**4. Inosine and pain**

634 Pharmacology

production (it can cause pain) and also K+ channels opening (it can inhibit pain) (Jacobson &

A2AR knockout animals are less sensitive to pain, suggesting that A2AR is a pain facilitator in acute (Hussey et al., 2007) and chronic pain (Bura et al., 2008). Bura and coworkers (2008) also demonstrated that microglia and astrocytes expression was higher in wild-type A2AR animals than in A2AR knockout animals. Also, A2AR located in glial cells is responsible for the release of inflammatory mediators that induce and maintain chronic pain (Boison et al., 2010). Thus, A2AR blockade might be an interesting approach for future treatments of neuropathic and chronic pain. Meantime, also in chronic pain exists distinct results about A2AR. A report showed that only one spinal injection of A2AR agonist was able to induce analgesia during several days in rats undergoing neuropathic pain (Loram et al., 2009). After all, it is clear that A2AR is involved in pain modulation. However, more studies are necessary to precisely explain how this receptor works in distinct situations, only then it will

Few studies have been evaluating the A2BR role in pain. Most part of these studies showed that A2BR facilitates pain transmission, because A2BR antagonists have reduced pain (Abo-Salem et al., 2004; Bilkei-Gorzo et al., 2008; GodFrey et al., 2006). A2BR antagonist reduced thermal hyperalgesia and was able to potentiate the analgesic effect caused by morphine and acetaminophen (Abo-Salem et al., 2004; Godfrey et al., 2006). Also, the blockade of A2BR

Similar to A2BR, adenosine A3R is not an interesting target to pain relief. However, A3R is implicated in pathological conditions such as ischemic diseases and in inflammation (for review see Borea et al., 2009). Regarding pain, there are few studies evaluating A3R role. Sawynok and colleagues (1997) showed that A3R activation causes pain and paw oedema through release of histamine and serotonin. A3R knockout animals presented an increased pain threshold in some models of pain but not difference in others (Fedorova et al., 2003; Wu et al., 2002). A3R might be an interesting target to inflammatory and autoimmune

The first report showing that adenosine kinase (AK) inhibition reduces behaviour associated to pain was published by Keil and DeLander, 1992. AK inhibitors are able to decrease pain levels when given peripherally or sistemically (Kowaluk et al., 1999; Lynch et al., 1999; Sawynok, 1998). Moreover, these inhibitors are efficacious against acute and chronic pain (Kowaluk et al., 2000; Lynch et al., 1999; McGaraughty et al., 2005; Poon & Sawynok, 1998, 1999; Suzuki et al., 2001). Another enzyme that regulates adenosine level is adenosine deaminase (ADA), that converts adenosine to inosine (Sawynok, 1998). However, the analgesic effect caused by ADA inhibition is not so clear yet. It has been showed that

presented an analgesic effect in inflammatory pain (Bilkei-Gorzo et al., 2008).

**3.8 Novel approaches in pain management involving adenosine receptors** 

**3.8.1 Management of adenosine receptors by metabolism modulation** 

Gao, 2006; Regaya et al., 2004; Sawynok, 1998).

be possible to make clinical approaches.

**3.6 A2B receptors and pain** 

**3.7 A3 receptors and pain** 

diseases, but not to pain states.

#### **4.1 Inosine within of purinergic system**

ATP is the main molecule of purinergic system. Inside the cell, ATP may be bi-directionally converted into AMP. AMP is broken down into adenosine. Adenosine may be converted back into AMP through phosphorylation by AK. Moreover, adenosine might leave the cell by nucleoside transporter (NT). Inside the cell, adenosine deaminase is responsible for the conversion from adenosine to inosine. Outside the cell, this conversion is performed by ectoadenosine kinase or even adenosine deaminase. Inosine is a substrate to purine nucleoside phosphorylase (PNP), leading to hypoxanthines as its products. Hypoxanthines are converted into xanthines and afterwards to uric acid by xanthine oxidase (Figure 4) (See review Sawynok & Liu, 2003).

The Involvement of Purinergic System in Pain:

paw of mice (Nascimento et al., 2010).

**4.4 Inosine effects on chronic pain** 

Adenosine Receptors and Inosine as Pharmacological Tools in Future Treatments 637

specific model. Therefore, these results indicate that inosine is able to prevent and reduce pain induced by inflammatory mediators. In this way, inosine may be inhibiting the synthesis or release of several neurotransmitters and mediators involved in pain conditions (Nascimento et al., 2010). We can also conclude that inosine is able to inhibit pain induced by central facilitation. Therefore, inosine increases pain threshold and it may be useful to treat some kinds of pain injury that result from a central sensitization. Adenosine receptors distribution on substantia gelatinosa, mainly A1R and A2AR could explain how inosine acts in this case (Sawynok, 1998; Sawynok & Liu, 2003). Inosine also presents a significant and dose-related inhibition of pain induced by glutamate (acute pain model) injection into the

The data described in literature strongly suggests that inosine may have an important effect in controlling chronic pain, since it has anti-inflammatory effect and can reduce acute pain. In fact, Nascimento and colleagues (2010) demonstrated that acute administration of inosine, intraperitoneally, was able to inhibit chronic inflammatory pain induced by CFA in mice, being effective up to 4 hours after administration. The CFA is responsible for inducing chronic inflammation by stimulating the body's immune response, this response is mediated by the synthesis and release of cytokines and inflammatory mediators (Zhang et al., 2011). In the study published by Nascimento and colleagues (2010), inosine was effective against mechanical and thermal allodynia induced by partial sciatic nerve ligation (PSNL) up to 4 hours after treatment by intraperitoneal route. Further, in another experiment, inosine was given daily for until 22 days and it also presented significant analgesic effect. Pain induced by PSNL is very strong and may last for weeks (Ueda, 2006). Animal models of neuropathic pain induce many functional and biochemical changes in local injury site. After the surgery there is the release of multiple inflammatory and pain mediators which in turn, may also be present in other areas involved and affected by sciatic nerve, as spinal cord and brain (Bridges et al., 2001; Ji & Woolf, 2001; Inoue et al., 2004; Ueda, 2006). Inosine activity in this kind of pain may indicate a promising molecule to new studies, because inosine might have a longer half-life

A1R has been considered the main receptor responsible for analgesic effect among adenosine receptors (Burnstock, 2007; Sawynok, 1998). A1R is also the main receptor involved in inosine analgesic effect. Both A1R antagonists DPCPX and 8-PT were able to reverse the inosine action. Inosine in a direct or indirect way activates A1R to induce analgesia (Nascimento et al., 2010). Other studies have showed that adenosine receptor antagonists block *in vivo* and *in vitro* inosine effects (Haskó et al., 2000) and adenosine receptor knockout animals do not present immunoprotective effects of inosine (Gomez & Sitkovsky, 2003). Thus, it is clear that

Involvement of A2AR in pain is quite controversial. Some studies show that A2AR blockade leads to analgesic effect (Borghi et al., 2002; Yoon et al., 2005) while other studies demonstrate that the blockade or deletion of A2AR causes pain relief (Bastia et al., 2002; Ledent et al., 1997). Inosine activates A2AR to induce analgesia, at least in the acetic acid

than adenosine and admittedly does not have toxic or side effects.

**4.5 Adenosine receptors involved in analgesic effects of inosine** 

the A1R activation is essential for inosine to exert its effect (Figure 5).

Fig. 4. Purinergic metabolism and its main molecules and enzymes. A1, adenosine A1 receptor; A2A, adenosine A2A receptor; A2B, adenosine A2B receptor; A3, adenosine A3 receptor ADA, adenosine deaminase; AK, adenosine kinase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; ENT, equilibrative nucleoside transporter; HXT, hypoxanthines; *NT5E, ecto-5'nucleotidase*; P2X, purine P2X receptor; P2Y, purine P2Y receptor; PNP, purine nucleoside phosphorilase.

Thus, inosine is one among many molecules in purinergic system. However, inosine has specific roles in several physiological states, we will show some functions and discuss the role of inosine in pain control in next sections.

#### **4.2 Inosine physiological roles**

In recent decades, many physiological roles for inosine have been shown. During the 70's, Aviado demonstrated that inosine exerts cardiotonic actions, such as preventing negative inotropic effect and increasing coronary vasodilatation (Aviado, 1978). Also, inosine presents several effects on axonal growth, such as axon growth induction and damaged neurons stimulation (Benowitz et al., 1999, 2002; Chen et al., 2002). Inosine also induces a regrowth in axotomized retinal ganglion cells in rats (Wu et al., 2003). These data indicate that inosine may constitute a new approach to treat the injured or degenerated nerves in central or peripheral nervous system. Despite of cardiovascular and axonal growth effects, the inflammatory effects of inosine are the most studied. Inosine has significant antiinflammatory effects in several *in vivo* and *in vitro* models of inflammation (Gomez & Sitkovsky, 2003; Haskó et al., 2000; Marton et al., 2001; Schneider et al., 2006). These effects seem to be mediated by A1R, A2R and A3R (Gomez & Sitkovsky, 2003; Haskó et al., 2000, 2004).

#### **4.3 Inosine effect on acute pain**

Inosine has analgesic action when administered by different routes (i.e. intraperitoneal, oral, intrathecal or intracerebroventricular) against pain induced by acetic acid (Nascimento et al., 2010). Of note, inosine also inhibits pain induced by formalin. Formalin test induces 2 distinct types of pain, neurogenic phase (acute pain) and inflammatory phase (inflammatory pain). Inosine is not able to relieve pain in neurogenic phase. However, inosine reduces nearly totally the inflammatory pain in formalin test (Nascimento et al., 2010). The effects of inosine in this model of pain extended the acetic acid data because formalin is a more specific model. Therefore, these results indicate that inosine is able to prevent and reduce pain induced by inflammatory mediators. In this way, inosine may be inhibiting the synthesis or release of several neurotransmitters and mediators involved in pain conditions (Nascimento et al., 2010). We can also conclude that inosine is able to inhibit pain induced by central facilitation. Therefore, inosine increases pain threshold and it may be useful to treat some kinds of pain injury that result from a central sensitization. Adenosine receptors distribution on substantia gelatinosa, mainly A1R and A2AR could explain how inosine acts in this case (Sawynok, 1998; Sawynok & Liu, 2003). Inosine also presents a significant and dose-related inhibition of pain induced by glutamate (acute pain model) injection into the paw of mice (Nascimento et al., 2010).

## **4.4 Inosine effects on chronic pain**

636 Pharmacology

Fig. 4. Purinergic metabolism and its main molecules and enzymes. A1, adenosine A1 receptor; A2A, adenosine A2A receptor; A2B, adenosine A2B receptor; A3, adenosine A3 receptor ADA, adenosine deaminase; AK, adenosine kinase; AMP, adenosine

receptor; PNP, purine nucleoside phosphorilase.

role of inosine in pain control in next sections.

**4.2 Inosine physiological roles** 

**4.3 Inosine effect on acute pain**

2004).

monophosphate; ATP, adenosine triphosphate; ENT, equilibrative nucleoside transporter; HXT, hypoxanthines; *NT5E, ecto-5'nucleotidase*; P2X, purine P2X receptor; P2Y, purine P2Y

Thus, inosine is one among many molecules in purinergic system. However, inosine has specific roles in several physiological states, we will show some functions and discuss the

In recent decades, many physiological roles for inosine have been shown. During the 70's, Aviado demonstrated that inosine exerts cardiotonic actions, such as preventing negative inotropic effect and increasing coronary vasodilatation (Aviado, 1978). Also, inosine presents several effects on axonal growth, such as axon growth induction and damaged neurons stimulation (Benowitz et al., 1999, 2002; Chen et al., 2002). Inosine also induces a regrowth in axotomized retinal ganglion cells in rats (Wu et al., 2003). These data indicate that inosine may constitute a new approach to treat the injured or degenerated nerves in central or peripheral nervous system. Despite of cardiovascular and axonal growth effects, the inflammatory effects of inosine are the most studied. Inosine has significant antiinflammatory effects in several *in vivo* and *in vitro* models of inflammation (Gomez & Sitkovsky, 2003; Haskó et al., 2000; Marton et al., 2001; Schneider et al., 2006). These effects seem to be mediated by A1R, A2R and A3R (Gomez & Sitkovsky, 2003; Haskó et al., 2000,

Inosine has analgesic action when administered by different routes (i.e. intraperitoneal, oral, intrathecal or intracerebroventricular) against pain induced by acetic acid (Nascimento et al., 2010). Of note, inosine also inhibits pain induced by formalin. Formalin test induces 2 distinct types of pain, neurogenic phase (acute pain) and inflammatory phase (inflammatory pain). Inosine is not able to relieve pain in neurogenic phase. However, inosine reduces nearly totally the inflammatory pain in formalin test (Nascimento et al., 2010). The effects of inosine in this model of pain extended the acetic acid data because formalin is a more The data described in literature strongly suggests that inosine may have an important effect in controlling chronic pain, since it has anti-inflammatory effect and can reduce acute pain. In fact, Nascimento and colleagues (2010) demonstrated that acute administration of inosine, intraperitoneally, was able to inhibit chronic inflammatory pain induced by CFA in mice, being effective up to 4 hours after administration. The CFA is responsible for inducing chronic inflammation by stimulating the body's immune response, this response is mediated by the synthesis and release of cytokines and inflammatory mediators (Zhang et al., 2011).

In the study published by Nascimento and colleagues (2010), inosine was effective against mechanical and thermal allodynia induced by partial sciatic nerve ligation (PSNL) up to 4 hours after treatment by intraperitoneal route. Further, in another experiment, inosine was given daily for until 22 days and it also presented significant analgesic effect. Pain induced by PSNL is very strong and may last for weeks (Ueda, 2006). Animal models of neuropathic pain induce many functional and biochemical changes in local injury site. After the surgery there is the release of multiple inflammatory and pain mediators which in turn, may also be present in other areas involved and affected by sciatic nerve, as spinal cord and brain (Bridges et al., 2001; Ji & Woolf, 2001; Inoue et al., 2004; Ueda, 2006). Inosine activity in this kind of pain may indicate a promising molecule to new studies, because inosine might have a longer half-life than adenosine and admittedly does not have toxic or side effects.

#### **4.5 Adenosine receptors involved in analgesic effects of inosine**

A1R has been considered the main receptor responsible for analgesic effect among adenosine receptors (Burnstock, 2007; Sawynok, 1998). A1R is also the main receptor involved in inosine analgesic effect. Both A1R antagonists DPCPX and 8-PT were able to reverse the inosine action. Inosine in a direct or indirect way activates A1R to induce analgesia (Nascimento et al., 2010). Other studies have showed that adenosine receptor antagonists block *in vivo* and *in vitro* inosine effects (Haskó et al., 2000) and adenosine receptor knockout animals do not present immunoprotective effects of inosine (Gomez & Sitkovsky, 2003). Thus, it is clear that the A1R activation is essential for inosine to exert its effect (Figure 5).

Involvement of A2AR in pain is quite controversial. Some studies show that A2AR blockade leads to analgesic effect (Borghi et al., 2002; Yoon et al., 2005) while other studies demonstrate that the blockade or deletion of A2AR causes pain relief (Bastia et al., 2002; Ledent et al., 1997). Inosine activates A2AR to induce analgesia, at least in the acetic acid

The Involvement of Purinergic System in Pain:

**4.7 Perspectives** 

Adenosine Receptors and Inosine as Pharmacological Tools in Future Treatments 639

effect. As fully mentioned previously, A1R is coupled to Gi/0 protein. A1R signaling causes downstream inhibition of adenylyl cyclase and induction of PLC activity. Further, when A1R is activated it causes potassium channel opening, PI3K and MAPK stimulation. All these pathways might participate in the inosine analgesic effect (Ansari et al., 2009; Jacobson & Gao, 2006; Sawynok, 1998; Schulte & Fredholm, 2003). Inosine inhibits the pain caused by PKC activator. Thus, at least in part, the analgesic action of inosine depends on PKC inhibition (Nascimento et al., 2010), even though it is not clear yet how it happens (Figure 5). Costenla and coworkers showed that an adenosine analog that has preference for A2AR is able to inhibit sodium current in NMDA receptors (Costenla et al., 1999). This signaling occurring *in vivo*

As previously described in this chapter, purinergic system is an important endogenous modulator of pain. Hundreds of pre-clinical studies targeting the adenosine receptors showed analgesic effect in distinct pain models. Inosine, an endogenous modulator of several physiological functions also presents a role in pain transmission. Inosine might be a natural activator of adenosine receptors. Also, it can indirectly increase the release or reduce the uptake of adenosine, potentiating the effect of its precursor. Thus, understanding how inosine acts to induce analgesia may help discover new ways to inhibit pain or new therapeutic targets. Moreover, inosine may be a potential molecule to treat pain, and it has a great advantage to be devoid of toxic or side effects because it has been used clinically for many years for other purposes. Another interesting approach would be to attempt to prolong and potentiate the effect of inosine. For this, a great understanding of purinergic

metabolism is necessary in order to correctly and effectively approach this matter.

**5. Active drugs on adenosine receptors and their clinical applications** 

and many adenosine compounds have been evaluated.

**5.1 Adenosine receptor ligands and their potential as novel drugs** 

Nowadays there is increasing interest in the therapeutic potential of adenosinergic compounds (including receptor agonists and antagonists, enzyme inhibitors and others),

Adenosine itself, for a long time, was the only adenosine agonist used in humans. It is widely used in the treatment of paroxysmal supraventricular tachycardia (Adenocard®) due to its activation of A1R, and as a diagnostic for myocardial perfusion imaging (Adenoscan®) utilizing its A2AR-activating effects resulting in vasodilation (Müller & Jacobson, 2011). However, other A1R-selective agonists such as Selodenoson, Capadenoson e Tecadenoson have been clinically evaluated for the treatment of paroxysmal supraventricular tachycardia, atrial fibrillation, or angina pectoris (Müller & Jacobson, 2011). Still talking about cardiovascular disorders, selective A2AR agonist, Apadenoson, Binodenoson and Sonedenoson appears as candidates for clinical use (Awad et al., 2006; Desai et al., 2005; Udelson et al. 2004). These agonists are of interest as vasodilator agents in cardiac imaging (Cerqueira, 2006) and inflammation suppressors. Accordingly, Regadenoson is already approved for diagnostic imaging (Iskandrian et al., 2007). A3R selective agonists are also currently in clinical trials and exhibit nanomolar affinity at the receptor, CF101 (Can-Fite Biopharma) and Cl-IB-MECA (CF102) are in trials for autoimmune inflammatory disorders

could partially explain how A2AR works in analgesia induced by inosine.

model (Nascimento et al., 2010). However, the participation of A2AR receptor in inosine analgesia in other pain animal models might be different or does not exist. Moreover, the A2AR involvement on inosine effect can occur due to its anti-inflammatory profile (Milne & Palmer, 2011) and due to activation of the K+ channels (Regaya et al., 2004).

Fig. 5. Principal mechanisms of inosine antinociception. A1R, adenosine A1 receptor; A2AR, adenosine A2A receptor, cAMP, cyclic adenosine monophosphate; K+, potassium channels; PKC, protein kinase C.

A2BR and A3R adenosine receptors do not have significant role in pain transmission or modulation (Sawynok, 1998). Although it has been shown that inosine binds to A3R, it seems that A2BR and A3R receptors are not involved in inosine analgesic effect (Nascimento et al., 2010).

#### **4.5.1 Does inosine binds to adenosine receptor?**

Few studies have evaluated if inosine binds to adenosine receptor. Jin and co-workers demonstrated that inosine binds to A3R in mast cells, but not to A1R or A2AR (Jin et al., 1997). In 2001, Fredholm's group observed that inosine weakly bound to A1R and A3R, but not to A2R (Fredholm et al., 2001). Fredholm concluded that inosine could not be considered a natural ligand of adenosine receptors. However, because of these few studies and considering *in vivo* studies where adenosine antagonists are able to block inosine effects, it is not is possible to affirm whether inosine is or not a natural ligand or a partial agonist of adenosine receptors. More studies are necessary to elucidate this issue.

#### **4.6 Intracellular signalling involved in analgesic effects of inosine**

The intracellular signaling involved in analgesic action of inosine is yet not entirely elucidated. Assuming that inosine effects depend on adenosine receptors, A1R and A2AR, we can consider that adenosine receptors signaling pathways are the major effectors of this effect. As fully mentioned previously, A1R is coupled to Gi/0 protein. A1R signaling causes downstream inhibition of adenylyl cyclase and induction of PLC activity. Further, when A1R is activated it causes potassium channel opening, PI3K and MAPK stimulation. All these pathways might participate in the inosine analgesic effect (Ansari et al., 2009; Jacobson & Gao, 2006; Sawynok, 1998; Schulte & Fredholm, 2003). Inosine inhibits the pain caused by PKC activator. Thus, at least in part, the analgesic action of inosine depends on PKC inhibition (Nascimento et al., 2010), even though it is not clear yet how it happens (Figure 5). Costenla and coworkers showed that an adenosine analog that has preference for A2AR is able to inhibit sodium current in NMDA receptors (Costenla et al., 1999). This signaling occurring *in vivo* could partially explain how A2AR works in analgesia induced by inosine.

## **4.7 Perspectives**

638 Pharmacology

model (Nascimento et al., 2010). However, the participation of A2AR receptor in inosine analgesia in other pain animal models might be different or does not exist. Moreover, the A2AR involvement on inosine effect can occur due to its anti-inflammatory profile (Milne &

Fig. 5. Principal mechanisms of inosine antinociception. A1R, adenosine A1 receptor; A2AR, adenosine A2A receptor, cAMP, cyclic adenosine monophosphate; K+, potassium channels;

A2BR and A3R adenosine receptors do not have significant role in pain transmission or modulation (Sawynok, 1998). Although it has been shown that inosine binds to A3R, it seems that A2BR and A3R receptors are not involved in inosine analgesic effect (Nascimento et al.,

Few studies have evaluated if inosine binds to adenosine receptor. Jin and co-workers demonstrated that inosine binds to A3R in mast cells, but not to A1R or A2AR (Jin et al., 1997). In 2001, Fredholm's group observed that inosine weakly bound to A1R and A3R, but not to A2R (Fredholm et al., 2001). Fredholm concluded that inosine could not be considered a natural ligand of adenosine receptors. However, because of these few studies and considering *in vivo* studies where adenosine antagonists are able to block inosine effects, it is not is possible to affirm whether inosine is or not a natural ligand or a partial agonist of

The intracellular signaling involved in analgesic action of inosine is yet not entirely elucidated. Assuming that inosine effects depend on adenosine receptors, A1R and A2AR, we can consider that adenosine receptors signaling pathways are the major effectors of this

PKC, protein kinase C.

**4.5.1 Does inosine binds to adenosine receptor?** 

adenosine receptors. More studies are necessary to elucidate this issue.

**4.6 Intracellular signalling involved in analgesic effects of inosine** 

2010).

Palmer, 2011) and due to activation of the K+ channels (Regaya et al., 2004).

As previously described in this chapter, purinergic system is an important endogenous modulator of pain. Hundreds of pre-clinical studies targeting the adenosine receptors showed analgesic effect in distinct pain models. Inosine, an endogenous modulator of several physiological functions also presents a role in pain transmission. Inosine might be a natural activator of adenosine receptors. Also, it can indirectly increase the release or reduce the uptake of adenosine, potentiating the effect of its precursor. Thus, understanding how inosine acts to induce analgesia may help discover new ways to inhibit pain or new therapeutic targets. Moreover, inosine may be a potential molecule to treat pain, and it has a great advantage to be devoid of toxic or side effects because it has been used clinically for many years for other purposes. Another interesting approach would be to attempt to prolong and potentiate the effect of inosine. For this, a great understanding of purinergic metabolism is necessary in order to correctly and effectively approach this matter.
