices, some spinal cord stimulation systems utilize integrated technology to perform "electronic fluoroscopy" to assess device orientation (i.e., the leads) without irradiation (Kosek et al., 2006). Recently, however, a first look at radiation exposure from spinal cord stimulation [trialing] procedures was published to help benchmark radiation exposure reference levels for this procedure (Wininger et al., 2010). Although estimated exposure was negligible, data on patient size was unavailable and the source-to-skin distance (SSD) was not taken into account due to

**Objective:** To address the aforementioned limitations, radiation exposure was reexamined

**Methods:** 106 dual parallel lead spinal cord stimulation trialing procedures [using either multiple-independent current-controlled systems or constant-voltage systems] in the nonuniversity, outpatient setting, from October 2008 to October 2009, were studied prospectively. Body mass index (BMI) measurements were retrieved. The \*fluoroscopy system automatically tabulated total fluoro-time (in seconds) per case, and partitioned the absolute time- and the percentage of time allocated to- pulsed and continuous-mode imaging. High dose fluoroscopy, or "boost" mode, was not used. A study specific ‡personal dosimeter was worn by the physician. For the dose model, radiation output was measured with a §dosimeter/ion chamber located 30 cm from the image intensifier, along the central axis of an anteroposterior projected beam, and calculated based on the following equation.

> ���� ����

Where *ESEpat* and *ESEpha* are skin exposure to the patient and †phantom; *Opha* and *Opat* are radiation output for phantom and patient exposure (in Röentgens); *SSDpat* and *SSDpha* are the distances from the x-ray source to the skin for the patient and phantom; and *tflu* is fluorotime (converted to minutes). Note: incident air kerma is measured in milligray (mGy) and is converted from *ESEpat* by applying a factor of 8.76 mGy to 1 Röentgen. Incident air kerma

estimates were stratified according to SSD and low dose mode engaged/disengaged.

 • �

������ ������ � 2

� • ����

������ � ������ • �

scatter."

**data set** 

simplistic modeling.

**Results**:

Total fluoroscopy time: Mean: 71.7 seconds

 (standard deviation: 34.9 seconds) Range: 19.5 seconds to 166.6 seconds

by the author for a new patient population.

	- Figure 4 compares the grouped subsets—based on one minute intervals.
	- Note: trending increase in body weight among the U.S. population paired with concern about cumulative dose trends (Yanch et al., 2009; Fazel et al., 2009) underscore the need to obtain accurate reference levels on radiation exposure, as such exposure will, in general, be higher for patients with greater body mass.

#### **Conclusions:**

Radiation exposure from spinal cord stimulation trialing procedures remains negligible despite the likelihood, as suggested here, for this therapy to be used in a patient population with a greater risk for increased irradiation based on BMI valuations.

#### **Acknowledgements:**

The author thanks Siva Gopal, PhD, Otterbein University, for his constructive instruction in descriptive statistical methods.

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 137

Fig. 4. Bar chart comparing the prior and new data sets with respect to fluoroscopy time

Fig. 5. *ESEpat* to anterior chest: 50 kg. adult patient accounting for one dose reduction feature, i.e., the low dose mode, either "on" or "off" for stratified SSDs (either 43 cm or 50 cm) in percutaneous spinal cord stimulation mapping. (Note: valuations represent continuous-

mode imaging, no beam collimation.)

during percutaneous spinal cord stimulation mapping procedures.

#### **Footnotes:**

\*OEC 9800 Super-C, GE Healthcare, Salt Lake City, UT, USA.

†Phantom: 3.8 cm of aluminum.




Fig. 3. Box plots comparing previous and current fluoroscopy time data sets for spinal cord stimulation mapping procedures. Note: Because all data (both sets) were obtained from the same interventional spine team, inter- and intra- procedural variability was minimized.

‡Badge report, study specific: Luxel, optically stimulated luminescence dosimetry,

§Radiation meter – Model 1515 with converter model 1050U and ion chamber model 10X6-

Previous data set (n=110)

Outliers 321.1 208.9 329.3 217.7 336.2 236.3 343.8 239.4 373.1 294.0 387.2 299.7 387.4 304.0 Fig. 3. Box plots comparing previous and current fluoroscopy time data sets for spinal cord stimulation mapping procedures. Note: Because all data (both sets) were obtained from the same interventional spine team, inter- and intra- procedural variability was minimized.

Interquartile Range 98.9 52.0 Lower Fence -76.6 -30.8 Upper Fence 319.1 177.2

Current data set (n=106)

\*OEC 9800 Super-C, GE Healthcare, Salt Lake City, UT, USA.

**Footnotes:** 

†Phantom: 3.8 cm of aluminum.

LANDAUER, Glenwood, IL, USA.

6M, Radcal Co., Monrovia, CA, USA.

Fig. 4. Bar chart comparing the prior and new data sets with respect to fluoroscopy time during percutaneous spinal cord stimulation mapping procedures.

Fig. 5. *ESEpat* to anterior chest: 50 kg. adult patient accounting for one dose reduction feature, i.e., the low dose mode, either "on" or "off" for stratified SSDs (either 43 cm or 50 cm) in percutaneous spinal cord stimulation mapping. (Note: valuations represent continuousmode imaging, no beam collimation.)

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 139

Balter Why (Continue to) Study Physics? Radiographics 1992;12:609 Rothenberg Radiation Dose in CT Radiographics 1992;12:1225 McNitt-Gray Topics in CT: Radiation Dose in CT? Radiographics 2002;22:1541

Table 7. Select references on the physics of CT appearing in the journals of the Radiological

Although the subject matter on this topic is diverse, no truly rigorous mathematical justification of a tomographic algorithm exists (Shepp & Kruskal, 1978). For this reason a generalized derivation (that of inverting the Radon transform, as this is the widely accepted technique to describe how we recapture the information lost to attenuated x-ray photons) will be described in plain mathematical language. In addition, where noted, Wolfram *Mathematica*—the online computational engine, Wolfram|Alpha™—was used to plot the traditional representative line equations of the x-ray photons. It is also important to note that in order to simplify the derivation the following three constraints will be made. First, we will ignore the playoff between Cartesian and polar coordinate representations, i.e., the 2 dimensional xy-plane versus spherical or circular symmetry. Second, we will not account for adjustments in the derivation for cone beam and/or fan-beam CT constructs due to their mathematical complexities (Note: the fan-beam third generation CT scanner, see Figure 7, is the most commonly utilized type of scanner.) Finally, discrete numerical analysis will not be

Techniques and Devices Radiographics 2008;28:245

Dose at CT Radiology 2008;248:995

**Journal Year;Volume:Page** 

**First Author Title of Article Citation:** 

Bauhs CT Dosimetry: Comparison of Measurement

Huda Converting Dose-Length Products to Effective

Fig. 7. Third generation "fan-beam" CT scanner.

Society of North America.

#### **3. Computed Tomography (CT) and interventional pain medicine**

#### **3.1 The CT imaging chain**

Although the underlying physical concepts are, for the most part, the same (such as x-ray production), the CT imaging chain offers a higher level of sophistication compared to the imaging chain of fluoroscopy. This is exemplified by the application of mathematical filters selected for a desired level of image reconstruction to control signal/quantum noise to optimize image quality (Sprawls, 1992), and most commonly applied using high-pass filters to control edge artifacts. According to Barnes (1992), while the CT scanner is capable of dividing its measurement of tissue attenuation into a range of 4,096 CT numbers, the eye is not capable of distinguishing this much detail in an image. The image display of a CT scanner represents only 256 levels of gray, which must therefore be mapped onto the portion of the Hounsfield scale that is to be displayed. Adjustments called "window level" and "window width" are used to define this mapping. Selection of the window level (i.e., brightness) specifies the CT number for centering the gray scale, and choice of the window width (i.e., contrast) defines the range of CT numbers over which the gray scale is to extend. These adjustments can be thought of as defining the "slope" of the gray scale. When the gray scale is placed at a window level of 100 and the window width is set at 500, the gray scale permits display of CT numbers from -150 to +350. All CT numbers below the lower limit of the window width are displayed as black, and all those above the upper limit are displayed as white on the image (see Figure 6).

Fig. 6. CT Numbers = Hounsfield Units.

#### **3.2 The mathematics of CT physics**

The language of mathematics not only permeates all scientific study, but the very application of mathematics itself allows exploration to occur at the limits-of-discovery to find answers to questions that vex human nature. To this end, it is through a mathematical framework that physicists talk about dosimetry—with various selected examples on dosimetric methods in CT given in Table 7. Moreover, computational models for CT scanning enable testing of quality control algorithms to ultimately help reduce overexposure errors (Ferreira et al., 2010). This section will serve as a mathematical primer to highlight the physics behind image acquisition/signal processing of CT scanning to allow interventional pain physicians to gain deeper insight into this modality, and through this appreciation, demystify the process of CT imaging.

Although the underlying physical concepts are, for the most part, the same (such as x-ray production), the CT imaging chain offers a higher level of sophistication compared to the imaging chain of fluoroscopy. This is exemplified by the application of mathematical filters selected for a desired level of image reconstruction to control signal/quantum noise to optimize image quality (Sprawls, 1992), and most commonly applied using high-pass filters to control edge artifacts. According to Barnes (1992), while the CT scanner is capable of dividing its measurement of tissue attenuation into a range of 4,096 CT numbers, the eye is not capable of distinguishing this much detail in an image. The image display of a CT scanner represents only 256 levels of gray, which must therefore be mapped onto the portion of the Hounsfield scale that is to be displayed. Adjustments called "window level" and "window width" are used to define this mapping. Selection of the window level (i.e., brightness) specifies the CT number for centering the gray scale, and choice of the window width (i.e., contrast) defines the range of CT numbers over which the gray scale is to extend. These adjustments can be thought of as defining the "slope" of the gray scale. When the gray scale is placed at a window level of 100 and the window width is set at 500, the gray scale permits display of CT numbers from -150 to +350. All CT numbers below the lower limit of the window width are displayed as black, and all those above the upper limit are

The language of mathematics not only permeates all scientific study, but the very application of mathematics itself allows exploration to occur at the limits-of-discovery to find answers to questions that vex human nature. To this end, it is through a mathematical framework that physicists talk about dosimetry—with various selected examples on dosimetric methods in CT given in Table 7. Moreover, computational models for CT scanning enable testing of quality control algorithms to ultimately help reduce overexposure errors (Ferreira et al., 2010). This section will serve as a mathematical primer to highlight the physics behind image acquisition/signal processing of CT scanning to allow interventional pain physicians to gain deeper insight into this modality, and through this appreciation,

**3. Computed Tomography (CT) and interventional pain medicine** 

**3.1 The CT imaging chain** 

displayed as white on the image (see Figure 6).

Fig. 6. CT Numbers = Hounsfield Units.

**3.2 The mathematics of CT physics** 

demystify the process of CT imaging.


Table 7. Select references on the physics of CT appearing in the journals of the Radiological Society of North America.

Although the subject matter on this topic is diverse, no truly rigorous mathematical justification of a tomographic algorithm exists (Shepp & Kruskal, 1978). For this reason a generalized derivation (that of inverting the Radon transform, as this is the widely accepted technique to describe how we recapture the information lost to attenuated x-ray photons) will be described in plain mathematical language. In addition, where noted, Wolfram *Mathematica*—the online computational engine, Wolfram|Alpha™—was used to plot the traditional representative line equations of the x-ray photons. It is also important to note that in order to simplify the derivation the following three constraints will be made. First, we will ignore the playoff between Cartesian and polar coordinate representations, i.e., the 2 dimensional xy-plane versus spherical or circular symmetry. Second, we will not account for adjustments in the derivation for cone beam and/or fan-beam CT constructs due to their mathematical complexities (Note: the fan-beam third generation CT scanner, see Figure 7, is the most commonly utilized type of scanner.) Finally, discrete numerical analysis will not be

Fig. 7. Third generation "fan-beam" CT scanner.

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 141

ݕ ൌ ݉ݔ ܾ െ λ ൏ ܾ ൏ λǡ Ͳ ݉ ൏ λ In this form, the coordinates of the lines in the xy-plane are the points (m,b), "m" the slope of the line and "b" the y-intercept. However, this coordinate system breaks down as "m" and "b" vary because the formula is not valid for vertical lines, such that a vertical slope is not defined (Larson et al., 2007). Therefore, a more suitable coordinate system is required to parameterize a line (and all families of parallel lines), and therefore, it is interesting to look

Fig. 8. (Top left) Gantry of CT scanner showing trajectories of x-rays [long arrows] emitted as lines/family of parallel lines. (Top right) Illustrated clockwise rotation of the x-ray tube inside the gantry. (Bottom left/right) Representative, corresponding line equations written in the slope-intercept form y=mx+b in the xy-plane. The (m,b) coordinates are given by the

Referring to Figure 9, one such identified commonality is that each line has the same angle to the horizontal axis, the x1-axis. Thus, we will call this angle, the angle φ (phi). Specifically, it is the normal vectors of these lines that have the same angle to the x1-axis. However, to better identify locations of lines, we need more than just the angle to the x1-axis. To singleout a line we look at its distance ρ (rho) from the line passing through the origin (see Figure 9). Thus, with these parameters, the distance ρ (rho) and the angle φ (phi), we have successfully established an unambiguous coordinate system that is not flawed by the non-

line equations.

at what a family of parallel lines may have in common (see Figure 9).

addressed. To this end, the steps necessary to invert the Radon transform with respect to the parallel beam model (and thus most enthusiastically applicable to first and second generation CT scanners will constitute the balance of this section. It is the hope that such insight will complement the physician's knowledge-base when carrying out CT-guided pain procedures.

#### **The set up**

The underlying theme in this mathematical application is a signal processing challenge, and the set up for the analysis is straightforward. We have a 2-dimensional slice of a region of variable density (the patient), and the goal as applied to CT scanning is to reconstruct the resulting x-ray signal (the image) after repeatedly passing x-rays through the region at different angles of initial projection (the CT gantry). More concisely stated, we are measuring the resultant signal at different trajectory lines by accumulating (integrating) the signal after projecting x-ray photons through the region. Hence, the approach reconstructs the densities of the materials interacting with the x-ray photons (Johns & Cunningham, 1983), to ultimately assign density values according to the Hounsfield unit scale of CT numbers for data acquisition/image processing (Jackson & Thomas, 2004). Such modeling serves as an engineering template for trouble-shooting in the event of errors, such as equipment failure or computer algorithm failures, which may lead to radiation overdose of the patient.

Given that the approach resolves signal processing by means of calculating line integrals to recover the intensity of the x-ray signal (i.e., capture the data lost to attenuated or scattered x-rays), a comparison may be made to the inverse square law which estimates beam intensity from known initial conditions, the intensity of- and distance from- the beam (Carlton & Adler, 2006). However, the comparison is rudimentary at best because the central and interesting feature of the model applicable here, i.e., the Radon transform and its inverse, lies in the fact that we are *strictly* calculating the intensity of the exit/secondary beam based *solely* on a known intensity of the primary beam.

It is important to understand that the Radon transform refers to a special case of the Fourier transform; and the Fourier transform is a limiting case of the Fourier series (Boyce & DiPrima, 2005; Bracewell, 1986). This means whereas a Fourier series is the mathematical instrument used when evaluating periodic phenomena (Boyce & DiPrima, 2005), a Fourier transform is reserved for the study of phenomena that is nonperiodic (Bracewell, 1986). Thus, the choice of the application of a "transform" is an intuitively simple decision, given that x-ray photons in the exit beam strike the image receptor in burst-like impulses that are mostly nonperiodic rather than periodic in fashion. In mathematical terms, burst-like physical phenomena that are almost periodic are known as line impulses. *The concept of the line impulse will be a key point expanded upon below*.

The derivation of the mathematical model can be relatively easy to follow since the steps involved are pragmatic to imaging tasks carried out in the CT suite. We begin by a detailed inspection of representative x-ray trajectories relative to the CT gantry (i.e., the family of parallel lines), and then compare the suitability of two different proposed coordinate systems for the model.

#### **Lines/family of lines**

Refer to Figure 8 for a depiction of the CT gantry with the x-ray beam drawn as a family of parallel lines though the region. Each representative x-ray trajectory (i.e., the parallel lines) can be written in the slope-intercept form of a line.

addressed. To this end, the steps necessary to invert the Radon transform with respect to the parallel beam model (and thus most enthusiastically applicable to first and second generation CT scanners will constitute the balance of this section. It is the hope that such insight will complement the physician's knowledge-base when carrying out CT-guided pain

The underlying theme in this mathematical application is a signal processing challenge, and the set up for the analysis is straightforward. We have a 2-dimensional slice of a region of variable density (the patient), and the goal as applied to CT scanning is to reconstruct the resulting x-ray signal (the image) after repeatedly passing x-rays through the region at different angles of initial projection (the CT gantry). More concisely stated, we are measuring the resultant signal at different trajectory lines by accumulating (integrating) the signal after projecting x-ray photons through the region. Hence, the approach reconstructs the densities of the materials interacting with the x-ray photons (Johns & Cunningham, 1983), to ultimately assign density values according to the Hounsfield unit scale of CT numbers for data acquisition/image processing (Jackson & Thomas, 2004). Such modeling serves as an engineering template for trouble-shooting in the event of errors, such as equipment failure or computer algorithm failures, which may lead to radiation overdose of

Given that the approach resolves signal processing by means of calculating line integrals to recover the intensity of the x-ray signal (i.e., capture the data lost to attenuated or scattered x-rays), a comparison may be made to the inverse square law which estimates beam intensity from known initial conditions, the intensity of- and distance from- the beam (Carlton & Adler, 2006). However, the comparison is rudimentary at best because the central and interesting feature of the model applicable here, i.e., the Radon transform and its inverse, lies in the fact that we are *strictly* calculating the intensity of the exit/secondary

It is important to understand that the Radon transform refers to a special case of the Fourier transform; and the Fourier transform is a limiting case of the Fourier series (Boyce & DiPrima, 2005; Bracewell, 1986). This means whereas a Fourier series is the mathematical instrument used when evaluating periodic phenomena (Boyce & DiPrima, 2005), a Fourier transform is reserved for the study of phenomena that is nonperiodic (Bracewell, 1986). Thus, the choice of the application of a "transform" is an intuitively simple decision, given that x-ray photons in the exit beam strike the image receptor in burst-like impulses that are mostly nonperiodic rather than periodic in fashion. In mathematical terms, burst-like physical phenomena that are almost periodic are known as line impulses. *The concept of the* 

The derivation of the mathematical model can be relatively easy to follow since the steps involved are pragmatic to imaging tasks carried out in the CT suite. We begin by a detailed inspection of representative x-ray trajectories relative to the CT gantry (i.e., the family of parallel lines), and then compare the suitability of two different proposed coordinate

Refer to Figure 8 for a depiction of the CT gantry with the x-ray beam drawn as a family of parallel lines though the region. Each representative x-ray trajectory (i.e., the parallel lines)

beam based *solely* on a known intensity of the primary beam.

*line impulse will be a key point expanded upon below*.

can be written in the slope-intercept form of a line.

systems for the model. **Lines/family of lines** 

procedures. **The set up** 

the patient.

ݕ ൌ ݉ݔ ܾ െ λ ൏ ܾ ൏ λǡ Ͳ ݉ ൏ λ

In this form, the coordinates of the lines in the xy-plane are the points (m,b), "m" the slope of the line and "b" the y-intercept. However, this coordinate system breaks down as "m" and "b" vary because the formula is not valid for vertical lines, such that a vertical slope is not defined (Larson et al., 2007). Therefore, a more suitable coordinate system is required to parameterize a line (and all families of parallel lines), and therefore, it is interesting to look at what a family of parallel lines may have in common (see Figure 9).

Fig. 8. (Top left) Gantry of CT scanner showing trajectories of x-rays [long arrows] emitted as lines/family of parallel lines. (Top right) Illustrated clockwise rotation of the x-ray tube inside the gantry. (Bottom left/right) Representative, corresponding line equations written in the slope-intercept form y=mx+b in the xy-plane. The (m,b) coordinates are given by the line equations.

Referring to Figure 9, one such identified commonality is that each line has the same angle to the horizontal axis, the x1-axis. Thus, we will call this angle, the angle φ (phi). Specifically, it is the normal vectors of these lines that have the same angle to the x1-axis. However, to better identify locations of lines, we need more than just the angle to the x1-axis. To singleout a line we look at its distance ρ (rho) from the line passing through the origin (see Figure 9). Thus, with these parameters, the distance ρ (rho) and the angle φ (phi), we have successfully established an unambiguous coordinate system that is not flawed by the non-

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 143

Accordingly, it is necessary to account for the nonperiodic nature of the signal concentrated along each trajectory taken by the x-ray photons, and this is accomplished by considering the line impulse (Bracewell, 1986). The line impulse describes the physical phenomena of xray photons striking the image receptor in the CT gantry. To define the line impulse mathematically, we first need to set the *Cartesian equation of the line for the model* to zero as

The resultant equation, specifically the left hand side of the new equation above, then

The delta function δ, is the classical way to approach the line impulse, and has advantageous implications for dimensionality and integration of a line (Figure 10) (Bracewell, 1986). Such line integrals have a domain of infinity on the line and zero off the

௦

becomes a function of delta, denoted by δ, on the right hand side (Bracewell, 1986).

Fig. 10. Expansion of the line integral to an integral of a plane (2-dimensional space) containing the line impulse. The above notations of the integrals, L and R2, are understood

Equipped with a suitable coordinate system and having addressed the line integral with respect to the line impulse, we are ready to introduce the computational steps central to the mathematical model, inverting the Radon transform. As we do this, it is important to first point out what is varying as we work through the computations, i.e., to identify the

As shown below in Figure 11, superimposition of the useful/suitable coordinate system (as described earlier) onto a representative cross-sectional image (the region of interest) will

to have domains or "boundaries" from negative infinity (-∞) to infinity (∞).

variables associated with the integrand (those terms being integrated).

ሱۛۛۛۛۛۛሮ ߩെݔଵ ߮ െ ݔଶ ߮ ൌ Ͳ

ሱۛۛۛۛۛሮ ߜሺߩ െ ݔଵ ߮ െ ݔଶ ߮ሻ

ߩൌݔଵ ߮ ݔଶ ߮ ௦௧௧௭

ߩെݔଵ ߮ െ ݔଶ ߮

**Line impulse** 

shown.

line (Bracewell, 1986).

**The Radon transform** 

help identify the variable.

existence issue of a vertical slope. The *Cartesian equation of the line for the model*, is now specified by a given coordinate pair (ρ,φ) in the form:

$$\mathbf{x} \cdot \mathbf{n} = \mathbf{x}\_1 \cos \varphi + \mathbf{x}\_2 \sin \varphi = \rho$$

where both **x** and **n** are vectors, each defined in the following way, **x**=‹x1,x2› and **n**=‹cos(φ),sin(φ)›, and the line equation is derived by vector multiplication, in this case by using the dot product method, where it is said that **x** is dotted with **n**.

Fig. 9. A better-suited coordinate system (ρ,φ) for the model. (Top panel) Arrow demonstrating the unit normal vector associated with the line passing through the origin, and oriented with an angle phi (φ) to the x1-axis in the x1-x2 plane. (Bottom panel) A family of 3-parallel lines and their unit normal vectors [arbitrarily placed on the lines] showing signed distances rho (ρ) from the origin [double ended arrows]. By convention, distances are positive [i.e., positive rho (ρ)] when measured *in the direction of the normal vector from the line passing through the origin to associated parallel lines*. In a similar fashion, distances are negative [i.e., negative rho (-ρ)] when measured *from the line passing through the origin to parallel lines spatially existing opposite to the direction established for the normal vector*. Rho (ρ) is zero at the line passing through the origin. (Note: the unit normal vectors are not drawn to scale, and when compared to Figure 8, the xy-plane has been renamed the x1-x2 plane.)

#### **Line impulse**

142 Pain Management – Current Issues and Opinions

existence issue of a vertical slope. The *Cartesian equation of the line for the model*, is now

ߩ ൌ ߮ ଶݔ ߮ ଵݔൌڄ where both **x** and **n** are vectors, each defined in the following way, **x**=‹x1,x2› and **n**=‹cos(φ),sin(φ)›, and the line equation is derived by vector multiplication, in this case by

specified by a given coordinate pair (ρ,φ) in the form:

using the dot product method, where it is said that **x** is dotted with **n**.

Fig. 9. A better-suited coordinate system (ρ,φ) for the model. (Top panel) Arrow

the x1-x2 plane.)

demonstrating the unit normal vector associated with the line passing through the origin, and oriented with an angle phi (φ) to the x1-axis in the x1-x2 plane. (Bottom panel) A family of 3-parallel lines and their unit normal vectors [arbitrarily placed on the lines] showing signed distances rho (ρ) from the origin [double ended arrows]. By convention, distances are positive [i.e., positive rho (ρ)] when measured *in the direction of the normal vector from the line passing through the origin to associated parallel lines*. In a similar fashion, distances are negative [i.e., negative rho (-ρ)] when measured *from the line passing through the origin to parallel lines spatially existing opposite to the direction established for the normal vector*. Rho (ρ) is zero at the line passing through the origin. (Note: the unit normal vectors are not drawn to scale, and when compared to Figure 8, the xy-plane has been renamed

Accordingly, it is necessary to account for the nonperiodic nature of the signal concentrated along each trajectory taken by the x-ray photons, and this is accomplished by considering the line impulse (Bracewell, 1986). The line impulse describes the physical phenomena of xray photons striking the image receptor in the CT gantry. To define the line impulse mathematically, we first need to set the *Cartesian equation of the line for the model* to zero as shown.

$$\rho = \mathbf{x}\_1 \cos \varphi + \mathbf{x}\_2 \sin \varphi \xrightarrow[\text{set to zero}]{} \rho - \mathbf{x}\_1 \cos \varphi - \mathbf{x}\_2 \sin \varphi = \mathbf{0}$$

The resultant equation, specifically the left hand side of the new equation above, then becomes a function of delta, denoted by δ, on the right hand side (Bracewell, 1986).

$$\left(\rho - \mathfrak{x}\_1 \cos \varphi - \mathfrak{x}\_2 \sin \varphi \xrightarrow{ becomes} \delta(\rho - \mathfrak{x}\_1 \cos \varphi - \mathfrak{x}\_2 \sin \varphi)\right)$$

The delta function δ, is the classical way to approach the line impulse, and has advantageous implications for dimensionality and integration of a line (Figure 10) (Bracewell, 1986). Such line integrals have a domain of infinity on the line and zero off the line (Bracewell, 1986).

Fig. 10. Expansion of the line integral to an integral of a plane (2-dimensional space) containing the line impulse. The above notations of the integrals, L and R2, are understood to have domains or "boundaries" from negative infinity (-∞) to infinity (∞).

#### **The Radon transform**

Equipped with a suitable coordinate system and having addressed the line integral with respect to the line impulse, we are ready to introduce the computational steps central to the mathematical model, inverting the Radon transform. As we do this, it is important to first point out what is varying as we work through the computations, i.e., to identify the variables associated with the integrand (those terms being integrated).

As shown below in Figure 11, superimposition of the useful/suitable coordinate system (as described earlier) onto a representative cross-sectional image (the region of interest) will help identify the variable.

$$\mathcal{R}\mu(\rho,\varphi) = \int\limits\_{\mathcal{L}(\rho,\varphi)} \mu = \int\limits\_{-\infty}^{\infty} \int\limits\_{-\infty}^{\infty} \mu(\mathfrak{x}\_1, \mathfrak{x}\_2) \,\delta\left(\rho - \mathfrak{x}\_1 \cos \varphi - \mathfrak{x}\_2 \sin \varphi\right) \,d\mathfrak{x}\_1 \,d\mathfrak{x}\_2$$

$$\mathcal{F}\_{\rho}\Big(\mathcal{R}\mu(\rho,\varphi)\big) = \int\_{-\infty}^{\infty} e^{-2\pi ir\rho} \left(\mathcal{R}\mu(\rho,\varphi)\right) d\rho$$

$$\int\_{-\infty}^{\omega} e^{-2\pi lr\rho} \,\delta\left(\rho - (\mathbf{x}\_1 \cos\varphi + \mathbf{x}\_2 \sin\varphi)\right) d\rho = e^{-2\pi lr(\mathbf{x}\_1 \cos\varphi + \mathbf{x}\_2 \sin\varphi)}$$

$$
\xi\_1 = r \cos \varphi \quad \text{and} \quad \xi\_2 = r \sin \varphi
$$

$$=e^{-2\pi l(\boldsymbol{x}\_1\xi\_1+\boldsymbol{x}\_2\xi\_2)}$$

$$\mathcal{F}\_{\rho} \left( \mathcal{R} \mu (\rho, \varphi) \right) = \int\_{-\infty}^{\infty} \int\_{-\infty}^{\infty} \mu (\mathbf{x}\_1, \mathbf{x}\_2) \, \mathbf{e}^{-2\pi l (\mathbf{x}\_1 \xi\_1 + \mathbf{x}\_2 \xi\_2)} \, d\mathbf{x}\_1 \, d\mathbf{x}\_2 \, d\mathbf{x}\_1$$

	-

$$\mathcal{F}\mu(\xi\_1, \xi\_2) = \mathbb{G}(\xi\_1, \xi\_2) \xrightarrow{recovered \ \mu} \mu = \mathcal{F}^{-1}\mathbb{G}(\xi\_1, \xi\_2)$$

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 149

increases in order to compensate for the corresponding loss of signal intensity. It is also interesting to note that *attenuation is inversely related to waveform frequency*, and that this relationship is nontrivial with respect to image resolution (i.e., recorded detail or the ability to distinguish between objects) and ultrasound physics. In the following subsection, which highlights the physics behind ultrasound imaging, we will further explore this

Based on waveform physics, that is, frequency, amplitude, and wavelength, the principles of ultrasound are unified by the foregoing description. A pulse of sound is emitted from a source (i.e., the transducer) and travels outward through a medium. If an object reflects the wave, then acoustic energy travels back to the source and is detected as an echo at the source. Thus, at a known speed (the speed of sound of the surrounding medium), the waveform travels a distance equal to twice the distance from the source to the reflected

� �� ��� �� ��⁄2 where *L* is twice the distance from the source to the object, *VS* is the speed of sound of the surrounding medium, and *T* is time. Note: the average value of *VS* in soft tissue is 1540 m/s. The ultrasound scanner records the time required for each pulse to return, and then uses the speed of sound to calculate the distance of the object. See Figure 13. Echo intensity is indicated by plotting a variety of intensities on the monitor subsequent to a gray-scale (white to gray to black). Thus, brightness is a consequence of a mapping of echo intensity versus position; hence

relation to better understand the clinical impact of sound wave attenuation.

this viewing algorithm/mode is named B-scan, where "B" means brightness.

Fig. 13. Panel-A shows a target nerve (or scattering reflector) and direction of travel of incident sound waves (white) and echoes (blue). Panel-B shows a zoomed-in view of the same nerve to more closely exhibit the reflection of sound waves from near and far tissue

**4.2 Waveform propagation in tissue: The physics of ultrasound** 

object (Kane, 2009). The basic equation follows:

borders (with respect to the transducer).

some of the effects of attenuation. First, most machines allow the operator to artificially increase (or decrease) the signal intensity of the return echoes from all points in the displayed field. This is accomplished by adjusting the gain control higher to increase the overall brightness. Second, most machines offer the operator the ability to control gain independently at specified depth intervals. This is known as time gain compensation. The time gain compensation should be progressively increased as the depth of penetration

intermittent exposure techniques and/or exposure parameters, such as lower tube current (Meleka et al., 2005). Moreover, strategies have also emerged to help reduce occupational radiation dose. For example, the use of lead shields, or as previously discussed, the use of lead aprons. In addition, the use of needle holders, when feasible during the procedure, avoids physician hand placement directly into the x-ray beam (Kato, 1996).


Key: CT-Fluoroscopy time (in seconds); effective dose (in microSieverts).

Table 8. CT-Fluoroscopy exposure metrics.

#### **4. Ultrasound and interventional pain medicine**

#### **4.1 The ultrasound imaging chain**

Continued research in the area of medical imaging has led to the development of compact and durable ultrasound scanners with improved imaging capabilities. Nevertheless, the basic instrumentation and underlying principles of this modality remain the same. The ultrasound imaging chain is considered a "closed" loop made up of the following links: a transmitter, a transducer, a receiver, and the image viewing system (Aldrich, 2007). Note that the physical phenomenon behind image creation is the piezoelectric effect, or stated more explicitly it is the effect on and induced by deformations of piezoelectric crystals embedded within materials housed inside the transducer which enables mechanical energy to be transformed into ultrasonic impulses—and vice versa for signal processing.

Upon interaction with tissue, ultrasonic waveforms may be 1) transmitted through the tissue, 2) undergo reflection (echo) or refraction (bending) at tissue boundaries, or 3) the acoustic energy may be attenuated. Surfaces that **reflect** these sound waves are classified as either *specular reflectors* or *scattering reflectors*. An example of the former is the needle, whereas an example of the latter is the interface between neural and adjacent tissues. Thus, it is the reflected sound waves (i.e., the available energy contained in the echoes collected at the transducer) which contribute to a meaningful image. **Refracted** sound waves are those which change direction due to slight differences at the boundary (i.e., edge) between two tissue types. We note that such waves may not contribute to successful imaging if a significant amount of the propagated waveform is lost. Finally, with similarities which evoke comparisons to the attenuated x-ray beam, the attenuation of sound beams conveys a loss of energy as the ultrasonic waveforms are absorbed by the tissue. According to Sites et al. (2007):

While attenuation can have a profound negative impact on image quality, there are two important adjustments that can be made on the ultrasound machine that help to overcome

intermittent exposure techniques and/or exposure parameters, such as lower tube current (Meleka et al., 2005). Moreover, strategies have also emerged to help reduce occupational radiation dose. For example, the use of lead shields, or as previously discussed, the use of lead aprons. In addition, the use of needle holders, when feasible during the procedure,

**2004b** CT-Fluoroscopy

Lumbar Epidural

Continued research in the area of medical imaging has led to the development of compact and durable ultrasound scanners with improved imaging capabilities. Nevertheless, the basic instrumentation and underlying principles of this modality remain the same. The ultrasound imaging chain is considered a "closed" loop made up of the following links: a transmitter, a transducer, a receiver, and the image viewing system (Aldrich, 2007). Note that the physical phenomenon behind image creation is the piezoelectric effect, or stated more explicitly it is the effect on and induced by deformations of piezoelectric crystals embedded within materials housed inside the transducer which enables mechanical energy

Upon interaction with tissue, ultrasonic waveforms may be 1) transmitted through the tissue, 2) undergo reflection (echo) or refraction (bending) at tissue boundaries, or 3) the acoustic energy may be attenuated. Surfaces that **reflect** these sound waves are classified as either *specular reflectors* or *scattering reflectors*. An example of the former is the needle, whereas an example of the latter is the interface between neural and adjacent tissues. Thus, it is the reflected sound waves (i.e., the available energy contained in the echoes collected at the transducer) which contribute to a meaningful image. **Refracted** sound waves are those which change direction due to slight differences at the boundary (i.e., edge) between two tissue types. We note that such waves may not contribute to successful imaging if a significant amount of the propagated waveform is lost. Finally, with similarities which evoke comparisons to the attenuated x-ray beam, the attenuation of sound beams conveys a loss of energy as the

While attenuation can have a profound negative impact on image quality, there are two important adjustments that can be made on the ultrasound machine that help to overcome

to be transformed into ultrasonic impulses—and vice versa for signal processing.

ultrasonic waveforms are absorbed by the tissue. According to Sites et al. (2007):

Lumbar Selective Nerve Root Block

CT-Fluoroscopy Time o Patient Effective Dose

CT-Fluoroscopy Time

Effective Dose (given in microSieverts per second )

o Physician Hand

o Effective Dose (Total) Physician

o Effective Dose (Physician/Procedure) o Effective Dose (Total) Physician

avoids physician hand placement directly into the x-ray beam (Kato, 1996).

Wagner

Not specified

— — —

> 1 75

Key: CT-Fluoroscopy time (in seconds); effective dose (in microSieverts).

**4. Ultrasound and interventional pain medicine** 

Kato **1996**

> — — —

> — —

Wagner **2004a**

> 2 7.3 390

> > — —

1140 — —

**4.1 The ultrasound imaging chain** 

Table 8. CT-Fluoroscopy exposure metrics.

some of the effects of attenuation. First, most machines allow the operator to artificially increase (or decrease) the signal intensity of the return echoes from all points in the displayed field. This is accomplished by adjusting the gain control higher to increase the overall brightness. Second, most machines offer the operator the ability to control gain independently at specified depth intervals. This is known as time gain compensation. The time gain compensation should be progressively increased as the depth of penetration increases in order to compensate for the corresponding loss of signal intensity.

It is also interesting to note that *attenuation is inversely related to waveform frequency*, and that this relationship is nontrivial with respect to image resolution (i.e., recorded detail or the ability to distinguish between objects) and ultrasound physics. In the following subsection, which highlights the physics behind ultrasound imaging, we will further explore this relation to better understand the clinical impact of sound wave attenuation.

#### **4.2 Waveform propagation in tissue: The physics of ultrasound**

Based on waveform physics, that is, frequency, amplitude, and wavelength, the principles of ultrasound are unified by the foregoing description. A pulse of sound is emitted from a source (i.e., the transducer) and travels outward through a medium. If an object reflects the wave, then acoustic energy travels back to the source and is detected as an echo at the source. Thus, at a known speed (the speed of sound of the surrounding medium), the waveform travels a distance equal to twice the distance from the source to the reflected object (Kane, 2009). The basic equation follows:

$$L = (\mathcal{V}\_{\mathbb{S}} \cdot T) / 2$$

where *L* is twice the distance from the source to the object, *VS* is the speed of sound of the surrounding medium, and *T* is time. Note: the average value of *VS* in soft tissue is 1540 m/s. The ultrasound scanner records the time required for each pulse to return, and then uses the speed of sound to calculate the distance of the object. See Figure 13. Echo intensity is indicated by plotting a variety of intensities on the monitor subsequent to a gray-scale (white to gray to black). Thus, brightness is a consequence of a mapping of echo intensity versus position; hence this viewing algorithm/mode is named B-scan, where "B" means brightness.

Fig. 13. Panel-A shows a target nerve (or scattering reflector) and direction of travel of incident sound waves (white) and echoes (blue). Panel-B shows a zoomed-in view of the same nerve to more closely exhibit the reflection of sound waves from near and far tissue borders (with respect to the transducer).

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 151

imaging has been investigated (Feinglass et al., 2007; Foxall, et al., 2007). Moreover, cutting edge ultrasonic technological advances have introduced image-enhanced tissue staining to remotely palpate the target nerve of interest using acoustic radiation force imaging to both improve accuracy and limit variability in regional anesthesia (Palmeri et al., 2008;

While there are no known absolute contraindications in ultrasound imaging, the position of The U.S. Food and Drug Administration (FDA) (n.d.) concerning ultrasound safety follows. Ultrasound imaging has been used for over 20 years and has an excellent safety record. It is non-ionizing radiation, so it does not have the same risks as x-rays or other types of ionizing radiation. Even though there are no known risks of ultrasound imaging, it can produce effects on the body. When ultrasound enters the body, it heats the tissues slightly. In some cases, it can also produce small pockets of gas in body fluids or tissues (cavitation). The long-term effects of tissue heating and cavitation are not known. For a more in-depth discussion on tissue sensitivity relative to interpreting risk from exposure to ultrasound imaging, the reader is encouraged to study the report on this topic issued by members of the World Federation for Ultrasound in Medicine and Biology Safety

While CT-fluoroscopy offers unique viewing perspectives with overall imaging capabilities (as discussed in the earlier section, "Triplanar Imaging in Pain Medicine Procedures: Conventional-CT Guidance and CT-Fluoroscopy"), interestingly, real-time 3D imaging has not been reported. Conversely, although real-time 3D ultrasound has been show to be beneficial in regional anesthesia (as cited in the above section, "Ultrasound guidance in Pain Medicine: Regional Anesthesia"), ultrasound imaging does not offer the allure that

Technology is currently available to fit mobile C-arm fluoroscopy systems with 3D imaging capabilities (Stübig et al., 2009; Izadpanah et al., 2009; Villavicencio et al., 2005). (Note: current fluoroscopy systems creating 3D imagery are limited to 150°—360° with mechanical orientation or manipulation or post-processing.) Notably, the limiting factor in the utility of these systems under most interventional pain protocols is image construction time. However, one company, Imaging3 Inc., claims to have developed signal processing algorithms to produce 3D high-resolution fluoroscopic images in *real-time* via its "Dominion" platform. (Note: at the time of this publication, this device has *not* received FDA clearance.) The design is built around a dedicated O-arm—a gantry similar to that used in CT—to allow continuous 360° rotation of the x-ray tube and scintillation detector with ondemand imaging of the patient under continuous or pulsed fluoroscopy. (See the earlier discussion on radiation risk management in fluoroscopy to review the advantages of pulsed fluoroscopy.) This approach is expected to allow imaging of the patient from any frame of reference or angulation. Intended use is anticipated by the company to be procedures in which multiple frames of reference are required. From a pain medicine perspective, realtime 3D fluoroscopic guidance may be possible for discography, vertebral augmentation, percutaneous lumbar decompression, facet rhizotomy, or intradiscal electrothermal therapy.

fluoroscopy enjoys with respect to image resolution, particularly of bony anatomy.

Nightingale et al., 2001).

Committee (Barnett et al., 1997).

**5. Future directions in interventional pain medicine** 

**5.1 \*The promise of real-time three-dimensional (3D) fluoroscopy** 

**4.4 Risk management: Ultrasound safety** 

As emphasized in the above subsection, a nontrivial inverse relationship exists between attenuation and waveform frequency. We will now look more closely at this relation.

The most important aspects of ultrasound image resolution are those which govern axial, lateral, and temporal resolution—*in which all three together comprise spatial resolution*. **Axial resolution** is the ability to distinguish two structures at different depths, parallel to the direction of the ultrasound beam. Furthermore, it is approximately equal to one half of the ultrasound pulse length. In other words, if the distance between two objects is greater than one half of the pulse length, then the objects will appear as two distinct structures. It follows that higher waveform frequencies (short pulse lengths) produce the best axial resolution. However, because of the existing inverse relationship, higher frequency waveforms are more readily attenuated, and thus, tissue penetration is sacrificed. **Lateral resolution** is the ability to distinguish two structures at the same depth, perpendicular to the direction of the ultrasound beam. High frequency and focused ultrasound beams produce the narrowest beams, thus maximizing lateral resolution, but once again tissue penetration is sacrificed. Finally, **temporal resolution** relates to frame rate, and therefore, the ability to distinguish between real-time imaging and motion artifacts. During nerve blocks in regional anesthesia for example, motion artifacts occur with movement of the probe/transducer, or during needle insertion, or with injection of the anesthetic agent. Ultimately, temporal resolution is limited by the sweep speed (activation of the piezoelectric crystals) of the ultrasound beam, which in turn is limited by the speed of sound in tissue. Attempts to control temporal resolution consist of 1) increasing the sweep speed or 2) decreasing the scanning angle (applicable to phased array probes only). The first option decreases the lateral resolution, and the second option decreases the field of view. Thus, not only do we see an interconnected relationship with respect to image resolution, but at present, adjustments available to try to improve resolution are restrained by the laws of waveform physics. That is to say, despite the progress made in ultrasound equipment and technology (which we will highlight in the accompanying discussion on regional anesthesia), ultrasound imaging is, in reality, a tradeoff between spatial resolution and achievable depth of imaging (Sites et al., 2007).

#### **4.3 Ultrasound guidance in pain medicine: Regional anesthesia**

The use of ultrasound for image guidance in regional anesthesia has several practical benefits (Sites et al., 2009), the foremost being that ionizing radiation is not necessary for image production enabling ultrasound machines to be highly portable. Case series and small-scaled outcomes studies with respect to nerve blocks have purported shortened procedure times and faster block onset; increased patient satisfaction; and fewer blockrelated complications (Marhofer & Chan, 2007). As far as limitations, there are two primary considerations: 1) resolution and image quality vary inversely with depth of penetration, as previously discussed; and 2) needle tracking for in-plane needle entry is a challenge in part because the needle is not visible on the monitor (Marhofer & Chan, 2007). However, with respect to in-plane needle tracking, one company (SonoSite, Inc.) has developed an ultrasound system to remedy this problem. By sending out a "secondary beam" at a 45° angle from the transducer for perpendicular beam-to-needle alignment established outside the region of interest, needle visualization is optimized. This enables the physician to see the needle's approach to the target nerve within the field of view. In addition, to help improve therapeutic accuracy of ultrasound-guided pain medicine, the ultrasound characteristics of needles have been described (Maecken et al., 2007), "echo-friendly" needle designs have been developed (Deam, 2007), and the benefits of three-dimensional (3D) ultrasound imaging has been investigated (Feinglass et al., 2007; Foxall, et al., 2007). Moreover, cutting edge ultrasonic technological advances have introduced image-enhanced tissue staining to remotely palpate the target nerve of interest using acoustic radiation force imaging to both improve accuracy and limit variability in regional anesthesia (Palmeri et al., 2008; Nightingale et al., 2001).

#### **4.4 Risk management: Ultrasound safety**

150 Pain Management – Current Issues and Opinions

As emphasized in the above subsection, a nontrivial inverse relationship exists between

The most important aspects of ultrasound image resolution are those which govern axial, lateral, and temporal resolution—*in which all three together comprise spatial resolution*. **Axial resolution** is the ability to distinguish two structures at different depths, parallel to the direction of the ultrasound beam. Furthermore, it is approximately equal to one half of the ultrasound pulse length. In other words, if the distance between two objects is greater than one half of the pulse length, then the objects will appear as two distinct structures. It follows that higher waveform frequencies (short pulse lengths) produce the best axial resolution. However, because of the existing inverse relationship, higher frequency waveforms are more readily attenuated, and thus, tissue penetration is sacrificed. **Lateral resolution** is the ability to distinguish two structures at the same depth, perpendicular to the direction of the ultrasound beam. High frequency and focused ultrasound beams produce the narrowest beams, thus maximizing lateral resolution, but once again tissue penetration is sacrificed. Finally, **temporal resolution** relates to frame rate, and therefore, the ability to distinguish between real-time imaging and motion artifacts. During nerve blocks in regional anesthesia for example, motion artifacts occur with movement of the probe/transducer, or during needle insertion, or with injection of the anesthetic agent. Ultimately, temporal resolution is limited by the sweep speed (activation of the piezoelectric crystals) of the ultrasound beam, which in turn is limited by the speed of sound in tissue. Attempts to control temporal resolution consist of 1) increasing the sweep speed or 2) decreasing the scanning angle (applicable to phased array probes only). The first option decreases the lateral resolution, and the second option decreases the field of view. Thus, not only do we see an interconnected relationship with respect to image resolution, but at present, adjustments available to try to improve resolution are restrained by the laws of waveform physics. That is to say, despite the progress made in ultrasound equipment and technology (which we will highlight in the accompanying discussion on regional anesthesia), ultrasound imaging is, in reality, a tradeoff between spatial resolution and achievable depth of

attenuation and waveform frequency. We will now look more closely at this relation.

imaging (Sites et al., 2007).

**4.3 Ultrasound guidance in pain medicine: Regional anesthesia** 

The use of ultrasound for image guidance in regional anesthesia has several practical benefits (Sites et al., 2009), the foremost being that ionizing radiation is not necessary for image production enabling ultrasound machines to be highly portable. Case series and small-scaled outcomes studies with respect to nerve blocks have purported shortened procedure times and faster block onset; increased patient satisfaction; and fewer blockrelated complications (Marhofer & Chan, 2007). As far as limitations, there are two primary considerations: 1) resolution and image quality vary inversely with depth of penetration, as previously discussed; and 2) needle tracking for in-plane needle entry is a challenge in part because the needle is not visible on the monitor (Marhofer & Chan, 2007). However, with respect to in-plane needle tracking, one company (SonoSite, Inc.) has developed an ultrasound system to remedy this problem. By sending out a "secondary beam" at a 45° angle from the transducer for perpendicular beam-to-needle alignment established outside the region of interest, needle visualization is optimized. This enables the physician to see the needle's approach to the target nerve within the field of view. In addition, to help improve therapeutic accuracy of ultrasound-guided pain medicine, the ultrasound characteristics of needles have been described (Maecken et al., 2007), "echo-friendly" needle designs have been developed (Deam, 2007), and the benefits of three-dimensional (3D) ultrasound While there are no known absolute contraindications in ultrasound imaging, the position of The U.S. Food and Drug Administration (FDA) (n.d.) concerning ultrasound safety follows.

Ultrasound imaging has been used for over 20 years and has an excellent safety record. It is non-ionizing radiation, so it does not have the same risks as x-rays or other types of ionizing radiation. Even though there are no known risks of ultrasound imaging, it can produce effects on the body. When ultrasound enters the body, it heats the tissues slightly. In some cases, it can also produce small pockets of gas in body fluids or tissues (cavitation). The long-term effects of tissue heating and cavitation are not known.

For a more in-depth discussion on tissue sensitivity relative to interpreting risk from exposure to ultrasound imaging, the reader is encouraged to study the report on this topic issued by members of the World Federation for Ultrasound in Medicine and Biology Safety Committee (Barnett et al., 1997).

#### **5. Future directions in interventional pain medicine**

#### **5.1 \*The promise of real-time three-dimensional (3D) fluoroscopy**

While CT-fluoroscopy offers unique viewing perspectives with overall imaging capabilities (as discussed in the earlier section, "Triplanar Imaging in Pain Medicine Procedures: Conventional-CT Guidance and CT-Fluoroscopy"), interestingly, real-time 3D imaging has not been reported. Conversely, although real-time 3D ultrasound has been show to be beneficial in regional anesthesia (as cited in the above section, "Ultrasound guidance in Pain Medicine: Regional Anesthesia"), ultrasound imaging does not offer the allure that fluoroscopy enjoys with respect to image resolution, particularly of bony anatomy.

Technology is currently available to fit mobile C-arm fluoroscopy systems with 3D imaging capabilities (Stübig et al., 2009; Izadpanah et al., 2009; Villavicencio et al., 2005). (Note: current fluoroscopy systems creating 3D imagery are limited to 150°—360° with mechanical orientation or manipulation or post-processing.) Notably, the limiting factor in the utility of these systems under most interventional pain protocols is image construction time. However, one company, Imaging3 Inc., claims to have developed signal processing algorithms to produce 3D high-resolution fluoroscopic images in *real-time* via its "Dominion" platform. (Note: at the time of this publication, this device has *not* received FDA clearance.) The design is built around a dedicated O-arm—a gantry similar to that used in CT—to allow continuous 360° rotation of the x-ray tube and scintillation detector with ondemand imaging of the patient under continuous or pulsed fluoroscopy. (See the earlier discussion on radiation risk management in fluoroscopy to review the advantages of pulsed fluoroscopy.) This approach is expected to allow imaging of the patient from any frame of reference or angulation. Intended use is anticipated by the company to be procedures in which multiple frames of reference are required. From a pain medicine perspective, realtime 3D fluoroscopic guidance may be possible for discography, vertebral augmentation, percutaneous lumbar decompression, facet rhizotomy, or intradiscal electrothermal therapy.

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 153

electromyographic guidance (Botwin et al., 2008). One advantage of this technique is that in the cervicothoracic area, the physician can see the lungs in order to guard against inadvertent procedure-induced pneumothorax. In other applications, the use of ultrasound was also recently documented as the modality of choice to facilitate placement of percutaneous leads in two peripheral nerve stimulation trialing procedures to treat ilioinguinal neuralgia (Carayannopoulos et al., 2009). This marked a new era in ultrasoundguided pain medicine, as well as technical improvement and refinement of a surgical technique. Likewise, image guidance with ultrasound in pain procedures traditionally reserved for x-ray producing modalities (fluoroscopy) have been reported. The evidence for this is gleaned from reports on real-time ultrasound-guided epidural injections (Karmakar, 2009), and even the introduction of current procedural terminology codes for transforaminal

However, more compelling evidence for the versatility of ultrasound in pain medicine is found in its utility in pain-related physical rehabilitation (a role for this modality which is in its infancy) (Peolsson & Brodin, 2009; Primack, 2010; Wininger, 2010). It is well recognized that when musculoskeletal injuries/syndromes induce pain, use of the involved body part (such as the shoulder for example) becomes limited, which may in turn cause more pain and more limited use and a vicious cycle is started, possibly leading to physical limitation that negatively influences the quality of life of the patient. Alternatively, qualitative and quantitative description of musculoskeletal tissue dynamics and coordination during realtime procedures is possible through ultrasound imaging via tissue velocity imaging. This technique may be used to scrutinize both intra-muscular and inter-muscular coordination patterns (Peolsson & Brodin, 2009). Dynamic imaging using ultrasound to assess shoulder impingement syndromes is another example of pain-related physical rehabilitation ultrasound use (Bureau et al., 2006). To this point, it is noteworthy to mention that researchers have looked into how the overall utility of musculoskeletal ultrasound imaging impacts MRI. It was discovered that selective substitution of musculoskeletal ultrasound for MRI can result in significant cost savings to the health care system, but issues related to accuracy, variability, education and competence need to be further addressed (Jacobson,

AAPM. Cardiac catheterization equipment performance. (2001). American Association of

Aguirre DA, Bermudez S, & Diaz OM. (2005). Spinal CT-guided interventional procedures

Aldrich JE. (2007). Basic physics of ultrasound imaging. *Crit Care Med*, vol. 35, no. 5, pp.

Aufrichtig R, Xue P, Thomas CW, Gilmore GC, & Wilson DL. (1994). Perceptual comparison of pulsed and continuous fluoroscopy. *Med Phys*, vol. 21, no. 2, pp. 245-256. Back DL, Hilton AI, Briggs TW, Scott J, Burns M, & Warren P. (2005). Radiation protection

Barnes JE. (1992). Characteristics and control of contrast in CT. *Radiographics*, vol. 12, no. 4,

Barnett SB, Rott HD, ter Haar GR. Ziskin MC, Maeda K. (1997). The sensitivity of biological

tissue to ultrasound. *Ultrasound Med Biol*, vol. 23, no. 6, pp. 805-812.

for management of chronic back pain. *J Vasc Interv Radiol*, vol. 16, no. 5, pp. 689-697.

Physicists in Medicine. Report Series No. 70.

for your hands. *Injury*, vol. 36, no. 12, pp. 1416-1420.

epidural injections and paravertebral/facet injections.

2009).

**6. References** 

S131-S137.

pp. 825-837.

In addition, it is projected that the Dominion will offer a multi-modal feature to give physicians the ability to view cross-sectional anatomy by emulating CT, using a cone-beam CT model.

\*Note: neither the author nor anyone known to the author has any relationship with any of the companies mentioned in this subsection. This includes but is not limited to financial, consulting, and business relationships. All information was obtained through company filings and/or press releases and/or marketing literature.

#### **5.2 \*Interventional magnetic resonance imaging (MRI)**

This section presents a brief discussion on the concept of MRI-guided procedures. Whereas the use of MRI for this purpose is in its infancy, the intent of MRI-guided/interventional scanning is to assist physicians during intra-operative, and diagnostic and therapeutic procedures using magnetic-compatible instrumentation. The Fonar 360™ MRI unit represents the cutting edge in technology for this vision. The Fonar 360™ is a specialized MRI design with the field and gradient magnets encapsulated in the ceiling and floor of a dedicated, monolithic room. In this capacity, the enlarged room-sized magnet and the 360° access to the patient permits full-fledged medical teams to walk into the room (i.e., "inside the magnet") to interact with the patient. The first unit was installed at the Nuffield Orthopaedic Centre in Oxford, United Kingdom.

Another developer, MRI Interventions Inc., a medical device maker focusing on interventional MRI applications, has obtained FDA clearance on their ClearPoint® system which is designed to enable minimally invasive procedures in the brain—namely to facilitate image guidance for the introduction of deep brain stimulation leads—utilizing a hospital's existing MRI suite. On this note, although to date the use of deep brain stimulation for pain is limited due to lackluster outcomes (Levy et al., 1987; Coffey, 2001; Hamani et al., 2006), research is ongoing to find appropriate surgical candidates and areas in the brain conducive to long-term efficacy for conditions with a central pain aspect. Such targeted brain centers currently under investigation are the ventral capsule/ventral striatum in thalamic pain syndrome (U.S. National Institutes of Health [NIH], NCT 01072656). On another note, the introduction of deep brain stimulation systems with "steerable" field currents (the VANTAGE trial) (NIH, NCT 01221948) may ultimately prove clinically advantageous in such approaches to pain management.

Finally, our discussion on interventional MRI would be incomplete without reference made to patient care and concerns about biologic effects relative to exposure to MRI. Hence, we refer the reader to the collection of work by Frank Shellock. On this point, we encourage the reader to begin with Shellock and Cruse (2004) to gain an overview on tissue sensitivity associated with gradient magnetic fields, acoustic noise, and radiofrequency fields/radiation in MRI.

\*Note: neither the author nor anyone known to the author has any relationship with any of the companies mentioned in this subsection. This includes but is not limited to financial, consulting, and business relationships. All information was obtained through company filings and/or press releases and/or marketing literature.

#### **5.3 Ultrasound imaging: Beyond regional anesthesia**

With ultrasound guidance for nerve blocks in regional anesthesia established, intriguing applications of this modality in pain medicine are beginning to surface. For example, ultrasound guidance for trigger point injection therapy has been shown to be comparable to electromyographic guidance (Botwin et al., 2008). One advantage of this technique is that in the cervicothoracic area, the physician can see the lungs in order to guard against inadvertent procedure-induced pneumothorax. In other applications, the use of ultrasound was also recently documented as the modality of choice to facilitate placement of percutaneous leads in two peripheral nerve stimulation trialing procedures to treat ilioinguinal neuralgia (Carayannopoulos et al., 2009). This marked a new era in ultrasoundguided pain medicine, as well as technical improvement and refinement of a surgical technique. Likewise, image guidance with ultrasound in pain procedures traditionally reserved for x-ray producing modalities (fluoroscopy) have been reported. The evidence for this is gleaned from reports on real-time ultrasound-guided epidural injections (Karmakar, 2009), and even the introduction of current procedural terminology codes for transforaminal epidural injections and paravertebral/facet injections.

However, more compelling evidence for the versatility of ultrasound in pain medicine is found in its utility in pain-related physical rehabilitation (a role for this modality which is in its infancy) (Peolsson & Brodin, 2009; Primack, 2010; Wininger, 2010). It is well recognized that when musculoskeletal injuries/syndromes induce pain, use of the involved body part (such as the shoulder for example) becomes limited, which may in turn cause more pain and more limited use and a vicious cycle is started, possibly leading to physical limitation that negatively influences the quality of life of the patient. Alternatively, qualitative and quantitative description of musculoskeletal tissue dynamics and coordination during realtime procedures is possible through ultrasound imaging via tissue velocity imaging. This technique may be used to scrutinize both intra-muscular and inter-muscular coordination patterns (Peolsson & Brodin, 2009). Dynamic imaging using ultrasound to assess shoulder impingement syndromes is another example of pain-related physical rehabilitation ultrasound use (Bureau et al., 2006). To this point, it is noteworthy to mention that researchers have looked into how the overall utility of musculoskeletal ultrasound imaging impacts MRI. It was discovered that selective substitution of musculoskeletal ultrasound for MRI can result in significant cost savings to the health care system, but issues related to accuracy, variability, education and competence need to be further addressed (Jacobson, 2009).

#### **6. References**

152 Pain Management – Current Issues and Opinions

In addition, it is projected that the Dominion will offer a multi-modal feature to give physicians the ability to view cross-sectional anatomy by emulating CT, using a cone-beam

\*Note: neither the author nor anyone known to the author has any relationship with any of the companies mentioned in this subsection. This includes but is not limited to financial, consulting, and business relationships. All information was obtained through company

This section presents a brief discussion on the concept of MRI-guided procedures. Whereas the use of MRI for this purpose is in its infancy, the intent of MRI-guided/interventional scanning is to assist physicians during intra-operative, and diagnostic and therapeutic procedures using magnetic-compatible instrumentation. The Fonar 360™ MRI unit represents the cutting edge in technology for this vision. The Fonar 360™ is a specialized MRI design with the field and gradient magnets encapsulated in the ceiling and floor of a dedicated, monolithic room. In this capacity, the enlarged room-sized magnet and the 360° access to the patient permits full-fledged medical teams to walk into the room (i.e., "inside the magnet") to interact with the patient. The first unit was installed at the Nuffield

Another developer, MRI Interventions Inc., a medical device maker focusing on interventional MRI applications, has obtained FDA clearance on their ClearPoint® system which is designed to enable minimally invasive procedures in the brain—namely to facilitate image guidance for the introduction of deep brain stimulation leads—utilizing a hospital's existing MRI suite. On this note, although to date the use of deep brain stimulation for pain is limited due to lackluster outcomes (Levy et al., 1987; Coffey, 2001; Hamani et al., 2006), research is ongoing to find appropriate surgical candidates and areas in the brain conducive to long-term efficacy for conditions with a central pain aspect. Such targeted brain centers currently under investigation are the ventral capsule/ventral striatum in thalamic pain syndrome (U.S. National Institutes of Health [NIH], NCT 01072656). On another note, the introduction of deep brain stimulation systems with "steerable" field currents (the VANTAGE trial) (NIH, NCT 01221948) may ultimately prove clinically

Finally, our discussion on interventional MRI would be incomplete without reference made to patient care and concerns about biologic effects relative to exposure to MRI. Hence, we refer the reader to the collection of work by Frank Shellock. On this point, we encourage the reader to begin with Shellock and Cruse (2004) to gain an overview on tissue sensitivity associated with gradient magnetic fields, acoustic noise, and radiofrequency

\*Note: neither the author nor anyone known to the author has any relationship with any of the companies mentioned in this subsection. This includes but is not limited to financial, consulting, and business relationships. All information was obtained through company

With ultrasound guidance for nerve blocks in regional anesthesia established, intriguing applications of this modality in pain medicine are beginning to surface. For example, ultrasound guidance for trigger point injection therapy has been shown to be comparable to

filings and/or press releases and/or marketing literature.

**5.2 \*Interventional magnetic resonance imaging (MRI)** 

Orthopaedic Centre in Oxford, United Kingdom.

advantageous in such approaches to pain management.

filings and/or press releases and/or marketing literature.

**5.3 Ultrasound imaging: Beyond regional anesthesia** 

fields/radiation in MRI.

CT model.


Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 155

Davies AG, Cowen AR, Kengyelics SM, et al. (2006). X-ray dose reduction in fluoroscopically guided electrophysiology procedures. *PACE*, vol. 29, no. 3, pp. 262-271. Deam RK, Kluger R, Barrington MJ, & McCutcheon CA. (2007). Investigation of a new

Deshpande KK, Wininger KL. (2011). Feasibility of combined epicranial temporal and

Dowd SB, Tilson ER. (1999). *Practical Radiation Protection and Applied Radiobiology*. 2nd ed.

Fazel R, Krumholz HM, Wang Y, et al. (2009). Exposure to low-dose ionizing radiation from medical imaging procedures. *N Eng J Med*, vol. 361, no. 9, pp. 849-857. Feinglass NG, Clendenen SR, Torp KD, Wang RD, Castello R, & Greengrass RA. (2007).

report and image description. *Anesth Analg*, vol. 105, no. 1, pp. 272-274. Ferreira CC, Galvão LA, Veira JW, Maia AF. (2010). Validation of an exposure

Finnerty M, Brennan PC. (2005). Protective aprons in imaging departments: manufacturer

Fish DE, Kim A, Ornelas C, Song S, & Pangarkar S. The risks of radiation exposure to the

Foxall GL, Hardman JG, Bedforth NM. (2007). Three-dimensional, multiplanar, ultrasoundguided, radial nerve block. *Reg Anesth Pain Med*, vol. 32, no. 6, pp. 516-521. Gangi A, Dietemann JL, Mortazavi R, Pfleger D, Kauff C, & Roy C. (1998). CT-guided

Geleijns J, Wondergem J. (2005). X-ray imaging and the skin: Radiation biology, patient dosimetry and observed effects. *Rad Prot Dos*, vol. 114, no. 1-3, pp. 121-125. Kapural L, Goyle A. (2007). Imaging for provocative discography and minimally invasive

Gruber H, Bodner G. (2004). Why CT guided? [comment]. *AJR Am J Roentgenol*, vol. 182, no.

Hamani C, Schwalb JM, Rezai AR, Dostrovsky JO, Davis KD, Lozano AM. (2006). Deep

Izadpanah K, Konrad G, Südkamp NP, & Oberst M. (2009). Computer navigation in balloon

Jackson S, Thomas R. (2004). Introduction to CT physics. *Cross-Sectional Imaging Made Easy*.

Jacobson JA. (2009). Musculoskeletal ultrasound: focused impact on MRI. *AJR Am J* 

Johns HE, Cunningham JR. (1983). The interaction of ionizing radiation with matter. In: *The* 

incidence of insertional effect. *Pain*, vol. 125, no. 1, pp. 188-196.

*Physics of Radiology*. 4th ed. pp. 133-164, Thomas, Springfield, IL.

p. 7, Churchill Livingston; Edinburgh, Scotland. 2004.

*Roentgenol*, vol. 193, no. 3, pp. 619-627.

Article ID 609537, 5 pages, 2011. doi:10.1155/2011/609537.

*Radiographics*, vol. 18, no. 3, pp. 621-633.

*Anesth Pain Manag*, vol. 11, no. 2, pp. 73-80.

*Care*, vol. 35, no. 4, pp. 582-586.

*Physician*, vol. 14, no. 1, pp. 37-44.

Philadelphia, Pa: Saunders.

vol. 4, no. 1, pp. 19-22.

1477-1484.

3, p. 824.

pp. 1325-1329.

echogenic needle for use with ultrasound peripheral nerve blocks. *Anaesth Intensive* 

occipital neurostimulation: treatment of a challenging case of headache. *Pain* 

Real-time three-dimensional ultrasound for continuous popliteal blockade: a case

computational model to computed tomography. *Brazilian Journal of Physics Médica*,

stated lead equivalence values require validation. *Eur Radiol*, vol. 15, no. 7, pp.

eyes of the interventional pain physician. *Radiology Research and Practice*, vol. 2011.

Interventional procedures for pain management in the lumbosacral spine.

percutaneous procedures for treatment of discogenic lower back pain. *Tech Reg* 

brain stimulation for chronic neuropathic pain: long-term outcome and the

kyphoplasty reduces the intraoperative radiation exposure. *Spine*, vol. 34, no. 12,


Balter S. (2006). Methods for measuring fluoroscopic skin dose. *Pediatr Radiol*, vol. 36, suppl.

Balter S. (2008). Capturing patient doses from fluoroscopically based diagnostic and

Boszczyk BM, Bierschneider M, Panzer S, et al. (2006). Fluoroscopic radiation exposure of

Botwin KP, Freeman ED, Gruber RD, et al. (2001). Radiation exposure to a physician

Botwin KP, Thomas S, Gruber RD, et al. (2002). Radiation exposure of the spinal

Botwin KP, Fuoco GS, Torres FM, et al. (2003). Radiation exposure to the spinal interventionalist performing lumbar discography. *Pain Physician*, vol. 6, no. 3, pp. 295-300. Botwin KP, Sharma K, Saliba R, & Patel BC. (2008). Ultrasound-guided trigger point

Boyce WE, DiPrima RC. (2005). *Elementary Differential Equations*. 8th ed. John Wiley & Sons,

Bracewell RN. (1986). *The Fourier Transform and Its Applications*. 3rd ed. McGraw Hill.

Bureau NJ, Beauchamp M, Cardinal E, & Brassard P. (2006). Dynamic sonography

Bushong SC. (2004). Radiation protection procedures. In: Bushong SC, ed. *Radiologic Science* 

Carayannopoulos A, Beasley R, & Sites B. (2009). Facilitation of percutaneous trial lead

Clasper JC, Pinks T. (1995). Technical note: an assessment of x-ray protective gloves. *Br J* 

Christodoulou EG, Goodsitt MM, Larson SC, Darner KL, Satti J, & Chan HP. (2003).

Coffey RJ. (2001). Deep brain stimulation for chronic pain: results of two multicenter trials

Cousin AJ, Lawdahl RB, Chakraborty DP, & Koehler RE. (1987). The case for radioprotective

Daly B, Templeton PA. (1999). Real-time CT fluoroscopy: evolution of an interventional tool.

Datir A, Connell D. (2010). CT-guided injection for ganglion impar blockade: a radiological approach to the management of coccydynia. *Clin Radiol*, vol. 65, no. 1, pp. 21-25.

and a structured review. *Pain Med*, vol. 2, no. 3, pp. 183-192.

steroid injections. *Arch Phys Med Rehabil*, vol. 83, no. 5, pp. 697-701.

performing fluoroscopically guided caudal epidural steroid injections. *Pain* 

interventionalist performing fluoroscopically guided transforaminal epidural

injections in the cervicothoracic musculature: a new and unreported technique. *Pain* 

evaluation of shoulder impingement syndrome. *AJR Am J Roentgenol*, vo. 187, no. 1,

*for Technologists: Physics, Biology, and Protection*. 8th ed. pp. 583-601, Mosby Inc., St.

placement with ultrasound guidance for peripheral nerve stimulation trial of ilioinguinal neuralgia: a technical note. *Neuromodulation*, vol. 12, no. 4, pp. 296-301. Carlton RR, Adler AM. (2006). *Principles of Radiographic Imaging: An Art and a Science*. 4th ed.

Evaluation of the transmitted exposure through lead equivalent aprons in a radiology department, including the contribution from backscatter. *Med Phys*, vol.

eyewear/facewear. Practical implications and suggestions. *Invest Radiol*, vol. 22, no.

interventional systems. *Health Phys*, vol. 95, no. 5, pp. 535-540.

the kyphoplasty patient. *Eur Spine J*, vol. 15, no. 3, pp. 347-355.

2, pp. 136-140.

*Physician*, vol. 4, no. 4, pp. 343-348.

*Physician*, vol. 11, no. 6, pp. 885-889.

Clifton Park, NY: Thomson Delmar.

*Radiol*, vol. 68, no. 812, pp. 917-919.

*Radiology*, vol. 211, no. 2, pp. 309-315.

30, no. 6, pp. 1033-1038.

8, pp. 688-692.

Inc; Hoboken, NJ.

Singapore.

pp. 216-220.

Louis, Mo.


Jacobson JA. (2009). Musculoskeletal ultrasound: focused impact on MRI. *AJR Am J Roentgenol*, vol. 193, no. 3, pp. 619-627.

Johns HE, Cunningham JR. (1983). The interaction of ionizing radiation with matter. In: *The Physics of Radiology*. 4th ed. pp. 133-164, Thomas, Springfield, IL.

Applied Radiologic Science in the Treatment of Pain: Interventional Pain Medicine 157

Oritz AO, Natarajan V, Gregorius DR, & Pollack S. (2006). Significantly reduced radiation

Palmeri ML, Dahl JJ, MacLeod D, Grant S, & Nightingale KR. Regional anesthesia guidance

Peolsson M, Brodin LA. (2009). Functional musculoskeletal ultrasound. *European* 

Perisinakis K, Damilakis J, Theocharopoulos N, Papadokostakis G, Hadjipavlou A, &

Primack SJ. (2010). A physiatrist's perspective on musculoskeletal ultrasound. *Phys Med* 

Schmid MR, Kissling RO, Curt A, Jaschko G, & Hodler J. (2006). Sympathetic skin response:

Schueler BA. (2000). The AAPM/RSNA physics tutorial for residents: general overview of

Shahabi S. Radiation safety/protection and health physics. (1999). In: Dowd SB, Tilson ER,

Shellock FA, Crues JV. (2004). MR procedures: biologic effects, safety, and patient care.

Shepp LA, Kruskal JB. (1978). Computerized tomography: the new medical x-ray

Sites BD, Brull R, Chan VWS, et al. (2007). Artifacts and pitfall errors associated with

Sites BD, Chan VW, Neal JM, et al. (2009). The American Society of Regional Anesthesia and

Smiddy PF, Quinn AD, Freyne PJ, Marsh D, & Murphy JM. (1996). Dose reduction in double

Sprawls P. AAPM tutorial. (1992). CT image detail and noise. *Radiographics*, vol. 12, no. 5,

Stam W, Pillay M. (2008). Inspection of lead aprons: a practical rejection model. *Health Phys*,

Stübig T, Kendoff D, Citak M, et al. (2009). Comparative study of different intraoperative 3-

regional anesthesia. *Reg Anesth Pain Med*, vol. 34, no. 1, pp. 40-46.

fluoroscopic imaging. *Radiographics*, vol. 20, no. 4, pp. 1115-1126.

http://www.jointcommission.org/assets/1/18/Radiation\_Overdose.pdf.

Sentinel Event Policy and Procedures. The Joint Commission website.

technology. *Am Math Mon*, vol. 85, no. 6, pp. 420-439.

and techniques. *AJNR Am J Neuroradiol*, vol. 27, no. 5, pp. 989-994.

Elasticity. Oct. 27-30, 2008.

pp. 701-701.

595-602.

pp. 412-418.

825, pp. 852-854.

vol. 95, suppl. 2, pp. S133-S136.

pp. 1041-1046.

Accessed June 21, 2008.

Saunders, Philadelphia, Pa.

*Radiology*, vol. 232, no. 3, pp. 635-652.

*Musculoskeletal Review*, vol. 4, no. 2, pp. 102-107.

*Rehabil Clin N Am*, vol. 21, no. 3, pp. 645-650.

exposure to operators during kyphoplasty and vertebroplasty procedures: methods

using acoustic radiation force imaging. [abstract]. Proceedings of the Seventh International Conference on the Ultrasonic Measurement and Imaging of Tissue

Gourtsoylannis N. (2004). Patient exposure and associated radiation risks from fluoroscopically-guided vertebroplasty or kyphoplasty. *Radiology*, vol. 232, no. 3,

monitoring of CT-guided lumbar sympathetic blocks. *Radiology*, vol. 241, no. 2, pp.

eds. *Practical Radiation Protection and Applied Radiobiology*. 2nd ed. pp. 167-196,

ultrasound-guided regional anesthesia. Part I: Understanding the basic principles of ultrasound physics and machine operations. *Reg Anesth Pain Med*, vol. 32, no. 5,

Pain Medicine and the European Society of Regional Anaesthesia and Pain Therapy joint committee recommendations for education and training in ultrasound-guided

contrast barium enema by use of low fluoroscopic current. *Br J Radiol*, vol. 69, no.

D image intensifiers in orthopedic trauma care. *J Trauma*, vol. 66, no. 3, pp. 821-830.


Johnston J, Killion JB, Vealé B, & Comello R. (2011). U.S. technologists' radiation exposure

Kallmes DF, O E, Roy SS, et al. (2003). Radiation dose to the operator during vertebroplasty:

Kane SA. (2009). *Introduction to Physics in Modern Medicine*. 2nd ed. Boca Raton, Fl: CRC Press. Karmaka MK, Li X, Ho AM, Kwok WH, & Chui PT. (2009). Real-time ultrasound-guided

Kato R, Katada K, Anno H, Suzuki S, Ida Y, Koga S. (1996). Radiation dosimetry at CT

Kosek P, Morgan D, Dunn J, et al. Electronically generated lead (EGL) scan: report of first clinical use. [abstract]. *North American Neuromodulation Society*. Dec. 7-9, 2006. Levy RM, Lamb S, & Adams JE. (1987). Treatment of chronic pain by deep brain stimulation:

Larson R, Hostetler B, & Edwards BH. (2007). Calculus: Early Transcendental Functions. 4th

Little MP, Wakeford R, Tawn EJ, Bouffler SD, & Berrington de Gonzalez A. (2009). Risks

Mahesh M. (2001). Fluoroscopy: patient radiation exposure issues. *Radiographics*, vol. 21, no.

Manchikanti L, Cash KA, Moss TL, & Pampati V. (2002). Radiation exposure to the physician in interventional pain management. *Pain Physician*, vol. 5, no. 4, pp. 385-393. Manchikanti L, Cash KA, Moss TL, & Pampati V. (2003a). Effectiveness of protective

management: a prospective study. *Pain Physician*, vol. 6, no. 3, pp. 301-305. Manchikanti L, Cash KA, Moss TL, Rivera J, & Pampati V. (2003b). Risk of whole body

10-year evaluation from 1997 to 2006. *Pain Physician*, vol. 12, no. 1, pp. 9-34. Marhofer P, Chan VWS. (2007). Ultrasound-guided regional anesthesia: current concepts

McKetty MH. (1998). The AAPM/RSNA physics tutorial for residents. X-ray attenuation.

Meleka S, Patra A, Minkoff E, & Murphy K. (2005). Value of CT fluoroscopy for lumbar facet

Nightingale KR, Palmeri ML, Nightingale RW, & Trahey GE. (2001). On the feasibility of

remote palpation using acoustic radiation force. *J Acoust Soc Am*, vol. 110, no. 1, pp.

techniques: a prospective evaluation. *BMC Anesthesiol*, vol. 3, no. 1, p. 2. Manchikanti L, Singh V, Pampati V, Smith HS, & Hirsch J. (2009). Analysis of growth of

and future trends. *Anesth Analg*, vol. 104, no. 5, pp. 1265-1269.

blocks. *AJNR Am J Neuroradiol*, vol. 26, no. 5, pp. 1001-1003.

*Radiographics*, vol. 18, no. 1, pp. 151-163.

may be (almost) the best we can do. *Radiology*, vol. 251, no. 1, pp. 6-12. Maecken T, Zenz M, & Grau T. (2007). Ultrasound characteristics of needles for regional

anesthesia. *Reg Anesth Pain Med*, vol. 32, no. 5, pp. 440-447.

prospective comparison of the use of 1-cc syringes versus an injection device. *AJNR* 

paramedian epidural access: evaluation of a novel in-plane technique. *Br J Anaesth*,

fluoroscopy: physician's hand dose and development of needle holders. *Radiology*,

long term follow-up and review of the literature. *Neurosurgery*, vol. 21, no. 6, pp.

associated with low doses and low dose rates of ionizing radiation: why linearity

measures in reducing risk of radiation exposure in interventional pain

radiation exposure and protection measures in fluoroscopically guided interventional

interventional techniques in managing chronic pain in the Medicare population: A

perceptions and practices. *Radiol Technol*, vol. 82, no. 4, pp. 311-320.

*Am J Neuroradiol*, vol. 24, no. 6, pp. 1257-1260.

ed. Houghton Mifflin Company; Boston, MA.

vol. 102, no. 6, pp. 845-854.

vol. 201, no. 2, pp. 576-578.

885-893.

4, pp. 1033-1045.

625-634.


**Part 2** 

**Acute Pain** 


http://apps.who.int/bmi/index.jsp?introPage=intro\_3.html. n.d.


**Part 2** 

**Acute Pain** 

158 Pain Management – Current Issues and Opinions

Thoumas D, Leroi AM, Mauillon J, et al. (1999). Pudendal neuralgia: CT-guided pudendal

Tuohy B, Marsh DM, O'Reilly G, Dowling A, Cooney P, & Malone JF. (1997). Quality

U.S. Food and Drug Administration Radiation Emitting Products: Radiation Emitting

U.S. National Academy of Sciences, National Research Council, Committee to Assess Health

U.S. National Institutes of Health. Safety study of deep brain stimulation to manage

U.S. National Institutes of Health. Vercise implantable stimulator for treating Parkinson's

Vano E, Gonzalez L, Ten JI, et al. (2001). Skin dose and dose-area product values for interventional cardiology procedures. *Br J Radiol*, vol. 74, no. 877, pp. 48-55. Villavicencio AT, Burneikiene S, Bulsara KR, & Thramann JJ. (2005). Intraoperative three-

Wagner AL. (2004a). Selective lumbar nerve root blocks with CT fluoroscopic guidance:

Wagner AL. (2004b). CT fluoroscopy-guided epidural injections: techniques and results.

Wang J, Blackburn TJ. (2000). The AAPM/RSNA physics tutorial for residents: x-ray image intensifiers for fluoroscopy. *Radiographics*, vol. 20, no. 5, pp. 1471-1477. Whitworth ML. Fluoroscopy scatter radiation studies of the lumbar spine. In: Interventional Spine. Volume 5, Issue 5. Kentfield, Ca: International Spine Intervention Society, n.d. Wininger KL. (2010). The lumbosacral spine: kinesiology, physical rehabilitation, and interventional pain medicine. *Clinical Kinesiology*, vol. 64, no. 3, pp. 22-50. Wininger KL, Deshpande KK, & Deshpande KK. (2010). Radiation exposure in percutaneous

World Health Organization. The WHO Global Database on Body Mass Index. BMI

Yanch JC, Behrman RH, Hendricks MJ, & McCall JH. (2009). Increased radiation dose to

Zhou Y, Singh N, Abdi S, Wu J, Crawford J, Furgang FA. (2005). Fluoroscopy radiation

percutaneous kyphoplasty. *Neurosurg Focus*, vol. 18, no. 3, p. E3.

assurance programme applied to mobile C-arm fluoroscopy systems. *Eur Radiol*,

Products and Procedures: Medical Imaging: Ultrasound Imaging:

EmittingProducts/RadiationEmittingProductsandProcedures/MedicalImaging/uc

Risks from Exposure to Low Levels of Ionizing Radiation. Health Risks from Exposure to Low Levels of Ionizing Radiation. BEIR VII Phase 2. Washington, DC:

thalamic pain syndrome. Identifier: NCT01072656. Clinical Trials website.

disease (VANTAGE). Identifier: NCT01221948. Clinical Trials website.

dimensional fluoroscopy-based computerized tomography guidance for

technique, results, procedure time, and radiation dose. *AJNR Am J Neuroradiol*, vol.

spinal cord stimulation mapping: a preliminary report. Pain Physician, vol. 13, no.

overweight and obese patients from radiographic examinations. *Radiology*, 2009,

safety for spine interventional pain procedures in university teaching hospitals.

nerve block technique. *Abdom Imaging*, vol. 24, no. 3, pp. 309-312.

vol. 7, no. 4, pp. 534-541.

m115357.htm. n.d.

http://www.fda.gov/Radiation-

National Academies Press, 2006.

25, no. 9, pp. 1592-1594.

1, pp. 7-18.

Classification website.

vol. 252, no. 1, pp. 128-139.

*Pain Physician*, vol. 8, no. 1, pp. 49-53.

http://clinicaltrials.gov. Accessed May 16, 2011

http://clinicaltrials.gov. Accessed May 16, 2011.

*AJNR Am J Neuroradiol*, vol. 25, no. 10, pp. 1821-1823.

http://apps.who.int/bmi/index.jsp?introPage=intro\_3.html. n.d.

**8** 

Joseph Baker

*Ireland* 

*Cappagh National Orthopaedic Hospital* 

**Local Anesthetic Agents in Arthroscopy** 

Arthroscopy is performed with increasing frequency on a number of joints. In the lower limb the role of knee arthroscopy is well established with procedures enabling more accurate diagnosis and treatment of a myriad of conditions including but not being limited to meniscal injury and articular surface defects. Hip and ankle arthroscopy are less widely performed. However, despite this their use can be expected to increase as indications are

While surgical technique often determines outcome in the long-term, analgesic control can significantly affect the patient's satisfaction following a procedure as well as the overall acceptability of a procedure. Arthroscopic procedures in particular have enabled many procedures to be performed on a day case basis where as more traditional surgical interventions may have required at least an overnight hospital stay. This trend toward day

Traditionally intra-articular analgesic agents have been used following arthroscopic procedures as an augment to post-operative pain control. Classically these include the typical local anesthetic agents but also alternatives such as morphine. Recently however, the potential for deleterious effects of the intra-articular analgesics on the articular cartilage has been reported in a number of experimental studies, which has caused concern among practicing arthroscopic surgeons. The purpose of this chapter is to review the potential intra-articular analgesic agents used for pain control following lower limb arthroscopy and to also provide an up-to-date review of the evidence for the potential chondrotoxic effect of

Classical local anesthetic agents can be classified into the esters and amides. Amides including lignocaine and bupivacaine among others have commonly been used in arthroscopy. Local anesthetics block action potential initiation and propagation along sensory pathways by blocking the sodium channel transmembrane pores. Their activity is increased in alkaline conditions and this enables them to penetrate the nerve sheath and

Other agents to have been trialed as intra-articular agents include opiates or opiate related substances (e.g. morphine, tramadol), non-steroidal anti-inflammatory medications, benzodiazepines and NMDA-receptor antagonists (e.g. magnesium sulfate) among others.

case surgery also emphasizes the importance of optimum analgesic control.

**1. Introduction** 

these agents.

**2. Analgesic agents** 

axonal membrane.

better developed and techniques honed.

### **Local Anesthetic Agents in Arthroscopy**

Joseph Baker *Cappagh National Orthopaedic Hospital Ireland* 

#### **1. Introduction**

Arthroscopy is performed with increasing frequency on a number of joints. In the lower limb the role of knee arthroscopy is well established with procedures enabling more accurate diagnosis and treatment of a myriad of conditions including but not being limited to meniscal injury and articular surface defects. Hip and ankle arthroscopy are less widely performed. However, despite this their use can be expected to increase as indications are better developed and techniques honed.

While surgical technique often determines outcome in the long-term, analgesic control can significantly affect the patient's satisfaction following a procedure as well as the overall acceptability of a procedure. Arthroscopic procedures in particular have enabled many procedures to be performed on a day case basis where as more traditional surgical interventions may have required at least an overnight hospital stay. This trend toward day case surgery also emphasizes the importance of optimum analgesic control.

Traditionally intra-articular analgesic agents have been used following arthroscopic procedures as an augment to post-operative pain control. Classically these include the typical local anesthetic agents but also alternatives such as morphine. Recently however, the potential for deleterious effects of the intra-articular analgesics on the articular cartilage has been reported in a number of experimental studies, which has caused concern among practicing arthroscopic surgeons. The purpose of this chapter is to review the potential intra-articular analgesic agents used for pain control following lower limb arthroscopy and to also provide an up-to-date review of the evidence for the potential chondrotoxic effect of these agents.

#### **2. Analgesic agents**

Classical local anesthetic agents can be classified into the esters and amides. Amides including lignocaine and bupivacaine among others have commonly been used in arthroscopy. Local anesthetics block action potential initiation and propagation along sensory pathways by blocking the sodium channel transmembrane pores. Their activity is increased in alkaline conditions and this enables them to penetrate the nerve sheath and axonal membrane.

Other agents to have been trialed as intra-articular agents include opiates or opiate related substances (e.g. morphine, tramadol), non-steroidal anti-inflammatory medications, benzodiazepines and NMDA-receptor antagonists (e.g. magnesium sulfate) among others.

Local Anesthetic Agents **in** Arthroscopy 163

When compared to opiate type analgesics ropivacaine was shown in one study to provide quicker onset of analgesia but was not significantly better at 24 hours after surgery(Franceschi et al., 2001). The benefit of morphine as an intra-articular analgesic is

Combinations of amide local anesthetics with other agents have been tried and this may represent the optimum way to control pain although at this point in time it is unknown. A combination of magnesium sulfate and bupivacaine was shown to be superior to either agent in isolation, which were again superior to placebo with regard pain scores following knee arthroscopy(Elsharnouby et al., 2008). These findings are supported by another study that also included morphine in the intra-articular cocktail but again found that a combination of agents was superior to any of the agents given in isolation(Farouk and Aly, 2009). A combination of bupivacaine with fentanyl was shown to be superior to bupivacaine in isolation following knee arthroscopy in a randomized trial including 33 patients(Jawish et al., 1996). Despite these promising reports in combinations of an amide local anesthetic and an opiate type agent others have failed to find this multimodal approach any better than placebo alone(Aasbo et al., 1996). While pain intensity or pain scale score is a frequent measure in these studies, the actual need for additional analgesia is a limiting factor with regard the ability to perform a procedure as a day case or not and may reflect a more

Despite a small number of studies suggesting that morphine provides adequate analgesic control following knee a recent review of these studies has suggested that of the higher quality studies, most had a negative finding not in favor of its use as an intra-articular analgesic agent(Rosseland, 2005, Drosos et al., 2002). The key point of this review was that post-operative pain intensity was no less in the morphine treated groups than the placebo treated groups in the well-designed studies. This review is supported by a study by the same author group that found only those with intense pain after arthroscopy had any

Other agents have been studied in with some success including midazolam (increased the time to first analgesia after surgery compared to placebo), clonidine (additive effect with bupivacaine compared to bupivacaine alone) and neostigmine (more effective when compared to morphine)(Batra et al., 2008, Tamosiunas et al., 2005, Yang et al., 1998). Unfortunately these agents have been studies in a very limited capacity and a clear

> Patients randomised to receive either: **bupivacaine** (20ml of 2.5mg/ml) + **morphine** (3mg); **bupivacaine** (20ml of 2.5mg/ml) alone; **morphine** (3mg) alone; or isotonic saline – no differences between the groups with regard analgesic requirement post-surgery

> Intra-articular **dexmedetomidine** (-2-adrenergic agonist) given via the intra-articular route resulted in less post-operative pain and analgesic requirement than either **dexmedetomidine** given intravenously or intra-articulat and intravenous placebo (saline)

Intra-articular **tramadol** at doses 50-100mg provided good postoperative analgesia with the higher doe more effectuve. The intraarticular route was more effective than the intravenous route.

Intra-articular **midazolam** (50 or 75g/kg) provided superior, albeit briefly, analgesic control compared to saline placebo. Time to first analgesic requirement was 4.7 and 4.6 hours compared to 0.7.

questionable however as noted later and this perhaps reflects poorly on ropivacaine.

practical end-point for further research.

benefit from intra-articular morphine(Rosseland et al., 1999).

conclusion in unable to be drawn as their effectiveness.

(Aasbo et al.,

(Al-Metwalli

(Alagol et al.,

(Batra et al.,

1996) RCT 107

et al., 2008) RCT 60

2004) RCT 210

2008) RCT 60

**Author Setting Number Key findings** 

#### **2.1 Hip and ankle arthroscopy**

Numerous studies have assessed the ability of local anesthetic agents to provide pain control following arthroscopic procedures in the lower limb. A vast majority of these have focused on the knee and only a few have reported the use of local anaesthetic following hip and ankle arthroscopy(Middleton et al., 2006, Baker et al., 2011c).

Of these two studies both found that intra-articular local anaesthetic was superior to either placebo or local anesthetic infiltrated around the arthroscopic portals (Table 1). The paucity of data here reflects the relative infancy of hip and ankle arthroscopy compared to knee arthroscopy and highlights the need for further work – hip arthroscopy in particular requires significant force to overcome the intra-articular negative pressures and can result in significant post-operative pain(Baker et al., 2011a).


Table 1. Studies assessing the benefit of intra-articular analgesic agents following hip and ankle arthroscopy

#### **2.2 Knee arthroscopy**

Numerous studies have attempted to establish the ideal intra-articular analgesic for pain control following knee arthroscopy (for a summary of these studies see Table2). The studies selected for inclusion here predominantly include those that use an intra-articular analgesic following surgery in a bolus dose fashion. Some studies that use it prior to surgery are also included for comparison sake particularly where comparison is later made with a bolus given following surgery. This section focuses on intra-articular analgesia given as an augment following surgery performed under general anesthesia or spinal anesthesia.

Although many studies have found that classical local anesthetic agents are of benefit following knee arthroscopy a randomized controlled trial reported by Townsend et al noted that intra-articular bupivacaine was no more effective that bupivacaine infiltrated around the portal sites(Townshend et al., 2009). This equivalence takes on even more importance with the reported potential for the toxic effect on articular cartilage of bupivacaine and other similar agents.

In general local anesthetics have been shown to be effective compared to placebo although this is not necessarily the case if the surgery is performed under spinal anesthetic when it appears the additional use of an intra-articular agent is negated by the spinal block(Santanen et al., 2001).

Non-steroidal anti-inflammatory medications have been trialed as intra-articular agents but are not in wide spread use. A single study has found that tenoxicam was superior to bupivacaine following surgery but this was only with regards analgesic consumption – the reported pain scores were still similar(Cook et al., 1997). It was similarly found that lornoxicam resulted in lower pain scores than did bupivacaine in a randomized controlled trial of 40 patients(Fagan et al., 2003). The use of an anti-inflammatory into the joint cavity may play a role in pain control particularly when a significant inflammatory component to the intra-articular pathology is found(Izdes et al., 2003).

Numerous studies have assessed the ability of local anesthetic agents to provide pain control following arthroscopic procedures in the lower limb. A vast majority of these have focused on the knee and only a few have reported the use of local anaesthetic following hip

Of these two studies both found that intra-articular local anaesthetic was superior to either placebo or local anesthetic infiltrated around the arthroscopic portals (Table 1). The paucity of data here reflects the relative infancy of hip and ankle arthroscopy compared to knee arthroscopy and highlights the need for further work – hip arthroscopy in particular requires significant force to overcome the intra-articular negative pressures and can result in

2011c) RCT 73 Intra-articular bupivacaine superior to peri-portal bupivacaine at

Table 1. Studies assessing the benefit of intra-articular analgesic agents following hip and

Numerous studies have attempted to establish the ideal intra-articular analgesic for pain control following knee arthroscopy (for a summary of these studies see Table2). The studies selected for inclusion here predominantly include those that use an intra-articular analgesic following surgery in a bolus dose fashion. Some studies that use it prior to surgery are also included for comparison sake particularly where comparison is later made with a bolus given following surgery. This section focuses on intra-articular analgesia given as an

Although many studies have found that classical local anesthetic agents are of benefit following knee arthroscopy a randomized controlled trial reported by Townsend et al noted that intra-articular bupivacaine was no more effective that bupivacaine infiltrated around the portal sites(Townshend et al., 2009). This equivalence takes on even more importance with the reported potential for the toxic effect on articular cartilage of bupivacaine and other

In general local anesthetics have been shown to be effective compared to placebo although this is not necessarily the case if the surgery is performed under spinal anesthetic when it appears the additional use of an intra-articular agent is negated by the spinal

Non-steroidal anti-inflammatory medications have been trialed as intra-articular agents but are not in wide spread use. A single study has found that tenoxicam was superior to bupivacaine following surgery but this was only with regards analgesic consumption – the reported pain scores were still similar(Cook et al., 1997). It was similarly found that lornoxicam resulted in lower pain scores than did bupivacaine in a randomized controlled trial of 40 patients(Fagan et al., 2003). The use of an anti-inflammatory into the joint cavity may play a role in pain control particularly when a significant inflammatory component to

augment following surgery performed under general anesthesia or spinal anesthesia.

controlling pain following HIP arthroscopy

Intra-articular bupivacaine was superior to saline placebo in reducing post-operative VAS pain scores and need for supplemental analgesia following ANKLE arthroscopy

and ankle arthroscopy(Middleton et al., 2006, Baker et al., 2011c).

**Author Setting Number Key findings** 

significant post-operative pain(Baker et al., 2011a).

**2.1 Hip and ankle arthroscopy** 

(Baker et al.,

(Middleton

ankle arthroscopy

similar agents.

block(Santanen et al., 2001).

the intra-articular pathology is found(Izdes et al., 2003).

**2.2 Knee arthroscopy** 

et al., 2006) RCT 35

When compared to opiate type analgesics ropivacaine was shown in one study to provide quicker onset of analgesia but was not significantly better at 24 hours after surgery(Franceschi et al., 2001). The benefit of morphine as an intra-articular analgesic is questionable however as noted later and this perhaps reflects poorly on ropivacaine.

Combinations of amide local anesthetics with other agents have been tried and this may represent the optimum way to control pain although at this point in time it is unknown. A combination of magnesium sulfate and bupivacaine was shown to be superior to either agent in isolation, which were again superior to placebo with regard pain scores following knee arthroscopy(Elsharnouby et al., 2008). These findings are supported by another study that also included morphine in the intra-articular cocktail but again found that a combination of agents was superior to any of the agents given in isolation(Farouk and Aly, 2009). A combination of bupivacaine with fentanyl was shown to be superior to bupivacaine in isolation following knee arthroscopy in a randomized trial including 33 patients(Jawish et al., 1996). Despite these promising reports in combinations of an amide local anesthetic and an opiate type agent others have failed to find this multimodal approach any better than placebo alone(Aasbo et al., 1996). While pain intensity or pain scale score is a frequent measure in these studies, the actual need for additional analgesia is a limiting factor with regard the ability to perform a procedure as a day case or not and may reflect a more practical end-point for further research.

Despite a small number of studies suggesting that morphine provides adequate analgesic control following knee a recent review of these studies has suggested that of the higher quality studies, most had a negative finding not in favor of its use as an intra-articular analgesic agent(Rosseland, 2005, Drosos et al., 2002). The key point of this review was that post-operative pain intensity was no less in the morphine treated groups than the placebo treated groups in the well-designed studies. This review is supported by a study by the same author group that found only those with intense pain after arthroscopy had any benefit from intra-articular morphine(Rosseland et al., 1999).

Other agents have been studied in with some success including midazolam (increased the time to first analgesia after surgery compared to placebo), clonidine (additive effect with bupivacaine compared to bupivacaine alone) and neostigmine (more effective when compared to morphine)(Batra et al., 2008, Tamosiunas et al., 2005, Yang et al., 1998). Unfortunately these agents have been studies in a very limited capacity and a clear conclusion in unable to be drawn as their effectiveness.


Local Anesthetic Agents **in** Arthroscopy 165

the injection pre-operatively.

RCT Combinations of **bupivacaine, morphine** and **epinephrine** given pre- or post-surgery resulted in similar pain control.

respect to post-opertive pain control

**Ropivacaine** (75mg in 20ml saline) had quicker onset of effective analgesia post-opeatively than **morphine** (2mg in 20ml saline) with lower VAS pain scores in the first 4 hours and equivalent control in

**Bupivacaine with epinephrine** and **morphine** or **bupivacaine with epinephrine** alone given either pre- or post-operatively resulted in lower pain scores and narcotic onsumption than with epinephrine alone. There was a trend toward superior control in those receiving

Patients received either 10ml 0.5% **bupivacaine** or 5mg **morphine** in normal saline. Mean time to rescue analgesia was shorter in the bupivacaine group but there was no difference in reported VAS pain

Intra-articular analgesia given at completion of arthroscopy was equivalent to pre-operative intravenous regional analgesia with

Knee arthroscopy performed under LA (prilocaine (5mg/ml). **Morphine** (3mg), **ketorolac** (30mg) or a combination of the two was given at completion of surgery. A combination of morphine and ketorolac provided significantly superior analgesia than morphine

Patients receiving intra-articular **piroxicam** (20mg) and 25ml of 0.25% **bupivacaine** had longer analgesic duration in cases where synovial inflammation was confirmed present than when not

**Levobupivacaine** (5mg/ml) significantly reduced the need for analgesia in the first 24 hours post-surgery compared to **levobupivacaine** (2.5mg/ml) and **lidocaine** (10mg/ml) with

Knee arthroscopy [performed under LA. Patients receiving intraarticular **morphine** (4mg) had lower VAS pain scores than those receiving 0.25% **bupivacaine** or saline placebo. Less supplemental

Patients receiving a combination of 0.25% **bupivacaine** with 50g of **fentanyl** had reduced post-operative pain for at least 9 hours postsurgery when compared to patients receiving 0.25% **bupivacaine**

Patients receiving intra-articular **morphine** (5mg) following knee arthroscopy had lower VAS pain scores and needed less rescue analgesia than those recevinign saline placebo. Low serum morphine

Intra-articular **morphine** (5mg) either in isolation or incombination with 25ml of 0.25% **bupivacaine** resulted in significantly lower pain scores and need for supplementary analgesia than **bupivacaine** in

metabolites suggested that the morphine was acting locally.

pain medication was needed by the morphine group.

RCT 59 **Morphine** (1mg) given either intra-articularly or intravenously at the end of arthroscopy had equivalent analgesic benefit.

**Author Setting Number Key findings** 

scores.

alone or placebo.

present.

**adrenaline**.

alone or saline placebo.

isolation or saline placebo.

the first 24 hours.

(Franceschi

(Goodwin et al., 2005, Goodwin and Parker, 2005)

(Goodwin and Parker, 2005)

(Grabowska-Gawel et al., 2003)

(Graham et

(Gupta et al.,

(Izdes et al.,

(Jacobson et

(Jaureguito

(Jawish et al.,

(Joshi et al.,

(Joshi et al.,

et al., 1995) RCT

(Hege-Scheuing et al., 1995)

et al., 2001) RCT 90

al., 2000) RCT 36

1999) RCT 100

2003) RCT 90

al., 2006) RCT 120

1996) RCT 33

1992) RCT 20

1993) RCT 40

RCT 50

56


Patients given **morphine** (1mg) and **clonidine** (150g) intraarticularly in combination had lower VAS pain scores at 2 hours post-surgery and lower need for rescue analgesia compared to groups given either agent in isolation or saline placebo

Following arthroscopic meniscectomy, patients receiving intraarticular **ketorolac** (60mg) had better post-operative pain control and less need for rescue analgesia compared to those receiving 10ml of 0.25% **bupivacaine**, 1mg of **morphine** or normal saline placebo.

Patients receiving intra-articular **tenoxicam** had lower pain scores at 30-180 minutes post-surgery and required less analgesia later than

Patients given 5mg **ketorolac** with 20ml of 0.25% **bupivacaine** into the joint after surgery provided similar analgesic control to 10mg ketorolac given intravenously with 20ml of 0.25% **bupivacaine** given

Patients received either 40ml solution containing only normal saline,

arthroscopy. Less analgesia was needed by the tenoxicam group but

Patients receiving 1mg of **morphine** intrarticularly at the end of surgery had lower pain scores at 8- and 24-hours after surgery and used less paracetamol compared to those receiving saline placebo.

No significant difference seen in VAS pain scores between patients receiving intra-articular saline, 5mg **morphine** or 15mg **morphine** following diagnostic arthroscopy or arthroscopic meniscectomy.

Patients randomised to receive either saline placebo; 2% **lidocaine** and 10mg **pethidine**, or; 2% **lidocaine**, 10mg **pethidine** and 20mg **tenoxicam**. Combination of all three agents resulted in lower VAS

Patients receiving 1g **magnesium sulfate** and 0.25% **bupivacaine**  (20ml total) had significantly lower VAS pain scores and longer time to first analgesic use than thos receiving either agent in isolation or

Patients receving either 8mg **lornoxicam** or 50mg **bupivacaine** had less analgesic consumption after surgery than those receiving plcebo. Pain rating were lower for those receinving the lornoxicam than thos

A combination of **magnesium** (150mg) and **morphine** (2mg) with 20 ml of 0.25% **bupivacaine** provided superior analgesic control (lower VAS scores and longer time to first analgesic) than either agent alone

Patients receiving pre-emptive injection of **bupivacaine with adrenaline** showed a trend toward needing less analgesia in the recovery room than those receiving the injection at completion of

0.25% **bupivacaine** or 20mg **tenoxicam** at the end of knee

subjective pain reporting was similar in all groups.

pain scores for longer and less need for analgesic use.

Intra-articular and subcuticular **morphine** (10mg) and intraarticuular **bupivacaine** (20ml 0.5%) were compared with noraml saline placebo. Single dose morphine by either route provided superior pain conrol with lower pain scores at 6- and 3-

thos receiving the same drug intravenously.

**Author Setting Number Key findings** 

hours post-surgery.

into the joint.

placebo.

surgery.

receiving the bupivacaine.

with bupivacaine or bupivacaine alone.

(Buerkle et

(Calmet et

(Cepeda et

(Colbert et

(Convery et

(Cook et al.,

(Dalsgaard et

(Drosos et

(Elhakim et

(Elsharnouby

(Eren et al.,

(Fagan et al.,

(Farouk and

al., 2000) RCT 60

al., 2004) RCT 80

al., 1997) RCT 112

al., 1999) RCT 88

al., 1998) RCT 60

1997) RCT 63

al., 1993) RCT 52

al., 2002) RCT 30

al., 1999) RCT 60

et al., 2008) RCT 108

2008) RCT 90

2003) RCT 40

Aly, 2009) RCT 80


Local Anesthetic Agents **in** Arthroscopy 167

2mg) was equivalent to saline placebo.

analgesia when compared to saline control.

than **bupivacaine** in isolation or placebo.

articular **buprenorphine** or **bupivacaine.**

with neostigmine 125 or 250g.

Table 2. Studies assessing the benefit of intra-articular analgesic agents following knee

hour post-surgery.

**with epinephrine**..

in pain scores.

supplementary analgesia.

Only patients with more intense pain after arthroscopy had beneift from intra-articular **morphine** (2mg) with regard reduced pain intensity and analgesia requirement. In most patients **morphine** (1 or

Patientes received either 10 or 20ml of 7.5mg/ml **ropivacaine**. Both provided excellent pain control for two hours, however after that the

Knee arthroscopy performed under spinal anaesthesia. 20ml of 0.5% **ropivacaine** failed to reduce VAS pain scores or need for rescue

Patients received intra-articular **morphine** (5mg) or saline placebo via intra-articular catheter 1 hour post-surgery if they developed at least moderate pain. Morphine was of no greater benefit than saline.

Patients receiving either saline placebo, 10ml 0.25% **bupivacaine**, 2mg **morphine** or 100g **fentanyl** intra-articularly at the end of arthroscopy did not differ significantly in reported pain intensity.

Patients receiving 20ml of 0.5% **bupivacaine** with the addition of 1g/kg of **clonidine** controlled post-operative pain more effectivley

Patients receiving 20ml of 0.5% bupivacaine either intra-articulalry or infiltrated around the portals reported equivalent pain scores at 1-

**bupivacaine** (50mg) resulted in lower VAS pain scores in the 6-hours

**Sufentanil** (5 or 10g) given intra-articulalry resulted in lower VAS pain scores than in control (intravenous sufentanil). Post-operative

Patients treated with **prilocaine with adenaline** reported prolonged time to first dose of oral analgesia but overall there was no difference

Patients receiving intra-articular **neostigmine** (500g) had lower VAS pain scores 1-hour after surgery and had longer lasting duration of analgesia compared to those receiving intra-articular **morphine** (2mg) or saline placebo. No significant effects were seen

Intra-articular administration of **tramadol** (100mg) and 0.25% **bupivacaine** to 20ml vloume had lower VAS scores, longer time to rescue analgesia and less analgesic use in the first 24-hours compared to when either agent was given in isolation.

Patients receiving intra-artticular **morphine** (2mg) reported significantly less pain and lower analgesic requirements in the 24 hours after surgery than those receiving 30ml of 0.25% **bupivacaine** 

Intra-articular **buprenorphine** (100g) and intra-articular

after surgery than intra-articular saline or intra-muscular buprenorphine. Analgesic use was less in thoe treated with intra-

analgesic use was also lower in the treatment group.

Timing of catheter removal did not influence the outcome.

lower dose group reported increased pain and need for

**Author Setting Number Key findings** 

(Rosseland et

(Samoladas

(Santanen et

(Solheim et

(Souza et al.,

(Tamosiunas

(Townshend

(VanNess and Gittins, 1994)

(Varrassi et

(Vranken et

(White et al., 1990) RCT

(Yang et al.,

(Zeidan et

al., 1999) RCT 90

et al., 2006) RCT 60

al., 2001) RCT 100

al., 2006) RCT 40

2002) RCT 60

et al., 2005) RCT 48

et al., 2009) RCT 137

al., 1999) RCT 48

al., 2001) RCT 60

1998) RCT 60

al., 2008) RCT 90

arthroscopy. Treatments are in bold.

RCT 81


There was no difference in reposrted VAS pain scores or

those receiving 1mg **morphine** or saline placebo.

**levobupivacaine** and 20ml of 0.5% **bupivacaine.**

(1mg) was given by the intra-articular route.

acetaminophen use in the 48 hours after knee arthroscopy in patients receiving either 2 or 4mg of intra-articular **morphine** after surgery

Patients receiving 5mg **morphine** intra-articularly after arthroscopy had superior pain control in the 24 hours after surgery compared to

No significant difference was found in control of post-operative pain and analgesic requirement between patients receiving 20ml of 0.5%

Infiltration of **morphine** (1mg) into the synovial tissue or outer third of meniscal tissue resulted in better pain control (lower VAS pain scores and less analgesic use) post-arthroscopy than if morphine

Patients underwent knee arthroscopy under either spinal or LA (1% lidocaine with adrenaline) blockade. Intra-articular **morphine** (1mg) given at the end of the procedure resulted in reduced rescue analgesia requirement in the group that had LA block.

Patients randomized to receive either intra-articular **bupivacaine** or **fantanyl** after knee arthroscopy reported similar pain scores except

Patients receinving 10mg **morphine** intra-articularly repoted lower pain scores between 4- and 24-hours post-surgery and consumed less analgesia than patients receiving 10mg morphine via the

Pre-operative per oral diclofenac reduced post-operative pain scores compared to the intra-articular **ropivacaine** given at the time of surgery. Arthoscopy performed under spinal anaesthesia.

Intra-articular morphine (1mg) was superior to bupivacaine (100mg) at reducing pain scores and need for supplementary anlgesia at 6-

An intra-articular dose of 5mg morphine was more effective thatn 1mg intra-articular or 5mg intravenous at reducing VAS pain scores.

Intra-articular **saline** (1 or 10ml) was given following surgery via an intra-articular catheter in patients with at least moderate pain. Within 1 hour VAS pain scores reduced from 50 to 27 on a 100mm

Intra-articular **saline** (10ml) or **morphin**e (2mg in 10ml saline) was given following surgery via an intra-articular catheter in patients with at least moderate pain. Equivalent improvements in pain

at 2-hours post-surgery when bupivacaine was superior

Patients randomized to receive either saline placebo; 150mg **bupivacaine** and 4mg **morphine,** or; 150mg **bupivacaine**, 4mg **morphine** and 40mg **methylprednisolone** intra-articulalry at the end of surgery.Bupivacaine with mprhine was effective at reducing pain and duration of immobilization, the addition of methylprednisolone

futher reduce dpain and use of analgesics.

intramuscular route.

and 24-hours post-surgery.

scale with both volumes.

intesnity were found in both groups.

Intra-articular **bupivacaine** 0.25% (40ml) with the addition of **morphine** 1mg compared to **bupivacaine** alone. Lower VAS pain scores noted with the addition of morphine in the 24 hours after surgery but no difference in supplementary analgesic use.

**Author Setting Number Key findings** 

(Juelsgaard

(Kanbak et

(Karaman et

(Kligman et

(Lundin et

(Niemi et al.,

(Pooni et al.,

(Raj et al.,

(Rasmussen

(Rautoma et

(Richardson et al., 1997) RCT

(Rosseland et

(Rosseland et

al., 1997) RCT

et al., 1993) RCT 47

al., 2009) RCT 40

al., 2002) RCT 60

al., 1998) RCT 50

1994) RCT 80

1999) RCT 107

2004) RCT 40

et al., 2002) RCT 60

al., 2000) RCT 200

al., 2004) RCT 60

al., 2003) RCT 40


Table 2. Studies assessing the benefit of intra-articular analgesic agents following knee arthroscopy. Treatments are in bold.

Local Anesthetic Agents **in** Arthroscopy 169

The toxic effects of bupivacine (0.125, 0.25 and 0.5%) on the the articular chondrocyte from a bovine cell line were well demonstrated (Chu et al., 2008). Cells were cultured in a 3 dimensional alginate-bead culture. Specimens were exposed for 15, 30 or 60 minutes and analysis was performed at 1 and 24 hours and at 1 week. A clear time and concentration dependent respose to the local anaetshetic treatments was observed. Treatment with 0.125% bupivacaine for 15 minutes was not significantly different to the saline control. Almost complete loss of cell viability was noted with 0.5% bupivacaine. Analysis of osteochondral cores with an intact superficial cell layer suggested that an the superficial layer of the articular cartilage provided some protective benefit when intact. This may be significant in deciding during surgery whether or not intra-articular analgesic agents are safe to

To test the respective toxic effects on chondrocytes of lidocaine, mepivacaine and bupivacaine Park et al used an equine model (Park et al., 2011). Bupivacaine (0.5%) was the most toxic of the agents used with cell viability reduced to 29 +/- 8% after 30 minutes. Cell viability after treatment with saline was 96%. Lidocaine and mepivacaine were both less

A number of studies have used human cell lines which is arguably more useful for the extrapolation of results into clinical pratice. Dragoo et al used a custom made bioreactor to mimic the metabolism of synovial fluid to simulate the use of a pain pump following arthroscopic surgery(Dragoo et al., 2008). They found that both lignocaine (1%) and bupivacaine (0.25 or 0.5%) resulted in reduced cell viability but that the rates of necrosis were noted with the presence of epinephrine. Cell viability was similar at 24 and 48 hours in the bupivaine group, but there was a greater toxic effect seen at 72 hours. Further work using the same bioreactor model demonstrated that epineprhine, at levels of 1:100000- 200000, conferred no significant increase in cell death compared to acidic media with a pH of 4.5-5.0 and local anaesthetics in combination with epinephrine (Dragoo et al., 2010). The authors suggest that local anaesthetic agents containing epinephrine should be used with

Syed et al reported significant toxic effects of bupivacaine either alone or in combination with triamcinolone in a monlayer culture model using human articular chondrocytes (Syed et al., 2011). When the treatments were administered to the osteochondral plug with an intact surface however, the toxic effect of bupivacaine in isolation was no more than that of the control – again suggesting there is a benefit to an intact articular surface with regard

Using chondrocytes harvested from osteoarthritic human knees it was demonstrated that exposure to lidocaine, bupivacaine or ropivacaine for 24 or 120 hours resulted in significant levels of cell death(Grishko et al., 2010). In the lignocaine 2% group massive necrosis was seen at 24 hours. After 120 hours exposure there were significant dereases in cell viability in all treatments groups with the exception of those cells treated with 0.2% ropivacaine. As

Jacobs et al harvested human articular chondrocytes from the knees of human tissue donors or patients undergoing total knee arthroplasty(Jacobs et al., 2011). They treated the articular chondrcytes with either 1% or 2% lidocaine with or without epinephrine and used saline as a control. Cell death between 91-99% was seen for each of the three treatments. A prolonged

Ropivacaine 0.5% was found to be significantly less toxic to human chondrocytes than bupivacaine 0.5% (Piper and Kim, 2008). Normal human articular cartilage was harvested

toxic with mepivacine exerting the least toxic effect of the three.

viability decreased a concomitant rise in cell apoptosis was noted.

exposure time was also associated with higher rates of cell death.

caution as these are often titrated to a low pH.

exposure to potentially toxic agents.

administer.

In summary a myriad of agents have been studied for their potential use in attenuating postoperative pain following knee arthroscopy. While the amide local anesthetic agents are the most widely studied their continuing benefit and use is questionable as portal infiltration has been shown to be as effective at providing pain control for a procedure that is generally very well tolerated. Knee arthroscopy performed under local anesthetic is a different entity although far less frequent.

Hip and ankle arthroscopy are far less studied and the ideal intra-articular agent is uncertain in these joints. A multi-model intra-articular analgesic bolus may be the best approach in these joints that require significant traction and subsequent injury to the capsule that can cause greater discomfort after surgery.

#### **2.3 The potential for articular chondrocyte toxicity**

The potential for deleterious effects of local anesthetic agents on articular chondrocytes was increasingly noted with the use of arthroscopic pain pumps following glenohumeral arthroscopy(Busfield and Romero, 2009, Hansen et al., 2007, Solomon et al., 2009). Increasingly however studies are alerting the practicing clinician to the potential for toxic effects secondary to amide local anesthetics given as a single bolus injection. Most of these are laboratory-based studies. Although some have questioned the relevance given the long use of intra-articular local anesthetic without seemingly any complication the serve as a caution.

The aim of this section is to provide an over view of the basic science evidence on the potential for local anesthetic agents to cause articular chondrocyte toxicity.

#### **2.3.1 In vitro reports**

A number of different laboratory modesl utilizing cell lines from a variety of animal species have been used in the study of local anaesthetic toxicity. A toxic effect in canine chondrocytes exposed to bupivacaine 0.5% using a proven in vitro model by Anz et al. They reported an almost 100% reduction in cell viability after two days exposure to bupivacaine. Bupivacaine conferred an anti-inflammatory effect in their study, evidenced by reduced nitric oxide and PGE rise in the presence of interleukin-1, but their conclusion maintained that continuous exposure to bupivacine resulted in a clear toxic effect toward the canine chondrocytes(Anz et al., 2009). Again using a canine osteochondral model the toxic effect of bupivacaine was again confirmed, with or without the addition of methylparaben(Hennig et al., 2010). Exposure to the local anaesthetic alone for 5 or 30 minutes caused significant cell death, although this was only significant statistically at the 30 minute exposure.

Miyazaki et al demonstrated a concentration dependent reduction in bovine chondrocyte viability after treatment with lidocaine (0.125, 0.25, 0.5 or 1%) (Miyazaki et al., 2011). Glycosaminoglycan (GAG)content of the cells was also noted to be reduced as the concentration of the local anaesthetic was increased. GAG and lactate production were higher in the cells treated with 0.5 and 1% lidocaine. The authors felt that this finding conferred a reparative response by the cells.

Using bovine articular chondrocytes in alginate bead cultures Karpie et al exposed these to 1 or 2% lidocaine for 15 to 60 minutes(Karpie and Chu, 2007). A dose and time dependent increase in cell toxicity was reported. An intact surface on the osteochondral core or variation in the pH of the treatments (pH 7.4, 7.0, 5.0) failed to confer any protective effect (this is in contrast to other studies – see below). Others have also reported time and concentration dependent reductions in cell viability using a bovine disc model(Lo et al., 2009). In this case osteochondral cores were harvested from the radiocarpal joint of cows and these were treated with either lidocaine (1%), bupivacaine (0.25%) or ropivacaine (0.5%).

In summary a myriad of agents have been studied for their potential use in attenuating postoperative pain following knee arthroscopy. While the amide local anesthetic agents are the most widely studied their continuing benefit and use is questionable as portal infiltration has been shown to be as effective at providing pain control for a procedure that is generally very well tolerated. Knee arthroscopy performed under local anesthetic is a different entity

Hip and ankle arthroscopy are far less studied and the ideal intra-articular agent is uncertain in these joints. A multi-model intra-articular analgesic bolus may be the best approach in these joints that require significant traction and subsequent injury to the capsule

The potential for deleterious effects of local anesthetic agents on articular chondrocytes was increasingly noted with the use of arthroscopic pain pumps following glenohumeral arthroscopy(Busfield and Romero, 2009, Hansen et al., 2007, Solomon et al., 2009). Increasingly however studies are alerting the practicing clinician to the potential for toxic effects secondary to amide local anesthetics given as a single bolus injection. Most of these are laboratory-based studies. Although some have questioned the relevance given the long use of intra-articular

The aim of this section is to provide an over view of the basic science evidence on the

A number of different laboratory modesl utilizing cell lines from a variety of animal species have been used in the study of local anaesthetic toxicity. A toxic effect in canine chondrocytes exposed to bupivacaine 0.5% using a proven in vitro model by Anz et al. They reported an almost 100% reduction in cell viability after two days exposure to bupivacaine. Bupivacaine conferred an anti-inflammatory effect in their study, evidenced by reduced nitric oxide and PGE rise in the presence of interleukin-1, but their conclusion maintained that continuous exposure to bupivacine resulted in a clear toxic effect toward the canine chondrocytes(Anz et al., 2009). Again using a canine osteochondral model the toxic effect of bupivacaine was again confirmed, with or without the addition of methylparaben(Hennig et al., 2010). Exposure to the local anaesthetic alone for 5 or 30 minutes caused significant cell

Miyazaki et al demonstrated a concentration dependent reduction in bovine chondrocyte viability after treatment with lidocaine (0.125, 0.25, 0.5 or 1%) (Miyazaki et al., 2011). Glycosaminoglycan (GAG)content of the cells was also noted to be reduced as the concentration of the local anaesthetic was increased. GAG and lactate production were higher in the cells treated with 0.5 and 1% lidocaine. The authors felt that this finding

Using bovine articular chondrocytes in alginate bead cultures Karpie et al exposed these to 1 or 2% lidocaine for 15 to 60 minutes(Karpie and Chu, 2007). A dose and time dependent increase in cell toxicity was reported. An intact surface on the osteochondral core or variation in the pH of the treatments (pH 7.4, 7.0, 5.0) failed to confer any protective effect (this is in contrast to other studies – see below). Others have also reported time and concentration dependent reductions in cell viability using a bovine disc model(Lo et al., 2009). In this case osteochondral cores were harvested from the radiocarpal joint of cows and these were treated

local anesthetic without seemingly any complication the serve as a caution.

potential for local anesthetic agents to cause articular chondrocyte toxicity.

death, although this was only significant statistically at the 30 minute exposure.

with either lidocaine (1%), bupivacaine (0.25%) or ropivacaine (0.5%).

although far less frequent.

**2.3.1 In vitro reports** 

that can cause greater discomfort after surgery.

conferred a reparative response by the cells.

**2.3 The potential for articular chondrocyte toxicity** 

The toxic effects of bupivacine (0.125, 0.25 and 0.5%) on the the articular chondrocyte from a bovine cell line were well demonstrated (Chu et al., 2008). Cells were cultured in a 3 dimensional alginate-bead culture. Specimens were exposed for 15, 30 or 60 minutes and analysis was performed at 1 and 24 hours and at 1 week. A clear time and concentration dependent respose to the local anaetshetic treatments was observed. Treatment with 0.125% bupivacaine for 15 minutes was not significantly different to the saline control. Almost complete loss of cell viability was noted with 0.5% bupivacaine. Analysis of osteochondral cores with an intact superficial cell layer suggested that an the superficial layer of the articular cartilage provided some protective benefit when intact. This may be significant in deciding during surgery whether or not intra-articular analgesic agents are safe to administer.

To test the respective toxic effects on chondrocytes of lidocaine, mepivacaine and bupivacaine Park et al used an equine model (Park et al., 2011). Bupivacaine (0.5%) was the most toxic of the agents used with cell viability reduced to 29 +/- 8% after 30 minutes. Cell viability after treatment with saline was 96%. Lidocaine and mepivacaine were both less toxic with mepivacine exerting the least toxic effect of the three.

A number of studies have used human cell lines which is arguably more useful for the extrapolation of results into clinical pratice. Dragoo et al used a custom made bioreactor to mimic the metabolism of synovial fluid to simulate the use of a pain pump following arthroscopic surgery(Dragoo et al., 2008). They found that both lignocaine (1%) and bupivacaine (0.25 or 0.5%) resulted in reduced cell viability but that the rates of necrosis were noted with the presence of epinephrine. Cell viability was similar at 24 and 48 hours in the bupivaine group, but there was a greater toxic effect seen at 72 hours. Further work using the same bioreactor model demonstrated that epineprhine, at levels of 1:100000- 200000, conferred no significant increase in cell death compared to acidic media with a pH of 4.5-5.0 and local anaesthetics in combination with epinephrine (Dragoo et al., 2010). The authors suggest that local anaesthetic agents containing epinephrine should be used with caution as these are often titrated to a low pH.

Syed et al reported significant toxic effects of bupivacaine either alone or in combination with triamcinolone in a monlayer culture model using human articular chondrocytes (Syed et al., 2011). When the treatments were administered to the osteochondral plug with an intact surface however, the toxic effect of bupivacaine in isolation was no more than that of the control – again suggesting there is a benefit to an intact articular surface with regard exposure to potentially toxic agents.

Using chondrocytes harvested from osteoarthritic human knees it was demonstrated that exposure to lidocaine, bupivacaine or ropivacaine for 24 or 120 hours resulted in significant levels of cell death(Grishko et al., 2010). In the lignocaine 2% group massive necrosis was seen at 24 hours. After 120 hours exposure there were significant dereases in cell viability in all treatments groups with the exception of those cells treated with 0.2% ropivacaine. As viability decreased a concomitant rise in cell apoptosis was noted.

Jacobs et al harvested human articular chondrocytes from the knees of human tissue donors or patients undergoing total knee arthroplasty(Jacobs et al., 2011). They treated the articular chondrcytes with either 1% or 2% lidocaine with or without epinephrine and used saline as a control. Cell death between 91-99% was seen for each of the three treatments. A prolonged exposure time was also associated with higher rates of cell death.

Ropivacaine 0.5% was found to be significantly less toxic to human chondrocytes than bupivacaine 0.5% (Piper and Kim, 2008). Normal human articular cartilage was harvested

Local Anesthetic Agents **in** Arthroscopy 171

neostigmine. Histological analysis performed at 1, 2 and 10 days confirmed more toxic

Despite a number of studies reporting the potential toxic effects the mechanism by which these agents exert their effect is uncertain. Mitochondrial dysfunction is thought to be a key factor in articular chondrocyte death(Grishko et al., 2010). Grishko et al demonstrated mitochondrial DNA damage and a reduction in ATP and mitochondrial protein levels in

In another study cells exposed to lidocaine or bupivacine in isolation had rates of cell death just over 10%(Bogatch et al., 2010). When the local anaesthetics were mixed with the cell culture medium this rate rose to over 96% in each instance. Crystal formation was seen when the bupivacine was mixed with culture medium. Acididc phosphate buffered saline resulted in increased cell death only when the acidity was increased to a pH less than 3.4. Based on these results the authors propose an incompatibility between the synovial fluid and the local anaesthetic is responsible for the majority of chondrocyte death rather than the

A number of different agents have been trialed as intra-articular analgesic agents following arthroscopy in the lower limb. Many of the reported trials have focussed on the use of amide local anaesthetic sgents as these are the most widely used in clinical practice. Depsite multiple studies there is no agent that appears clearly superior to the rest. Bupivacaine or ropivacaine appear the most likely to offer the greatest analgesic control by this route and a small number of studies are supportive of a multi-modal infiltration. Magnesium sulfate for

Although Townsend et al have offered evidence that intra-articular local anaesthetc can be avoided in knee arthroscopy without compromising analgesic control, the ideal mode of analgesic control in hip and ankle arthroscopy is still uncertain. Recent reports of chondrolysis in shoulder arthroscopy prompted a number of investigations in the potential

Ropivacaine appears to less toxic than bupivacaine and a combination of ropivacaine and magnesium has also been suggested as a more acceptable alternative approach to intraarticular local anaesthesia(Baker et al., 2011b, Webb and Ghosh, 2009). The potential diffculties in applying laboratory findings to the clinical setting has been noted(Webb and Ghosh, 2009). In the arthroscopic setting, a number of variables including the articular surface disease state, the dilutional effect of the arthroscopic fluid and absorbance of injected agents into surrounding synovium and adjacent soft tissues could all modify the effect the local anaesthetic has on the articular chondrocyte. The potential for toxic effects on articular

Aasbo, V., Raeder, J. C., Grogaard, B. & Roise, O. 1996. No additional analgesic effect of

intra-articular morphine or bupivacaine compared with placebo after elective knee

toxic effect that amide local anaesthetcis may have on articular chondrocytes.

chondrocytes by local anaesthetic needs to be further investigated.

arthroscopy. *Acta Anaesthesiol Scand,* 40, 585-8.

response to treatment with a variety of local anaesthetics at varying concentrations.

changes in both treatment groups compared to saline control.

local anaesthetic agent itself.

one may be an ideal synergist.

**3. Summary** 

**4. References** 

**2.3.3 The mechaisn of local anaesthetic mediated chondroctoxicity** 

from the femoral head or tibial plateau in in patients underoing surgical procedures. Full thickness explants and cultured chondrocytes were treated with either ropivacaine or bupivacaine for 30 minutes. Cell viability in the explant cultures fell to 95% and 78% after treatment with ropivacaine and bupivacaine respectively. Viability in the cell cultures fell to 64% and 37%. The viability of the cells in the explant cultures treated with ropivacaine did not differ significantly to that in the controls treated with saline. Ropivacaine may therefore confer a much more acceptable risk than bupivacaine – an important consideration if using it as an intra-articular agent following arthroscopy.

However, others failed to find a difference between these two agents using a simple monolayer culture model. Both ropivacaine and bupivacaine conferred simlar toxic effects to the articular chondrocytes either in isolation or if they were used in combination with magnesium sulfate(Baker et al., 2011b, Baker et al., 2011d). Lignocaine combined with magnesium sulfate was less toxic than either ropivacaine, bupivacaine or levobupivacaine combined with magnesium suflate after an exposure time of only 15 minutes(Baker et al., 2011b).

A useful finding for the practicing surgeon in the studies that have assessed human cells in in vitro settings is the recurrent finding that ropivacaine is less toxic than bupivacaine(Baker et al., 2011b, Baker et al., 2011d, Piper and Kim, 2008). If ropivacaine confers a less toxic effect, then as long as it provides equally efficacious analgesic control, then these studies support its use. Notably, Piper et al also found ropivacaine to be less toxic in the explant culture with cells embedded in an intact matrix, potentially a better representation of the in vivo state.

#### **2.3.2 In vivo studies**

In vivo models shoulde in theory provide the best simulation of what may happen in pratcice. However, consideration needs to be given to the culture model used and also the species studied. Arguably the ideal model is unknown to date and no human in vivo studies at the time of writing have been able to demonstrate a lasting deleterious effect of local anaesthetic on articular chondrocytes.

The effect of a single intra-articular injection of 0.5% bupivacaine into a stifle joint compared to 0.9% saline control was studied(Chu et al., 2010). Six months following injection gross and histological appearances showed that the chondral surfaces remained intact. They diod note howeevr, that there was a reduction in chondrocyte density of up to 50% in the joint treated with local anaesthetic compared to the saline control.

In an in vivo rabbit study three groups recieved continuous infusions of either saline, bupivacaine or bupivacaine with epinephrine over 48 hours(Gomoll et al., 2006). One week after treatment the animals were sacrificed and osteochondral and synovial samples analysed. Bupivacaine with or without epinephrine resulted in cell viability reduction by 20 to 32%. Histological analysis was worse in both treatment gourps compared to saline control.

A similar treatment regime did not result in long term changes in articular cartilage(Gomoll et al., 2009). When the rabbits were sacrificed three months after the infusion of the saline of local anaesthetic there was no significant difference found between treatment and control groups. An increase in cartilage metabolism in the treatment groups was noted suggesting that the cartilage was undergoing a reparative process. This study provides conflicting information to the earlier one noted by Chu et al creating more difficulty in ascertaining the true chronic effect of intra-articular local anaesthetic use.

In another, histological changes in rabbit knee joint articular cartilage have been reported (Dogan et al., 2004). Knees were injected with either 0.9% saline, bupivacaine or

from the femoral head or tibial plateau in in patients underoing surgical procedures. Full thickness explants and cultured chondrocytes were treated with either ropivacaine or bupivacaine for 30 minutes. Cell viability in the explant cultures fell to 95% and 78% after treatment with ropivacaine and bupivacaine respectively. Viability in the cell cultures fell to 64% and 37%. The viability of the cells in the explant cultures treated with ropivacaine did not differ significantly to that in the controls treated with saline. Ropivacaine may therefore confer a much more acceptable risk than bupivacaine – an important consideration if using

However, others failed to find a difference between these two agents using a simple monolayer culture model. Both ropivacaine and bupivacaine conferred simlar toxic effects to the articular chondrocytes either in isolation or if they were used in combination with magnesium sulfate(Baker et al., 2011b, Baker et al., 2011d). Lignocaine combined with magnesium sulfate was less toxic than either ropivacaine, bupivacaine or levobupivacaine combined with magnesium suflate after an exposure time of only 15 minutes(Baker et al.,

A useful finding for the practicing surgeon in the studies that have assessed human cells in in vitro settings is the recurrent finding that ropivacaine is less toxic than bupivacaine(Baker et al., 2011b, Baker et al., 2011d, Piper and Kim, 2008). If ropivacaine confers a less toxic effect, then as long as it provides equally efficacious analgesic control, then these studies support its use. Notably, Piper et al also found ropivacaine to be less toxic in the explant culture with cells

In vivo models shoulde in theory provide the best simulation of what may happen in pratcice. However, consideration needs to be given to the culture model used and also the species studied. Arguably the ideal model is unknown to date and no human in vivo studies at the time of writing have been able to demonstrate a lasting deleterious effect of local

The effect of a single intra-articular injection of 0.5% bupivacaine into a stifle joint compared to 0.9% saline control was studied(Chu et al., 2010). Six months following injection gross and histological appearances showed that the chondral surfaces remained intact. They diod note howeevr, that there was a reduction in chondrocyte density of up to 50% in the joint treated

In an in vivo rabbit study three groups recieved continuous infusions of either saline, bupivacaine or bupivacaine with epinephrine over 48 hours(Gomoll et al., 2006). One week after treatment the animals were sacrificed and osteochondral and synovial samples analysed. Bupivacaine with or without epinephrine resulted in cell viability reduction by 20 to 32%.

A similar treatment regime did not result in long term changes in articular cartilage(Gomoll et al., 2009). When the rabbits were sacrificed three months after the infusion of the saline of local anaesthetic there was no significant difference found between treatment and control groups. An increase in cartilage metabolism in the treatment groups was noted suggesting that the cartilage was undergoing a reparative process. This study provides conflicting information to the earlier one noted by Chu et al creating more difficulty in ascertaining the

In another, histological changes in rabbit knee joint articular cartilage have been reported (Dogan et al., 2004). Knees were injected with either 0.9% saline, bupivacaine or

Histological analysis was worse in both treatment gourps compared to saline control.

embedded in an intact matrix, potentially a better representation of the in vivo state.

it as an intra-articular agent following arthroscopy.

2011b).

**2.3.2 In vivo studies** 

anaesthetic on articular chondrocytes.

with local anaesthetic compared to the saline control.

true chronic effect of intra-articular local anaesthetic use.

neostigmine. Histological analysis performed at 1, 2 and 10 days confirmed more toxic changes in both treatment groups compared to saline control.

#### **2.3.3 The mechaisn of local anaesthetic mediated chondroctoxicity**

Despite a number of studies reporting the potential toxic effects the mechanism by which these agents exert their effect is uncertain. Mitochondrial dysfunction is thought to be a key factor in articular chondrocyte death(Grishko et al., 2010). Grishko et al demonstrated mitochondrial DNA damage and a reduction in ATP and mitochondrial protein levels in response to treatment with a variety of local anaesthetics at varying concentrations.

In another study cells exposed to lidocaine or bupivacine in isolation had rates of cell death just over 10%(Bogatch et al., 2010). When the local anaesthetics were mixed with the cell culture medium this rate rose to over 96% in each instance. Crystal formation was seen when the bupivacine was mixed with culture medium. Acididc phosphate buffered saline resulted in increased cell death only when the acidity was increased to a pH less than 3.4. Based on these results the authors propose an incompatibility between the synovial fluid and the local anaesthetic is responsible for the majority of chondrocyte death rather than the local anaesthetic agent itself.

#### **3. Summary**

A number of different agents have been trialed as intra-articular analgesic agents following arthroscopy in the lower limb. Many of the reported trials have focussed on the use of amide local anaesthetic sgents as these are the most widely used in clinical practice. Depsite multiple studies there is no agent that appears clearly superior to the rest. Bupivacaine or ropivacaine appear the most likely to offer the greatest analgesic control by this route and a small number of studies are supportive of a multi-modal infiltration. Magnesium sulfate for one may be an ideal synergist.

Although Townsend et al have offered evidence that intra-articular local anaesthetc can be avoided in knee arthroscopy without compromising analgesic control, the ideal mode of analgesic control in hip and ankle arthroscopy is still uncertain. Recent reports of chondrolysis in shoulder arthroscopy prompted a number of investigations in the potential toxic effect that amide local anaesthetcis may have on articular chondrocytes.

Ropivacaine appears to less toxic than bupivacaine and a combination of ropivacaine and magnesium has also been suggested as a more acceptable alternative approach to intraarticular local anaesthesia(Baker et al., 2011b, Webb and Ghosh, 2009). The potential diffculties in applying laboratory findings to the clinical setting has been noted(Webb and Ghosh, 2009). In the arthroscopic setting, a number of variables including the articular surface disease state, the dilutional effect of the arthroscopic fluid and absorbance of injected agents into surrounding synovium and adjacent soft tissues could all modify the effect the local anaesthetic has on the articular chondrocyte. The potential for toxic effects on articular chondrocytes by local anaesthetic needs to be further investigated.

#### **4. References**

Aasbo, V., Raeder, J. C., Grogaard, B. & Roise, O. 1996. No additional analgesic effect of intra-articular morphine or bupivacaine compared with placebo after elective knee arthroscopy. *Acta Anaesthesiol Scand,* 40, 585-8.

Local Anesthetic Agents **in** Arthroscopy 173

Cook, T. M., Tuckey, J. P. & Nolan, J. P. 1997. Analgesia after day-case knee arthroscopy:

Dalsgaard, J., Felsby, S., Juelsgaard, P. & Frokjaer, J. 1993. [Analgesic effect of low-dose

Dogan, N., Erdem, A. F., Erman, Z. & Kizilkaya, M. 2004. The effects of bupivacaine and

Dragoo, J. L., Korotkova, T., Kanwar, R. & Wood, B. 2008. The effect of local anesthetics administered via pain pump on chondrocyte viability. *Am J Sports Med,* 36, 1484-8. Dragoo, J. L., Korotkova, T., Kim, H. J. & Jagadish, A. 2010. Chondrotoxicity of low pH,

Drosos, G. I., Vlachonikolis, I. G., Papoutsidakis, A. N., Gavalas, N. S. & Anthopoulos, G.

Elhakim, M., Nafie, M., Eid, A. & Hassin, M. 1999. Combination of intra-articular tenoxicam,

Elsharnouby, N. M., Eid, H. E., Abou Elezz, N. F. & Moharram, A. N. 2008. Intraarticular

Fagan, D. J., Martin, W. & Smith, A. 2003. A randomized, double-blind trial of pre-emptive

Farouk, S. & Aly, A. 2009. A comparison of intra-articular magnesium and/or morphine

Franceschi, F., Rizzello, G., Cataldo, R. & Denaro, V. 2001. Comparison of morphine and

Gomoll, A. H., Kang, R. W., Williams, J. M., Bach, B. R. & Cole, B. J. 2006. Chondrolysis after

investigating chondrotoxicity in the rabbit shoulder. *Arthroscopy,* 22, 813-9. Gomoll, A. H., Yanke, A. B., Kang, R. W., Chubinskaya, S., Williams, J. M., Bach, B. R. &

Goodwin, R. C., Amjadi, F. & Parker, R. D. 2005. Short-term analgesic effects of intra-

Goodwin, R. C. & Parker, R. D. 2005. Comparison of the analgesic effects of intra-articular

Grabowska-Gawel, A., Gawel, K., Hagner, W. & Bilinski, P. J. 2003. Morphine or

Graham, N. M., Shanahan, M. D., Barry, P., Burgert, S. & Talkhani, I. 2000. Postoperative

articular injections after knee arthroscopy. *Arthroscopy,* 21, 307-12.

local anesthesia in day-case knee arthroscopy. *Arthroscopy,* 19, 50-3.

ropivacaine following knee arthroscopy. *Arthroscopy,* 17, 477-80.

placebo. *Br J Anaesth,* 78, 163-8.

*Am J Sports Med,* 38, 1154-9.

arthroscopy]. *Agri,* 20, 17-22.

model. *Am J Sports Med,* 37, 72-7.

arthroscopy. *Ortop Traumatol Rehabil,* 5, 758-62.

*Anesth,* 23, 508-12.

*Knee Surg,* 18, 17-24.

4166-9.

*Res,* 32, 513-9.

*Knee,* 9, 335-40.

43, 803-8.

double-blind study of intra-articular tenoxicam, intra-articular bupivacaine and

intra-articular morphine after ambulatory knee arthroscopy]. *Ugeskr Laeger,* 155,

neostigmine on articular cartilage and synovium in the rabbit knee joint. *J Int Med* 

epinephrine, and preservatives found in local anesthetics containing epinephrine.

2002. Intra-articular morphine and postoperative analgesia after knee arthroscopy.

lidocaine, and pethidine for outpatient knee arthroscopy. *Acta Anaesthesiol Scand,*

injection of magnesium sulphate and/or bupivacaine for postoperative analgesia after arthroscopic knee surgery. *Anesth Analg,* 106, 1548-52, table of contents. Eren, M., Koltka, K., Koknel Talu, G., Asik, M. & Ozyalcin, S. 2008. [Comparison of analgesic

activity of intraarticular lornoxicam, bupivacaine and saline after knee

with bupivacaine for postoperative analgesia after arthroscopic knee surgery. *J* 

continuous intra-articular bupivacaine infusion: an experimental model

Cole, B. J. 2009. Long-term effects of bupivacaine on cartilage in a rabbit shoulder

injections administered preoperatively and postoperatively in knee arthroscopy. *J* 

bupivacaine in controlling postoperative pain in patients subjected to knee joint

analgesia after arthroscopic knee surgery: a randomized, prospective, double-blind


Al-Metwalli, R. R., Mowafi, H. A., Ismail, S. A., Siddiqui, A. K., Al-Ghamdi, A. M., Shafi, M.

Anz, A., Smith, M. J., Stoker, A., Linville, C., Markway, H., Branson, K. & Cook, J. L. 2009.

Baker, J. F., Byrne, D. P., Hunter, K. & Mulhall, K. J. 2011a. Post-operative opiate requirements after hip arthroscopy. *Knee Surg Sports Traumatol Arthrosc,* 19, 1399-402. Baker, J. F., Byrne, D. P., Walsh, P. M. & Mulhall, K. J. 2011b. Human chondrocyte viability

Baker, J. F., McGuire, C. M., Byrne, D. P., Hunter, K., Eustace, N. & Mulhall, K. J. 2011c.

Baker, J. F., Walsh, P. M., Byrne, D. P. & Mulhall, K. J. 2011d. In vitro assessment of human

Batra, Y. K., Mahajan, R., Kumar, S., Rajeev, S. & Singh Dhillon, M. 2008. A dose-ranging

Bogatch, M. T., Ferachi, D. G., Kyle, B., Popinchalk, S., Howell, M. H., Ge, D., You, Z. &

Buerkle, H., Huge, V., Wolfgart, M., Steinbeck, J., Mertes, N., Van Aken, H. & Prien, T. 2000.

Busfield, B. T. & Romero, D. M. 2009. Pain pump use after shoulder arthroscopy as a cause

Calmet, J., Esteve, C., Boada, S. & Gine, J. 2004. Analgesic effect of intra-articular ketorolac in

Cepeda, M. S., Uribe, C., Betancourt, J., Rugeles, J. & Carr, D. B. 1997. Pain relief after knee

Chu, C. R., Coyle, C. H., Chu, C. T., Szczodry, M., Seshadri, V., Karpie, J. C., Cieslak, K. M. &

Chu, C. R., Izzo, N. J., Coyle, C. H., Papas, N. E. & Logar, A. 2008. The in vitro effects of bupivacaine on articular chondrocytes. *J Bone Joint Surg Br,* 90, 814-20. Colbert, S. T., Curran, E., O'Hanlon, D. M., Moran, R. & McCarroll, M. 1999. Intra-articular

Convery, P. N., Milligan, K. R., Quinn, P., Scott, K. & Clarke, R. C. 1998. Low-dose intra-

bupivacaine on articular cartilage. *J Bone Joint Surg Am,* 92, 599-608.

normal saline. *Knee Surg Sports Traumatol Arthrosc*.

induced by local anaesthetics? *Am J Sports Med,* 38, 520-6.

of glenohumeral chondrolysis. *Arthroscopy,* 25, 647-52.

*Traumatol Arthrosc,* 12, 184-8.

study. *Arthroscopy,* 27, 213-7.

*Arthroscopy,* 25, 225-31.

377.

653-7.

*Anaesthesia,* 53, 1125-9.

*Analg,* 107, 669-72.

*Traumatol Arthrosc,* 12, 552-5.

morphine? *Reg Anesth,* 22, 233-8.

A. & El-Saleh, A. R. 2008. Effect of intra-articular dexmedetomidine on postoperative analgesia after arthroscopic knee surgery. *Br J Anaesth,* 101, 395-9. Alagol, A., Calpur, O. U., Kaya, G., Pamukcu, Z. & Turan, F. N. 2004. The use of

intraarticular tramadol for postoperative analgesia after arthroscopic knee surgery: a comparison of different intraarticular and intravenous doses. *Knee Surg Sports* 

The effect of bupivacaine and morphine in a coculture model of diarthrodial joints.

after treatment with local anesthetic and/or magnesium: results from an in vitro

Analgesic control after hip arthroscopy: a randomised, double-blinded trial comparing portal with intra-articular infiltration of bupivacaine. *Hip Int,* 21, 373-

chondrocyte viability after treatment with local anaesthetic, magnesium sulphate or

study of intraarticular midazolam for pain relief after knee arthroscopy. *Anesth* 

Savoie, F. H. 2010. Is chemical incompatibility responsible for chondrocyte death

Intra-articular clonidine analgesia after knee arthroscopy. *Eur J Anaesthesiol,* 17, 295-9.

knee arthroscopy: comparison of morphine and bupivacaine. *Knee Surg Sports* 

arthroscopy: intra-articular morphine, intra-articular bupivacaine, or subcutaneous

Pringle, E. K. 2010. In vivo effects of single intra-articular injection of 0.5%

tenoxicam improves postoperative analgesia in knee arthroscopy. *Can J Anaesth,* 46,

articular ketorolac for pain relief following arthroscopy of the knee joint.


Local Anesthetic Agents **in** Arthroscopy 175

Kligman, M., Bruskin, A., Sckliamser, J., Vered, R. & Roffman, M. 2002. Intra-synovial,

Lo, I. K., Sciore, P., Chung, M., Liang, S., Boorman, R. B., Thornton, G. M., Rattner, J. B. &

Middleton, F., Coakes, J., Umarji, S., Palmer, S., Venn, R. & Panayiotou, S. 2006. The efficacy

Miyazaki, T., Kobayashi, S., Takeno, K., Yayama, T., Meir, A. & Baba, H. 2011. Lidocaine

Niemi, L., Pitkanen, M., Tuominen, M., Bjorkenheim, J. M. & Rosenberg, P. H. 1994.

Park, J., Sutradhar, B. C., Hong, G., Choi, S. H. & Kim, G. 2011. Comparison of the cytotoxic

Piper, S. L. & Kim, H. T. 2008. Comparison of ropivacaine and bupivacaine toxicity in

Pooni, J. S., Hickmott, K., Mercer, D., Myles, P. & Khan, Z. 1999. Comparison of intra-

Raj, N., Sehgal, A., Hall, J. E., Sharma, A., Murrin, K. R. & Groves, N. D. 2004. Comparison

intramuscular morphine for knee arthroscopy. *Eur J Anaesthesiol,* 21, 932-7. Rasmussen, S., Lorentzen, J. S., Larsen, A. S., Thomsen, S. T. & Kehlet, H. 2002. Combined

Richardson, M. D., Bjorksten, A. R., Hart, J. A. & McCullough, K. 1997. The efficacy of intra-

Rosseland, L. A. 2005. No evidence for analgesic effect of intra-articular morphine after knee arthroscopy: a qualitative systematic review. *Reg Anesth Pain Med,* 30, 83-98. Rosseland, L. A., Helgesen, K. G., Breivik, H. & Stubhaug, A. 2004. Moderate-to-severe pain

Rosseland, L. A., Stubhaug, A., Grevbo, F., Reikeras, O. & Breivik, H. 2003. Effective pain

Rosseland, L. A., Stubhaug, A., Skoglund, A. & Breivik, H. 1999. Intra-articular morphine for pain relief after knee arthroscopy. *Acta Anaesthesiol Scand,* 43, 252-7.

and proteoglycan metabolism. *Knee Surg Sports Traumatol Arthrosc*.

human articular chondrocytes. *J Bone Joint Surg Am,* 90, 986-91.

following day-case knee arthroscopy. *Can J Anaesth,* 47, 220-4.

regional anaesthesia. *Acta Anaesthesiol Scand,* 38, 402-5.

knee arthroscopy. *Eur J Anaesthesiol,* 16, 708-11.

trial. *Anesth Analg,* 98, 1546-51, table of contents.

controlled clinical study. *Acta Anaesthesiol Scand,* 47, 732-8.

arthroscopy menisectomy. *Can J Anaesth,* 49, 380-3.

*Arthroscopy,* 14, 192-6.

*Bone Joint Surg Br,* 88, 1603-5.

*Vet Anaesth Analg,* 38, 127-33.

compared to intra-articular morphine provides better pain relief following knee

Muldrew, K. 2009. Local anesthetics induce chondrocyte death in bovine articular cartilage disks in a dose- and duration-dependent manner. *Arthroscopy,* 25, 707-15. Lundin, O., Rydgren, B., Sward, L. & Karlsson, J. 1998. Analgesic effects of intra-articular

morphine during and after knee arthroscopy: a comparison of two methods.

of intra-articular bupivacaine for relief of pain following arthroscopy of the ankle. *J* 

cytotoxicity to the bovine articular chondrocytes in vitro: changes in cell viability

Intraarticular morphine for pain relief after knee arthroscopy performed under

effects of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes.

articular fentanyl and intra-articular bupivacaine for post-operative pain relief after

of the analgesic efficacy and plasma concentrations of high-dose intra-articular and

intra-articular glucocorticoid, bupivacaine and morphine reduces pain and convalescence after diagnostic knee arthroscopy. *Acta Orthop Scand,* 73, 175-8. Rautoma, P., Santanen, U., Avela, R., Luurila, H., Perhoniemi, V. & Erkola, O. 2000.

Diclofenac premedication but not intra-articular ropivacaine alleviates pain

articular morphine for postoperative knee arthroscopy analgesia. *Arthroscopy,* 13, 584-9.

after knee arthroscopy is relieved by intraarticular saline: a randomized controlled

relief from intra-articular saline with or without morphine 2 mg in patients with moderate-to-severe pain after knee arthroscopy: a randomized, double-blind

study of intravenous regional analgesia versus intra-articular analgesia. *Arthroscopy,* 16, 64-6.


Grishko, V., Xu, M., Wilson, G. & Pearsall, A. W. t. 2010. Apoptosis and mitochondrial

Gupta, A., Axelsson, K., Allvin, R., Liszka-Hackzell, J., Rawal, N., Althoff, B. & Augustini, B.

Hansen, B. P., Beck, C. L., Beck, E. P. & Townsley, R. W. 2007. Postarthroscopic

Hege-Scheuing, G., Michaelsen, K., Buhler, A., Kustermann, J. & Seeling, W. 1995. [Analgesia

Izdes, S., Orhun, S., Turanli, S., Erkilic, E. & Kanbak, O. 2003. The effects of preoperative

Jacobs, T. F., Vansintjan, P. S., Roels, N., Herregods, S. S., Verbruggen, G., Herregods, L. L. &

human cartilage cells: an in vitro study. *Knee Surg Sports Traumatol Arthrosc*. Jacobson, E., Assareh, H., Cannerfelt, R., Anderson, R. E. & Jakobsson, J. G. 2006. The

Jaureguito, J. W., Wilcox, J. F., Cohn, S. J., Thisted, R. A. & Reider, B. 1995. A comparison of

Jawish, D., Antakly, M. C., Dagher, F., Nasser, E. & Geahchan, N. 1996. [Intra-articular

Joshi, G. P., McCarroll, S. M., Cooney, C. M., Blunnie, W. P., O'Brien, T. M. & Lawrence, A. J.

Joshi, G. P., McCarroll, S. M., O'Brien, T. M. & Lenane, P. 1993. Intraarticular analgesia

Juelsgaard, P., Dalsgaard, J., Felsby, S. & Frokjaer, J. 1993. [Analgesic effect of 2 different

randomized, prospective, double-blind study]. *Ugeskr Laeger,* 155, 4169-72. Kanbak, M., Akpolat, N., Ocal, T., Doral, M. N., Ercan, M. & Erdem, K. 1997. Intraarticular

Karaman, Y., Kayali, C., Ozturk, H., Kaya, A. & Bor, C. 2009. A comparison of analgesic

Karpie, J. C. & Chu, C. R. 2007. Lidocaine exhibits dose- and time-dependent cytotoxic effects on bovine articular chondrocytes in vitro. *Am J Sports Med,* 35, 1621-7.

analgesia after arthroscopy of the knee]. *Cah Anesthesiol,* 44, 415-7.

randomized study with patient-controlled analgesia]. *Anaesthesist,* 44, 351-8. Hennig, G. S., Hosgood, G., Bubenik-Angapen, L. J., Lauer, S. K. & Morgan, T. W. 2010.

and ropivacaine. *J Bone Joint Surg Am,* 92, 609-18.

ketorolac and/or morphine. *Reg Anesth Pain Med,* 24, 225-30.

glenohumeral chondrolysis. *Am J Sports Med,* 35, 1628-34.

0.5% solution of bupivacaine. *Am J Vet Res,* 71, 875-83.

study. *Knee Surg Sports Traumatol Arthrosc,* 14, 120-4.

following knee arthroscopy. *Anesth Analg,* 76, 333-6.

knee arthroscopy. *Anesth Analg,* 97, 1016-9, table of contents.

*Arthroscopy,* 16, 64-6.

23, 350-3.

*Surg Br,* 74, 749-51.

*Anaesthesiol,* 14, 153-6.

arthroscopy. *Saudi Med J,* 30, 629-32.

study of intravenous regional analgesia versus intra-articular analgesia.

dysfunction in human chondrocytes following exposure to lidocaine, bupivacaine,

G. 1999. Postoperative pain following knee arthroscopy: the effects of intra-articular

with intra-articular morphine following knee joint arthroscopy? A double-blind,

Evaluation of chondrocyte death in canine osteochondral explants exposed to a

inflammation on the analgesic efficacy of intraarticular piroxicam for outpatient

Almqvist, K. F. 2011. The effect of Lidocaine on the viability of cultivated mature

postoperative analgesic effects of intra-articular levobupivacaine in elective daycase arthroscopy of the knee: a prospective, randomized, double-blind clinical

intraarticular morphine and bupivacaine for pain control after outpatient knee arthroscopy. A prospective, randomized, double-blinded study. *Am J Sports Med,*

1992. Intra-articular morphine for pain relief after knee arthroscopy. *J Bone Joint* 

doses of intra-articular morphine after ambulatory knee arthroscopy. A

morphine administration provides pain relief after knee arthroscopy. *Eur J* 

effect of intra-articular levobupivacaine with bupivacaine following knee


**9** 

*Turkey* 

**Multimodal Analgesia for** 

*Sisli Etfal Training and Research Hospital,* 

G. Ulufer Sivrikaya

**Postoperative Pain Management** 

*Department of 2nd Anesthesiology and Reanimation, Istanbul,* 

The experience of pain is complex, multifaceted, and "an unpleasant sensory and emotional experience," as defined in part by the International Association for the Study of Pain. It is a personal, subjective experience that involves sensory, emotional and behavioral factors associated with actual or potential tissue injury (Rawal). The differential behavior response to surgical incision can be influenced by many variables including global (i.e., personality, gender, age, cultural background, pre-existing pain syndromes, genetic makeup, kind and type of surgical approach, cultural background) and specific (i.e., fear, anxiety, depression, anger, and coping) psychological factors (Eccleston, 2001). Only by considering all

Millions of surgeries are performed on an annual basis, necessitating the frequent use of acute postoperative pain management. There are many types of surgery and, with few exceptions, all are painful. Fear of uncontrolled pain is among the primary concerns of many

One of the most important factors in determining when a patient can be safely discharged from a surgical facility, and that also has a major influence on the patient's ability to resume his/her normal activities of daily living, is the adequacy of postoperative pain control. Pain is a predictable part of the postoperative experience. Unrelieved postoperative pain may result in clinical and psychological changes that increase morbidity and mortality as well as

The guidelines for acute pain management in the perioperative setting published in 1992 and 1995 (Acute Pain, 1992; American Pain, 1995; Practice Guidelines, 1995) promoted aggressive treatment of acute pain and educate patients about the need to communicate unrelieved pain. Nonetheless these guidelines appear to have had little influence on practice patterns or on improved pain control for patients. In a study of Warfield and Kahn (Warfield&Kahn, 1995) they found three of four patients reported experiencing pain after surgery, and 80% of these patients rated pain after surgery as moderate to extreme. Since their study, newer drugs, techniques and protocols for postoperative pain management have been developed, and minimally invasive surgical techniques, such as endoscopic procedures, are used more frequently. These changes in practice patterns thought that they could affect the management of postoperative pain and patient attitudes about pain. But in a recent study (Apfelbaum, 2003) that assessed patients' postoperative pain experience and

concomitant factors can physicians provide optimal treatment.

costs and that decrease quality of life (Carr& Goudas, 1999).

patients who are about to undergo surgery.

**1. Introduction** 


### **Multimodal Analgesia for Postoperative Pain Management**

G. Ulufer Sivrikaya *Sisli Etfal Training and Research Hospital, Department of 2nd Anesthesiology and Reanimation, Istanbul, Turkey* 

#### **1. Introduction**

176 Pain Management – Current Issues and Opinions

Samoladas, E. P., Chalidis, B., Fotiadis, H., Terzidis, I., Ntobas, T. & Koimtzis, M. 2006. The

Santanen, U., Rautoma, P., Luurila, H. & Erkola, O. 2001. Intra-articular ropivacaine

Solheim, N., Rosseland, L. A. & Stubhaug, A. 2006. Intra-articular morphine 5 mg after knee

Souza, R. H., Issy, A. M. & Sakata, R. K. 2002. [Intra-articular analgesia with morphine,

Syed, H. M., Green, L., Bianski, B., Jobe, C. M. & Wongworawat, M. D. 2011. Bupivacaine

Tamosiunas, R., Brazdzionyte, E., Tarnauskaite-Augutiene, A. & Tranauskaite-Keraitiene, G.

Townshend, D., Emmerson, K., Jones, S., Partington, P. & Muller, S. 2009. Intra-articular

VanNess, S. A. & Gittins, M. E. 1994. Comparison of intra-articular morphine and

Varrassi, G., Marinangeli, F., Ciccozzi, A., Iovinelli, G., Facchetti, G. & Ciccone, A. 1999.

Vranken, J. H., Vissers, K. C., de Jongh, R. & Heylen, R. 2001. Intraarticular sufentanil

Webb, S. T. & Ghosh, S. 2009. Intra-articular bupivacaine: potentially chondrotoxic? *Br J* 

White, A. P., Laurent, S. & Wilkinson, D. J. 1990. Intra-articular and subcutaneous prilocaine

Yang, L. C., Chen, L. M., Wang, C. J. & Buerkle, H. 1998. Postoperative analgesia by intra-

Zeidan, A., Kassem, R., Nahleh, N., Maaliki, H., El-Khatib, M., Struys, M. M. & Baraka, A.

bupivacaine following knee arthroscopy. *Orthop Rev,* 23, 743-7.

double-blind study. *Acta Anaesthesiol Scand,* 43, 51-5.

*Orthop Surg Res,* 1, 17.

52, 570-80.

92, 625-8.

107, 292-9.

*Anaesth,* 102, 439-41.

*Surg Engl,* 72, 350-2.

*Orthop Relat Res*.

spinal anaesthesia. *Ann Chir Gynaecol,* 90, 47-50.

contributors and causal pathways. *Arthroscopy,* 25, 1329-42.

intra-articular use of ropivacaine for the control of post knee arthroscopy pain. *J* 

injection does not alleviate pain after day-case knee arthroscopy performed under

arthroscopy does not produce significant pain relief when administered to patients with moderate to severe pain via an intra-articular catheter. *Reg Anesth Pain Med,* 31, 506-13. Solomon, D. J., Navaie, M., Stedje-Larsen, E. T., Smith, J. C. & Provencher, M. T. 2009.

Glenohumeral chondrolysis after arthroscopy: a systematic review of potential

bupivacaine or fentanyl after knee video-arthroscopy surgery.]. *Rev Bras Anestesiol,*

and Triamcinolone May Be Toxic To Human Chondrocytes: A Pilot Study. *Clin* 

2005. [Postoperative analgesia with intraarticular local anesthetic bupivacaine and alpha2-agonist clonidine after arthroscopic knee surgery]. *Medicina (Kaunas),* 41, 547-52.

injection versus portal infiltration of 0.5% bupivacaine following arthroscopy of the knee: a prospective, randomised double-blinded trial. *J Bone Joint Surg Br,* 91, 601-3.

Intra-articular buprenorphine after knee arthroscopy. A randomised, prospective,

administration facilitates recovery after day-case knee arthroscopy. *Anesth Analg,*

with adrenaline for pain relief in day case arthroscopy of the knee joint. *Ann R Coll* 

articular neostigmine in patients undergoing knee arthroscopy. *Anesthesiology,* 88, 334-9.

2008. Intraarticular tramadol-bupivacaine combination prolongs the duration of postoperative analgesia after outpatient arthroscopic knee surgery. *Anesth Analg,*

The experience of pain is complex, multifaceted, and "an unpleasant sensory and emotional experience," as defined in part by the International Association for the Study of Pain. It is a personal, subjective experience that involves sensory, emotional and behavioral factors associated with actual or potential tissue injury (Rawal). The differential behavior response to surgical incision can be influenced by many variables including global (i.e., personality, gender, age, cultural background, pre-existing pain syndromes, genetic makeup, kind and type of surgical approach, cultural background) and specific (i.e., fear, anxiety, depression, anger, and coping) psychological factors (Eccleston, 2001). Only by considering all concomitant factors can physicians provide optimal treatment.

Millions of surgeries are performed on an annual basis, necessitating the frequent use of acute postoperative pain management. There are many types of surgery and, with few exceptions, all are painful. Fear of uncontrolled pain is among the primary concerns of many patients who are about to undergo surgery.

One of the most important factors in determining when a patient can be safely discharged from a surgical facility, and that also has a major influence on the patient's ability to resume his/her normal activities of daily living, is the adequacy of postoperative pain control. Pain is a predictable part of the postoperative experience. Unrelieved postoperative pain may result in clinical and psychological changes that increase morbidity and mortality as well as costs and that decrease quality of life (Carr& Goudas, 1999).

The guidelines for acute pain management in the perioperative setting published in 1992 and 1995 (Acute Pain, 1992; American Pain, 1995; Practice Guidelines, 1995) promoted aggressive treatment of acute pain and educate patients about the need to communicate unrelieved pain. Nonetheless these guidelines appear to have had little influence on practice patterns or on improved pain control for patients. In a study of Warfield and Kahn (Warfield&Kahn, 1995) they found three of four patients reported experiencing pain after surgery, and 80% of these patients rated pain after surgery as moderate to extreme. Since their study, newer drugs, techniques and protocols for postoperative pain management have been developed, and minimally invasive surgical techniques, such as endoscopic procedures, are used more frequently. These changes in practice patterns thought that they could affect the management of postoperative pain and patient attitudes about pain. But in a recent study (Apfelbaum, 2003) that assessed patients' postoperative pain experience and

Multimodal Analgesia for Postoperative Pain Management 179

Increased oxygen consumption (with negative impact in the case of coronary artery

 Impaired gastrointestinal motility (while opioids induce constipation or nausea, untreated pain may also be an important cause of impaired bowel movement or

Delays mobilisation and promotes thromboembolism (postoperative pain is one of the

 increased release of catecholamines (resulting increase in systemic vascular resistance, cardiac work and myocardial oxygen consumption associated negative

reduced blood flow in lower extremities (resulting a higher risk of deep vein

Risk of behavioural changes (frequently in children for a prolonged period after

Chronic pain is a potential adverse outcome from surgery. It is costly to society in terms of suffering and disability. For humanitarian and economic reasons, the problem of chronic pain after surgery should be addressed. In a review of Perkins et al. (Perkins&Kehlet, 2000) they showed there was a significant variability in the incidence of chronic pain among surgical procedures (i.e. 3-56% for cholecystectomy, 0-37% for inguinal hernia surgery, 11- 57% for breast surgery). They concluded that chronic pain after surgery was common as that has been confirmed with another review (Camann, 1998). Another conclusion of this study is; the intensity of acute postoperative pain was one of the most striking predictive factor for chronic pain, especially following breast surgery (Elia, 2005), thorasic surgery

Advances in the knowledge of molecular mechanisms have led to the development of

Postoperative pain treatment may not be enough to provide major improvements in some outcomes because it is unlikely that a unimodal intervention can be effective in addressing a complex problem such as perioperative outcomes (Boisseau, 2001; Kehlet&Nolte, 2001). The analgesic benefits of controlling postoperative pain are generally maximized when a multimodal strategy to facilitate the patient's convalescence is implemented (Kehlet, 1997). Pain involves multiple mechanisms that ideally require treatment using a multimodal (or 'balanced') analgesic technique (White&Kehlet, 2010) Principles of a multimodal strategy include control of postoperative pain to allow early mobilization, early enteral nutrition, education, and attenuation of the perioperative stress response through the use of regional anesthetic techniques and a combination of analgesic

multimodal analgesia and new pharmaceutical products to treat postoperative pain.

disease, leading to coronary ischemia and myocardial infarction)

effects in patients with coronary artery diseases)

(Carli F,2002; Viscusi, 2004) and hernia repair (Birnbach, 1989).

**3. Multimodal approach to postoperative pain** 

 Severe acute pain is a risk factor for the development of chronic pain Sleep disturbance (with negative impact on mood and mobilisation)

postoperative nausea and vomiting-PONV)

major causes for delayed mobilisation)

Increased sympathetic activity

thrombosis)

Delay in long-term recovery

agents (i.e., multimodal analgesia).

**2.2 Chronic effects** 

surgical pain) Poor wound healing

the status of acute pain management in a random sample, approximately 80 percent of patients said (not very different the previous study mentioned above) they experienced acute pain after surgery. The authors concluded that; despite an increased focus on pain management programs and the development of new standards for pain management, many patients continue to experience intense pain after surgery.

Effective and appropriate pain management requires a proactive approach using a variety of treatment modalities to obtain an optimal outcome with respect to facilitating rapid recovery and returning to full function, allowing early discharge from hospital, improving quality of life for the patient and reducing morbidity (Rawal). Protocols for postoperative pain treatment should be made with considering patients' needs, surgical indications, and institutional resources. It is important to use effective state-of-the-art techniques combined with hospital protocols for early rehabilitation and recovery.

Many options are available for the treatment of postoperative pain, including systemic (i.e., opioid and nonopioid) analgesics and regional (i.e., neuraxial and peripheral) analgesic techniques. Multimodal analgesia is achieved by combining different analgesics that act by different mechanisms and at different sites in the nervous system, resulting in additive or synergistic analgesia with lowered adverse effects of sole administration of individual analgesics (Kehlet&Dahl, 1993). It also refers to concurrent application of analgesic pharmacotherapy in combination with regional analgesia (Elvir Lazo&White, 2010).

This chapter's aim is to overview on the topic of multimodal analgesia for postoperative pain management and to provide an update on the drugs and techniques used for this approach.

#### **2. Consequences of postoperative pain**

When an appropriate analgesic treatment is not given for postoperative pain, various adverse effects might occur in the respiratory, cardiovascular, gastrointestinal, urinary, endocrinological systems, as well as in patient's metabolisms and mentality. These changes were relievable with application of appropriate types of analgesic regimens.

Postoperative pain, especially when poorly controlled, may produce a range of detrimental acute (i.e., adverse physiologic responses) (Vadivelu et al, 2010) and chronic effects (i.e., delayed long-term recovery and chronic pain) (Perkins&Kehlet, 2000). Good pain control after surgery is important to prevent negative outcomes such as tachycardia, hypertension, myocardial ischemia, decrease in alveolar ventilation, immobility, deep venous thrombosis and poor wound healing (Vadivelu et al, 2010; Nett, 2010).

Pathophysiology of acute pain, includes of changes in neuroendocrine, respiratory and renal function, gastrointestinal activity, circulatory and autonomic nervous system activity.

Unsufficient pain management can cause acute and chronic effects:

#### **2.1 Acute effects**

	- Decreased respiratory motion
	- Inhibition of cough and sputum excretion
	- increased release of catecholamines (resulting increase in systemic vascular resistance, cardiac work and myocardial oxygen consumption associated negative effects in patients with coronary artery diseases)
	- reduced blood flow in lower extremities (resulting a higher risk of deep vein thrombosis)

#### **2.2 Chronic effects**

178 Pain Management – Current Issues and Opinions

the status of acute pain management in a random sample, approximately 80 percent of patients said (not very different the previous study mentioned above) they experienced acute pain after surgery. The authors concluded that; despite an increased focus on pain management programs and the development of new standards for pain management, many

Effective and appropriate pain management requires a proactive approach using a variety of treatment modalities to obtain an optimal outcome with respect to facilitating rapid recovery and returning to full function, allowing early discharge from hospital, improving quality of life for the patient and reducing morbidity (Rawal). Protocols for postoperative pain treatment should be made with considering patients' needs, surgical indications, and institutional resources. It is important to use effective state-of-the-art techniques combined

Many options are available for the treatment of postoperative pain, including systemic (i.e., opioid and nonopioid) analgesics and regional (i.e., neuraxial and peripheral) analgesic techniques. Multimodal analgesia is achieved by combining different analgesics that act by different mechanisms and at different sites in the nervous system, resulting in additive or synergistic analgesia with lowered adverse effects of sole administration of individual analgesics (Kehlet&Dahl, 1993). It also refers to concurrent application of analgesic

This chapter's aim is to overview on the topic of multimodal analgesia for postoperative pain management and to provide an update on the drugs and techniques used for this

When an appropriate analgesic treatment is not given for postoperative pain, various adverse effects might occur in the respiratory, cardiovascular, gastrointestinal, urinary, endocrinological systems, as well as in patient's metabolisms and mentality. These changes

Postoperative pain, especially when poorly controlled, may produce a range of detrimental acute (i.e., adverse physiologic responses) (Vadivelu et al, 2010) and chronic effects (i.e., delayed long-term recovery and chronic pain) (Perkins&Kehlet, 2000). Good pain control after surgery is important to prevent negative outcomes such as tachycardia, hypertension, myocardial ischemia, decrease in alveolar ventilation, immobility, deep venous thrombosis

Pathophysiology of acute pain, includes of changes in neuroendocrine, respiratory and renal function, gastrointestinal activity, circulatory and autonomic nervous system activity.

Respiratory system side effects (leading to atelectasis, retention of secretions and

pharmacotherapy in combination with regional analgesia (Elvir Lazo&White, 2010).

were relievable with application of appropriate types of analgesic regimens.

patients continue to experience intense pain after surgery.

with hospital protocols for early rehabilitation and recovery.

**2. Consequences of postoperative pain** 

and poor wound healing (Vadivelu et al, 2010; Nett, 2010).

Emotional and physical suffering for the patient

Inhibition of cough and sputum excretion

Decreased respiratory motion

Unsufficient pain management can cause acute and chronic effects:

Sleep disturbance (with negative impact on mood and mobilisation)

Cardiovascular side effects (such as hypertension and arrhythmias)

approach.

**2.1 Acute effects** 

pneumonia)


Chronic pain is a potential adverse outcome from surgery. It is costly to society in terms of suffering and disability. For humanitarian and economic reasons, the problem of chronic pain after surgery should be addressed. In a review of Perkins et al. (Perkins&Kehlet, 2000) they showed there was a significant variability in the incidence of chronic pain among surgical procedures (i.e. 3-56% for cholecystectomy, 0-37% for inguinal hernia surgery, 11- 57% for breast surgery). They concluded that chronic pain after surgery was common as that has been confirmed with another review (Camann, 1998). Another conclusion of this study is; the intensity of acute postoperative pain was one of the most striking predictive factor for chronic pain, especially following breast surgery (Elia, 2005), thorasic surgery (Carli F,2002; Viscusi, 2004) and hernia repair (Birnbach, 1989).

#### **3. Multimodal approach to postoperative pain**

Advances in the knowledge of molecular mechanisms have led to the development of multimodal analgesia and new pharmaceutical products to treat postoperative pain.

Postoperative pain treatment may not be enough to provide major improvements in some outcomes because it is unlikely that a unimodal intervention can be effective in addressing a complex problem such as perioperative outcomes (Boisseau, 2001; Kehlet&Nolte, 2001). The analgesic benefits of controlling postoperative pain are generally maximized when a multimodal strategy to facilitate the patient's convalescence is implemented (Kehlet, 1997). Pain involves multiple mechanisms that ideally require treatment using a multimodal (or 'balanced') analgesic technique (White&Kehlet, 2010) Principles of a multimodal strategy include control of postoperative pain to allow early mobilization, early enteral nutrition, education, and attenuation of the perioperative stress response through the use of regional anesthetic techniques and a combination of analgesic agents (i.e., multimodal analgesia).

Multimodal Analgesia for Postoperative Pain Management 181

A lower incidence of adverse effects and improved analgesia has been demonstrated with multimodal analgesia techniques, which may provide for shorter hospitalization times, improved recovery and function, and possibly decreased healthcare costs (Buvanendran &

To achieve a maximum short-term and long-term benefits from multimodal analgesic therapies, the pain management would be initiated as a preventive in the preoperative period continued in the early postoperative period and extended into the postcharge period for 3-7 days (Bisgaard, 2006; White et al, 2007). A deficiency in the design of many of the published studies involving multimodal analgesic therapies is that the drug regimens were not continued into the postdischarge period (Ma, 2004). For example, only immediate pre- and postoperative administration of the cyclooxygenase 2 (COX-2) inhibitor rofecoxib as part of a multimodal analgesic regimen in outpatients undergoing inguinal hernia repair provided limited benefits beyond the early postoperative period (White&Kehlet, 2007). However, when the COX-2 inhibitors are administered for 3 to 5 days after ambulatory surgery, (Gan, 2004; Joshi, 2004) the greater benefits were achieved with respect to clinically relevant patient outcomes (eg, resumption of normal activities) and improvements in pain control. Bisgaard et al (Bisgaard, 2006) concluded that a multimodal analgesic regimen consisting of a preoperative single dose of dexamethasone, incisional local anesthetics (at the beginning and/or end of surgery), and continuous treatment with NSAIDs (or COX-2 inhibitors) during the first 3 to 4 days provided the best clinical outcome. Moreover, recent clinical studies suggest that when classical NSAIDs or more selective COX-2 inhibiting drugs were administered for 3–5 days after ambulatory surgery, a significant benefit was achieved with respect to clinically relevant patient outcomes (e.g., resumption of normal activities) and improvements in short-term

A multimodal analgesic regimen should be adjusted to meet the needs of the individual patient by taking into consideration their pre-existing medical conditions, types of surgery, and previous experiences related to both acute and chronic pain management. Critical multimodal protocols must be designed based on surgical procedures and structural organization to warrant improved outcome including having minimum side effects related to the treatment and rapid returning to social life and daily activities

Several multimodal approaches have been advocated based on different combinations of anti-inflammatory drugs, and regional anesthesia (epidural, peripheral nerve blocks, paravertebral blocks, and local injection/infusion of local anesthetics) (Buvanendran, 2010; Mathiesen, 2009). Although each of these drugs and/or techniques has been demonstrated as being effective in reducing the need for postoperative intravenous opioids alone, the evidence supporting specific combinations of drugs and/or regional techniques is still

Faster recovery, reduced hospital stay, and decreased length of convalescence can occur if multimodal analgesia is combined with a rehabilitation program that is multidisciplinary

Treatment of postoperative pain requires good multi-disciplinary and multi-professional cooperation. Multidisciplinary team consists of the anesthesiologist himself has overall

Kroin, 2009).

pain control (Gan, 2004; White, 2007).

**3.1 Multidisciplinary approach** 

and multimodal (Gajraj&Joshi, 2005).

(Fanelli, 2008).

limited.

The concept of multimodal analgesia was introduced more than a decade ago as a technique to improve analgesia and reduce the incidence of opioid-related adverse events. Multimodal analgesia is achieved by combining different analgesics that act by different mechanisms at different sites in the nervous system, reducing the incidence of side effects owing to the lower doses of the individual drugs (Buvanendran&Kroin 2009). For example, epidural opioids can be administered in combination with epidural local anesthetics; intravenous opioids can be administered in combination with Nonsteroidal Antiinflammatory Drug (NSAID)s, which have a dose sparing effect for systemically administered opioids. It also refers to concurrent application of analgesic pharmacotherapy in combination with regional analgesia (Elvir Lazo&White, 2010; Rawal).

In the literature some different definitions of multimodal analgesia exists. In some contexts, multimodal analgesia refers to systemic administration of analgesic drugs with different mechanisms of action, whereas in other situations it refers to concurrent application of analgesic pharmacotherapy in combination with regional analgesia. Multimodal analgesia is based on to choice paracetamol and NSAIDs for low intensity pain with opioid analgesics and/or local analgesia techniques being used for moderate and high intensity pain as indicated. (Elvir Lazo&White, 2010; Rawal) (Table 1)


Table 1. Treatment options in relation to magnitude of postoperative pain expected following different types of surgery (by permission from Publisher AstraZeneca)

The concept of multimodal analgesia was introduced more than a decade ago as a technique to improve analgesia and reduce the incidence of opioid-related adverse events. Multimodal analgesia is achieved by combining different analgesics that act by different mechanisms at different sites in the nervous system, reducing the incidence of side effects owing to the lower doses of the individual drugs (Buvanendran&Kroin 2009). For example, epidural opioids can be administered in combination with epidural local anesthetics; intravenous opioids can be administered in combination with Nonsteroidal Antiinflammatory Drug (NSAID)s, which have a dose sparing effect for systemically administered opioids. It also refers to concurrent application of analgesic pharmacotherapy in combination with regional analgesia (Elvir Lazo&White, 2010;

In the literature some different definitions of multimodal analgesia exists. In some contexts, multimodal analgesia refers to systemic administration of analgesic drugs with different mechanisms of action, whereas in other situations it refers to concurrent application of analgesic pharmacotherapy in combination with regional analgesia. Multimodal analgesia is based on to choice paracetamol and NSAIDs for low intensity pain with opioid analgesics and/or local analgesia techniques being used for moderate and high intensity pain as

**Mild intensity pain Moderate intensity pain Severe intensity pain** 

*For example:*  Thoracotomy

Aortic surgery Knee replacement

(IV PCA)

(i) Paracetamol and wound infiltration with local anesthetic

(single shot or continuous infusion) or opioid injection (IV

(ii) NSAIDs (unless contraindicated) and

(iii) Peripheral nerve block

Add weak opioid or rescue analgesia with small increments of intravenous strong opioid

Table 1. Treatment options in relation to magnitude of postoperative pain expected following different types of surgery (by permission from Publisher AstraZeneca)

PCA) (i) Paracetamol and wound infiltration with local anesthetic

(ii) NSAIDs (unless contraindicated) and

(iii) Regional block analgesia

if necessary

(ii) NSAIDs (unless contraindicated) and

Upper abdominal surgery

(i) Paracetamol and wound infiltration with local anesthetic

(iii) Epidural local analgesia or major peripheral nerve or plexus block or opioid injection

*For example:*  Hip replacement Hysterectomy Jaw surgery

indicated. (Elvir Lazo&White, 2010; Rawal) (Table 1)

Rawal).

*For example:*  Inguinal hernia

Varices Laparoscopy A lower incidence of adverse effects and improved analgesia has been demonstrated with multimodal analgesia techniques, which may provide for shorter hospitalization times, improved recovery and function, and possibly decreased healthcare costs (Buvanendran & Kroin, 2009).

To achieve a maximum short-term and long-term benefits from multimodal analgesic therapies, the pain management would be initiated as a preventive in the preoperative period continued in the early postoperative period and extended into the postcharge period for 3-7 days (Bisgaard, 2006; White et al, 2007). A deficiency in the design of many of the published studies involving multimodal analgesic therapies is that the drug regimens were not continued into the postdischarge period (Ma, 2004). For example, only immediate pre- and postoperative administration of the cyclooxygenase 2 (COX-2) inhibitor rofecoxib as part of a multimodal analgesic regimen in outpatients undergoing inguinal hernia repair provided limited benefits beyond the early postoperative period (White&Kehlet, 2007). However, when the COX-2 inhibitors are administered for 3 to 5 days after ambulatory surgery, (Gan, 2004; Joshi, 2004) the greater benefits were achieved with respect to clinically relevant patient outcomes (eg, resumption of normal activities) and improvements in pain control. Bisgaard et al (Bisgaard, 2006) concluded that a multimodal analgesic regimen consisting of a preoperative single dose of dexamethasone, incisional local anesthetics (at the beginning and/or end of surgery), and continuous treatment with NSAIDs (or COX-2 inhibitors) during the first 3 to 4 days provided the best clinical outcome. Moreover, recent clinical studies suggest that when classical NSAIDs or more selective COX-2 inhibiting drugs were administered for 3–5 days after ambulatory surgery, a significant benefit was achieved with respect to clinically relevant patient outcomes (e.g., resumption of normal activities) and improvements in short-term pain control (Gan, 2004; White, 2007).

A multimodal analgesic regimen should be adjusted to meet the needs of the individual patient by taking into consideration their pre-existing medical conditions, types of surgery, and previous experiences related to both acute and chronic pain management. Critical multimodal protocols must be designed based on surgical procedures and structural organization to warrant improved outcome including having minimum side effects related to the treatment and rapid returning to social life and daily activities (Fanelli, 2008).

Several multimodal approaches have been advocated based on different combinations of anti-inflammatory drugs, and regional anesthesia (epidural, peripheral nerve blocks, paravertebral blocks, and local injection/infusion of local anesthetics) (Buvanendran, 2010; Mathiesen, 2009). Although each of these drugs and/or techniques has been demonstrated as being effective in reducing the need for postoperative intravenous opioids alone, the evidence supporting specific combinations of drugs and/or regional techniques is still limited.

#### **3.1 Multidisciplinary approach**

Faster recovery, reduced hospital stay, and decreased length of convalescence can occur if multimodal analgesia is combined with a rehabilitation program that is multidisciplinary and multimodal (Gajraj&Joshi, 2005).

Treatment of postoperative pain requires good multi-disciplinary and multi-professional cooperation. Multidisciplinary team consists of the anesthesiologist himself has overall

Multimodal Analgesia for Postoperative Pain Management 183

This approach does not seem to offer any clinically significant advantages over so-called preventative multimodal analgesic regimens when an effective pro-active approach to pain management is initiated in the early postoperative period and extended into the postdischarge period (Sun, 2008). Starting with intensive pain therapy at the beginning and analgesia must be continued, using step-down techniques that involve a change in drugs or route of administration (*i.e.*, from the epidural and intravenous routes to *per os* 

The main goals of preventive analgesia are: to decrease pain after tissue injury, to prevent spinal sensitization and to reduce the incidence of inflammatory or chronic pain (Senturk, 2002).

Multimodal (or balanced) analgesia represents an increasingly popular approach to preventing postoperative pain. The approach involves administering a combination of opioid and nonopioid analgesics (and adjuvant agents) that act at different sites within the central and peripheral nervous systems in an effort to improve pain control, with fewer opioid-related side effects mainly sedation, nausea, vomiting pruritis, constipation (Elvir

The development of newer agents available for postoperative pain control opens up possibilities for newer combinations in multimodal analgesia. Multi-pharmacological therapy based on synergistic effects of two or more drugs gives better results than a mono-

Opioid analgesics continue to play an important role in the acute treatment of moderate-tosevere pain in the early postoperative period. The problem about these drugs is the variety of perioperative complications eg, drowsiness and sedation, PONV, pruritus, urinary retention, ileus, constipation, ventilatory depression of them. These opioid-related adverse effects inhibit rapid recovery and rehabilitation (Buvanendran&Kroin, 2009; Vadivelu, 2010). Their effects can be summarized as hyperpolarization of first- and second-order sensory neurons, with inhibition of synaptic transmission. They act by binding to μ receptors, which initially results in increased G protein activity; this, in turn, leads to K+ efflux and inhibition of Ca2+ influx into the cell. Opioids also stimulate the supraspinal descending inhibitory system, which further increases the hyperpolarization of second-order neurons by releasing 5-HT and glycine. Opioid receptors have been demonstrated *in vitro* in peripheral nerve terminals, but they are unable to influence the inflammatory reaction, resulting lack of

Opioids can be used in different ways; i.e intravenous, intramuscular, subcutaneous, transmucosal, epidural, intrathecal, transdermal. The most common route of postoperative systemic opioid analgesic administration is intravenous. When the most important source of nociceptive stimuli is visceral pain, good results may be achieved by intrathecal administration

Patient controlled analgesia (PCA) optimizes delivery of analgesic opioids and minimizes the effects of pharmacokinetic and pharmacodynamics variability in individual patients. It can be programmed for several variables: demand (bolus) dose, lockout interval, continuous or basal infusion, and 4 h limit (Table 2). PCA provides superior postoperative analgesia and improves patient satisfaction when compared with traditional PRN analgesic regimens.

effectiveness on postoperative pain during movement (Christie, 2000).

administration) (Fanelli, 2008).

Lazo&White, 2010; Vadivelu, 2010).

**4.1 Opioids** 

pharmacologic approach (Vadivelu, 2010).

of small doses of opioids (Rathmell, 2005).

**4. Drugs in postoperative pain management** 

responsibility, pain nurse and specialist surgeon, sometimes pharmacist. In the ward the patient's physician and nurse, physiotherapist when needed are responsible for all care, in partnership with the pain team. The nurse is responsible to report the patient's intensity of pain to the physician and to treat the pain within the defined rules of the local guidelines. Also should pay attention to the effects and side effects of the pain treatment. The pain team nurse is the first point of contact while the anesthesiologist and pharmacist are available to provide specialist advice (Rawal).

All staff involved in the treatment of postoperative pain require regularly updated training emphasising the importance of team-working and co-operation. In this training programme the main headings should be included as; a. Physiology and pathophysiology of pain, b. Pharmacology of analgesics, c. Locally available treatment methods d. Monitoring routines with regard to treatment of pain and e. Local document for treatment and assessment of pain (Rawal).

It is important to understand of the postoperative pain experience from a patient's perspective, if health care professionals are to identify ways to improve care. In Apfelbaum et al study (Apfelbaum et al, 2003); when asked about attitudes regarding pain and pain medications, 75% of patients believed that it was necessary to experience some pain after surgery, and 8% of patients had postponed surgery because they were worried about the possibility of experiencing pain. Although most patients claimed to receive preoperative education on postoperative pain management, that study's findings suggested that a patient's real concern is not adequately addressed.

The patient himself and family members are also to be undertaken as the members of the multidisciplinary team. Education is an important role in this point. Patients are unlikely to be aware of postoperative pain treatment techniques and as the success of pain relief is influenced by their knowledge and beliefs, it is helpful to give patients (and parents in case of cognitively impaired, severely emotionally disturbed, children) detailed information about postoperative pain and pain treatment. Adequate information gives the patient realistic expectations of the care that can be provided (pain relief, not a "pain free status"). Patients who do not speak the local language, and patients whose level of education or cultural background differs significantly from that of their health care team need special concern. A preoperative discussion with the patient and relatives can be helpful about an effective postoperative pain management (Rawal).

It is important to emphasize the need for collaboration between the various health care providers involved in the patient's perioperative care (eg, anesthesiologists, surgeons, nurses, and physiotherapists) to integrate improved perioperative pain management strategies with the recently described fast-track recovery paradigms (White, 2007). This type of multi-disciplinary approach has been documented to improve the quality of the recovery process and reduce the hospital stay and postoperative morbidity, leading to a shorter period of convalescence after surgery (White&Kehlet, 2010).

#### **3.2 Pre-emptive – Preventive analgesia**

The concept of pre-emptive analgesia has its origins in the idea that painful stimuli, if not prevented by administration of preoperative analgesic drugs, could lead to spinal sensitization and neuroplasticity processes, resulting in increased pain intensity and duration after surgery. Many authors have studied the effects of different timing of administration of single drugs (*e.g*., pre-, intra- or postoperative) and have reported no differences in efficacy (Moiniche, 2002).

This approach does not seem to offer any clinically significant advantages over so-called preventative multimodal analgesic regimens when an effective pro-active approach to pain management is initiated in the early postoperative period and extended into the postdischarge period (Sun, 2008). Starting with intensive pain therapy at the beginning and analgesia must be continued, using step-down techniques that involve a change in drugs or route of administration (*i.e.*, from the epidural and intravenous routes to *per os*  administration) (Fanelli, 2008).

The main goals of preventive analgesia are: to decrease pain after tissue injury, to prevent spinal sensitization and to reduce the incidence of inflammatory or chronic pain (Senturk, 2002).

#### **4. Drugs in postoperative pain management**

Multimodal (or balanced) analgesia represents an increasingly popular approach to preventing postoperative pain. The approach involves administering a combination of opioid and nonopioid analgesics (and adjuvant agents) that act at different sites within the central and peripheral nervous systems in an effort to improve pain control, with fewer opioid-related side effects mainly sedation, nausea, vomiting pruritis, constipation (Elvir Lazo&White, 2010; Vadivelu, 2010).

The development of newer agents available for postoperative pain control opens up possibilities for newer combinations in multimodal analgesia. Multi-pharmacological therapy based on synergistic effects of two or more drugs gives better results than a monopharmacologic approach (Vadivelu, 2010).

#### **4.1 Opioids**

182 Pain Management – Current Issues and Opinions

responsibility, pain nurse and specialist surgeon, sometimes pharmacist. In the ward the patient's physician and nurse, physiotherapist when needed are responsible for all care, in partnership with the pain team. The nurse is responsible to report the patient's intensity of pain to the physician and to treat the pain within the defined rules of the local guidelines. Also should pay attention to the effects and side effects of the pain treatment. The pain team nurse is the first point of contact while the anesthesiologist and pharmacist are available to

All staff involved in the treatment of postoperative pain require regularly updated training emphasising the importance of team-working and co-operation. In this training programme the main headings should be included as; a. Physiology and pathophysiology of pain, b. Pharmacology of analgesics, c. Locally available treatment methods d. Monitoring routines with regard to treatment of pain and e. Local document for treatment and assessment of pain

It is important to understand of the postoperative pain experience from a patient's perspective, if health care professionals are to identify ways to improve care. In Apfelbaum et al study (Apfelbaum et al, 2003); when asked about attitudes regarding pain and pain medications, 75% of patients believed that it was necessary to experience some pain after surgery, and 8% of patients had postponed surgery because they were worried about the possibility of experiencing pain. Although most patients claimed to receive preoperative education on postoperative pain management, that study's findings suggested that a

The patient himself and family members are also to be undertaken as the members of the multidisciplinary team. Education is an important role in this point. Patients are unlikely to be aware of postoperative pain treatment techniques and as the success of pain relief is influenced by their knowledge and beliefs, it is helpful to give patients (and parents in case of cognitively impaired, severely emotionally disturbed, children) detailed information about postoperative pain and pain treatment. Adequate information gives the patient realistic expectations of the care that can be provided (pain relief, not a "pain free status"). Patients who do not speak the local language, and patients whose level of education or cultural background differs significantly from that of their health care team need special concern. A preoperative discussion with the patient and relatives can be helpful about an

It is important to emphasize the need for collaboration between the various health care providers involved in the patient's perioperative care (eg, anesthesiologists, surgeons, nurses, and physiotherapists) to integrate improved perioperative pain management strategies with the recently described fast-track recovery paradigms (White, 2007). This type of multi-disciplinary approach has been documented to improve the quality of the recovery process and reduce the hospital stay and postoperative morbidity, leading to a shorter

The concept of pre-emptive analgesia has its origins in the idea that painful stimuli, if not prevented by administration of preoperative analgesic drugs, could lead to spinal sensitization and neuroplasticity processes, resulting in increased pain intensity and duration after surgery. Many authors have studied the effects of different timing of administration of single drugs (*e.g*., pre-, intra- or postoperative) and have reported no

provide specialist advice (Rawal).

patient's real concern is not adequately addressed.

effective postoperative pain management (Rawal).

**3.2 Pre-emptive – Preventive analgesia** 

differences in efficacy (Moiniche, 2002).

period of convalescence after surgery (White&Kehlet, 2010).

(Rawal).

Opioid analgesics continue to play an important role in the acute treatment of moderate-tosevere pain in the early postoperative period. The problem about these drugs is the variety of perioperative complications eg, drowsiness and sedation, PONV, pruritus, urinary retention, ileus, constipation, ventilatory depression of them. These opioid-related adverse effects inhibit rapid recovery and rehabilitation (Buvanendran&Kroin, 2009; Vadivelu, 2010). Their effects can be summarized as hyperpolarization of first- and second-order sensory neurons, with inhibition of synaptic transmission. They act by binding to μ receptors, which initially results in increased G protein activity; this, in turn, leads to K+ efflux and inhibition of Ca2+ influx into the cell. Opioids also stimulate the supraspinal descending inhibitory system, which further increases the hyperpolarization of second-order neurons by releasing 5-HT and glycine. Opioid receptors have been demonstrated *in vitro* in peripheral nerve terminals, but they are unable to influence the inflammatory reaction, resulting lack of effectiveness on postoperative pain during movement (Christie, 2000).

Opioids can be used in different ways; i.e intravenous, intramuscular, subcutaneous, transmucosal, epidural, intrathecal, transdermal. The most common route of postoperative systemic opioid analgesic administration is intravenous. When the most important source of nociceptive stimuli is visceral pain, good results may be achieved by intrathecal administration of small doses of opioids (Rathmell, 2005).

Patient controlled analgesia (PCA) optimizes delivery of analgesic opioids and minimizes the effects of pharmacokinetic and pharmacodynamics variability in individual patients. It can be programmed for several variables: demand (bolus) dose, lockout interval, continuous or basal infusion, and 4 h limit (Table 2). PCA provides superior postoperative analgesia and improves patient satisfaction when compared with traditional PRN analgesic regimens.

Multimodal Analgesia for Postoperative Pain Management 185

stay. Nonopioid analgesics will likely assume a greater role as preventive analgesics in the future as the number of minimally invasive (keyhole) surgery cases continues to expand. Recent studies have confirmed that a rational combination of different nonopioid analgesics when given as part of multimodal analgesia reduces postoperative pain. The use of traditional NSAIDs, COX-2 inhibitors, acetaminophen, ketamine, dexmedetomidine, dextromethorphan, alpha2-agonists, gabapentin, pregabalin, and glucocorticoid steroids can provide beneficial effects when administered in appropriate doses as part of a multimodal

analgesic regimen in the perioperative setting (Elvir Lazo&White, 2010). Nonopioid drugs used in postoperative pain management can be classified as:

b. N-methyl-D-aspartate antagonists (Antihyperalgesic drugs)

1. NSAIDs and COX-2 inhibitors

i. Clonidine

i. Ketamine

iii. Magnesium c. Gabapentin-type drugs i. Gabapentin ii. Pregabalin d. Glucocorticoids

a. Alpha-2 adrenergic agonists

ii. Dexmedetomidine

ii. Dextramethorphan

i. Dexamethasone

Nicotine

**4.2.1 NSAIDs and cyclooxygenase-2-selective inhibitors** 

NSAIDs are administered orally, parenterally or by the rectal route.

NSAIDs are known to achieve pain relief by their effect on COX-1 and 2 with the various NSAIDs differing in the proportion to which they inhibit COX-1 and COX-2. They are acid compounds with analgesic, antipyretic and anti-inflammatory properties via inhibition of prostaglandin (PG) synthesis. Prostaglandins, including PG-E2, are responsible for reducing the pain threshold at the site of injury, resulting in central sensitization and a lower pain threshold in the surrounding uninjured tissue. The primary site of action of NSAIDs is believed to be in the periphery though recent research indicates that central inhibition of COX-2 may also play an important role in modulating nociception. NSAIDs inhibit the synthesis of prostaglandins both in the spinal cord and at the periphery, thus diminishing the hyperalgesic state after surgical trauma (Buvanendran&Kroin, 2009; Fanelli, 2008;

NSAIDs are useful as the sole analgesic after minor surgical procedures. They provide moderate postoperative analgesia and thereby have a significant opioid-sparing effect of 20-

e. Newer drugs i. Capsaicin ii. Glyceryl trinitrate iii. Cholinergic drugs

5. Local Anesthetics

McClane, 2010).

2. Acetaminophen 3. Paracetamol 4. Adjuvants


\* Individual patient requirements vary widely. Titrated loading doses can be used if necessary to establish initial analgesia.

\*\* Continuous infusions are not initially recommended for opioid-naive adult patients

Table 2. Intravenous PCA Regimens

Opioid analgesics will likely remain the primary treatment option for patients who require rescue analgesic therapy in the postoperative period until more potent and rapid-acting nonopioid analgesics become available for routine clinical use.

#### **4.1.1 Controlled-release opiods**

Controlled-release opioids are not traditionally considered useful in the immediate postoperative period, but some studies have demonstrated that controlled-release oxycodone may be used for postoperative pain control when remifentanil is used for maintenance of anesthesia. Its preoperative administration leads to adequate plasma concentrations for postoperative analgesia and hyperalgesia treatment following short surgery (1-2 h) (Nishimori, 2006). In addition, controlled-release opioids are an optimal choice for step-down analgesia in the late postoperative and rehabilitation periods following orthopedic surgical procedures (de Beer J de, 2005).

#### **4.1.2 Tramadol**

Tramadol enhances inhibitory effects on pain transmission at the spinal level blocking nociceptive signal transduction both by opioid and monoaminergic mechanisms. Its opioid and nonopioid modes of action appear to act synergistically. The drug is available in formulations suitable for oral, rectal and parenteral administration. Tramadol has been shown to provide effective analgesia after intravenous and oral (in a few of newer clinical studies) administration for postoperative pain management. The main advantage of tramadol in postoperative analgesia is a relative lack of respiratory depression. The potential of abuse is also negligible.

#### **4.2 Nonopioids**

Opioid analgesics, once considered the standard approach to preventing acute postoperative pain, are being replaced by a combination of nonopioid analgesic drugs with diverse modes of action as part of a multimodal approach to preventing pain after ambulatory surgery.

Nonopioid analgesics are increasingly being used before, during, and after surgery to facilitate the recovery process especially after ambulatory surgery because of their anesthetic-and analgesic-sparing effects and their ability to reduce postoperative pain (with movement), opioid analgesic requirement, and side effects, thereby shortening the duration of the hospital stay. Nonopioid analgesics will likely assume a greater role as preventive analgesics in the future as the number of minimally invasive (keyhole) surgery cases continues to expand.

Recent studies have confirmed that a rational combination of different nonopioid analgesics when given as part of multimodal analgesia reduces postoperative pain. The use of traditional NSAIDs, COX-2 inhibitors, acetaminophen, ketamine, dexmedetomidine, dextromethorphan, alpha2-agonists, gabapentin, pregabalin, and glucocorticoid steroids can provide beneficial effects when administered in appropriate doses as part of a multimodal analgesic regimen in the perioperative setting (Elvir Lazo&White, 2010).

Nonopioid drugs used in postoperative pain management can be classified as:


184 Pain Management – Current Issues and Opinions

Morphine (1 mg/ml) 0.5-2 mg 5-10 0-2 mg/h Fentanyl (0.01 mg/ml) 10-20 µg 5-10 0-60 µg/h

Sufentanil (0.002 mg/ml) 2-5 µg 4-10 0-8 µg/h Meperidine (10 mg/ml) 5-25 mg 5-10 0-20 mg/h Tramadol (4-5 mg/ml) 10-20 mg 6-10 0-20 mg/ml \* Individual patient requirements vary widely. Titrated loading doses can be used if necessary to

Opioid analgesics will likely remain the primary treatment option for patients who require rescue analgesic therapy in the postoperative period until more potent and rapid-acting

Controlled-release opioids are not traditionally considered useful in the immediate postoperative period, but some studies have demonstrated that controlled-release oxycodone may be used for postoperative pain control when remifentanil is used for maintenance of anesthesia. Its preoperative administration leads to adequate plasma concentrations for postoperative analgesia and hyperalgesia treatment following short surgery (1-2 h) (Nishimori, 2006). In addition, controlled-release opioids are an optimal choice for step-down analgesia in the late postoperative and rehabilitation periods following

Tramadol enhances inhibitory effects on pain transmission at the spinal level blocking nociceptive signal transduction both by opioid and monoaminergic mechanisms. Its opioid and nonopioid modes of action appear to act synergistically. The drug is available in formulations suitable for oral, rectal and parenteral administration. Tramadol has been shown to provide effective analgesia after intravenous and oral (in a few of newer clinical studies) administration for postoperative pain management. The main advantage of tramadol in postoperative analgesia is a relative lack of respiratory depression. The potential

Opioid analgesics, once considered the standard approach to preventing acute postoperative pain, are being replaced by a combination of nonopioid analgesic drugs with diverse modes of action as part of a multimodal approach to preventing pain after ambulatory surgery. Nonopioid analgesics are increasingly being used before, during, and after surgery to facilitate the recovery process especially after ambulatory surgery because of their anesthetic-and analgesic-sparing effects and their ability to reduce postoperative pain (with movement), opioid analgesic requirement, and side effects, thereby shortening the duration of the hospital

\*\* Continuous infusions are not initially recommended for opioid-naive adult patients

(min)

**Basal-Continuous infusion \*\*** 

**Drug \*- Concentration Bolus dose Lockout interval** 

Alfentanil (0.1 mg/ml) 0.1-0.2 mg 5-8

nonopioid analgesics become available for routine clinical use.

orthopedic surgical procedures (de Beer J de, 2005).

establish initial analgesia.

**4.1.2 Tramadol** 

**4.2 Nonopioids** 

of abuse is also negligible.

Table 2. Intravenous PCA Regimens

**4.1.1 Controlled-release opiods** 

	- i. Clonidine
	- ii. Dexmedetomidine
	- i. Ketamine
	- ii. Dextramethorphan
	- iii. Magnesium
	- i. Gabapentin
	- ii. Pregabalin
	- i. Dexamethasone
	- i. Capsaicin
	- ii. Glyceryl trinitrate
	- iii. Cholinergic drugs

Nicotine

5. Local Anesthetics

#### **4.2.1 NSAIDs and cyclooxygenase-2-selective inhibitors**

NSAIDs are known to achieve pain relief by their effect on COX-1 and 2 with the various NSAIDs differing in the proportion to which they inhibit COX-1 and COX-2. They are acid compounds with analgesic, antipyretic and anti-inflammatory properties via inhibition of prostaglandin (PG) synthesis. Prostaglandins, including PG-E2, are responsible for reducing the pain threshold at the site of injury, resulting in central sensitization and a lower pain threshold in the surrounding uninjured tissue. The primary site of action of NSAIDs is believed to be in the periphery though recent research indicates that central inhibition of COX-2 may also play an important role in modulating nociception. NSAIDs inhibit the synthesis of prostaglandins both in the spinal cord and at the periphery, thus diminishing the hyperalgesic state after surgical trauma (Buvanendran&Kroin, 2009; Fanelli, 2008; McClane, 2010).

NSAIDs are administered orally, parenterally or by the rectal route.

NSAIDs are useful as the sole analgesic after minor surgical procedures. They provide moderate postoperative analgesia and thereby have a significant opioid-sparing effect of 20-

Multimodal Analgesia for Postoperative Pain Management 187

Multimodal analgesia incorporates the use of analgesic adjuncts with different mechanisms of action to enhance postoperative pain management. Adjuvants are important in postoperative pain management due to side effects of opioid analgesics, which hinder recovery, especially in the increasingly utilized ambulatory surgical procedures (Buvanendran&Kroin, 2007). Multiple adjuvants recently have been developed for the

Alpha-2 adrenergic activation represents an intrinsic pain control network of the central nervous system. The alpha-2 adrenergic receptor has high density in the substantia gelatinosa of the dorsal horn in humans and that is believed to be the primary site of action by which alpha-2 adrenergic agonists can reduce pain (Buvanendran&Kroin,

Clonidine is originally classified as an anti-hypertensive drug with negative chronotropic activity, but has antinociceptive properties as well. In the spinal cord, clonidine acts at alpha-2 adrenergic receptors to stimulate acetylcholine release, which acts at both

Clonidine can be administered orally, intravenously, neuraxially or perineurally in combination with local anesthetics. However, the side effects could be significant. The most important ones are hypotension, bradycardia and sedation (Rawal). Data about the systemic administration of clonidine could support the usefulness of low-dose IV administration. Nonetheless due to the many side effects of systemic clonidine administration, the spinal

Low doses of clonidine proved to be a useful adjunct analgesic when given neuraxially and in combination with peripheral nerve blocks (Habib et al, 2005). Significant results in terms of block duration were obtained when clonidine was added to local anesthetics for epidural or perineural analgesia. At low doses (2 μg/kg), it was shown to increase the duration of perineural blockade. Animal studies suggest that the mechanism of clonidine's potentiation of lidocaine nerve block is inhibition of the hyperpolarization-activated cation current, not

Dexmedetomidine is a relatively new, highly selective central alpha-2 agonist. Dexmedetomidine, when used as an adjunct, can reduce postoperative morphine consumption in various surgical settings using various routes such as intravenous (Dholakia et al, 2007; Gurbet et al, 2006; Lin et al, 2009). In a recent study the authors found that; the addition of dexmedetomidine to intravenous PCA morphine resulted in superior analgesia, significant morphine sparing, and less morphine-induced nausea,while it was devoid of additional sedation and untoward hemodynamic changes (Dholakia et al,

With the discovery of the N-methyl-D-aspartate (NMDA) receptor and its links to nociceptive pain transmission and central sensitization, there has been renewed interest in utilizing noncompetitive NMDA receptor antagonists, such as ketamine, dextromethorphan,

via its binding to alpha-2 adrenergic receptors (Jurna, 1995).

**4.2.4.2 N-methyl-D-aspartate antagonists (Antihyperalgesic drugs)** 

magnesium ions as potential antihyperalgesic agents.

muscarinic and nicotinic receptor subtypes with analgesic effects (Fanelli et al, 2008).

control of pain.

**4.2.4.1.1 Clonidine** 

route is preferred.

2007).

**4.2.4.1.2 Dexmedetomidine** 

2007).

**4.2.4.1 Alpha-2 adrenergic agonists** 

30% after major surgery (Power, 1999). This may be of clinical importance as NSAIDs may reduce the incidence of opioid-related side-effects (respiratory depression, sedation, nausea and vomiting, ileus, urinary bladder dysfunction and possibly sleep disturbances). Since the COX-2 enzyme, the primary target of NSAIDs, is inducible, it is not found in damaged tissues until a few hours following the onset of a noxious stimulus. This could explain the lack of efficacy of preemptive administration of these drugs (Ness, 2001).

NSAID use is not appropriate in all patients because of their age or renal or hematological status or because of previous dyspeptic symptoms (McClane, 2010). COX-2-selective inhibitors (celecoxib, etoricoxib, rofecoxib- is no longer in use due to adverse cardiovascular events-) have the advantage over NSAIDs in the perioperative setting of not increasing the risk of bleeding (Buvanendran&Kroin, 2009).

Many patients now receive a NSAID as a routine part of their postoperative analgesic management. Recent practice guidelines for acute pain management in the perioperative setting specifically state 'unless contraindicated, all patients should receive around-the-clock regimen of NSAIDs, COX-2 inhibitors, or acetaminophen' (Ashburn, et al, 2004).

#### **4.2.2 Acetaminophen**

Acetaminophen is antipyretic and analgesic but has little, if any, anti-inflammatory action. Its analgesic efficacy is not more than that of traditional analgesics; however, it has fewer side effects. Preparation of intravenous acetaminophen recently has been released in Europe. A 100 ml solution is presented as 10 mg/ml for administration over a period of 15 minutes. The onset of action is within five to 10 minutes, with the peak at one to two hours. Optimal analgesia for moderate to severe postoperative pain cannot be achieved using a single agent alone, but a balanced approach in combination with non-steroidal agents can result in up to a 40 to 50 percent reduction in opioid requirements (Vadivelu, 2010).

#### **4.2.3 Paracetamol**

Paracetamol has antipyretic and analgesic properties, but it is devoid of anti-inflammatory effects. It has an inhibitory action on central COX-2 and COX-3 enzymes, which would explain its antipyretic activity. The analgesic effect seems to be due to activation of descending serotonergic inhibitory pathways as well as inhibition of central NO synthases (Graham&Scott, 2005). Similar to other analgesic drugs, paracetamol shows differential properties in terms of pain control. Paracetamol may be more effective in treating episiotomy or abdominal pain rather than pain following orthopedic surgery or tooth extraction (Gray, et al, 2005; macario&Lipman, 2001). The different relative roles of peripheral COX enzymes in postoperative pain may explain these differing efficacies.

When paracetamol and NSAIDs are administered by an intravenous route, they show sparing effects on opioid consumption (about 25% and 30%, respectively); this effect begins 4 h after their first administration and is synergistic (Elia et al, 2005; Mirande, 2006).

#### **4.2.4 Adjuvants**

Adjuvant drugs are defined as substances that may improve pain treatment and pain control, but they are not commonly defined as analgesics. Adjuvants are compounds, which by themselves have undesirable side effects or low potency but in combination with opioids allow a reduction of narcotic dosing for postoperative pain control. Thus they can provide beneficial effects when administered in appropriate doses as part of a multimodal analgesic regimen in the perioperative setting (Fanelli et al, 2008; Vadivelu et al, 2010).

Multimodal analgesia incorporates the use of analgesic adjuncts with different mechanisms of action to enhance postoperative pain management. Adjuvants are important in postoperative pain management due to side effects of opioid analgesics, which hinder recovery, especially in the increasingly utilized ambulatory surgical procedures (Buvanendran&Kroin, 2007). Multiple adjuvants recently have been developed for the control of pain.

#### **4.2.4.1 Alpha-2 adrenergic agonists**

Alpha-2 adrenergic activation represents an intrinsic pain control network of the central nervous system. The alpha-2 adrenergic receptor has high density in the substantia gelatinosa of the dorsal horn in humans and that is believed to be the primary site of action by which alpha-2 adrenergic agonists can reduce pain (Buvanendran&Kroin, 2007).

#### **4.2.4.1.1 Clonidine**

186 Pain Management – Current Issues and Opinions

30% after major surgery (Power, 1999). This may be of clinical importance as NSAIDs may reduce the incidence of opioid-related side-effects (respiratory depression, sedation, nausea and vomiting, ileus, urinary bladder dysfunction and possibly sleep disturbances). Since the COX-2 enzyme, the primary target of NSAIDs, is inducible, it is not found in damaged tissues until a few hours following the onset of a noxious stimulus. This could explain the

NSAID use is not appropriate in all patients because of their age or renal or hematological status or because of previous dyspeptic symptoms (McClane, 2010). COX-2-selective inhibitors (celecoxib, etoricoxib, rofecoxib- is no longer in use due to adverse cardiovascular events-) have the advantage over NSAIDs in the perioperative setting of not increasing the

Many patients now receive a NSAID as a routine part of their postoperative analgesic management. Recent practice guidelines for acute pain management in the perioperative setting specifically state 'unless contraindicated, all patients should receive around-the-clock

Acetaminophen is antipyretic and analgesic but has little, if any, anti-inflammatory action. Its analgesic efficacy is not more than that of traditional analgesics; however, it has fewer side effects. Preparation of intravenous acetaminophen recently has been released in Europe. A 100 ml solution is presented as 10 mg/ml for administration over a period of 15 minutes. The onset of action is within five to 10 minutes, with the peak at one to two hours. Optimal analgesia for moderate to severe postoperative pain cannot be achieved using a single agent alone, but a balanced approach in combination with non-steroidal agents can

Paracetamol has antipyretic and analgesic properties, but it is devoid of anti-inflammatory effects. It has an inhibitory action on central COX-2 and COX-3 enzymes, which would explain its antipyretic activity. The analgesic effect seems to be due to activation of descending serotonergic inhibitory pathways as well as inhibition of central NO synthases (Graham&Scott, 2005). Similar to other analgesic drugs, paracetamol shows differential properties in terms of pain control. Paracetamol may be more effective in treating episiotomy or abdominal pain rather than pain following orthopedic surgery or tooth extraction (Gray, et al, 2005; macario&Lipman, 2001). The different relative roles of peripheral COX enzymes in postoperative pain may explain these differing efficacies. When paracetamol and NSAIDs are administered by an intravenous route, they show sparing effects on opioid consumption (about 25% and 30%, respectively); this effect begins

regimen of NSAIDs, COX-2 inhibitors, or acetaminophen' (Ashburn, et al, 2004).

result in up to a 40 to 50 percent reduction in opioid requirements (Vadivelu, 2010).

4 h after their first administration and is synergistic (Elia et al, 2005; Mirande, 2006).

regimen in the perioperative setting (Fanelli et al, 2008; Vadivelu et al, 2010).

Adjuvant drugs are defined as substances that may improve pain treatment and pain control, but they are not commonly defined as analgesics. Adjuvants are compounds, which by themselves have undesirable side effects or low potency but in combination with opioids allow a reduction of narcotic dosing for postoperative pain control. Thus they can provide beneficial effects when administered in appropriate doses as part of a multimodal analgesic

lack of efficacy of preemptive administration of these drugs (Ness, 2001).

risk of bleeding (Buvanendran&Kroin, 2009).

**4.2.2 Acetaminophen** 

**4.2.3 Paracetamol** 

**4.2.4 Adjuvants** 

Clonidine is originally classified as an anti-hypertensive drug with negative chronotropic activity, but has antinociceptive properties as well. In the spinal cord, clonidine acts at alpha-2 adrenergic receptors to stimulate acetylcholine release, which acts at both muscarinic and nicotinic receptor subtypes with analgesic effects (Fanelli et al, 2008).

Clonidine can be administered orally, intravenously, neuraxially or perineurally in combination with local anesthetics. However, the side effects could be significant. The most important ones are hypotension, bradycardia and sedation (Rawal). Data about the systemic administration of clonidine could support the usefulness of low-dose IV administration. Nonetheless due to the many side effects of systemic clonidine administration, the spinal route is preferred.

Low doses of clonidine proved to be a useful adjunct analgesic when given neuraxially and in combination with peripheral nerve blocks (Habib et al, 2005). Significant results in terms of block duration were obtained when clonidine was added to local anesthetics for epidural or perineural analgesia. At low doses (2 μg/kg), it was shown to increase the duration of perineural blockade. Animal studies suggest that the mechanism of clonidine's potentiation of lidocaine nerve block is inhibition of the hyperpolarization-activated cation current, not via its binding to alpha-2 adrenergic receptors (Jurna, 1995).

#### **4.2.4.1.2 Dexmedetomidine**

Dexmedetomidine is a relatively new, highly selective central alpha-2 agonist. Dexmedetomidine, when used as an adjunct, can reduce postoperative morphine consumption in various surgical settings using various routes such as intravenous (Dholakia et al, 2007; Gurbet et al, 2006; Lin et al, 2009). In a recent study the authors found that; the addition of dexmedetomidine to intravenous PCA morphine resulted in superior analgesia, significant morphine sparing, and less morphine-induced nausea,while it was devoid of additional sedation and untoward hemodynamic changes (Dholakia et al, 2007).

#### **4.2.4.2 N-methyl-D-aspartate antagonists (Antihyperalgesic drugs)**

With the discovery of the N-methyl-D-aspartate (NMDA) receptor and its links to nociceptive pain transmission and central sensitization, there has been renewed interest in utilizing noncompetitive NMDA receptor antagonists, such as ketamine, dextromethorphan, magnesium ions as potential antihyperalgesic agents.

Multimodal Analgesia for Postoperative Pain Management 189

pharmacokinetics. The gabapentinoid compounds have been used as part of multimodal analgesic in the postoperative period. Earlier clinical trials with gabapentin for early

Gabapentin, a third-generation anti-epileptic drug, is a structural analogue of Gaba Aminobutyric Acid (GABA), an important neurotransmitter in the central nervous system. Its main action, however, is to inhibit the alpha2δ subunit of Ca2+ channels with a resultant decrease in neuronal hyperexcitability. During the immediate postoperative period, however, its activation of descending inhibitory pathways may be more relevant and might

Most of the reviews and meta-analyses concur that perioperative gabapentin helps to produce a significant opioid-sparing effect and probably also improves postoperative pain

Pregabalin a structural analog of GABA and a derivative of gabapentin (S+ 3-isobutyl GABA). It is a novel drug with a heightened research interest in the analgesic, sedative, anxiolytic, and opioid-sparing effects, in various pain settings, including postoperative

Its main advantages may be faster onset and reduced adverse side effects. Some studies suggest pregabalin to have effective sedative and opioid-sparing effects (Hartrick et al, 2009; Mathiesen et al, 2008), useful characteristics for the control of acute pain. Research on its established role as an analgesic adjuvant as a part of multimodal analgesia for acute pain

Glucocorticoids, including dexamethasone, have been used to reduce inflammation and postoperative pain in surgical procedures (Salerno et al, 2006). Glucocorticoid steroids can provide beneficial effects when administered in appropriate doses as part of a multimodal

Dexamethasone is a synthetic glucocorticoid with high potency and a long duration of action (half-life: 2 days), and has low mineralocorticoid activity. Although dexamethasone reduces PG synthesis, its possible analgesic effects have not yet been demonstrated. In patients undergoing total hip arthroplasty under spinal anesthesia with propofol sedation a single preoperative intravenous dose of dexamethasone decreased the pain upon standing at 24 h compared to placebo (Kardash et al, 2008). In a recent study, it did not reduce postoperative pain scores and analgesic requirements after laparoscopic cholecystectomy. The main advantage of postoperative dexamethasone is its ability to reduce postoperative

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a non narcotic and acts peripherally. It can

postsurgical pain have recently been reviewed (Buvanendran&Kroin, 2009).

score relative to the control group (Hartrick et al, 2009; Tiipana et al, 2007).

explain its synergistic effect with opioids (Hurley et al, 2006).

analgesic regimen in the perioperative setting (White, 2005, 2007).

**4.2.4.3.1 Gabapentin** 

**4.2.4.3.2 Pregabalin** 

control is ongoing. **4.2.4.4 Glucocorticoids** 

**4.2.4.4.1 Dexamethasone** 

**4.2.4.5 Newer drugs 4.2.4.5.1 Capcaisin** 

nausea and vomiting (Feo et al, 2006).

be used as a cream and also as an injectable analgesic.

pain.

#### **4.2.4.2.1 Ketamine**

Ketamine has been a well known general anesthetic and analgesic for the past 3 decades. There is evidence that low-dose ketamine may play an important role in postoperative pain management when used as an adjunct to opioids, local anesthetics, and other analgesic agents.

Ketamine, is the most commonly used antihyperalgesic drug. There is a definite role of ketamine in preventing opioid-induced hyperalgesia in patients receiving high doses of opioid for their postoperative pain relief (Mitra, 2008). It acts as an antagonist of NMDA receptors and may reduce the intensity of hyperalgesia following rapid μ opioid receptor stimulation by short-acting agonists such as remifentanil and, to a lesser extent, sufentanil and fentanyl. Perioperative administration of 2-10 μg/kg/min following a loading dose of 0.5 mg/kg decreases hyperalgesia and allodynia after thoracic and abdominal surgery (Bilgin et al, 2005; Joly et al, 2005), although doses may vary depending on the overall duration and amount of exposure to short-acting opioids.

Routes of administration include oral, intravenous,intramuscular, subcutaneous, epidural, transdermal, and intra-articular.

Clinical use of ketamine can be limited due to psychotomimetic adverse effects such as hallucinations, excessive sedation and bad dreams. Other common adverse effects are dizziness, blurred vision, and nausea and vomiting (Bell et al, 2006). Although high doses of ketamine have been implicated in causing psychomimetic effects, subanesthetic or low doses of ketamine have demonstrated significant analgesic efficacy without these side effects (Buvanendran&Kroin, 2009). It can be used in sub-anesthetic doses as an adjunct to provide postoperative pain relief in opioid-dependent patients (Mitra et al, 2004).

#### **4.2.4.2.2 Dextramethorphan**

Dextromethorphan has a similar mechanism of action with a lower affinity for the NMDA receptor. Following oral administration, it is rapidly absorbed from the gut and crosses the blood-brain barrier. A systematic review of perioperative dextromethorphan treatment for acute post-surgical pain concluded that the drug was a safe potential adjunct to classical opioid-based analgesia, but the results were inconsistent (Duedahl et al, 2006).

#### **4.2.4.2.3 Magnesium**

The magnesium ion was the first agent discovered to be an NMDA channel blocker. Similarly to ketamine and dextromethorphan, magnesium ions act by blocking the NMDA receptor pore. Since magnesium crosses the blood-brain barrier with difficulty in humans, it is not clear whether its therapeutic effects are related to NMDA antagonism in the central nervous system.

Several clinical studies have shown that magnesium increases postoperative analgesia, but the best dosage regimen remains to be determined (Lysakowski et al, 2007). At very high doses, perioperative intravenous magnesium sulfate has been reported to reduce postoperative morphine consumption but not postoperative pain scores (Koinig et al, 1998; Tramer et al, 1996).

#### **4.2.4.3 Gabapentin-type drugs**

Pregabalin and gabapentin bind to voltage-gated calcium channels in the spinal cord and brain. Both drugs are used for seizures and neuropathic pain. One advantage of pregabalin in clinical use is that it has higher bioavailability than gabapentin and linear pharmacokinetics. The gabapentinoid compounds have been used as part of multimodal analgesic in the postoperative period. Earlier clinical trials with gabapentin for early postsurgical pain have recently been reviewed (Buvanendran&Kroin, 2009).

#### **4.2.4.3.1 Gabapentin**

188 Pain Management – Current Issues and Opinions

Ketamine has been a well known general anesthetic and analgesic for the past 3 decades. There is evidence that low-dose ketamine may play an important role in postoperative pain management when used as an adjunct to opioids, local anesthetics, and other analgesic

Ketamine, is the most commonly used antihyperalgesic drug. There is a definite role of ketamine in preventing opioid-induced hyperalgesia in patients receiving high doses of opioid for their postoperative pain relief (Mitra, 2008). It acts as an antagonist of NMDA receptors and may reduce the intensity of hyperalgesia following rapid μ opioid receptor stimulation by short-acting agonists such as remifentanil and, to a lesser extent, sufentanil and fentanyl. Perioperative administration of 2-10 μg/kg/min following a loading dose of 0.5 mg/kg decreases hyperalgesia and allodynia after thoracic and abdominal surgery (Bilgin et al, 2005; Joly et al, 2005), although doses may vary depending on the overall

Routes of administration include oral, intravenous,intramuscular, subcutaneous, epidural,

Clinical use of ketamine can be limited due to psychotomimetic adverse effects such as hallucinations, excessive sedation and bad dreams. Other common adverse effects are dizziness, blurred vision, and nausea and vomiting (Bell et al, 2006). Although high doses of ketamine have been implicated in causing psychomimetic effects, subanesthetic or low doses of ketamine have demonstrated significant analgesic efficacy without these side effects (Buvanendran&Kroin, 2009). It can be used in sub-anesthetic doses as an adjunct to

Dextromethorphan has a similar mechanism of action with a lower affinity for the NMDA receptor. Following oral administration, it is rapidly absorbed from the gut and crosses the blood-brain barrier. A systematic review of perioperative dextromethorphan treatment for acute post-surgical pain concluded that the drug was a safe potential adjunct to classical

The magnesium ion was the first agent discovered to be an NMDA channel blocker. Similarly to ketamine and dextromethorphan, magnesium ions act by blocking the NMDA receptor pore. Since magnesium crosses the blood-brain barrier with difficulty in humans, it is not clear whether its therapeutic effects are related to NMDA antagonism in the central

Several clinical studies have shown that magnesium increases postoperative analgesia, but the best dosage regimen remains to be determined (Lysakowski et al, 2007). At very high doses, perioperative intravenous magnesium sulfate has been reported to reduce postoperative morphine consumption but not postoperative pain scores (Koinig et al, 1998;

Pregabalin and gabapentin bind to voltage-gated calcium channels in the spinal cord and brain. Both drugs are used for seizures and neuropathic pain. One advantage of pregabalin in clinical use is that it has higher bioavailability than gabapentin and linear

provide postoperative pain relief in opioid-dependent patients (Mitra et al, 2004).

opioid-based analgesia, but the results were inconsistent (Duedahl et al, 2006).

duration and amount of exposure to short-acting opioids.

transdermal, and intra-articular.

**4.2.4.2.2 Dextramethorphan** 

**4.2.4.2.3 Magnesium** 

nervous system.

Tramer et al, 1996).

**4.2.4.3 Gabapentin-type drugs** 

**4.2.4.2.1 Ketamine** 

agents.

Gabapentin, a third-generation anti-epileptic drug, is a structural analogue of Gaba Aminobutyric Acid (GABA), an important neurotransmitter in the central nervous system. Its main action, however, is to inhibit the alpha2δ subunit of Ca2+ channels with a resultant decrease in neuronal hyperexcitability. During the immediate postoperative period, however, its activation of descending inhibitory pathways may be more relevant and might explain its synergistic effect with opioids (Hurley et al, 2006).

Most of the reviews and meta-analyses concur that perioperative gabapentin helps to produce a significant opioid-sparing effect and probably also improves postoperative pain score relative to the control group (Hartrick et al, 2009; Tiipana et al, 2007).

#### **4.2.4.3.2 Pregabalin**

Pregabalin a structural analog of GABA and a derivative of gabapentin (S+ 3-isobutyl GABA). It is a novel drug with a heightened research interest in the analgesic, sedative, anxiolytic, and opioid-sparing effects, in various pain settings, including postoperative pain.

Its main advantages may be faster onset and reduced adverse side effects. Some studies suggest pregabalin to have effective sedative and opioid-sparing effects (Hartrick et al, 2009; Mathiesen et al, 2008), useful characteristics for the control of acute pain. Research on its established role as an analgesic adjuvant as a part of multimodal analgesia for acute pain control is ongoing.

#### **4.2.4.4 Glucocorticoids**

Glucocorticoids, including dexamethasone, have been used to reduce inflammation and postoperative pain in surgical procedures (Salerno et al, 2006). Glucocorticoid steroids can provide beneficial effects when administered in appropriate doses as part of a multimodal analgesic regimen in the perioperative setting (White, 2005, 2007).

#### **4.2.4.4.1 Dexamethasone**

Dexamethasone is a synthetic glucocorticoid with high potency and a long duration of action (half-life: 2 days), and has low mineralocorticoid activity. Although dexamethasone reduces PG synthesis, its possible analgesic effects have not yet been demonstrated.

In patients undergoing total hip arthroplasty under spinal anesthesia with propofol sedation a single preoperative intravenous dose of dexamethasone decreased the pain upon standing at 24 h compared to placebo (Kardash et al, 2008). In a recent study, it did not reduce postoperative pain scores and analgesic requirements after laparoscopic cholecystectomy. The main advantage of postoperative dexamethasone is its ability to reduce postoperative nausea and vomiting (Feo et al, 2006).

#### **4.2.4.5 Newer drugs**

#### **4.2.4.5.1 Capcaisin**

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a non narcotic and acts peripherally. It can be used as a cream and also as an injectable analgesic.

Multimodal Analgesia for Postoperative Pain Management 191

slowly (risk of high incidence of nausea and

soon as possible.

(iii) Oral administration as

**Paracetamol** Oral 4 x 1 g paracetamol/day (2 g

administration should start at least 30-60 min before

**Acetaminophen** Intravenous 100 ml solution (10 mg/ml)

(iii) Combined with local anesthetics-neuraxially or

**Ketamine** Intravenous Loading dose of 0.5 mg/kg

Table 3. The doses and routes of administration of frequently used drugs \* (modified table

Sodium channel blocking drugs are usually used in the management of both acute and chronic pain. When dealing with postoperative pain, local anesthetics such as lidocaine and

\* The doses and routes of administration of drugs described above are general examples and each

(ii) Oral administration should start as soon as

Duration: 3-5 days.

(ii) Intravenous

perineurally

end of surgery.

possible.

(iii) Rectal

vomiting). (ii) Intramuscular.

**OPIOIDS** 

**NONOPIOIDS**

**Combination of** 

**ADJUVANTS** 

**paracetamol and codeine** 

**NSAIDs** (i) Intravenous

**Clonidine** (i) Oral

patient should be assessed individually before prescribing

by permission from Publisher AstraZeneca)

bupivacaine mostly preferred (McCleane, 2010).

**4.2.5 Local anesthetics** 

**Tramadol** (i) Intravenous: inject

**Administration Dosage** 

0.75-1.0 mg/kg 50-100 mg 6 hourly.

propacetamol/day).

insufficiency.

codeine 30 mg.

(only IV form)

of 15 minutes.

3- 5 µg/kg (oral) 1 µg/kg (intravenous) 1-2 µg/kg (epidural) or 75- 100 µg (intrathecal)

include:

Oral Paracetamol 500 mg +

Dose to be reduced (e.g. 3 x 1 g/day) in case of hepatic

4 x 1 g paracetamol/day.

Ketorolac: 3 x 30-40 mg/day

Diclofenac: 2 x 75 mg/day Ketoprofen: 4 x 50 mg/day (ii) Selective NSAIDs

Meloxicam 15 mg once daily Celecoxib: 200 mg/day.

administration over a period

followed by 2-10 µg/kg/min

Capsaicin cream is usually combined with narcotic analgesics and NSAIDs to relieve a variety of painful ailments such as back pain, arthritic joint pains, and strains and sprains. Injectable capsaicin is used for the control of post operative pain, such as after total knee replacement, total hip replacement, hernia repair, shoulder arthroscopy, and bunionectomy (Aasvang et al, 2008). Pre-administration of neural blockade before injection of capsaicin may greatly decrease the burning discomfort.

Capsaicin appears to be a relatively safe drug. In the elderly who are sensitive to respiratory depression that can occur with opioids, capsaicin can be particularly beneficial as an adjuvant. The only absolute contraindication being patient hypersensitivity. Relative contraindications include age less than 2 years, patients with elevated liver enzymes, patients on ACE inhibitors, and patients showing signs of septic arthritis and joint infections (Vadivelu et al, 2010).

#### **4.2.4.5.2 Glyceryl trinitrate**

The organic nitrates, such as glyceryl trinitrate (GTN), act as nitric oxide donors.

High dose nitroglyserin patches, such as 30 mg daily, are hyperalgesic, whereas doses less than 6 mg per day are analgesic under different circumstances. Previously it has been observed that patients with past histories of angina who had spinal block, in which the nitroglycerin transdermal patch was applied prophylactically, required fewer analgesic after operation (Lauretti et al, 1999).

#### **4.2.4.5.3 Cholinergic drugs**

Acetylcholine may cause analgesia through direct action on spinal cholinergic muscarinic receptors M1 and M3 and nicotinic receptors subtypes.

#### **4.2.4.5.3.1 Nicotine**

In a study in nonsmoker patients having radical retropubic prostatectomy under general anesthesia, the application of a 7 mg nicotine patch 30–60 min before surgery for postoperative 24 hrs, resulted with lower cumulative PCA morphine consumption versus placebo group. But the intensity of nausea was greater in the nicotine group (Habib et al, 2008).


Table 3 summarizes the doses and routes of administration of frequently used drugs.

Capsaicin cream is usually combined with narcotic analgesics and NSAIDs to relieve a variety of painful ailments such as back pain, arthritic joint pains, and strains and sprains. Injectable capsaicin is used for the control of post operative pain, such as after total knee replacement, total hip replacement, hernia repair, shoulder arthroscopy, and bunionectomy (Aasvang et al, 2008). Pre-administration of neural blockade before injection of capsaicin

Capsaicin appears to be a relatively safe drug. In the elderly who are sensitive to respiratory depression that can occur with opioids, capsaicin can be particularly beneficial as an adjuvant. The only absolute contraindication being patient hypersensitivity. Relative contraindications include age less than 2 years, patients with elevated liver enzymes, patients on ACE inhibitors, and patients showing signs of septic arthritis and joint infections

High dose nitroglyserin patches, such as 30 mg daily, are hyperalgesic, whereas doses less than 6 mg per day are analgesic under different circumstances. Previously it has been observed that patients with past histories of angina who had spinal block, in which the nitroglycerin transdermal patch was applied prophylactically, required fewer analgesic after

Acetylcholine may cause analgesia through direct action on spinal cholinergic muscarinic

In a study in nonsmoker patients having radical retropubic prostatectomy under general anesthesia, the application of a 7 mg nicotine patch 30–60 min before surgery for postoperative 24 hrs, resulted with lower cumulative PCA morphine consumption versus placebo group. But the intensity of nausea was greater in the nicotine group (Habib et al,

**Administration Dosage** 

IV PCA

Bolus: 1-2 mg, lockout: 5-15 min (usually 7-8 min), no background infusion. Subcutaneous

0.1-0.15 mg/kg 4-6 hourly, adapted in relation to pain score, sedation and respiratory rate.

with paracetamol. A minimum of 30 mg codeine/tablet is required

Table 3 summarizes the doses and routes of administration of frequently used drugs.

(ii) Subcutaneous by continuous infusion or intermittent boluses via indwelling cannula. (iii) Intramuscular (not recommended due to incidence of pain. 5-10 mg 3-4 hourly).

**Codeine** Oral 3 mg/kg/day combined

The organic nitrates, such as glyceryl trinitrate (GTN), act as nitric oxide donors.

may greatly decrease the burning discomfort.

(Vadivelu et al, 2010).

**4.2.4.5.2 Glyceryl trinitrate** 

operation (Lauretti et al, 1999). **4.2.4.5.3 Cholinergic drugs** 

**4.2.4.5.3.1 Nicotine** 

2008).

**OPIOIDS** 

receptors M1 and M3 and nicotinic receptors subtypes.

**Morphine** (i) Intravenous.


\* The doses and routes of administration of drugs described above are general examples and each patient should be assessed individually before prescribing

Table 3. The doses and routes of administration of frequently used drugs \* (modified table by permission from Publisher AstraZeneca)

#### **4.2.5 Local anesthetics**

Sodium channel blocking drugs are usually used in the management of both acute and chronic pain. When dealing with postoperative pain, local anesthetics such as lidocaine and bupivacaine mostly preferred (McCleane, 2010).

Multimodal Analgesia for Postoperative Pain Management 193

peripheral regional techniques for each patient, especially in light of some of the controversies

Neuraxial (primarily epidural) and peripheral regional analgesic techniques (e.g., brachial plexus, lumbar plexus, femoral, sciatic-popliteal, and scalp nerve blocks), also a variety of wound infiltration techniques may be used for the effective treatment of postoperative pain. In general, the analgesia provided by epidural and peripheral techniques (particularly when local anesthetics are used) is superior to that with systemic opioids, (i.e., superior analgesia and decreased opioid-related side effects) and use of these techniques may even reduce

Spinal or epidural analgesia techniques in single or continuous forms can be used in postoperative pain management. The use of epidural anesthesia and analgesia is an integral part of the multimodal approach because of the superior analgesia and physiologic benefits

Among the most commonly used pain-relieving techniques, there is evidence that the epidural local anesthetic or local anesthetic-opioid techniques are the most effective on providing dynamic pain relief after major surgical procedures (Kehlet et al, 1999). Epidural local anesthetic application comes in as the major component of multimodal analgesia. Postoperative epidural analgesia is usually accomplished with a combination of a longacting local anesthetic and an opioid, in dilute concentrations (Table 5). Long-acting local anesthetics are preferred because they are associated with less tachyphylaxis. Thoracic epidural analgesia with local anesthetics and opioids for abdominal, thoracic and vascular surgery improves bowel recovery times while decreasing the risks of cardiovascular adverse events and of developing persistent pain (Liu, 2004; Nishimori et al, 2006;). In an unpublished study of ours (Sivrikaya et al, 2000), preemptive analgesia with epidural tramadol has supressed the peroperative stress response and also reduced the pain intensity in the early postoperative period in patients had abdominal hysterectomy under

Continuous Infusion: An easy technique that requires little intervention. The cumulative dose of local anesthetic is likely to be higher and side effects are more likely than with the

Intermittent Top-up: Results in benefits due to frequent patient/staff contact but can

Patient-Controlled Epidural Analgesia (PCEA): This technique produces high patient satisfaction and reduced dose requirements compared with continuous infusion. However, sophisticated pumps are required and accurate catheter position is important for optimal

produce a high staff workload and patients may have to wait for treatment.

about the use of these techniques in the presence of various anticoagulants.

morbidity and mortality (Wu&Fleisher 2000).

2. Peripheral Regional Analgesia Techniques

1. Neuraxial Techniques

3. Infiltration Techniques a. Wound Infiltration b. Topical Application c. Local Infiltration Analgesia 4. Other – Nonpharmacological Techniques

**5.1 Neuraxial techniques** 

general anesthesia.

other two techniques.

efficacy (Rawal).

conferred by epidural analgesia.

Techniques used in postopetaive pain management are:

Maintenance techniques in epidural analgesia include:

Local anesthetics block sodium channels, thereby, preventing transmission of nerve impulse along the axonal fibre. This is a local effect at the site of injection. Tissue anesthesia occurs after the injection of the local anesthetics into tissue at appropriate concentration, but it lasts after the duration of the drug ended. However, local anesthetics are also absorbed into the systemic circulation from the site of injection and, depending on the dose and rate of absorption, may have systemic analgesic effects (Gupta,2010; McCleane, 2010).

Local anesthetic solutions delivered through an epidural or perineural route are the most important treatments for decreasing incident pain, hormonal stress and sympathetic responses during and after surgery (Chelly, 2001; Liu, 2007). Generally, higher doses are used intraoperatively and then reduced to reach differential motor-sensory block in the postoperative period. The best results are achieved when local anesthetic solutions are infused neuraxially with lipophilic opioids, such as sufentanil and fentanyl at adequate concentrations (George, 2006; de-Leon-Casaola&Lema, 1996).

In some studies the effectiveness of the intravenous infusion of lidocaine in reducing postoperative pain and facilitating the recovery process have been demonstrated (Kaba et al, 2007; Lauwick et al, 2008). Yardeni and colleagues (Yardeni et al, 2009) suggested that, perioperative administration of intravenous lidocaine could improve early postoperative pain control and reduce surgery-induced immune alterations. The injection of local anesthetic around wound edges has been proven to reduce postoperative pain, but only for the duration of that local anesthetic (Moinichi et al, 1998). Several concerns about these drugs have been expressed in the literature including the risk of infection, chondrolysis and systemic local anesthetics toxicity when they used locally.


The maximum doses for local anesthetics are summarized in Table 4.

Table 4. Maximum doses of frequently used local anesthetics

#### **5. Techniques in postoperative pain management**

One approach for multimodal analgesia is the use of regional anesthesia and analgesia to inhibit the neural conduction from the surgical site to the spinal cord and decrease spinal cord sensitization (Buvanendran&Kroin, 2009). A variety of neuraxial and peripheral regional analgesic techniques can provide analgesia superior to that with systemic opioids and may even result in improvement in various outcomes. However, there are some risks associated with the use of such techniques. The clinician should evaluate the risks and benefits of these techniques on an individual basis in determining the appropriateness of neuraxial or peripheral regional techniques for each patient, especially in light of some of the controversies about the use of these techniques in the presence of various anticoagulants.

Neuraxial (primarily epidural) and peripheral regional analgesic techniques (e.g., brachial plexus, lumbar plexus, femoral, sciatic-popliteal, and scalp nerve blocks), also a variety of wound infiltration techniques may be used for the effective treatment of postoperative pain. In general, the analgesia provided by epidural and peripheral techniques (particularly when local anesthetics are used) is superior to that with systemic opioids, (i.e., superior analgesia and decreased opioid-related side effects) and use of these techniques may even reduce morbidity and mortality (Wu&Fleisher 2000).

Techniques used in postopetaive pain management are:

1. Neuraxial Techniques

192 Pain Management – Current Issues and Opinions

Local anesthetics block sodium channels, thereby, preventing transmission of nerve impulse along the axonal fibre. This is a local effect at the site of injection. Tissue anesthesia occurs after the injection of the local anesthetics into tissue at appropriate concentration, but it lasts after the duration of the drug ended. However, local anesthetics are also absorbed into the systemic circulation from the site of injection and, depending on the dose and rate of

Local anesthetic solutions delivered through an epidural or perineural route are the most important treatments for decreasing incident pain, hormonal stress and sympathetic responses during and after surgery (Chelly, 2001; Liu, 2007). Generally, higher doses are used intraoperatively and then reduced to reach differential motor-sensory block in the postoperative period. The best results are achieved when local anesthetic solutions are infused neuraxially with lipophilic opioids, such as sufentanil and fentanyl at adequate

In some studies the effectiveness of the intravenous infusion of lidocaine in reducing postoperative pain and facilitating the recovery process have been demonstrated (Kaba et al, 2007; Lauwick et al, 2008). Yardeni and colleagues (Yardeni et al, 2009) suggested that, perioperative administration of intravenous lidocaine could improve early postoperative pain control and reduce surgery-induced immune alterations. The injection of local anesthetic around wound edges has been proven to reduce postoperative pain, but only for the duration of that local anesthetic (Moinichi et al, 1998). Several concerns about these drugs have been expressed in the literature including the risk of infection, chondrolysis and

7 mg/kg(with epinephrine) **or** 300 mg

9 mg/kg(with epinephrine) **or** 500 mg

2.5 mg/kg(with epinephrine) **or** 150 mg

One approach for multimodal analgesia is the use of regional anesthesia and analgesia to inhibit the neural conduction from the surgical site to the spinal cord and decrease spinal cord sensitization (Buvanendran&Kroin, 2009). A variety of neuraxial and peripheral regional analgesic techniques can provide analgesia superior to that with systemic opioids and may even result in improvement in various outcomes. However, there are some risks associated with the use of such techniques. The clinician should evaluate the risks and benefits of these techniques on an individual basis in determining the appropriateness of neuraxial or

absorption, may have systemic analgesic effects (Gupta,2010; McCleane, 2010).

concentrations (George, 2006; de-Leon-Casaola&Lema, 1996).

systemic local anesthetics toxicity when they used locally.

Lidocaine 4 mg/kg (without epinephrine)

Prilocaine 6 mg/kg (without epinephrine)

Bupivacaine 2 mg/kg (without epinephrine)

Table 4. Maximum doses of frequently used local anesthetics

**5. Techniques in postoperative pain management** 

Levobupivacaine 2.5-3 mg/kg (insufficient data) **or** 150 mg Ropivacaine 3-4 mg/kg (without or with epinephrine)

**Local anesthetic Maximum total dosage** 

Prokain 400 mg Chlorprocaine 800 mg

The maximum doses for local anesthetics are summarized in Table 4.

	- a. Wound Infiltration
		- b. Topical Application
	- c. Local Infiltration Analgesia

#### **5.1 Neuraxial techniques**

Spinal or epidural analgesia techniques in single or continuous forms can be used in postoperative pain management. The use of epidural anesthesia and analgesia is an integral part of the multimodal approach because of the superior analgesia and physiologic benefits conferred by epidural analgesia.

Among the most commonly used pain-relieving techniques, there is evidence that the epidural local anesthetic or local anesthetic-opioid techniques are the most effective on providing dynamic pain relief after major surgical procedures (Kehlet et al, 1999). Epidural local anesthetic application comes in as the major component of multimodal analgesia.

Postoperative epidural analgesia is usually accomplished with a combination of a longacting local anesthetic and an opioid, in dilute concentrations (Table 5). Long-acting local anesthetics are preferred because they are associated with less tachyphylaxis. Thoracic epidural analgesia with local anesthetics and opioids for abdominal, thoracic and vascular surgery improves bowel recovery times while decreasing the risks of cardiovascular adverse events and of developing persistent pain (Liu, 2004; Nishimori et al, 2006;). In an unpublished study of ours (Sivrikaya et al, 2000), preemptive analgesia with epidural tramadol has supressed the peroperative stress response and also reduced the pain intensity in the early postoperative period in patients had abdominal hysterectomy under general anesthesia.

Maintenance techniques in epidural analgesia include:

Continuous Infusion: An easy technique that requires little intervention. The cumulative dose of local anesthetic is likely to be higher and side effects are more likely than with the other two techniques.

Intermittent Top-up: Results in benefits due to frequent patient/staff contact but can produce a high staff workload and patients may have to wait for treatment.

Patient-Controlled Epidural Analgesia (PCEA): This technique produces high patient satisfaction and reduced dose requirements compared with continuous infusion. However, sophisticated pumps are required and accurate catheter position is important for optimal efficacy (Rawal).

Multimodal Analgesia for Postoperative Pain Management 195

the side effects associated with central neuraxial blockade, such as hypotension and wide motor blockade with reduced mobility and proprioception, and complications such as epidural Hematoma, epidural abscess and paraparesis can be avoided (Liu&Salinas,

Continuous peripheral nerve blocks are being increasingly used since they may provide more selective but still excellent postoperative analgesia with reduced need for opioids over an extended period (Table 6). The vailability of disposable local anesthetic infusion systems and the encouraging results from these early studies have led to the increasing popularity of these techniques for pain control in the postdischarge period (Elvir Lazo&White, 2010). This technique has become increasingly popular due to its ability to control moderate to severe pain and accelerate recovery especially after orthopedic surgery procedures (Capdevila et

Patient controlled regional analgesia (PCRA) can also be used to maintain peripheral nerve block. A low basal infusion rate (e.g. 3-5 ml/h) associated with small PCA boluses (e.g. 2.5-5

> Ropivacaine 0.2%-0.375% Bupivacaine 0.1-0.125% Levobupivacaine 0.1-0.2%

Bolus dose: 2,5-5 ml

Lockout interval: 30-60 min

**Site of catheter Local anesthetics and dosage\***

\*Sometimes, higher concentrations are required in individual patients. As a standard, starting with a

Table 6. Examples of local anesthetics and doses in continuous peripheral nerve analgesia

The evidence suggests that the use of paravertebral blocks provide effective postoperative pain control following breast and thoracic surgery as well as for inguinal hernia repair (Greengrass et al, 1996; Karmakar, 2011; Pusch et al, 1999). On their own, paravertebral blocks have been demonstrated to provide effective postoperative analgesia lasting up to

Chelly et al showed in their study that; a multimodal approach, including paravertebral blocks (prior to surgery), celecoxib (pre and post surgey), and ketamine (immediately prior to surgery), provides better postoperative pain control than PCA morphine alone in patients

and PCA (modified table after using by permission from Publisher AstraZeneca)

Interscalene 5-9 ml/h Infraclavicular 5-9 ml/h Axillary 5-10 ml/h Femoral 7-10 ml/h Popliteal 3-7 ml/h Patient controlled regional analgesia Background: 3-5 ml/h

low concentration/dose is recommended to avoid sensory loss or motor block.

**5.2.1 Paravertebral blocks** 

24 hrs (Chelly et al, 2011).

2003).

al, 2005; Ilfeld&Enneking, 2005; White, 2003).

ml - lockout: 30-60 min) is the preferred technique (Rawal).


\* The tip of the catheter should be placed as close as possible to the surgical dermatomes: T6-T10 for major intra-abdominal surgery, and L2-L4 for lower limb surgery.

\*\* There are many possible variations in local anesthetic/opioid concentration yielding good results, the examples given here should be taken as a guideline; higher concentrations than the ones mentioned here are sometimes required but cannot be recommended as a routine for postoperative pain relief.

Table 5. Examples of local anesthetics and opioids and doses in epidural analgesia \* (by permission from Publisher AstraZeneca)

Continuous central neuraxial blockade is one of the most effective forms of postoperative analgesia, but it is also one of the most invasive. However, this technique remains the first choice for a number of indications, such as abdominal, thoracic, and major orthopedic surgery, where adequate pain relief cannot be achieved with other analgesia techniques alone. Continuous central neuraxial blockade can be achieved via two routes: Continuous epidural analgesia - the recommended first choice and continuous spinal analgesia - should be limited to selected cases only, as there is less experience with this technique.

#### **5.2 Peripheral regional analgesia techniques**

It is clear that local anesthetic techniques, particularly peripheral nerve blockade, will be one of the cornerstones of postoperative pain management. A variety of peripheral regional analgesic techniques (e.g. brachial plexus, lumbar plexus, femoral, paravertebral nerve blocks) as a single injection or continuous infusion can be used to enhance postoperative analgesia. Peripheral regional techniques may have several advantages over systemic opioids (i.e., superior analgesia and decreased opioid-related side effects). Also

Sufentanil 0.5-1 μg/ml **or**  Fentanyl 2-4 μg/ml **or**

Morphine 0.05-0.1 mg/ml **or**  Clonidine 5-20 μg/ml (clinical application is limited by its side effects) **or**  Epinephrine 2- 5 μg/ml

Ropivacaine 2% (2 mg/ml) **or** 

Levobupivacaine **or** 

0.1-0.2% (1-2 mg/ml)

Background: 4-6 ml/h Bolus dose: 2 ml (2-4 ml) Minimum lockout interval 10

Recommended maximum hourly dose (bolus + background): 12 ml

\* The tip of the catheter should be placed as close as possible to the surgical dermatomes: T6-T10 for

\*\* There are many possible variations in local anesthetic/opioid concentration yielding good results, the examples given here should be taken as a guideline; higher concentrations than the ones mentioned here are sometimes required but cannot be recommended as a routine for postoperative pain relief. Table 5. Examples of local anesthetics and opioids and doses in epidural analgesia \*

Continuous central neuraxial blockade is one of the most effective forms of postoperative analgesia, but it is also one of the most invasive. However, this technique remains the first choice for a number of indications, such as abdominal, thoracic, and major orthopedic surgery, where adequate pain relief cannot be achieved with other analgesia techniques alone. Continuous central neuraxial blockade can be achieved via two routes: Continuous epidural analgesia - the recommended first choice and continuous spinal analgesia - should be limited to selected cases only, as there is less experience with this

It is clear that local anesthetic techniques, particularly peripheral nerve blockade, will be one of the cornerstones of postoperative pain management. A variety of peripheral regional analgesic techniques (e.g. brachial plexus, lumbar plexus, femoral, paravertebral nerve blocks) as a single injection or continuous infusion can be used to enhance postoperative analgesia. Peripheral regional techniques may have several advantages over systemic opioids (i.e., superior analgesia and decreased opioid-related side effects). Also

Bupivacaine

6-12 ml/h

min

major intra-abdominal surgery, and L2-L4 for lower limb surgery.

(by permission from Publisher AstraZeneca)

**5.2 Peripheral regional analgesia techniques** 

(10-30 min)

**Local anesthetics /** 

**Dosage for continuous** 

(thoracic or lumbar

**Dosage for patient controlled infusion**  (thorasic or lumbar

**opioids** 

**infusion**

level)

level) \*\*

technique.

the side effects associated with central neuraxial blockade, such as hypotension and wide motor blockade with reduced mobility and proprioception, and complications such as epidural Hematoma, epidural abscess and paraparesis can be avoided (Liu&Salinas, 2003).

Continuous peripheral nerve blocks are being increasingly used since they may provide more selective but still excellent postoperative analgesia with reduced need for opioids over an extended period (Table 6). The vailability of disposable local anesthetic infusion systems and the encouraging results from these early studies have led to the increasing popularity of these techniques for pain control in the postdischarge period (Elvir Lazo&White, 2010). This technique has become increasingly popular due to its ability to control moderate to severe pain and accelerate recovery especially after orthopedic surgery procedures (Capdevila et al, 2005; Ilfeld&Enneking, 2005; White, 2003).

Patient controlled regional analgesia (PCRA) can also be used to maintain peripheral nerve block. A low basal infusion rate (e.g. 3-5 ml/h) associated with small PCA boluses (e.g. 2.5-5 ml - lockout: 30-60 min) is the preferred technique (Rawal).


\*Sometimes, higher concentrations are required in individual patients. As a standard, starting with a low concentration/dose is recommended to avoid sensory loss or motor block.

Table 6. Examples of local anesthetics and doses in continuous peripheral nerve analgesia and PCA (modified table after using by permission from Publisher AstraZeneca)

#### **5.2.1 Paravertebral blocks**

The evidence suggests that the use of paravertebral blocks provide effective postoperative pain control following breast and thoracic surgery as well as for inguinal hernia repair (Greengrass et al, 1996; Karmakar, 2011; Pusch et al, 1999). On their own, paravertebral blocks have been demonstrated to provide effective postoperative analgesia lasting up to 24 hrs (Chelly et al, 2011).

Chelly et al showed in their study that; a multimodal approach, including paravertebral blocks (prior to surgery), celecoxib (pre and post surgey), and ketamine (immediately prior to surgery), provides better postoperative pain control than PCA morphine alone in patients

Multimodal Analgesia for Postoperative Pain Management 197

pumps are available in the market today, including mechanical (elastometric) and electronic

Infiltrating local anesthetics into the skin and subcutaneous tissue prior to making an incision may be the simplest approach to analgesia. It is a safe procedure with few side effects and low risk for toxicity. Particularly, local anesthetic toxicity, wound infection and

Although the benefit of local wound infiltration has been documented (Barr-Dayan et al, 2004; Legeby et al, 2009; Park et al, 2002), controversy exists as to the appropriate timing of administering local anesthesia for surgery. A single injection of local anesthetics into the wound is unlikely to have long-lasting effects. Therefore, new techniques for wound infiltration have evolved during the last 10 years and several of them are today used routinely during ambulatory surgery and even in the inpatient setting. One such technique is the use of catheters inserted into incision, fascia, intra-articularly and intraabdominally for the intermittent injection or continuous infusion of local anesthetics and adjuvants for pain

Continuous wound infusion of local anesthetics, which is mainly used in general surgery and orthopedics, is an interesting technique in postoperative pain therapy. Continuous wound infusion of local anesthetics is able to reduce postoperative opioid requirements and results in decreased pain scores (Gupta et al, 2004; Rasmussen et al, 2004). Recent studies indicate that rehabilitation seems to be enhanced and postoperative hospital stay may be shorter. Continuous wound infusion is an effective analgesic technique, which is simple to perform. Comparisons with other analgesic techniques, such as peripheral nerve blocks,

Hollmann and Durieux (Hollmann&Durieux, 2000) found that there was a reduction in ileus and hospital stay when lidocaine was given intravenously following major abdominal surgery. Therefore, when administered in larger doses during wound infiltration analgesia, it is possible that some of the analgesic effect seen is via systemic absorption and anti-

Wound infiltration with local anesthetics is a simple, effective and inexpensive way of regimen which can be used in a multimodal analgesic regime without major complications. Nonetheless this technique still open some questions to be answered as; to the site of catheter placement, catheter type to be placed, the drugs and concentrations recommended, the technique of administration and side-effects of the technique, including toxicity of local anesthetics. Also it remains unclear as to whether this technique is useful in all types of

Lidocaine patches were applied to the wound area in the next two studies, and the evidence shows that these are particularly effective for wound pain when the patient coughes and they reduce the postoperative pain score at discharge (Habib et al, 2009; Saber et al, 2009). To place lidocaine patches over or at least close to the wound is suggested as a safe and

promising modality to consider in the management of postoperative pain control.

epidural analgesia and other multimodal analgesic concepts are still required.

surgery or should preferably be used for specific operations (Gupta, 2000).

healing do not appear to be major problems (Buvanendran&Kroin, 2009).

(Gupta, 2010).

**5.3.1 Wound infiltration** 

management (Gupta, 2010).

inflammation.

**5.3.2 Topical application 5.3.2.1 Local anesthetics** 

undergoing open radical retropubic prostatectomy. This approach also allows a reduction in the postoperative need for opioids, lessens the related side effects (e.g., PONV, constipation, and bladder spasm), and facilitates earlier patient recovery which can be connoted that it fascilitates the patient's early recovery (Chelly et al, 2011).

#### **5.3 Infiltration techniques**

Local anesthetics can be administered for perioperative pain management via different routes (Table 7). It is crucial for improving the perioperative outcomes especially after day-case surgery (White&Kehlet, 2010).


Table 7. Local anesthetic infiltration (by permission from Publisher AstraZeneca)

There are a few techniques for the delivery of the drugs locally into the tissues: intermittent injection, continuous infusion or a combination of two: Intermittent injections (also sometimes referred to as patient-controlled regional analgesia) have the advantage that pain relief can be timed in order to achieve maximal effect during the painful periods such as during mobilization. However, the disadvantage is that sleep quality may be disturbed, as patients sometimes wake up at night due to severe pain, which may be annoying and can also be a cause of patient dissatisfaction. Continuous local anesthetic administration has its advantage in that the patient has adequate pain relief most of the time. However, during periods of activity, the pain could be more severe, which may hamper mobilization. Methods using pumps that have a dual function with low-dose continuous infusion combined with self-administered bolus doses during mobilization are ideal. Several such

pumps are available in the market today, including mechanical (elastometric) and electronic (Gupta, 2010).

#### **5.3.1 Wound infiltration**

196 Pain Management – Current Issues and Opinions

undergoing open radical retropubic prostatectomy. This approach also allows a reduction in the postoperative need for opioids, lessens the related side effects (e.g., PONV, constipation, and bladder spasm), and facilitates earlier patient recovery which can be connoted that it

Local anesthetics can be administered for perioperative pain management via different routes (Table 7). It is crucial for improving the perioperative outcomes especially after day-case

Knee arthroscopy 0.75% Ropivacaine 20 ml Morphine 1-2

0.25-0.5% Ropivacaine 30-40 ml

0.25-0.5% Bupivacaine Up to 30 ml

0.25-0.5% Bupivacaine Up to 20 ml

0.25-0.5% Levobupivacaine

0.25-0.5% Levobupivacaine

There are a few techniques for the delivery of the drugs locally into the tissues: intermittent injection, continuous infusion or a combination of two: Intermittent injections (also sometimes referred to as patient-controlled regional analgesia) have the advantage that pain relief can be timed in order to achieve maximal effect during the painful periods such as during mobilization. However, the disadvantage is that sleep quality may be disturbed, as patients sometimes wake up at night due to severe pain, which may be annoying and can also be a cause of patient dissatisfaction. Continuous local anesthetic administration has its advantage in that the patient has adequate pain relief most of the time. However, during periods of activity, the pain could be more severe, which may hamper mobilization. Methods using pumps that have a dual function with low-dose continuous infusion combined with self-administered bolus doses during mobilization are ideal. Several such

Table 7. Local anesthetic infiltration (by permission from Publisher AstraZeneca)

Shoulder arthroscopy 0.75% Ropivacaine 10-20 ml

Gynecological 0.75% Ropivacaine 20 ml Cholecystectomy 0.25% Ropivacaine 40-60 ml

Thyroid surgery 0.25-0.5% Ropivacaine 10-20 ml

**Local anesthetic Volume Additives** 

0.5% Bupivacaine 20 ml Morphine 1-2

30-40 ml

10-20 ml

mg

mg

fascilitates the patient's early recovery (Chelly et al, 2011).

**5.3 Infiltration techniques** 

surgery (White&Kehlet, 2010).

**Intraarticular instillation** 

**Intraperitoneal instillation** 

**Wound infiltration**  Inguinal hernia Perianal surgery

Infiltrating local anesthetics into the skin and subcutaneous tissue prior to making an incision may be the simplest approach to analgesia. It is a safe procedure with few side effects and low risk for toxicity. Particularly, local anesthetic toxicity, wound infection and healing do not appear to be major problems (Buvanendran&Kroin, 2009).

Although the benefit of local wound infiltration has been documented (Barr-Dayan et al, 2004; Legeby et al, 2009; Park et al, 2002), controversy exists as to the appropriate timing of administering local anesthesia for surgery. A single injection of local anesthetics into the wound is unlikely to have long-lasting effects. Therefore, new techniques for wound infiltration have evolved during the last 10 years and several of them are today used routinely during ambulatory surgery and even in the inpatient setting. One such technique is the use of catheters inserted into incision, fascia, intra-articularly and intraabdominally for the intermittent injection or continuous infusion of local anesthetics and adjuvants for pain management (Gupta, 2010).

Continuous wound infusion of local anesthetics, which is mainly used in general surgery and orthopedics, is an interesting technique in postoperative pain therapy. Continuous wound infusion of local anesthetics is able to reduce postoperative opioid requirements and results in decreased pain scores (Gupta et al, 2004; Rasmussen et al, 2004). Recent studies indicate that rehabilitation seems to be enhanced and postoperative hospital stay may be shorter. Continuous wound infusion is an effective analgesic technique, which is simple to perform. Comparisons with other analgesic techniques, such as peripheral nerve blocks, epidural analgesia and other multimodal analgesic concepts are still required.

Hollmann and Durieux (Hollmann&Durieux, 2000) found that there was a reduction in ileus and hospital stay when lidocaine was given intravenously following major abdominal surgery. Therefore, when administered in larger doses during wound infiltration analgesia, it is possible that some of the analgesic effect seen is via systemic absorption and antiinflammation.

Wound infiltration with local anesthetics is a simple, effective and inexpensive way of regimen which can be used in a multimodal analgesic regime without major complications. Nonetheless this technique still open some questions to be answered as; to the site of catheter placement, catheter type to be placed, the drugs and concentrations recommended, the technique of administration and side-effects of the technique, including toxicity of local anesthetics. Also it remains unclear as to whether this technique is useful in all types of surgery or should preferably be used for specific operations (Gupta, 2000).

#### **5.3.2 Topical application**

#### **5.3.2.1 Local anesthetics**

Lidocaine patches were applied to the wound area in the next two studies, and the evidence shows that these are particularly effective for wound pain when the patient coughes and they reduce the postoperative pain score at discharge (Habib et al, 2009; Saber et al, 2009). To place lidocaine patches over or at least close to the wound is suggested as a safe and promising modality to consider in the management of postoperative pain control.

Multimodal Analgesia for Postoperative Pain Management 199

nonpharmacologic techniques, such as transcutaneous electrical nerve stimulation, acupuncture, psychological approaches (cold) and relaxing therapy and distraction, can be

The use of TENS at paravertebral dermatomes corresponding to the surgical incision and/or acupoints has also been reported to improve postoperative pain management (Chen et al, 1998). Because this technique cause few if any adverse effects, its use as an adjunct to conventional pharmaceutical approaches should be considered as part of multimodal analgesic regimens in the future, particularly for patients in whom conventional analgesic techniques fail and/or are accompanied by severe medication-related adverse events (Chen

The term acupuncture describes a family of procedures involving the stimulation of anatomical points on the body using a variety of techniques. Acupuncture theory is based on two conditions: "yin," which is considered feminine, passive, dark, and cold, and "yang," which is masculine, aggressive, bright, and hot, as well as "qi," which is considered the vital energy that flows and cycles throughout the body. The acupuncture theory is to harmonize any imbalance in yin-yang and qi in a human body to restore the body to a healthy condition. Acupuncture is

Usichenko et al. (Usichenko, 2008) focused on randomized controlled trials of only auricular acupuncture (a popular method in which needles are placed in various parts of the earlobe) for postoperative pain control. They identified nine studies of acceptable quality (though none of the best quality), and concluded that the evidence that auricular acupuncture controls postoperative pain is promising but not compelling. Sun et al. (Sun et al, 2008) conducted a systematic review to quantitatively evaluate the efficacy of acupuncture and related techniques as adjunct analgesics for acute postoperative pain management. The authors concluded that perioperative acupuncture might be a useful adjunct for acute postoperative pain management. However, there are issues with applicability and

Iced-water or continuous flow cold therapy is used in orthopedic surgery after knee-surgery (Barber et al, 2000). It can be used both at hospital and at home. There are commercial systems, which are easy to use. The use of iced-water in other kinds of surgery needs further

Music, or imagery, or hypnosis may have a positive effect in individual cases. There are

The percentage of surgical procedures being performed on an outpatient basis continues to rise. Many more complex and potentially painful procedures in comorbid conditions of the

thought to unblock any obstruction to the flow of qi and, thereby, relieves pain.

used in an attempt to alleviate postoperative pain.

et al, 1998; Usichenko, 2007; Wang et al, 1997).

generalizability of the procedure (Lee&Chan, 2006).

commercial music CDs available for relaxation (Rawal).

**5.4.4 Relaxing therapy and distraction** 

**5.4.2 Acupuncture** 

**5.4.3 Cold** 

investigation.

**6 Special aspects** 

**6.1 Ambulatory procedures** 

**5.4.1 Transcutaneous electrical nerve stimulation (TENS)** 

#### **5.3.2.2 Clonidine**

Clonidine is an alpha adrenoreceptor agonist and these receptors are known to be located centrally. In a volunteer study (Pratab et al, 2007) clonidine had a significant peripheral action in enhancing duration of local anesthesia on superficial co-infiltration with lidocaine. Hence an opportunity with this co-administration to prolong the duration of pain relief apparent after postoperative wound infiltration could be possible.

#### **5.3.2.3 Nonsteroidal anti-inflammatory drugs**

The topical application of NSAIDs could produce significant pain relief as the systemic levels achieved by transdermal application. Topically use of NSAIDs has become popular in the ophthalmic field, in which it has been shown that topically applied NSAIDs can reduce postoperative pain and inflammation (Cho, 2009; Jones&Francis, 2009). In a study, the use of a topical diclofenac patch resulted with reduced wound pain and analgesic requirement in patients who have undergone laparoscopic gynecologic surgery (Alessandri et al, 2006). As a result NSAID patch formulations, to be placed directly over the wound, would have a useful pain-relieving effect. But there is still some studies needed to compare this application with systemic administration of the same drug and what the side effect frequency might be with such application (McCleane, 2010).

#### **5.3.2.4 Glyceryl trinitrate**

Experimental data suggest that the production of endogenous nitric oxide is necessary for tonic cholinergic inhibition of spinal pain transmission. In a study; transdermal nitroglycerin and the central cholinergic agent neostigmine have enhanced each other's antinociceptive effects at the dose studied (Lauretti et al, 2010). In two recent more studies transdermal nitroglycerin enhanced the analgesic effect of intrathecal neostigmine following abdominal hysterectomy (Ahmed et al, 2010)and intrathecal fentanyl with bupivacaine following gynecological surgery (Gang et al, 2010).

#### **5.3.3 Local infiltration analgesia**

The administration of large volumes of local anesthetics with or without adjuvants into different tissue planes perioperatively is called local infiltration analgesia (LIA) (Gupta, 2010).

It is a multimodal technique developed by Kerr et al. (Kerr at al, 2008) for the control of pain following knee and hip surgery. In their study it was based on systematic infiltration of a mixture of a long acting local anesthetics (ropivacaine), a NSAID (ketorolac), and adrenaline into the tissues around the surgical field (periarticularly intraoperatively and via an intraarticular catheter postoperatively) to achieve satisfactory pain control with little physiological disturbance. The technique allows virtually immediate mobilization and earlier discharge from hospital. A recent study by Essving et al (Essving et al, 2009) on unicompartmental knee arthroplasty performed with minimal invasive technique, using the LIA technique found significantly shorter hospital stay, lower morphine consumption and pain intensity compared with placebo.

#### **5.4 Other – Nonpharmacological techniques**

A number of non-pharmacological methods of pain management may be used in conjunction with pharmacological methods in the postoperative setting. These nonpharmacologic techniques, such as transcutaneous electrical nerve stimulation, acupuncture, psychological approaches (cold) and relaxing therapy and distraction, can be used in an attempt to alleviate postoperative pain.

#### **5.4.1 Transcutaneous electrical nerve stimulation (TENS)**

The use of TENS at paravertebral dermatomes corresponding to the surgical incision and/or acupoints has also been reported to improve postoperative pain management (Chen et al, 1998). Because this technique cause few if any adverse effects, its use as an adjunct to conventional pharmaceutical approaches should be considered as part of multimodal analgesic regimens in the future, particularly for patients in whom conventional analgesic techniques fail and/or are accompanied by severe medication-related adverse events (Chen et al, 1998; Usichenko, 2007; Wang et al, 1997).

#### **5.4.2 Acupuncture**

198 Pain Management – Current Issues and Opinions

Clonidine is an alpha adrenoreceptor agonist and these receptors are known to be located centrally. In a volunteer study (Pratab et al, 2007) clonidine had a significant peripheral action in enhancing duration of local anesthesia on superficial co-infiltration with lidocaine. Hence an opportunity with this co-administration to prolong the duration of pain relief

The topical application of NSAIDs could produce significant pain relief as the systemic levels achieved by transdermal application. Topically use of NSAIDs has become popular in the ophthalmic field, in which it has been shown that topically applied NSAIDs can reduce postoperative pain and inflammation (Cho, 2009; Jones&Francis, 2009). In a study, the use of a topical diclofenac patch resulted with reduced wound pain and analgesic requirement in patients who have undergone laparoscopic gynecologic surgery (Alessandri et al, 2006). As a result NSAID patch formulations, to be placed directly over the wound, would have a useful pain-relieving effect. But there is still some studies needed to compare this application with systemic administration of the same drug and what the side effect

Experimental data suggest that the production of endogenous nitric oxide is necessary for tonic cholinergic inhibition of spinal pain transmission. In a study; transdermal nitroglycerin and the central cholinergic agent neostigmine have enhanced each other's antinociceptive effects at the dose studied (Lauretti et al, 2010). In two recent more studies transdermal nitroglycerin enhanced the analgesic effect of intrathecal neostigmine following abdominal hysterectomy (Ahmed et al, 2010)and intrathecal fentanyl with bupivacaine following

The administration of large volumes of local anesthetics with or without adjuvants into different tissue planes perioperatively is called local infiltration analgesia (LIA) (Gupta,

It is a multimodal technique developed by Kerr et al. (Kerr at al, 2008) for the control of pain following knee and hip surgery. In their study it was based on systematic infiltration of a mixture of a long acting local anesthetics (ropivacaine), a NSAID (ketorolac), and adrenaline into the tissues around the surgical field (periarticularly intraoperatively and via an intraarticular catheter postoperatively) to achieve satisfactory pain control with little physiological disturbance. The technique allows virtually immediate mobilization and earlier discharge from hospital. A recent study by Essving et al (Essving et al, 2009) on unicompartmental knee arthroplasty performed with minimal invasive technique, using the LIA technique found significantly shorter hospital stay, lower morphine consumption and

A number of non-pharmacological methods of pain management may be used in conjunction with pharmacological methods in the postoperative setting. These

apparent after postoperative wound infiltration could be possible.

frequency might be with such application (McCleane, 2010).

**5.3.2.3 Nonsteroidal anti-inflammatory drugs** 

**5.3.2.2 Clonidine** 

**5.3.2.4 Glyceryl trinitrate** 

gynecological surgery (Gang et al, 2010).

**5.3.3 Local infiltration analgesia** 

pain intensity compared with placebo.

**5.4 Other – Nonpharmacological techniques** 

2010).

The term acupuncture describes a family of procedures involving the stimulation of anatomical points on the body using a variety of techniques. Acupuncture theory is based on two conditions: "yin," which is considered feminine, passive, dark, and cold, and "yang," which is masculine, aggressive, bright, and hot, as well as "qi," which is considered the vital energy that flows and cycles throughout the body. The acupuncture theory is to harmonize any imbalance in yin-yang and qi in a human body to restore the body to a healthy condition. Acupuncture is thought to unblock any obstruction to the flow of qi and, thereby, relieves pain.

Usichenko et al. (Usichenko, 2008) focused on randomized controlled trials of only auricular acupuncture (a popular method in which needles are placed in various parts of the earlobe) for postoperative pain control. They identified nine studies of acceptable quality (though none of the best quality), and concluded that the evidence that auricular acupuncture controls postoperative pain is promising but not compelling. Sun et al. (Sun et al, 2008) conducted a systematic review to quantitatively evaluate the efficacy of acupuncture and related techniques as adjunct analgesics for acute postoperative pain management. The authors concluded that perioperative acupuncture might be a useful adjunct for acute postoperative pain management. However, there are issues with applicability and generalizability of the procedure (Lee&Chan, 2006).

#### **5.4.3 Cold**

Iced-water or continuous flow cold therapy is used in orthopedic surgery after knee-surgery (Barber et al, 2000). It can be used both at hospital and at home. There are commercial systems, which are easy to use. The use of iced-water in other kinds of surgery needs further investigation.

#### **5.4.4 Relaxing therapy and distraction**

Music, or imagery, or hypnosis may have a positive effect in individual cases. There are commercial music CDs available for relaxation (Rawal).

#### **6 Special aspects**

#### **6.1 Ambulatory procedures**

The percentage of surgical procedures being performed on an outpatient basis continues to rise. Many more complex and potentially painful procedures in comorbid conditions of the

Multimodal Analgesia for Postoperative Pain Management 201

Nonopioid analgesics are increasingly being used as adjuvant before, during, and after surgery to facilitate the recovery process after ambulatory surgery because of their anesthetic and analgesic-sparing effects, their ability to reduce postoperative pain (with movement), and their opioid related side-effects (e.g., gastrointestinal and bladder dysfunction), thereby shortening the duration of the hospital stay and the convalescence

Patient-controlled regional analgesia (PCRA) encompasses a variety of techniques that provide effective postoperative pain relief without systemic exposure to opioids. Using PCRA, patients control the application of pre-programmed doses of local anesthetics, most frequently ropivacaine or bupivacaine (occasionally in combination with an opioid), via an indwelling catheter, which can be placed in different regions of the body depending upon the type of surgery. It is important to use suitable local anesthetics in low concentration and to inform patient adequately to avoid the risk of local anesthetic toxicity (Rawal, Vadivelu et

Many detrimental pathophysiologic effects occur in the perioperative period and are associated with activation of nociceptors and the stress response. Uncontrolled pain may result in activation of the sympathetic nervous system, which can cause a variety of potentially harmful physiologic responses that may adversely influence the extent of

As afferent neural stimuli and activation of the autonomic nervous system and other reflexes by pain may serve as a major release mechanism of the endocrine metabolic responses and thus contribute to various organ dysfunctions, pain relief may be a powerful

Systemic opioids (PCA or intermittent), NSAID, epidural opiod, lumbar and thorasic epidural local anesthetics are analgesic techniques are mostly used to supress the postoperative surgical stress responces but there is a pronounced differential effect of these various techniques on surgical stress responses (Kehlet&Holte, 2001). Any treatment with opioids, being epidural or PCA opioids, has very little effect on surgical stress responses and organ dysfunctions. Same applies to clonidine and also NSAIDs. Epidural anesthesia has the

Several studies investigating lower extremity surgery have shown continuous lumbar epidural local anesthetic techniques to be most effective, probably because of a more effective afferent blockade. In abdominal procedures, there is a somewhat smaller efficacy of thoracic epidural local anesthetic techniques in modulating endocrine-metabolic responses, probably due to insufficient afferent blockade as well as the presence of other release

The neuraxial application of local anesthetics and opioids combined to general anesthesia (especially in patients undergoing major abdominal or thoracic procedures) as a multimodal strategy can provide superior pain relief, reduced hormonal and metabolic stress, enhanced normalization of gastrointestinal function, and thus a shortened postoperative recovery time, facilitating mobilization and physiotherapy (Schug&Chong, 2009). In a study by Sivrikaya et al (Sivrikaya et al, 2008) general anesthesia combined lumbar epidural analgesia can only partially attenuate the peroperative stress response and has some limited effects on

period (White&Kehlet, 2010).

al, 2010).

**6.2 Stress response** 

morbidity and mortality (Vadivelu et al, 2010).

technique to modify surgical stress responses.

most profound inhibitory effect on surgical stress responses.

mechanisms in eliciting the surgical stress response.

surgical outpatients are being routinely performed in the ambulatory setting (White & Kehlet, 2010).

Postoperative pain management have some disadvantages in this population; a. pain after minor surgery or in ambulatory patients is more difficult to treat because many of the aforementioned techniques are not available or are too risky. b. The increasing number and complexity of elective operations that are being performed on an ambulatory (or short-stay) basis in which the use of conventional opioid-based intravenous patient controlled analgesia and central neuraxial (spinal and epidural) analgesia techniques are simply not practical for acute pain management. c. The pressure to discharge patients after surgery could limit the pain medications health care professionals are willing to prescribe and it may explain the inadequate management of acute pain after surgery.

Most common medical causes of delayed discharge after ambulatory surgery are; pain, drowsiness and nausea/vomiting (Vadivelu et al, 2010). Although many factors, in addition to pain, must be carefully controlled to minimize postoperative morbidity and facilitate the recovery process after elective surgery, the adequacy of pain control should remain a major focus of health care providers, caring for patients undergoing ambulatory surgical procedures (Elvir Lazo&White, 2010). Many patients undergoing ambulatory surgery continue to experience unacceptably high levels of pain after their operation. A survey by McGrath et al. showed that 30% of patients suffer moderate-to-severe pain following minor surgical procedures (McGrath et al, 2004).

To have a qualified postoperative pain control after ambulatory surgery, it is required that patient discharge is not delayed and that pain control remains effective once the patient is at home. It is important to avoid to use of long acting analgesics and to use regional anesthesia techniques for the anesthesia. Regional analgesia techniques offer a number of advantages for day case surgery patients such as: flexible duration of analgesia (with single shot techniques and/or with catheter infusions), flexible intensity of blockade (according to the type, concentration and volume of local anesthetic) and reduced need for opioids. Wound infiltration, intraperitoneal instillation, peripheral nerve blocks e.g. brachial plexus, paravertebral, femoral nerve blocks can be used in ambulatory surgery patients.

The adaptation of multimodal (or balanced) analgesic techniques as the standard approach for the prevention of pain in the ambulatory setting is one of the keys to improving the recovery process after day-case surgery (McGrath et al, 2004; White, 2007). Early studies evaluating approaches to facilitating the recovery process have demonstrated that the use of multimodal analgesic techniques can improve early recovery as well as other clinically meaningful outcomes after ambulatory surgery. These benefits have been confirmed in more recent studies (Elvir Lazo&White, 2010).

An aggressive multimodal perioperative analgesic regimen that provides effective pain relief, has minimal side-effects, is intrinsically safe, and can be managed by the patient and their family members away from a hospital or surgical center is the ideal one. Current evidence suggests that these improvements in patient outcome related to pain control can best be achieved by using a combination of preventive analgesic techniques involving both centrally and peripherally acting analgesic drugs, as well as novel approaches to administering drugs in locations remote from the hospital setting (White&Kehlet, 2010).

Nonopioid analgesics are increasingly being used as adjuvant before, during, and after surgery to facilitate the recovery process after ambulatory surgery because of their anesthetic and analgesic-sparing effects, their ability to reduce postoperative pain (with movement), and their opioid related side-effects (e.g., gastrointestinal and bladder dysfunction), thereby shortening the duration of the hospital stay and the convalescence period (White&Kehlet, 2010).

Patient-controlled regional analgesia (PCRA) encompasses a variety of techniques that provide effective postoperative pain relief without systemic exposure to opioids. Using PCRA, patients control the application of pre-programmed doses of local anesthetics, most frequently ropivacaine or bupivacaine (occasionally in combination with an opioid), via an indwelling catheter, which can be placed in different regions of the body depending upon the type of surgery. It is important to use suitable local anesthetics in low concentration and to inform patient adequately to avoid the risk of local anesthetic toxicity (Rawal, Vadivelu et al, 2010).

#### **6.2 Stress response**

200 Pain Management – Current Issues and Opinions

surgical outpatients are being routinely performed in the ambulatory setting (White &

Postoperative pain management have some disadvantages in this population; a. pain after minor surgery or in ambulatory patients is more difficult to treat because many of the aforementioned techniques are not available or are too risky. b. The increasing number and complexity of elective operations that are being performed on an ambulatory (or short-stay) basis in which the use of conventional opioid-based intravenous patient controlled analgesia and central neuraxial (spinal and epidural) analgesia techniques are simply not practical for acute pain management. c. The pressure to discharge patients after surgery could limit the pain medications health care professionals are willing to prescribe and it may explain the

Most common medical causes of delayed discharge after ambulatory surgery are; pain, drowsiness and nausea/vomiting (Vadivelu et al, 2010). Although many factors, in addition to pain, must be carefully controlled to minimize postoperative morbidity and facilitate the recovery process after elective surgery, the adequacy of pain control should remain a major focus of health care providers, caring for patients undergoing ambulatory surgical procedures (Elvir Lazo&White, 2010). Many patients undergoing ambulatory surgery continue to experience unacceptably high levels of pain after their operation. A survey by McGrath et al. showed that 30% of patients suffer moderate-to-severe pain following minor

To have a qualified postoperative pain control after ambulatory surgery, it is required that patient discharge is not delayed and that pain control remains effective once the patient is at home. It is important to avoid to use of long acting analgesics and to use regional anesthesia techniques for the anesthesia. Regional analgesia techniques offer a number of advantages for day case surgery patients such as: flexible duration of analgesia (with single shot techniques and/or with catheter infusions), flexible intensity of blockade (according to the type, concentration and volume of local anesthetic) and reduced need for opioids. Wound infiltration, intraperitoneal instillation, peripheral nerve blocks e.g. brachial plexus, paravertebral, femoral nerve blocks can be used in ambulatory surgery

The adaptation of multimodal (or balanced) analgesic techniques as the standard approach for the prevention of pain in the ambulatory setting is one of the keys to improving the recovery process after day-case surgery (McGrath et al, 2004; White, 2007). Early studies evaluating approaches to facilitating the recovery process have demonstrated that the use of multimodal analgesic techniques can improve early recovery as well as other clinically meaningful outcomes after ambulatory surgery. These benefits have been confirmed in more

An aggressive multimodal perioperative analgesic regimen that provides effective pain relief, has minimal side-effects, is intrinsically safe, and can be managed by the patient and their family members away from a hospital or surgical center is the ideal one. Current evidence suggests that these improvements in patient outcome related to pain control can best be achieved by using a combination of preventive analgesic techniques involving both centrally and peripherally acting analgesic drugs, as well as novel approaches to administering drugs in locations remote from the hospital setting (White&Kehlet,

Kehlet, 2010).

patients.

2010).

inadequate management of acute pain after surgery.

surgical procedures (McGrath et al, 2004).

recent studies (Elvir Lazo&White, 2010).

Many detrimental pathophysiologic effects occur in the perioperative period and are associated with activation of nociceptors and the stress response. Uncontrolled pain may result in activation of the sympathetic nervous system, which can cause a variety of potentially harmful physiologic responses that may adversely influence the extent of morbidity and mortality (Vadivelu et al, 2010).

As afferent neural stimuli and activation of the autonomic nervous system and other reflexes by pain may serve as a major release mechanism of the endocrine metabolic responses and thus contribute to various organ dysfunctions, pain relief may be a powerful technique to modify surgical stress responses.

Systemic opioids (PCA or intermittent), NSAID, epidural opiod, lumbar and thorasic epidural local anesthetics are analgesic techniques are mostly used to supress the postoperative surgical stress responces but there is a pronounced differential effect of these various techniques on surgical stress responses (Kehlet&Holte, 2001). Any treatment with opioids, being epidural or PCA opioids, has very little effect on surgical stress responses and organ dysfunctions. Same applies to clonidine and also NSAIDs. Epidural anesthesia has the most profound inhibitory effect on surgical stress responses.

Several studies investigating lower extremity surgery have shown continuous lumbar epidural local anesthetic techniques to be most effective, probably because of a more effective afferent blockade. In abdominal procedures, there is a somewhat smaller efficacy of thoracic epidural local anesthetic techniques in modulating endocrine-metabolic responses, probably due to insufficient afferent blockade as well as the presence of other release mechanisms in eliciting the surgical stress response.

The neuraxial application of local anesthetics and opioids combined to general anesthesia (especially in patients undergoing major abdominal or thoracic procedures) as a multimodal strategy can provide superior pain relief, reduced hormonal and metabolic stress, enhanced normalization of gastrointestinal function, and thus a shortened postoperative recovery time, facilitating mobilization and physiotherapy (Schug&Chong, 2009). In a study by Sivrikaya et al (Sivrikaya et al, 2008) general anesthesia combined lumbar epidural analgesia can only partially attenuate the peroperative stress response and has some limited effects on

Multimodal Analgesia for Postoperative Pain Management 203

Apfelbaum J, Chen C,Mehta S, Gan T (2003). Postoperative pain experience: results from a

Barber FA. A comparison of crushed ice and continuous flow cold therapy (2000). *Am J Knee* 

Bar-Dayan A, Natour M, Bar-Zakai B, Zmora O, *et al* (2004). Preperitoneal bupivacaine

Bell R, Dahl J, Moore R, Kalso E. Perioperative ketamine for acute postoperative pain (2006).

Bilgin H, Ozcan B, Bilgin T, Kerimoglu B, *et al* (2005). The influence of timing of systemic

Birnbach DJ, Johnson MD, Arcario T, Datta S, *et al* (1989). Effect of diluent volume on

Bisgaard T (2006). Analgesic treatment after laparoscopic cholecystectomy: a critical

Boisseau N, Rabary O, Padovani B, Staccini P, *et al* (2001). Improvement of 'dynamic

Buvanendran A, Kroin J (2007). Useful adjuvants for postoperative pain management. *Best Pract Res Clin Anaesthesiol*, Vol.21, No.1(Mar), pp.31-49, ISSN 1521-6896. Buvanendran A, Kroin JS (2009). Multimodal analgesia for controlling acute postoperative

Buvanendran A, Kroin JS, Della Valle CJ, Kari M, *et al* (2010). Perioperative oral pregabalin

Capdevila X, Pirat P, Bringuier S, Gaertner R, *et al* (2005). Continuous peripheral nerve

patients. *Anesthesiology*, Vol.103, No.5(Nov), pp.1035-45, ISSN 0003-3022. Carli F, Mayo N, Klubien K, Schricker T, *et al* (2002). Epidural analgesia enhances functional

*Analg*, Vol.97, No.2(Aug), pp.534-40, ISSN 0003-2999.

*Surg*, Vol.13, No.2(Spring), pp.97-101, ISSN 0899-7403.

No.7(Jul), pp.1079-81, ISSN 0930-2794.

Vol.17, No.8(Dec), pp.592-7, ISSN 0952-8180.

No.4(Oct), pp.564-9, ISSN 0007-0912.

493X(Electronic).

10, ISSN 0003-2999.

0003-3022.

7907.

Review, ISSN 0140-6736.

updated report by the American Society of Anesthesiologists task force on acute pain management. *Anesthesiology,* Vol.100, No.6(June), pp.1573-81, ISSN 0003-3022.

national survey suggest postoperative pain continues to be undermanaged. *Anesth* 

attenuates pain following laparoscopic inguinal hernia repair. *Surg Endosc*, Vol.18,

*Cochrane Database Syst Rev*, Vol.25, No.1(Jan), CD004603, ISSN 1469-

ketamine administration on postoperative morphine consumption. *J Clin Anesth*,

analgesia produced by epidural fentanyl. *Anesth Analg*, Vol.68, No.6(Jun), pp.808-

assessment of the evidence. *Anesthesiology*, Vol.104, No.4(Apr), pp.835-46, ISSN

analgesia' does not decrease atelectasis after thoracotomy. *Br J Anaesth*, Vol.87,

pain. *Curr Opin Anaesthesiol*, Vol.22, No.5(Oct), pp.588-93, Review, ISSN 0952-7907.

reduces chronic pain after total knee arthroplasty: a prospective, randomized, controlled trial. *Anesth Analg*, Vol.110, No.1(Jan), pp.199-207, ISSN 0003-2999. Camann W, Abouleish A, Eisenach J, Hood D, *et al* (1998). Intrathecal sufentanil and

epidural bupivacaine for labor analgesia: dose-response of individual agents and in combination. *Reg Anesth Pain Med*, Vol.23, No.5(Sep-Oct), pp.457-62, ISSN 0952-

blocks in hospital wards after orthopedic surgery: a multicenter prospective analysis of the quality of postoperative analgesia and complications in 1,416

exercise capacity and health-related quality of life after colonic surgery: results of a randomized trial. *Anesthesiology*, Vol.97, No.3(Sep), pp.540-9, ISSN 0003-3022. Carr DB, Goudas LC (1999). Acute pain. *Lancet*, Vol.353(9169), No.12(Jun), pp.2051-8,

recovery of gastrointestinal functions, nevertheless provided a better postoperative analgesia compared to general anesthesia alone. Epidural opioid techniques are less effective on the stress response, and are comparable with systemic opioid techniques and the use of NSAIDs. More data on the use of multimodal analgesic techniques with combinations of different analgesics are needed on this issue.

#### **7. Conclusion**

Postoperative pain is a complication of surgery, which, in turn, complicates recovery with functional impairment and drug-related adverse effects. Despite an increased focus on pain management programs and the development of new standards for pain management, many patients continue to experience intense pain after surgery.

Many factors must be considered before deciding on the type of pain therapy to be provided to the surgical patient. These include the patients' co-morbid conditions, psychological status, exposure to analgesic therapies, and the type of surgical procedure.

The multimodal approach may potentially decrease perioperative morbidity, reduce the length of hospital stay, and improve patient satisfaction without compromising safety. However, widespread implementation of these programs requires multidisciplinary collaboration, change in the traditional principles of postoperative care, additional resources, and expansion of the traditional acute pain service. Although a multipharmacologic approach may be universally recommended, drugs and their route of administration must be changed according to the type of surgery and hospital resources, and of course to the patient needs.

#### **8. References**


recovery of gastrointestinal functions, nevertheless provided a better postoperative analgesia compared to general anesthesia alone. Epidural opioid techniques are less effective on the stress response, and are comparable with systemic opioid techniques and the use of NSAIDs. More data on the use of multimodal analgesic techniques with

Postoperative pain is a complication of surgery, which, in turn, complicates recovery with functional impairment and drug-related adverse effects. Despite an increased focus on pain management programs and the development of new standards for pain management, many

Many factors must be considered before deciding on the type of pain therapy to be provided to the surgical patient. These include the patients' co-morbid conditions, psychological

The multimodal approach may potentially decrease perioperative morbidity, reduce the length of hospital stay, and improve patient satisfaction without compromising safety. However, widespread implementation of these programs requires multidisciplinary collaboration, change in the traditional principles of postoperative care, additional resources, and expansion of the traditional acute pain service. Although a multipharmacologic approach may be universally recommended, drugs and their route of administration must be changed according to the type of surgery and hospital resources,

Aasvang E, Hansen J, Malmstrøm J, Asmussen T, *et al* (2008). The effect of wound

Acute pain management: operative or medical procedures and trauma, part 1 (1992).

Ahmed F, Garg A, Chawla V, Khandelwal M (2010). Transdermal nitroglycerine enhances

hysterectomies. *Indian J Anaesth*, Vol.54, No. 1(Jan), pp.24-8, ISSN 0019-5049. Alessandri F, Lijoi D, Mistrangelo E, Nicoletti a, *et al* (2006). Topical diclofenac patch for

American Pain Society Quality of Care Committee. Quality improvement guidelines for the

American Society of Anesthesiologists Task Force on Acute Pain Management (2004).

instillation of a novel purified capsaicin formulation on postherniotomy pain: a double-blind, randomized, placebo-controlled study. *AnesthAnalg*, Vol.107,

Agency for Health Care Policy and Research. *Clin Pharm* , Vol.11, No.4(Apr),

postoperative analgesia of intrathecal neostigmine following abdominal

postoperative wound pain in laparoscopic gynaecologic surgery: a randomized study. *J Minim Invasive Gynecol*, Vol.13, No.3(May-June), pp.195-200, ISSN 1553-

treatment of acute pain and cancer pain (1995). *JAMA* , Vol.274, No.23(Dec),

Practice guidelines for acute pain management in the perioperative setting. An

combinations of different analgesics are needed on this issue.

patients continue to experience intense pain after surgery.

No.1(Jul), pp.282-91, ISSN 0003-2999.

pp.309-31. ISSN 0278-2677.

pp.1874-80, ISSN 0098-7484.

status, exposure to analgesic therapies, and the type of surgical procedure.

**7. Conclusion** 

and of course to the patient needs.

**8. References** 

4650.

updated report by the American Society of Anesthesiologists task force on acute pain management. *Anesthesiology,* Vol.100, No.6(June), pp.1573-81, ISSN 0003-3022.


Multimodal Analgesia for Postoperative Pain Management 205

Fanelli G, Berti M, Baciarello M (2008). Updating postoperative pain management: from

Feo CV, Sortini D, Ragazzi R, De Palma M, *et al* (2006). Randomized clinical trial of the effect

Gan TJ, Joshi GP, Viscusi E, Cheung RY, *et al* (2004). Preoperative parenteral parecoxib and

Garg A, Ahmed F, Khandelwal M, Chawla V, *et al* (2010). The effect of transdermal

George MJ (2006). The site of action of epidurally administered opioids and its relevance to

Graham GG, Scott KF (2005). Mechanism of action of paracetamol. *Am J Ther*, Vol.12,

Gray A, Kehlet H, Bonnet F, Rawal N (2005). Predicting postoperative analgesia outcomes:

Greengrass R, O'Brien F, Lyerly K, Hardman D, *et al* (1996). Paravertebral block for breast cancer surgery. *Can J Anaesth*, Vol.43, No.8(Aug), pp.858-61, ISSN 0832-610X. Gupta A, Perniola A, Axelsson K, Thörn SE, *et al* (2004). Postoperative pain after abdominal

Gupta A (2010). Wound infiltration with local anaesthetics in ambulatory surgery. *Curr Opin* 

Gurbet A, Basagan-Mogol E, Turker G, Ugun F, *et al* (2006). Intraoperative infusion of

Habib A, Gan T (2005). Role of analgesic adjuncts in postoperative pain management. *Anesthesiol Clin North America*, Vol.23, No.1(Mar), pp.85-107, ISSN 0889-8537. Habib AS, White WD, El Gasim MA, Saleh G, *et al* (2008). Transdermal nicotine for analgesia

Habib AS, Polascik TJ, Weizer AZ, White WD, *et al* (2009). Lidocaine patch for postoperative

Hartrick C, Van Hove I, Stegmann J, Oh C, *et al* (2009). Efficacy and tolerability of tapentadol

*Anaesthesiol*, Vol.23, No.6(dec), pp.708-13, Review, ISSN 0952-7907.

cholecystectomy. *Br J Surg*, Vol.93, No.3(Mar), pp.295-9, ISSN 0007-1323. Gajraj N, Joshi G (2005). Role of cyclooxygenase-2 inhibitors in postoperative pain

500, ISSN 0375-9393.

90, ISSN 0310-057X.

pp.710-4, ISSN 0007-0912.

1004, ISSN 0003-2999.

pp.1950-53, ISSN 0003-2999.

No.6(Jun), pp.1665-73, ISSN 0889-8537.

No.1(Jan-Feb), pp.46-55, ISSN 1075-2765.

Vol.53, No.7(Jul), pp.646-52, ISSN 0832-610X.

0889-8537.

0003-2409.

2999.

multimodal to context-sensitive treatment. *Minerva Anestesiol*, Vol.74, No.9, pp 489-

of preoperative dexamethasone on nausea and vomiting after laparoscopic

management. *Anesthesiol Clin North America*, Vol.23, No.1(Mar), pp.49-72, ISSN

follow-up oral valdecoxib reduce length of stay and improve quality of patient recovery after laparoscopic cholecystectomy surgery. *Anesth Analg*, Vol.98,

nitroglycerine on intrathecal fentanyl with bupivacaine for postoperative analgesia following gynaecological surgery. *Anaesth Intensive Care*, Vol.38, No.2(Mar), pp.285-

postoperative pain management. *Anaesthesia*, Vol.61, No.1(Jul), pp.659-64, ISSN

NNT league tables or procedure-specific evidence? *Br J Anaesth*, Vol.94, No.6(Jun),

hysterectomy: a double-blind comparison between placebo and local anesthetic infused intraperitoneally. *Anesth Analg*, Vol.99, No.4(Oct), pp.1173-9, ISSN 0003-

dexmedetomidine reduces perioperative analgesic requirements. *Can J Anaesth*,

after radical retropubic prostatectomy. *Anesth Analg*, Vol.107, No.3(Sep), pp.999-

analgesia after radical retropubic prostatectomy. *Anesth Analg*, Vol.108, No.6(Jun),

immediate release and oxycodone HCl immediate release in patients awaiting


Chelly JE (2001). General concepts and indications. In: Chelly JE, Casati A, Fanelli G, editors. Continuous peripheral nerve block techniques. London: Mosby, pp.11-21. Chelly JE, Ploskanych T, Dai F, Nelson JB (2011). Multimodal analgesic approach

Chen L, Tang J, White PF, Sloninsky A, *et al* (1998). The effect of location of transcutaneous

Cho H, Wolf KJ, Wolf EJ (2009). Management of ocular inflammation and pain following

Christie MJ, Connor M, Vaughan CW, Ingram SL, *et al* (2000). Cellular actions of opioids and

de Beer Jde V, Winemaker MJ, Donnelly GA, Miceli PC, *et al* (2005). Efficacy and safety of

de Leon-Casasola OA, Lema MJ (1996). Postoperative epidural opioid analgesia: what are the choices? *Anesth Analg*, Vol.83, No.4(Oct), pp.867-75, ISSN 0003-2999. Dholakia C, Beverstein G, Garren M, Nemergut C, *et al* (2007). The impact of perioperative

Dolin SJ, Cashman JN, Bland JM (2009). Effectiveness of acute postoperative pain

Duedahl TH, Romsing J, Moiniche S, Dahl JB (2006). A qualitative systematic review of peri-

Eccleston C (2001). Role of psychology in pain management. *Br J Anaesth*, Vol.87, No.1(Jul),

Elia N, Lysakowski C, Tramèr MR (2005). Does multimodal analgesia with acetaminophen,

Elvir-Lazo OL, White PF (2010). Postoperative pain management after ambulatory surgery:

Essving P, Axelsson K, Kjellberg J, Wallgren O, *et al* (2009). Reduced hospital stay, morphine

No.4(Apr), pp.371-8, ISSN 0832-610X.

*Physiol*, Vol.27, No.7(Jul), pp.520-3, ISSN 0305-1870.

pp.1129-34, ISSN 0003-2999.

pp.199-210, ISSN 1177-5467.

ISSN 1091-255X.

pp.409-23, ISSN 0007-0912.

304, ISSN 0003-3022.

1932-2275.

ISSN 1745-3674.

No.1(Jan), pp.1-13, ISSN 0001-5172.

pp.144-52, Review, ISSN 0007-0912.

incorporating paravertebral blocks for open radical retropubic prostatectomy: a randomized double-blind placebo-controlled study. *Can J Anaesth*, Vol.58,

electrical nerve stimulation on postoperative opioid analgesic requirement: acupoint versus nonacupoint stimulation. *Anesth Analg*, Vol.87, No.5(Nov),

cataract surgery: focus on bromfenac ophthalmic solution. *Clin Ophthalmol*, Vol.3,

other analgesics: implications for synergism in pain relief. *Clin Exp Pharmacol* 

controlled release oxycodone and standard therapies for postoperative pain after knee or hip replacement. *Can J Surg*, Vol.48, No.4(Aug), pp.277-83, ISSN 0008-428X.

dexmedetomidine infusion on postoperative narcotic use and duration of stay after laparoscopic bariatric surgery. *J Gastrointest Surg,* Vol.11, No.11(Nov), pp.1556-9,

management: I. Evidence from published data. *Br J Anaesth*, Vol.89, No.3(Sep),

operative dextromethorphan in post-operative pain. *Acta Anaesthesiol Scand*, Vol.50,

nonsteroidal antiinflammatory drugs, or selective cyclooxygenase-2 inhibitors and patient-controlled analgesia morphine offer advantages over morphine alone? Meta-analyses of randomized trials. *Anesthesiology*, Vol.103, No.6(Dec), pp.1296-

role of multimodal analgesia. *Anesthesiol Clin*, Vol.28, No.2(Jun), pp.217-24, ISSN

consumption, and pain intensity with local infiltration analgesia after unicompartmental knee arthroplasty. *Acta Orthop*, Vol.80, No.2(Apr), pp.213-9,


Multimodal Analgesia for Postoperative Pain Management 207

Koinig H, Wallner T, Marhofer P, Andel H, *et al* (1998). Magnesium sulfate reduces intra-

Lauretti GR, de Oliveira R, Reis MP, Mattos AL, *et al* (1999). Transdermal nitroglycerine

Lauretti GR, Oliveira AP, Julião MC, Reis MP, *et al* (2000). Transdermal nitroglycerine

Lee A, Chan S (2006). Acupuncture and anaesthesia. *Best Pract Res Clin Anaesthesiol*, Vol.20,

Legeby M, Jurell G, Beausang-Linder M, Olofsson C (2009). Placebo-controlled trial of local

Lin TF, Yeh YC, Lin FS, Wang YP, *et al* (2009). Effect of combining dexmedetomidine and

Liu SS, Salinas FV (2003). Continuous plexus and peripheral nerve blocks for postoperative analgesia. *Anesth Analg*, Vol.96, No.1(Jan), pp. 263-72, Review, ISSN 0003-2999. Liu SS (2004). Anesthesia and analgesia for colon surgery. *Reg Anesth Pain Med*, Vol.29,

Liu SS, Wu CL (2007). Effect of postoperative analgesia on major postoperative

Lysakowski C, Dumont L, Czarnetzki C, Tramer MR (2007). Magnesium as an adjuvant to

Ma H, Tang J, White PF, Zaentz A, *et al* (2004). Perioperative rofecoxib improves early

Macario A, Lipman AG (2001). Ketorolac in the era of cyclo-oxygenase-2 selective

Mathiesen O, Jacobsen L, Holm H, Randall S, *et al* (2008). Pregabalin and dexamethasone

Mathiesen O, Rasmussen ML, Dierking G, Leck H, *et al* (2009). Pregabalin and

*Br J Anaesth*, Vol.101, No.4(Oct), pp.535-41, ISSN 0007-0912.

surgery. *Anesthesiology*, Vol.93, No.4(Oct), pp.943-6, ISSN 0003-3022. Lauwick S, Kim DJ, Michelagnoli G, Mistraletti G, *et al* (2008). Intraoperative infusion of

*Anesthesiology*, Vol.90, No.3(Mar), pp.734-9, ISSN 0003-3022.

*Surg Hand Surg* , Vol.43, No.6, pp.315-9. ISSN 0284-4311.

10, ISSN 0003-2999.

0832-610X.

ISSN 0003-2999.

No.2(Jun), pp.303-14, ISSN 1521-6896.

Vol.1(Jan), pp. 117-22, ISSN 0007-0912.

No.1(Jan-Feb), pp.52-7, ISSN 1098-7339.

No.3(Mar), pp.689-702, ISSN 0003-2999.

Vol.7, No.7(Jul), pp.6, ISSN 1471-2253.

Vol.104, No.6(Jun), pp.1532-9, ISSN 0003-2999.

and postoperative analgesic requirements. *Anesth Analg*, Vol.87, No.1(Jul), pp.206-

enhances spinal sufentanil postoperative analgesia following orthopedic surgery.

enhances spinal neostigmine postoperative analgesia following gynecological

lidocaine reduces postoperative fentanyl requirements in patients undergoing laparoscopic cholecystectomy. *Can J Anaesth*, Vol.55, No.11(Nov), pp.754-60, ISSN

anaesthesia for treatment of pain after breast reconstruction. *Scand J Plast Reconstr* 

morphine for intravenous patient-controlled analgesia. *Br J Anaesth*, Vol.102,

complications: a systematic update of the evidence. *Anesth Analg*, Vol.104,

postoperative analgesia: a systematic review of randomized trials. *Anesth Analg*,

recovery after outpatient herniorrhaphy. *Anesth Analg*, Vol.98, No.4(Apr), pp.970-5,

nonsteroidal anti-inflammatory drugs: a systematic review of efficacy, side effects, and regulatory issues. *Pain Med* , Vol.2, No.4(Dec), pp.336-51, ISSN 1526-2375. Mathiesen O, Møiniche S, Dahl J (2007). Gabapentin and postoperative pain: a qualitative

and quantitative systematic review, with focus on procedure. *BMC Anesthesiol,* 

for postoperative pain control: a randomized controlled study in hip arthroplasty.

dexamethasone in combination with paracetamol for postoperative pain control

primary joint replacement surgery for end-stage joint disease: a 10-day, phase III, randomized, double-blind, active- and placebo-controlled study. *Clin Ther*, Vol.31, No.2(Feb), pp.260-71, ISSN 0149-2918.


Hollmann MW, Durieux ME (2000). Local anesthetics and the inflammatory response: a new

Hurley RW, Cohen SP, Williams KA, Rowlingson AJ, *et al* (2006). The analgesic effects of

Ilfeld BM, Enneking FK (2005). Continuous peripheral nerve blocks at home: a review. *Anesth Analg* , Vol.100, No.6(Jun), pp.1822-33, Review, ISSN 0003-2999. Joly V, Richebe P, Guignard B, Fletcher D, *et al* (2005). Remifentanil-induced postoperative

Jones J, Francis P (2009). Ophthalmic utility of topical bromfenac, a twice-daily nonsteroidal

Joshi GP, Viscusi ER, Gan TJ, Minkowitz H, *et al* (2004). Effective treatment of laparoscopic

Jurna I (1995). [Antinociceptive effects of alpha(2)-adrenoceptor agonists ("analgesic" actions

Kaba A, Laurent SR, Detroz BJ, Sessler DI, *et al* (2007). Intravenous lidocaine infusion

Kardash KJ, Sarrazin F, Tessler MJ, Velly AM (2008). Single-dose dexamethasone reduces

Karmakar MK (2001). Thoracic paravertebral block. *Anesthesiology*, Vol.95, No.3(Sep),

Kehlet H, Dahl JB (1993). The value of multimodal or balanced analgesia in the

Kehlet H (1997). Multimodal approach to control postoperative pathophysiology and rehabilitation. *Br J Anaesth*, Vol.78, No.5(May), pp.606-17, ISSN 0007-0912. Kehlet H, Werner M, Perkins F (1999). Balanced analgesia: what is it and what are its

Kehlet H, Holte K (2001). Effect of postoperative analgesia on surgical outcome. *Br J Anaesth*,

Kerr DR, Kohan L (2008). Local infiltration analgesia: a technique for the control of acute

*Med*, Vol.31, No.3(May-Jun), pp.237-47, ISSN 1098-7339.

*Anesth Analg*, Vol.98, No.2(Feb), pp.336-42, ISSN 0003-2999.

*Schmerz*, Vol.9, No.6(Nov), pp.286-92, ISSN 0932-433X.

Vol.87, No.1(Jul), pp.62-72, Review, ISSN 0007-0912.

*Acta Orthop*, Vol.79, No.2(Apr), pp.174-83, ISSN 1745-3674.

No.2(Feb), pp.260-71, ISSN 0149-2918.

No.1(Jul), pp.147-55, ISSN 0003-3022.

No.1(Jan), pp.11-8, ISSN 0003-3022.

pp.771-80, Review, ISSN 0003-3022.

pp.1253-57, ISSN 0003-2999.

Review, ISSN 0003-2999.

6667.

3022.

ISSN 1465-6566.

primary joint replacement surgery for end-stage joint disease: a 10-day, phase III, randomized, double-blind, active- and placebo-controlled study. *Clin Ther*, Vol.31,

therapeutic indication? *Anesthesiology*, Vol.93, No.3(Sep), pp.858-75, ISSN 0003-

perioperative gabapentin on postoperative pain: a meta-analysis. *Reg Anesth Pain* 

hyperalgesia and its prevention with small-dose ketamine. *Anesthesiology*, Vol.103,

anti-inflammatory agent. *Expert Opin Pharmacother*, Vol.10, No.14(Oct), pp.2379-85,

cholecystectomy pain with intravenous followed by oral COX-2 specific inhibitor.

in animal experiments)agonists ("analgesic" actions in animal experiments).].

facilitates acute rehabilitation after laparoscopic colectomy. *Anesthesiology*, Vol.106,

dynamic pain after total hip arthroplasty. *Anesth Analg*, Vol.106, No.4(Apr),

postoperative pain treatment. *Anesth Analg*, Vol.77, No.5(Nov), pp. 1048-56,

advantages in postoperative pain? *Drugs*, Vol.58, No.5(Nov), pp.793-7, ISSN 0012-

postoperative pain following knee and hip surgery: a case study of 325 patients.


Multimodal Analgesia for Postoperative Pain Management 209

154 patients. *Acta Orthop Scand,* Vol.75, No.5(Oct), pp.606-9, ISSN 0001-6470. Rathmell JP, Lair TR, Nauman B (2005). The role of intrathecal drugs in the treatment of acute pain. *Anesth Analg*, Vol.101, No.5 Suppl(Nov), pp.S30-43, ISSN 0003-2999. Rawal N (Co-Ordinator) Postoperative Pain Management – Good Clinical Practice, General

Saber AA, Elgamal AH, Rao AJ, Itawi EA, *et al* (2009). Early experience with lidocaine patch

Salerno A, Hermann R (2006). Efficacy and safety of steroid use for postoperative pain relief.

Schug S, Chong C (2009). Pain management after ambulatory surgery. *Curr Opin* 

Senturk M, Ozcan PE, Talu GK, Kiyan E, *et al* (2002). The effects of three different analgesia

Sivrikaya GU, Eksioglu B, Basgul A, Enhos H, *et al* (2000). The effects of preemptive

Sivrikaya GU, Koc Bekil EH, Hanci A, Kilinc LT, *et al* (2008). The effect of combined epidural-

Sun Y, Gan T, Dubose J, Habib A (2008). Acupuncture and related techniques for

Tiippana E, Hamunen K, Kontinen V, Kalso E (2007). Do surgical patients benefit from

Tramer MR, Schneider J, Marti R-A, Rifat K (1996). Role of magnesium sulfate in

Usichenko TI, Kuchling S, Witstruck T, Pavlovic D, et al (2007). Auricular acupuncture for

Usichenko T, Lehmann C, Ernst E (2008). Auricular acupuncture for postoperative pain

*Anaesth Int Care*, Vol.36, No.6(Nov-Dec), pp.358-65, ISSN 1304-0871. Sun T, Sacan O, White PF, Coleman J, et al (2008). Perioperative vs postoperative celecoxib

*Anaesthesiol*, Vol.22, No.6(Dec), pp.738-43, ISSN 0952-7907.

Italy. *The International Monitor (IMRAPT)*, Vol.12, No.3, pp.65.

*Anaesth*Vol.101, No.2(Aug), pp.151-60, ISSN 0007-0912.

*Anesth Analg*, Vol.104, No.6(Jun), pp.1545-56, ISSN 0003-2999.

http://www.esraeurope.org/PostoperativePain

Vol.7, No.1(Feb), pp.36-8, ISSN 1743-9191.

No.6(June), pp.1361-72, ISSN 0021-9355.

No.3(Mar), pp.950-8, ISSN 0003-2999.

No.2(Jan), pp.179-83, ISSN 1488-2329.

Vol.12(Dec), pp.1343-8, ISSN 0003-2409.

Management.pdf

pp.11-5, ISSN 0003-2999.

3022.

primary total knee replacement: open intervention study of efficacy and safety in

recommendations and principles for succesful pain management.

for postoperative pain control after laparoscopic ventral hernia repair. *Int J Surg*,

Update and review of the medical literature. *J Bone Joint Surg Am*, Vol.88,

techniques on long-term postthoracotomy pain. *Anesth Analg*, Vol.94, No.1(Jan),

epidural tramadol on peroperative stress response and postoperative analgesia (Oral communication), 19th Annual ESRA Congress, 20–23 November 2000, Rome,

general anaesthesia on intraoperative stress response and postoperative analgesic consumption and gastrointestinal function in lower abdominal surgery. *J Turk* 

on patient outcome after major plastic surgery procedures. *Anesth Analg*, Vol.106,

postoperative pain: a systematic review of randomized controlled trials. *Br J* 

perioperative gabapentin/pregabalin? Asystematic review of efficacy and safety.

postoperative analgesia. *Anesthesiology* , Vol.84, No.2(Feb), pp.340-7, ISSN 0003-

pain relief after ambulatory knee surgery: a randomized trial. *CMAJ*, Vol.176,

control: a systematic review of randomised clinical trials. *Anaesthesia*, Vol.63,

after abdominal hysterectomy. A randomized clinical trial. *Acta Anaesthesiol Scand*, Vol.53, No.2(Feb), pp.227-35, ISSN 0001-5172.


McCleane G (2010). Topical application of analgesics: a clinical option in day case anaesthesia? *Curr Opin Anaesthesiol*, Vol.23, No.6(Dec), pp.704-7, ISSN 0952-7907. McGrath B, Elgendy H, Chung F, Kamming D, *et al* (2004). Thirty percent of patients have

Miranda HF, Puig MM, Prieto JC, Pinardi G (2006). Synergism between paracetamol and

Mitra S, Sinatra R (2004). Perioperative management of acute pain in the opioid-dependent patient. *Anesthesiology*, Vol.101, No.1(Jul), pp.212-27, ISSN 0003-3022. Mitra S (2008). Opioid-induced hyperalgesia: pathophysiology and clinical implications. *J* 

Møiniche S, Mikkelsen S, Wetterslev J, Dahl JB (1998). A qualitative systematic review of

Moiniche S, Kehlet H, Dahl JB (2002). A qualitative and quantitative systematic review of

Ness TJ (2001). Pharmacology of peripheral analgesia. *Pain Pract*, Vol.1, No.3(Sep), pp.243-

Nett MP (2010). Postoperative pain management. *Orthopedics*, Vol.33, No.9 Suppl(Sep),

Nishimori M, Ballantyne JC, Low JH (2006). Epidural pain relief *versus* systemic opioid-

Park JY, Lee GW, Kim Y, Yoo MJ (2002). The efficacy of continuous intrabursal infusion with

Perkins FM, Kehlet H (2000). Chronic pain as an outcome of surgery. A review of predictive factors. *Anesthesiology*, Vol.93, No.4(Oct), pp.1123-33; ISSN 0003-3022. Power I, Barratt S. Analgesic agents for the postoperative period. Nonopioids (1999). *Surg* 

Practice guidelines for acute pain management in the perioperative setting: a report by the

Pusch F, Freitag H, Weinstabl C, Obwegeser R, *et al* (1999). Single-injection paravertebral

Rasmussen S, Kramhøft MU, Sperling KP, Pedersen JH.(2004) Increased flexion and reduced

*Opioid Manag*, Vol.4, No.3(May-Jun), pp. 123-30, ISSN 1551-7489.

*Br J Anaesth*, Vol. 81, No.3(Sep), pp.377-83, ISSN 0007-0912.

*Anesthesiology*, Vol.96, No.3(Mar), pp.725-41, ISSN 0003-3022.

No.3(Jul), CD005059, ISSN 1469-493X(Electronic).

*Clin N Am*, Vol.79, No.2(Apr), pp.275-95, ISSN 0039-6109.

Vol.104, No.4(Apr), pp.982-3, ISSN 0003-2999.

Vol.43, No.7(Aug), pp.770-4, ISSN 0001-5172.

Vol.53, No.2(Feb), pp.227-35, ISSN 0001-5172.

No.1-2(Mar), pp.22-8, ISSN 0304-3959.

54, ISSN 1530-7085.

7339.

pp.23-6, ISSN 0147-7447.

*Can J Anaesth*, Vol.51, No.9(Nov), 886-91, ISSN 0832-610X.

after abdominal hysterectomy. A randomized clinical trial. *Acta Anaesthesiol Scand*,

moderate to severe pain 24 hr after ambulatory surgery: a survey of 5,703 patients.

nonsteroidal anti-inflammatory drugs in experimental acute pain. *Pain*, Vol.121,

incisional local anaesthesia for postoperative pain relief after abdominal operations.

preemptive analgesia for postoperative pain relief: the role of timing of analgesia.

based pain relief for abdominal aortic surgery. *Cochrane Database Syst Rev*, Vol.19,

morphine and bupivacaine for postoperative analgesia after subacromial arthroscopy. *Reg Anesth Pain Med*, Vol.27, No.2(Mar-Apr), pp.145-9, ISSN 1098-

American Society of Anesthesiologists Task Force on Pain Management, Acute Pain Section (1995). *Anesthesiology*, Vol.82, No.4(Apr), pp.1071-81, ISSN 0003-3022. Pratap JN, Shankar RK, Goroszeniuk T (2007). Co-injection of clonidine prolongs the

anesthetic effect of lidocaine skin infiltration by a peripheral action. *Anesth Analg*,

block compared to general anesthesia in breast surgery. *Acta Anaesthesiol Scand*,

hospital stay with continuous intraarticular morphine and ropivacaine after

primary total knee replacement: open intervention study of efficacy and safety in 154 patients. *Acta Orthop Scand,* Vol.75, No.5(Oct), pp.606-9, ISSN 0001-6470.


**10** 

*Turkey* 

**The Effect of General Anesthesia and General** 

Levobupivacaine, a new long-acting local anesthetic, is reported to achieve an effective and safe epidural anesthesia, similar to the anesthesia achieved by bupivacaine. Levobupivacaine with a pharmacological structure similar to that of bupivacaine was shown to have a wider confidence interval, and less neurotoxic and cardiotoxic effects. A large number of trials have been conducted on determining the anesthetic methods that decrease the stress response of major surgery. These trials usually compared the effects of general, epidural and general + epidural anesthetic methods on the stress response occurring in major surgery with respect to mortality and morbidity. While some authors recommended general + epidural anesthesia, some only recommended the general

A combination of epidural and general anesthesia is reported to reduce the requirement for analgesic and anesthetic agents. Intraoperative hemodynamic stability can be better achieved and the metabolic, endocrine and immunologic responses better suppressed. Management of these responses is important in reducing postoperative morbidity and mortality. With the combination of epidural and general anesthesia, recovery is faster, a higher anesthetic quality can be achieved and patients can be mobilized earlier (1-4). There

This trial was designed to compare the epidural bupivacaine or levobupivacaine combined with general anesthesia and general anesthesia alone in patients who will undergo TAH-BSO, with respect to stress response to surgery, intraoperative hemodynamics, requirement for peroperative anesthetics and analgesic agents, the quality of the postoperative analgesia,

This trial included 54 ASA I-II group patients in the age range of 18-65 who were scheduled to undergo TAH-BSO and who gave written consent to participate in the trial. Those with

are no adequate trials on the novel agent, levobupivacaine.

recovery from anesthesia and postoperative side effects.

**1. Introduction** 

anesthesia.

**2. Methods** 

**Anesthesia Plus Epidural Levobupivacaine** 

**or Bupivacaine on Hemodynami Stress** 

**Response and Postoperative Pain** 

Atilla Erol, Aybars Tavlan and Seref Otelcioglu

Semra Calimli, Ahmet Topal,

*Selcuk university Meram Medical Faculty,* 


### **The Effect of General Anesthesia and General Anesthesia Plus Epidural Levobupivacaine or Bupivacaine on Hemodynami Stress Response and Postoperative Pain**

Semra Calimli, Ahmet Topal, Atilla Erol, Aybars Tavlan and Seref Otelcioglu *Selcuk university Meram Medical Faculty, Turkey* 

#### **1. Introduction**

210 Pain Management – Current Issues and Opinions

Vadivelu N, Mitra S, Narayan D (2010). Recent advances in postoperative pain

Viscusi ER, Reynolds L, Chung F, Atkinson LE, et al (2004). Patient-controlled transdermal

Wang B, Tang J, White PF, Naruse R, et al (1997). Effect of the intensity of transcutaneous

Warfield CA, Kahn CH (1995). Acute pain management: programs in U.S. hospitals and

White PF, Issioui T, Skrivanek GD, Early JS, et al (2003). Use of a continuous popliteal sciatic

White PF (2005). The changing role of nonopioid analgesic techniques in the management of

White PF (2007). Multimodal pain management: the future is now! *Curr Opin Investig Drugs*,

White PF, Sacan O, Tufanogullari B, Eng M, et al (2007). Effect of short-term postoperative

White PF, Kehlet H (2007). Postoperative pain management and patient outcome: time to

White PF, Kehlet H, Neal JM, Schricker T, et al (2007). Role of the anesthesiologist in fast-

White PF, Kehlet H (2010). Improving postoperative pain management: what are the unresolved issues? *Anesthesiology*, Vol.112, No.1(Jan), pp.220-5, ISSN 0003-3022. Wu CL, Fleisher LA (2000). Outcomes research in regional anesthesia and analgesia. *Anesth* 

Yardeni IZ, Beilin B, Mayburd E, Levinson Y, et al (2009). The effect of perioperative

*Can J Anaesth*, Vol.54, No.5(May), pp.342-8, ISSN 0832-610X.

*Analg*, Vol.104, No.6(Jun), pp.1380-96, ISSN 0003-2999.

*Analg*, Vol.91, No.5(Nov), pp.1232-42, ISSN 0003-2999.

Vol.109, No.5(Nov), 1464-9, ISSN 0003-2999.

*Analg*, Vol.85, No.2(Aug), pp.406-13, ISSN 0003-2999.

Vol.8, No.7(Jul), pp.517-8, ISSN 1472-4472.

7484.

0003-2999.

2999.

2999.

pp.1090-4, ISSN 0003-3022.

management. *Yale J Biol Med*, Vol.83, No.1(Mar), pp.11-25, Review, ISSN 0044-0086.

fentanyl hydrochloride vs intravenous morphine pump for postoperative pain: a randomized controlled trial. *JAMA,* Vol.17, No.11(Mar), pp.1333-41, ISSN 0098-

acupoint electrical stimulation on the postoperative analgesic requirement. *Anesth* 

experiences and attitudes among U.S adults. *Anesthesiology*Vol.83, No.5(Nov),

nerve block for the management of pain after major podiatric surgery: does it improve quality of recovery? *Anesth Analg*, Vol.97, No.5(Nov), pp.1303-9, ISSN

postoperative pain. *Anesth Analg*, Vol.101, No.5 Suppl(Nov), pp.S5-22, ISSN 0003-

celecoxib administration on patient outcome after outpatient laparoscopic surgery.

return to work! [editorial]. *Anesth Analg*, Vol.104, No.3(Mar), pp.487–90, ISSN 0003-

track surgery: from multimodal analgesia to perioperative medical care. *Anesth* 

intravenous lidocaine on postoperative pain and immune function. *Anesth Analg*,

Levobupivacaine, a new long-acting local anesthetic, is reported to achieve an effective and safe epidural anesthesia, similar to the anesthesia achieved by bupivacaine. Levobupivacaine with a pharmacological structure similar to that of bupivacaine was shown to have a wider confidence interval, and less neurotoxic and cardiotoxic effects.

A large number of trials have been conducted on determining the anesthetic methods that decrease the stress response of major surgery. These trials usually compared the effects of general, epidural and general + epidural anesthetic methods on the stress response occurring in major surgery with respect to mortality and morbidity. While some authors recommended general + epidural anesthesia, some only recommended the general anesthesia.

A combination of epidural and general anesthesia is reported to reduce the requirement for analgesic and anesthetic agents. Intraoperative hemodynamic stability can be better achieved and the metabolic, endocrine and immunologic responses better suppressed. Management of these responses is important in reducing postoperative morbidity and mortality. With the combination of epidural and general anesthesia, recovery is faster, a higher anesthetic quality can be achieved and patients can be mobilized earlier (1-4). There are no adequate trials on the novel agent, levobupivacaine.

This trial was designed to compare the epidural bupivacaine or levobupivacaine combined with general anesthesia and general anesthesia alone in patients who will undergo TAH-BSO, with respect to stress response to surgery, intraoperative hemodynamics, requirement for peroperative anesthetics and analgesic agents, the quality of the postoperative analgesia, recovery from anesthesia and postoperative side effects.

#### **2. Methods**

This trial included 54 ASA I-II group patients in the age range of 18-65 who were scheduled to undergo TAH-BSO and who gave written consent to participate in the trial. Those with

The Effect of General Anesthesia and General Anesthesia Plus Epidural

and requested amounts were recorded.

The level of significance was set at p<0.05.

the duration of surgery (p>0.05) (Table 1).

Table 1. Patient characteristics (Mean ± SD)

**3. Results** 

II (p>0.05).

(Table 2).

Levobupivacaine or Bupivacaine on Hemodynami Stress Response and Postoperative Pain 213

ephedrine were required. Pain intensity was evaluated by the visual analogue scale (VAS) and the motor block was assessed by the Bromage scale; the hemodynamic data and the side effects (hypotension, respiratory depression, motor block, nausea-vomiting, itching, tremor)

To relieve the postoperative pain, Group III was administered iv morphine and PCA at a concentration of 1 mg ml-1 concentration with a loading dose of 1 mg and a lock-out period of 6 minutes. In Group I, 0.125% bupivacaine + 0.025 mg ml-1 morphine, in Group II, 0.125% levobupivacaine + 0.025 mg ml-1 morphine and 5 ml of h-1 basal infusion were prepared for PCA with a 1 ml loading and a lock-out period of 20 minutes and PCA administration was initiated in the recovery room. The total amount of anesthetics used and the administered

Statistical analysis were performed using the SPSS 12.0 software. The data were summarized as mean ± standard deviation and percentage. Comparisons between the three groups were assessed by one way variance analysis (Anova) in cases where the parametric conditions could be met and by Kruskal Wallis variance analysis in nonparametric conditions. In the three-group comparisons, post-hoc Tukey-HSD test and Bonferroni correction Mann-Whitney U test were used for significantly differing parameters. The comparison between the two groups was made with a t test. The chisquare test was used for comparing categorical data. Variance analysis was used to analysis the parametric data and Wilcoxon Signed Ranks test Bonferroni correction was used to analyze the non-parametric data for the analysis of the repeated measurements.

The groups showed similarity in the mean values for age, weight, height, the ASA score and

Age (year) 46.55 ± 4.97 47.53 ± 6.87 48.44 ± 8.75 0.246 Weight (kg) 70.88 ± 8.58 75.50 ± 15.27 79.55 ± 8.05 0.075 Hight (cm) 160.55 ± 5.29 162.16 ± 5.95 160.27 ± 4.61 0.520 Surgery time (min) 74.88 ± 18.31 72.83 ± 20.47 80.94 ± 13.35 0.365 ASA I / II 11 / 7 13 / 5 10 / 8 0.574

Time to achieve sensory block at T6 dermatome was 18.72±4.41 and 21.27±4.48 in Group I and Group II, respectively; the sensory block upper levels were 5.66±0.68 and 5.88±0.32 dermatome, respectively (p>0.05). The pre-operative Bromage scores were 0 in Group I and

The total doses of the intra-operatively administered remifentanil and sevoflurane were similar between Group I and Group II, however, statistically higher in Group III (p<0.000) (Table 2). While there was no statistically significant difference between Group I and Group II in the postoperative recovery evaluated by spontaneous respiratory time, extubation time, eye opening time and the time to reach an Aldrete recovery score of ≥9, Group III had a significantly longer recovery time compared to Groups I and II (p<0.000)

GROUP I GROUP II GROUP III P

were recorded at 0 and 30 minutes, and 2, 6, 12 and 24 hours after the operation.

severe cardiac, pulmonary, hepatic diseases, renal failure, hemorrhagic diathesis, fever, infection and those with known hypersensitivity to investigational drugs were excluded from the trial. Non-premedicated cases were randomly assigned to three groups: general anesthesia + epidural bupivacaine (Group I, n=18), general anesthesia + epidural levobupivacaine (Group II, n=18) and general anesthesia (Group III, n=18). All the patients were monitored for EKG, non-invasive blood pressure, peripheral oxygen saturation (SpO2), end-tidal carbon dioxide pressure (EtCO2) and body temperature.

In Groups I and II, the epidural space was entered by a 16-gauge Tuohy epidural needle before the surgery using the loss of resistance method through the L3-L4 space while the patient was in the sitting position and an 18-gauge epidural catheter was inserted (Perifix, Braun, Germany). As a test dose, 2 ml of 2% lidocaine (Aritmal Ampul® Osel) was administered; five minutes later, Group I and Group II were administered 5 ml of 0.25% bupivacaine (Marcaine flacon® Eczacbaş, Turkey) and 0.25% levobupivacaine (Chirocaine flacon® Abbott, USA) respectively via epidural catheter, followed by administration of 10 ml of 0.25% bupivacaine to Group I and 10 ml of 0.25% levobupivacaine to Group II via epidural catheter five minutes later. The sensory block upper level*,* time to achieve sensory block at T6 dermatome and the Bromage Scale values were assessed.

Anesthetic induction was achieved in all patients (when reached the sensorial block level dermatome of T6 in Group I and Group II) by 2 mg kg-1 propofol (Propofol ampul® Fresenius Kabi) and 1 µg kg-1 remifentanil (Ultiva® Glaxo Wellcome) administered in 60 seconds. 0.6 mg kg-1 rocuronium (Esmeron® Organon) was used for achieving neuromuscular block. For all three groups, the maintenance of anesthesia was achieved using 1% sevoflurane (Sevorane® Abbott, USA) in 50% O2-air mixture and 0.1 µg kg-1 min-1 remifentanil infusion (Perfusor Compact-Braun). Regarding the patients who would require an anesthesia duration of more than two hours, Group I was scheduled to receive an additional 5 ml of 0.25% bupivacaine and Group II was scheduled to receive an additional 5 ml of 0.25% levobupivacaine from the epidural catheter.

When the heart beat rate (HBR) and the mean blood pressure (MBP) was reduced by 20% of the control value, the concentration of the inhalation agent was reduced by 50%. 250 ml of ringer lactate solution was rapidly administered. In case of absence of improvement, the dose of remifentanil was decreased by 50%. If the low level persisted, atropine or ephedrine was administered as required. When the HBR and MBP increased by more than 20% of the control value, the concentration of the inhalation agent was increased by 50%. In the case of persistence of the high level, the dose of remifentanil was increased by 50%. For maintenance of the neuromuscular blockage, 0.15 mg kg-1 rocuronium iv was administered, where necessary.

The hemodynamic parameters, systolic blood pressure (SBP), diastolic blood pressure (DBP), MBP, HBR, and SpO2 were recorded 2 and 5 minutes after the intubation, 2, 5, 10, 15, 30, 45, 60, 90 and 120 minutes after the skin incision and after the extubation. For measuring the glucose, cortisol, insulin and CRP levels, preoperative venous access was achieved followed by blood sampling in the first and 24th hours of operation. The glucose, glucose oxidase, cortisol and insulin values were measured by chemiluminescent immunoassay, CRP, and the immunoturbidimetric methods.

The postoperative recovery was evaluated by the spontaneous breathing time, extubation time, eye opening time and the time to reach an Aldrete recovery score of ≥9. Data were recorded on the amount of sevoflurane used (ml) (Datex Ohmeda, S5. Sweden), the total dose of remifentanil (mg), whether muscle relaxant was added and whether atropine or ephedrine were required. Pain intensity was evaluated by the visual analogue scale (VAS) and the motor block was assessed by the Bromage scale; the hemodynamic data and the side effects (hypotension, respiratory depression, motor block, nausea-vomiting, itching, tremor) were recorded at 0 and 30 minutes, and 2, 6, 12 and 24 hours after the operation.

To relieve the postoperative pain, Group III was administered iv morphine and PCA at a concentration of 1 mg ml-1 concentration with a loading dose of 1 mg and a lock-out period of 6 minutes. In Group I, 0.125% bupivacaine + 0.025 mg ml-1 morphine, in Group II, 0.125% levobupivacaine + 0.025 mg ml-1 morphine and 5 ml of h-1 basal infusion were prepared for PCA with a 1 ml loading and a lock-out period of 20 minutes and PCA administration was initiated in the recovery room. The total amount of anesthetics used and the administered and requested amounts were recorded.

Statistical analysis were performed using the SPSS 12.0 software. The data were summarized as mean ± standard deviation and percentage. Comparisons between the three groups were assessed by one way variance analysis (Anova) in cases where the parametric conditions could be met and by Kruskal Wallis variance analysis in nonparametric conditions. In the three-group comparisons, post-hoc Tukey-HSD test and Bonferroni correction Mann-Whitney U test were used for significantly differing parameters. The comparison between the two groups was made with a t test. The chisquare test was used for comparing categorical data. Variance analysis was used to analysis the parametric data and Wilcoxon Signed Ranks test Bonferroni correction was used to analyze the non-parametric data for the analysis of the repeated measurements. The level of significance was set at p<0.05.

#### **3. Results**

212 Pain Management – Current Issues and Opinions

severe cardiac, pulmonary, hepatic diseases, renal failure, hemorrhagic diathesis, fever, infection and those with known hypersensitivity to investigational drugs were excluded from the trial. Non-premedicated cases were randomly assigned to three groups: general anesthesia + epidural bupivacaine (Group I, n=18), general anesthesia + epidural levobupivacaine (Group II, n=18) and general anesthesia (Group III, n=18). All the patients were monitored for EKG, non-invasive blood pressure, peripheral oxygen saturation (SpO2),

In Groups I and II, the epidural space was entered by a 16-gauge Tuohy epidural needle before the surgery using the loss of resistance method through the L3-L4 space while the patient was in the sitting position and an 18-gauge epidural catheter was inserted (Perifix, Braun, Germany). As a test dose, 2 ml of 2% lidocaine (Aritmal Ampul® Osel) was administered; five minutes later, Group I and Group II were administered 5 ml of 0.25% bupivacaine (Marcaine flacon® Eczacbaş, Turkey) and 0.25% levobupivacaine (Chirocaine flacon® Abbott, USA) respectively via epidural catheter, followed by administration of 10 ml of 0.25% bupivacaine to Group I and 10 ml of 0.25% levobupivacaine to Group II via epidural catheter five minutes later. The sensory block upper level*,* time to achieve sensory

Anesthetic induction was achieved in all patients (when reached the sensorial block level dermatome of T6 in Group I and Group II) by 2 mg kg-1 propofol (Propofol ampul® Fresenius Kabi) and 1 µg kg-1 remifentanil (Ultiva® Glaxo Wellcome) administered in 60 seconds. 0.6 mg kg-1 rocuronium (Esmeron® Organon) was used for achieving neuromuscular block. For all three groups, the maintenance of anesthesia was achieved using 1% sevoflurane (Sevorane® Abbott, USA) in 50% O2-air mixture and 0.1 µg kg-1 min-1 remifentanil infusion (Perfusor Compact-Braun). Regarding the patients who would require an anesthesia duration of more than two hours, Group I was scheduled to receive an additional 5 ml of 0.25% bupivacaine and Group II was scheduled to receive an additional 5

When the heart beat rate (HBR) and the mean blood pressure (MBP) was reduced by 20% of the control value, the concentration of the inhalation agent was reduced by 50%. 250 ml of ringer lactate solution was rapidly administered. In case of absence of improvement, the dose of remifentanil was decreased by 50%. If the low level persisted, atropine or ephedrine was administered as required. When the HBR and MBP increased by more than 20% of the control value, the concentration of the inhalation agent was increased by 50%. In the case of persistence of the high level, the dose of remifentanil was increased by 50%. For maintenance of the neuromuscular blockage, 0.15 mg kg-1 rocuronium iv was administered,

The hemodynamic parameters, systolic blood pressure (SBP), diastolic blood pressure (DBP), MBP, HBR, and SpO2 were recorded 2 and 5 minutes after the intubation, 2, 5, 10, 15, 30, 45, 60, 90 and 120 minutes after the skin incision and after the extubation. For measuring the glucose, cortisol, insulin and CRP levels, preoperative venous access was achieved followed by blood sampling in the first and 24th hours of operation. The glucose, glucose oxidase, cortisol and insulin values were measured by chemiluminescent immunoassay,

The postoperative recovery was evaluated by the spontaneous breathing time, extubation time, eye opening time and the time to reach an Aldrete recovery score of ≥9. Data were recorded on the amount of sevoflurane used (ml) (Datex Ohmeda, S5. Sweden), the total dose of remifentanil (mg), whether muscle relaxant was added and whether atropine or

end-tidal carbon dioxide pressure (EtCO2) and body temperature.

block at T6 dermatome and the Bromage Scale values were assessed.

ml of 0.25% levobupivacaine from the epidural catheter.

CRP, and the immunoturbidimetric methods.

where necessary.


The groups showed similarity in the mean values for age, weight, height, the ASA score and the duration of surgery (p>0.05) (Table 1).

Table 1. Patient characteristics (Mean ± SD)

Time to achieve sensory block at T6 dermatome was 18.72±4.41 and 21.27±4.48 in Group I and Group II, respectively; the sensory block upper levels were 5.66±0.68 and 5.88±0.32 dermatome, respectively (p>0.05). The pre-operative Bromage scores were 0 in Group I and II (p>0.05).

The total doses of the intra-operatively administered remifentanil and sevoflurane were similar between Group I and Group II, however, statistically higher in Group III (p<0.000) (Table 2). While there was no statistically significant difference between Group I and Group II in the postoperative recovery evaluated by spontaneous respiratory time, extubation time, eye opening time and the time to reach an Aldrete recovery score of ≥9, Group III had a significantly longer recovery time compared to Groups I and II (p<0.000) (Table 2).

The Effect of General Anesthesia and General Anesthesia Plus Epidural
