Section 1 Drug Transport

**3**

**Chapter 1**

**Abstract**

physic-chemical properties.

**1. Introduction**

Liposome-A Comprehensive

*Mani Sharma, Jyoti Joshi, Neeraj Kumar Chouhan,* 

*Mamta N. Talati, Sandeep Vaidya and Abhiram Kumar*

Bangham was first to develop these spherical-shaped nano-vesicles called liposomes in the early 1960s. Today, liposomes have emerged as crucial tools for bettering the delivery of drugs that majorly includes-antifungal drug, peptide hormones, enzymes, vaccines antimicrobial agents, drugs against cancer, and genetic materials. Following the different manufacturing practices and versatile properties liposomes can be categorized in various parameters of size, charge, poly-dispersity index, encapsulation efficiency, solubility properties, and lamellarity. Alteration in such parameters elevates the loading and bioavailability of a drug by giving more clear target specification, desired or controlled release. This bibliographic chapter provides a comprehensive overview of methods for the preparation of liposomes with other perspectives that majorly includes—physio-chemical characteristics, dosage regimen, advantages over other delivery systems, approved liposomal based drugs and other ongoing drugs in clinical trials. It will help researchers to breakthrough more structurally successful delivery vehicles depending upon their various

**Keywords:** liposomes, particle size, zeta potential, polydispersity index, encapsulation efficiency, methods of preparation and bioavailability

Liposomes can be microscopically examined as the vesicle with spherical structure that comprises one or more bilayer lipid in the aqueous core part of a shell. Liposomes are widely used in the delivery of variety of drugs depending upon its various physic-chemical characteristics. Design and development of liposomes are classified in many ways among which thin film hydration method is the most globally accepted procedure. Liposmes formation occurs when lipids are incorporated into water or buffer solution under continuous stirring, that in return forms the spherically shaped vesicles termed as liposomes. There are many methods to develop liposomes among which thin film hydration method is most common. Recently, lipid film hydration method was used to develop a multilamellar vesicle (MLV) loaded with curcumin (CUR) and Rhodamine B (RhB), [1] as a successful drug delivery approach. Phospholipids and cholesterol are the major components used in the development of liposomes (**Figure 1**). Where bilayer lipid composes of a hydrophilic head group, i.e., phospholipid and a hydrophilic tail group. Where phospholipids can easily penetrate and localize in the skin thus increases the overall

Approach for Researchers

#### **Chapter 1**

### Liposome-A Comprehensive Approach for Researchers

*Mani Sharma, Jyoti Joshi, Neeraj Kumar Chouhan, Mamta N. Talati, Sandeep Vaidya and Abhiram Kumar*

#### **Abstract**

Bangham was first to develop these spherical-shaped nano-vesicles called liposomes in the early 1960s. Today, liposomes have emerged as crucial tools for bettering the delivery of drugs that majorly includes-antifungal drug, peptide hormones, enzymes, vaccines antimicrobial agents, drugs against cancer, and genetic materials. Following the different manufacturing practices and versatile properties liposomes can be categorized in various parameters of size, charge, poly-dispersity index, encapsulation efficiency, solubility properties, and lamellarity. Alteration in such parameters elevates the loading and bioavailability of a drug by giving more clear target specification, desired or controlled release. This bibliographic chapter provides a comprehensive overview of methods for the preparation of liposomes with other perspectives that majorly includes—physio-chemical characteristics, dosage regimen, advantages over other delivery systems, approved liposomal based drugs and other ongoing drugs in clinical trials. It will help researchers to breakthrough more structurally successful delivery vehicles depending upon their various physic-chemical properties.

**Keywords:** liposomes, particle size, zeta potential, polydispersity index, encapsulation efficiency, methods of preparation and bioavailability

#### **1. Introduction**

Liposomes can be microscopically examined as the vesicle with spherical structure that comprises one or more bilayer lipid in the aqueous core part of a shell. Liposomes are widely used in the delivery of variety of drugs depending upon its various physic-chemical characteristics. Design and development of liposomes are classified in many ways among which thin film hydration method is the most globally accepted procedure. Liposmes formation occurs when lipids are incorporated into water or buffer solution under continuous stirring, that in return forms the spherically shaped vesicles termed as liposomes. There are many methods to develop liposomes among which thin film hydration method is most common. Recently, lipid film hydration method was used to develop a multilamellar vesicle (MLV) loaded with curcumin (CUR) and Rhodamine B (RhB), [1] as a successful drug delivery approach. Phospholipids and cholesterol are the major components used in the development of liposomes (**Figure 1**). Where bilayer lipid composes of a hydrophilic head group, i.e., phospholipid and a hydrophilic tail group. Where phospholipids can easily penetrate and localize in the skin thus increases the overall

**Figure 1.** *Liposome molecule with lipid bilayer.*

bioavailability in case of many dermal formulations whereas, cholesterol not only increases microviscosity of the bilayer but also defines the stability and rigidity of the formulation [2].

There are many routes to administer liposomes containing drugs, i.e., pulmonary, ocular, intramuscular, intravenous, topical, nasal and oral. Liposomes can be delivered in many ways involving sprays, capsules, ointments, creams, solutions, etc. for curing various disseases: bacterial, fungal, ocular, vaccines, fibrinolysis, endocrine, arthritis, asthma, diabetes, diseases of immune system, herpes, analgesics, topical anesthesia and even cancer [3].

Based on different parameters, liposomes are further classified depending upon method of preparation, structural parameters, biochemistry, cosmetics and medicine composition, and application in biology. Phospholipids can be from natural sources such as soya bean, egg yolk and olive oil. Depending upon various characteristics liposomes can be categorized on the basis of various physical parameters such as—pH, temperature, ionic charges, immunogenicity and stability.

In a recent study performed in 2019, it is revealed that the concentration of phospholipids and cholesterol variates the protein binding of the formulation [4].

Most commonly employed phospholipids in the formulation of liposomes are: phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), dipalmitoyl phosphatidylserine1,2dioleoylsnglyc ero3phosphoserine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) [5].

**5**

*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

• Disloyal phosphatidylcholine

• Disloyal phosphatidylethanolamine

Cholesterol are optimized to be used in the formulation of liposomes up to a wide range with a molar ratios 1:1 or 2:1 against phospholipids. Cholesterol defines a strategic role in liposome composition; although, the adequate quantity to be used in the formulation has not been yet clarified. Thus, we can optimize lipids and cholesterol ratio, to prepare stable and controlled drug release vehicles (**Figures 2** and 3) [6].

2.Synthetic phospholipids:

**2. Physio-chemical properties**

*Inner and outer structure of liposome.*

*Hydrophilic and lipophilic terminals of lipid.*

**3. Applications of liposomes**

Role of liposome in drug delivery:

• It increases the overall therapeutic index and efficacy of a liposomal

• Selective & passive targeting.

See **Tables 1**–**3**.

formulation.

B. Cholesterol

**Figure 2.**

**Figure 3.**

#### **1.1 Composition of liposomes**

#### A. Phospholipids

	- Phosphatidylcholine
	- Phosphatidylserine
	- Phosphatidylethanolamine

*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

2.Synthetic phospholipids:


*Molecular Pharmacology*

the formulation [2].

*Liposome molecule with lipid bilayer.*

**Figure 1.**

stability.

anesthesia and even cancer [3].

**1.1 Composition of liposomes**

1.Derived from natural sources:

• Phosphatidylethanolamine

• Phosphatidylcholine

• Phosphatidylserine

A. Phospholipids

bioavailability in case of many dermal formulations whereas, cholesterol not only increases microviscosity of the bilayer but also defines the stability and rigidity of

Based on different parameters, liposomes are further classified depending upon method of preparation, structural parameters, biochemistry, cosmetics and medicine composition, and application in biology. Phospholipids can be from natural sources such as soya bean, egg yolk and olive oil. Depending upon various characteristics liposomes can be categorized on the basis of various physical parameters such as—pH, temperature, ionic charges, immunogenicity and

In a recent study performed in 2019, it is revealed that the concentration of phospholipids and cholesterol variates the protein binding of the formulation [4]. Most commonly employed phospholipids in the formulation of liposomes are: phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), dipalmitoyl phosphatidylserine1,2dioleoylsnglyc ero3phosphoserine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphati-

dylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) [5].

There are many routes to administer liposomes containing drugs, i.e., pulmonary, ocular, intramuscular, intravenous, topical, nasal and oral. Liposomes can be delivered in many ways involving sprays, capsules, ointments, creams, solutions, etc. for curing various disseases: bacterial, fungal, ocular, vaccines, fibrinolysis, endocrine, arthritis, asthma, diabetes, diseases of immune system, herpes, analgesics, topical

**4**

Cholesterol are optimized to be used in the formulation of liposomes up to a wide range with a molar ratios 1:1 or 2:1 against phospholipids. Cholesterol defines a strategic role in liposome composition; although, the adequate quantity to be used in the formulation has not been yet clarified. Thus, we can optimize lipids and cholesterol ratio, to prepare stable and controlled drug release vehicles (**Figures 2** and 3) [6].

**Figure 2.** *Hydrophilic and lipophilic terminals of lipid.*

**Figure 3.** *Inner and outer structure of liposome.*

### **2. Physio-chemical properties**

See **Tables 1**–**3**.

#### **3. Applications of liposomes**

Role of liposome in drug delivery:



#### **Table 1.**

*Physical characterization [6].*


#### **Table 2.**

*Chemical characterization [6].*


#### **Table 3.**

*Biological characterization [6].*


**7**

**Table 4.**

*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

philic environment in a single system

• It helps in protecting the encapsulated drug.

• Facilitates both active and passive targeting.

• No toxicity in heart as it does not accumulates in the heart.

• Chelation therapy in case of of heavy metal poisoning

• Liposomes are able to provide both aqueous "milieu internee" and the lipo-

• Method of preparation is easy and has no such complicated or expensive

In parasitic diseases After IV injection liposomes are comfortly digested by phagocytic cells in the

In bioengineering Fragments of siRNA and DNA are delivered with the help of modern genetic engineering and gene recombinant technology In vaccination Liposomes are considerably used in proper vaccination due its fine active targeting In agro-food industry Due to its versatile physio-chemical properties lipids are extensively manufactured

Anticancer therapy Liposomes are effective for the cells not only in tumors but also in the

body and hence considered as one of the best vehicle to dispatch cargo into

These liposomes are sterically stabilized vesicles and are long circulating microreservoirs or tumor (or site of inflammation and infection) targeting vehicles

• Biocompatibility

procedures involved

• Intercepts the oxidation of drug

• Diagnostic imaging of tumors

• In enzyme replacement therapy

• Study of membranes

• As drug delivery carriers

• In multidrug resistance

• In cosmetology (**Table 4**)

**Category Application utilized**

macrophages

gastrointestinal mucosa

and used in large scale up sectors

• In gene delivery

• In immunology

Other medical applications

*Applications of liposomes [6].*

• Biodegradable

• Active targeting can also be achieved by coupling with the site-specific ligands.

#### **3.1 Other advantages of using liposomes**


*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

• Biocompatibility

*Molecular Pharmacology*

Mean vesicle size and size distribution (submicron and micron range)

**Characterization parameters Analytical method/instrument** Phospholipid concentration Barlett assay, Stewart assay, HPLC

**Characterization parameters Analytical method/instrument**

Vesicle shape and surface morphology Transmission electron microscopy Electrical surface potential and surface pH Zetasizer & pH measurement device

Phase behavior Differential scanning calorimetry (DSC) Lamellarity Freeze-fracture electron microscopy Percent of free drug/percent capture Minicolumn centrifugation, ion-exchange

Surface charge free Flow electrophoresis

Zetasizer

chromatography, radiolabelling

Phopholipid peroxidation UV absorbance, iodometric, GLC

**Characterization parameters Analytical method/instrument** Sterility aerobic or anaerobic cultures Sterility aerobic or anaerobic cultures Pyrogenicity Limulus amebocyte lysate (LAL) test

Animal toxicity By pathology and histology

• Due to the encapsulation of drug, overall stability is increased and reduced the

• It helps to improve the pharmacokinetic processes by increasing the circulation lifetime, decreasing elimination and toxic effects thus elevating the overall

• Active targeting can also be achieved by coupling with the site-specific

Concentration of cholesterol By HPLC

adverse effects of encapsulated drug.

bioavailability of a drug [7].

**3.1 Other advantages of using liposomes**

• Efficient control of the release

• Increased targeting prospects

• Resemblance to natural membrane structures

ligands.

• Biodegradability

**6**

**Table 2.**

**Table 3.**

**Table 1.**

*Chemical characterization [6].*

*Biological characterization [6].*

*Physical characterization [6].*



#### **Table 4.** *Applications of liposomes [6].*

### **4. Methods of preparation**

See **Figure 4**.

#### **4.1 Thin film hydration method**

This is one of the widely used methods for the preparation of liposomes. As it has no such complicated steps involved in it. Multilamellar vesicles (MLV) are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vacuum at a temperature around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aqueous phase—phosphate buffer solution of pH 7.2 for 1–2 h at 60–70°C.

This kind of procedure can be applied to almost any kind of lipid mixtures, but has some drawbacks that majorly includes—low encapsulation space, a bit difficult to scale up and layer formed are not always homogeneous thus shows heterogeneous size distribution during later physio-chemical examination of liposomes through zetasizer.

#### **4.2 Injection methods**

#### *4.2.1 Ether injection method*

Here, the lipid mixture is dissolved in ether or diethyl ether under continuous stirring that is later injected into a PBS or aqueous phase. Which under injection pressure causes the removal of almost all organic solvent that ultimately forms liposomes. This method also suffers with the heterogeneous liposomal formulation defect.

**9**

*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

In ethanol injection method the lipid mixture is dissolved in ethanol under continuous stirring that is later injected into a preheated TRIS-HCl buffer or distilled water. Hydrophobicity and hydrophilicity of a drug accounts for drug intake in a liposomal vesicle. It has an advantage of using non-toxic and ethanol and is also easily scalable.

It is the most widely accepted method to develop small unilamellar vesicles (SLV). SLV are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vaccum at a temperature around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aquous phase—phosphate buffer solution of pH 7.2 for 1–2 h at 60–70°C. Further the bath sonicator is used to transform the size of vesicles. Lastly, liposomes are centrifuged in order to remove the titanium particles that might got added due to overheating in sonication process. Less encapsulation space is the major drawback of such vesicles.

Liposomes are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vacuum at a temperature around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aqueous phase—phosphate buffer solution of pH 7.2 for 1–2 h at 60–70°C. In addition, these lipoosmes are passes through high pressure extruder for 10 cycles in order to obtain

Here, the lipid mixture is dissolved in organic solvents ether or diethyl ether or a mixture of diethyl ether and chloroform (1:1 v/v); a mixture of methanolchloroform (1:2 v/v) under continuous stirring that is later injected into a PBS or aqueous phase comprising citric-Na2HPO4 to improve the overall efficacy of a formulation. Which under injection pressure causes the removal of organic solvent that ultimately leads to the formation of liposomes. This method also suffers with the heterogeneous liposomal formulation defect. Organic solvent is then dried using rota-vapor instrument thus forming homogeneous liposome. The major disadvantage of this procedure is the leftover of remaining organic solvent in the final

Here acidic phospholipids are used to prepare SUV by following the thin film hydration process followed on with the addition of calcium that causes fusion to form MLV. Final addition of ethylenediaminetetraacetic acid (EDTA) to MLV

*4.2.2 Ethanol injection method*

**4.3 Sonication method**

**4.4 High-pressure extrusion method**

more uniform and stable liposomes.

**4.5 Reverse-phase evaporation method**

**4.6 Calcium-induced fusion method**

formulation also faces difficulty in scale up procedures.

results in the formation of large unilamellar vesicles LUV.

#### **Figure 4.**

*General representation for method of preparation of liposome.*

#### *4.2.2 Ethanol injection method*

*Molecular Pharmacology*

See **Figure 4**.

zetasizer.

defect.

**4.2 Injection methods**

*4.2.1 Ether injection method*

**4. Methods of preparation**

**4.1 Thin film hydration method**

solution of pH 7.2 for 1–2 h at 60–70°C.

This is one of the widely used methods for the preparation of liposomes. As it has no such complicated steps involved in it. Multilamellar vesicles (MLV) are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vacuum at a temperature around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aqueous phase—phosphate buffer

This kind of procedure can be applied to almost any kind of lipid mixtures, but has some drawbacks that majorly includes—low encapsulation space, a bit difficult to scale up and layer formed are not always homogeneous thus shows heterogeneous size distribution during later physio-chemical examination of liposomes through

Here, the lipid mixture is dissolved in ether or diethyl ether under continuous stirring that is later injected into a PBS or aqueous phase. Which under injection pressure causes the removal of almost all organic solvent that ultimately forms liposomes. This method also suffers with the heterogeneous liposomal formulation

**8**

**Figure 4.**

*General representation for method of preparation of liposome.*

In ethanol injection method the lipid mixture is dissolved in ethanol under continuous stirring that is later injected into a preheated TRIS-HCl buffer or distilled water. Hydrophobicity and hydrophilicity of a drug accounts for drug intake in a liposomal vesicle. It has an advantage of using non-toxic and ethanol and is also easily scalable.

#### **4.3 Sonication method**

It is the most widely accepted method to develop small unilamellar vesicles (SLV). SLV are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vaccum at a temperature around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aquous phase—phosphate buffer solution of pH 7.2 for 1–2 h at 60–70°C. Further the bath sonicator is used to transform the size of vesicles. Lastly, liposomes are centrifuged in order to remove the titanium particles that might got added due to overheating in sonication process. Less encapsulation space is the major drawback of such vesicles.

#### **4.4 High-pressure extrusion method**

Liposomes are prepared by solubilizing natural or synthesized phospholipid in chloroform, dichloromethane, ethanol or in a mixture of chloroform and methanol in a ratio of 3:1 v/v; 2:1 v/v or 9:1 v/v. A homogeneous thin film forms when this mixture is revolved and dried in a rota-evaporator under vacuum at a temperature around 45–60°C. Layes is kept under nitrogen drying for overnight. Next, comes the hydration process where completely dried thin film is hydrated using aqueous phase—phosphate buffer solution of pH 7.2 for 1–2 h at 60–70°C. In addition, these lipoosmes are passes through high pressure extruder for 10 cycles in order to obtain more uniform and stable liposomes.

#### **4.5 Reverse-phase evaporation method**

Here, the lipid mixture is dissolved in organic solvents ether or diethyl ether or a mixture of diethyl ether and chloroform (1:1 v/v); a mixture of methanolchloroform (1:2 v/v) under continuous stirring that is later injected into a PBS or aqueous phase comprising citric-Na2HPO4 to improve the overall efficacy of a formulation. Which under injection pressure causes the removal of organic solvent that ultimately leads to the formation of liposomes. This method also suffers with the heterogeneous liposomal formulation defect. Organic solvent is then dried using rota-vapor instrument thus forming homogeneous liposome. The major disadvantage of this procedure is the leftover of remaining organic solvent in the final formulation also faces difficulty in scale up procedures.

#### **4.6 Calcium-induced fusion method**

Here acidic phospholipids are used to prepare SUV by following the thin film hydration process followed on with the addition of calcium that causes fusion to form MLV. Final addition of ethylenediaminetetraacetic acid (EDTA) to MLV results in the formation of large unilamellar vesicles LUV.

#### **4.7 Dehydration-rehydration method**

Liposomes are prepared by using the sonication method as explained in Section 4.3. Developed liposomes are freeze dried overnight where the formation of multilamellar vesicles occurs when dry powder gets controlled rehydration.

#### **4.8 Freeze-thaw method**

Liposomes are prepared by using thin film hydration method as explained in Section 4.1. Developed liposomes are freeze dried overnight and is then thawed


**11**

*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

**4.9 Microfluidization**

**4.10 Supercritical fluids (SCF)**

**5. Mechanism of liposomal formulation**

ment of a aqueous medium**.**

**of liposomal-formulation**

phology of a liposomal structure.

product as per ICH guidelines.

**6. Evaluation**

zetasizer.

liposomes.

in order to govern the ionic strength and phospholipid concentration of the final liposomal formation. Physical disruption of lamellar structure occurs due to freeze-

Boltic et al. was the first to introduce such method for the preparation of liposomes. Here liposomes are prepared using thin film hydration method as explained in Section 4.1, which is then sonicated and microfluidized in order to obtain partial homogenization. This method has its wide application in industrial formulation of

Supercritical fluids (SCF) were introduced to replace toxic organic solvents for the preparation of liposomes. Supercritical carbon dioxide is the most widely used supercritical fluid as it has many advantages over conventionally used organic solvents such as—it is not flammable, can be recycled, non-toxic, can be comparatively easily removed from the solvents, requires moderate temperature and also exclude the product degradation in inert surroundings. Karn et al. experimented and explained the comparative study between thin film hydration method and supercritical fluids using method evaluating the non toxicity and better field approaches in term of using super critical fluids for the formulation of liposomes (**Table 5**).

• Phospholipids shows affinity for polar molecules as well as for **aqueous** phase due to a hydrophobic tail, that has 2 fatty acids which are made up of 10–24 C

• In a phospholipid molecule the polar portion connects with a polar environ-

• Phospholipids arrange layers of lipids in close alignment in a planer bilayer sheet. Sufficient amount of energy is required for this planar arrangement

The average size, size distribution, and zeta potential shall be determined by

Transmission electron microscopy is used to study the shape and surface mor-

Stability studies: stability studies shall be conducted to assess the shelf-life of

atoms comprising of 0–6 double bonds in every chain [8]**.**

(sonication, homogenization, heating, etc**.**) (**Figure 5**)**.**

**6.1 Morphological and physicochemical characterization** 

**6.2 In vitro performance evaluation and stability studies**

thaw of liposomal formulation giving it a final ionic structure.

#### **Table 5.**

*Methods for the preparation of liposomal formulation to deliver drugs [2].*

in order to govern the ionic strength and phospholipid concentration of the final liposomal formation. Physical disruption of lamellar structure occurs due to freezethaw of liposomal formulation giving it a final ionic structure.

#### **4.9 Microfluidization**

*Molecular Pharmacology*

**4.8 Freeze-thaw method**

**Antifungal drugs**

**Analgesic drugs**

**Antibiotic drugs**

**Antifibrinolytic drugs**

**Drugs against cancer**

**Hormone drugs**

**Ophthalmic drugs**

hydrochloride

**Vaccines**

**Immunosuppressive drugs**

**Potential drugs as oral insulin** Sodium glycocholate and metformin

**4.7 Dehydration-rehydration method**

Liposomes are prepared by using the sonication method as explained in Section 4.3. Developed liposomes are freeze dried overnight where the formation of multi-

Liposomes are prepared by using thin film hydration method as explained in Section 4.1. Developed liposomes are freeze dried overnight and is then thawed

**Drugs liposome formulation Method Type of** 

Amphotericin B Thin-film hydration method MLV Clotrimazole Rotary evaporation method MLV Fluconazole Thin film hydration method MLV

Ketorolac tromethamine Thin-film hydration method MLV

Tranexamic acid Chloroform film and sonication method SUV

5-Fluorouracil Lipid-film hydration method, extrusion,

Amikacin Reverse phase evaporation method MLV, LUV Mafenide acetate Solvent evaporation and microencapsulation MLV SUV

evaporation method

Vinblastine sulphate Thin-film hydration method and sonication MLV SUV Tamoxifen Thin-film hydration method MLV Bis-demethoxy curcumin analogue Thin-film hydration method and sonication MLV SUV Doxorubicin Lipid-film hydration method and extrusion MLV

Cyproterone acetate Thin-film hydration method MLV

Sirolimus Thin-film hydration method MLV Tacrolimus (Fk-506) Thin-film hydration method MLV

Brimonidine tartrate Thin-film hydration method and sonication MLV SUV

hydration method

hydration method

Tetanus toxoid diphtheria toxoid Reverse phase evaporation method MLV, LUV

Reverse phase evaporation and thin-film

Acetazolamide Reverse phase evaporation and thin-film

*Methods for the preparation of liposomal formulation to deliver drugs [2].*

ethanol injection and reverse phase

**liposome**

MLV, LUV, SUV MLV, LUV

MLV, LUV MLV

MLV, LUV MLV

lamellar vesicles occurs when dry powder gets controlled rehydration.

**10**

**Table 5.**

Boltic et al. was the first to introduce such method for the preparation of liposomes. Here liposomes are prepared using thin film hydration method as explained in Section 4.1, which is then sonicated and microfluidized in order to obtain partial homogenization. This method has its wide application in industrial formulation of liposomes.

#### **4.10 Supercritical fluids (SCF)**

Supercritical fluids (SCF) were introduced to replace toxic organic solvents for the preparation of liposomes. Supercritical carbon dioxide is the most widely used supercritical fluid as it has many advantages over conventionally used organic solvents such as—it is not flammable, can be recycled, non-toxic, can be comparatively easily removed from the solvents, requires moderate temperature and also exclude the product degradation in inert surroundings. Karn et al. experimented and explained the comparative study between thin film hydration method and supercritical fluids using method evaluating the non toxicity and better field approaches in term of using super critical fluids for the formulation of liposomes (**Table 5**).

### **5. Mechanism of liposomal formulation**


### **6. Evaluation**

#### **6.1 Morphological and physicochemical characterization of liposomal-formulation**

The average size, size distribution, and zeta potential shall be determined by zetasizer.

Transmission electron microscopy is used to study the shape and surface morphology of a liposomal structure.

#### **6.2 In vitro performance evaluation and stability studies**

Stability studies: stability studies shall be conducted to assess the shelf-life of product as per ICH guidelines.

**Figure 5.** *Mechanism of liposome formation.*

MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to evaluate the in-vitro cytotoxicity of the developed formulation.

FACS (fluorescence assisted cell sorting) is used to quantify the cell uptake study.

#### **7. Marketed liposomal formulations**

See **Tables 6** and **7**.


**13**

**8. Conclusions**

Formule Liposome

Future Perfect Skin

Gel

**Table 6.**

Gel

**Table 7.**

**Acknowledgements**

institute or person.

**Conflict of interest**

that could have influenced its outcome.

Liposomes evolved as an extraordinary tool or micro-engineered membranes for the delivery of drugs because of their minimum toxicity and flexibility that can be tailored for various desirable intentions. This unparalleled delivery approach can be used for almost every drug or active pharmaceutical ingredient despite of its varied physicochemical properties and route of administration. Extensive uses of liposome in the delivery of drugs can be starched further by researchers, medical representatives and in scale-up processes in order to develop desired modification and better delivery approaches by holding the promising physio-chemical properties and pharmacokinetics (absorption, distribution, metabolism, and elimination)

**Product Manufacturer Liposomes and key ingredients**

**Marketed product Drug used Target diseases Company**

Symphatic 2000 Biopharm GmbH Thymus extract vitamin A palmitate Niosomes Lancome (L'Or'eal) Glyceropolyether with moisturizers Inovita Pharm/Apotheke Thymus extract, hyaluronic

Estee Lauder TMF, Vitamins E, A palmitate,

leishmaniasis

(Thymoxin) hyaluronic acid

SEQUUS, USA

cerebroside ceramide

lancome

Formule Liposome Gel Payot (Ferdinand Muehlens)

AmphotecTM Amphotericin B fungal infections,

Flawless finish Elizabeth Arden Liquid make up

Nactosomes Lancome (L'Or'eal) Vitamins

Eye Perfector Avon Soothing cream to reduce eye

Natipide II Nattermann PL Liposomal gel for do-it yourself

Efect du Soleil L'Or'eal Tanning agents in liposomes niosomes

I thank all my coauthors who are listed, and the work was not funded by any

We wish to declare that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work

involved with liposomes, as described in the chapter.

*Liposomal cosmetic formulations present in the market [10]***.**

*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

*Liposomal formulations present in the market [9].*


#### **Table 6.**

*Molecular Pharmacology*

uptake study.

DoxilTM or CaelyxTM

EvacetTM Doxorubicin

**Figure 5.**

MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to

evaluate the in-vitro cytotoxicity of the developed formulation.

**7. Marketed liposomal formulations**

AlecTM Dry protein free powder

of DPPC PG

VentusTM Prostaglandin E1 Systemic inflammatory

Doxil® Doxorubicin HCl Refractory ovarian

Topex Br Terbutaline sulphate Asthma ozone USA

Novasome Smallpox vaccine Smallpox Novavax USA

Doxorubicin Metastatic breast

Fungizone® Amphotericin B Fungal infections Leishmaniasis Depocyt Cytarabine Cancer therapy Skye Pharm USA

See **Tables 6** and **7**.

*Mechanism of liposome formation.*

FACS (fluorescence assisted cell sorting) is used to quantify the cell

**Marketed product Drug used Target diseases Company**

Expanding lung diseases in babies

diseases

cancer

cancer

Doxorubicin Kaposi's sarcoma SEQUUS, USA

Britannia Pharm, UK

The liposome company,

USA

The Liposome Company, USA

ALZA, USA

**12**

*Liposomal formulations present in the market [9].*


#### **Table 7.**

*Liposomal cosmetic formulations present in the market [10]***.**

#### **8. Conclusions**

Liposomes evolved as an extraordinary tool or micro-engineered membranes for the delivery of drugs because of their minimum toxicity and flexibility that can be tailored for various desirable intentions. This unparalleled delivery approach can be used for almost every drug or active pharmaceutical ingredient despite of its varied physicochemical properties and route of administration. Extensive uses of liposome in the delivery of drugs can be starched further by researchers, medical representatives and in scale-up processes in order to develop desired modification and better delivery approaches by holding the promising physio-chemical properties and pharmacokinetics (absorption, distribution, metabolism, and elimination) involved with liposomes, as described in the chapter.

#### **Acknowledgements**

I thank all my coauthors who are listed, and the work was not funded by any institute or person.

#### **Conflict of interest**

We wish to declare that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.

*Molecular Pharmacology*

#### **Author details**

Mani Sharma1 \*, Jyoti Joshi1 , Neeraj Kumar Chouhan2 , Mamta N. Talati<sup>2</sup> , Sandeep Vaidya<sup>2</sup> and Abhiram Kumar1

1 Uttarakhand Technical University [UKTU], Uttarakhand, India

2 National Institute of Pharmaceutical Education and Research [NIPER], Telangana, India

\*Address all correspondence to: mninup2015@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**15**

*Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

> Development and evaluation of stability and ultrasound response of DSPC-DPSG-based freeze-dried microbubbles.

Journal of Liposome Research.

[9] Sackmann E. Physical basis of self-organization and function of membranes: Physics of vesicles. In: Handbook of Biological Physics. Vol. 1.

[10] Lasic DD, Frederik PM, Stuart MC, Barenholz Y, McIntosh TJ. Gelation of liposome interior A novel method for drug encapsulation. FEBS Letters.

2019;**29**(4):368-374

1995. pp. 213-304

1992;**312**(2-3):255-258

[1] Anishiya Chella ER, Rajendran NK, Jeyaraj M, Ramu A, Rajan M. Retinal photoreceptors targeting SA-g-AA coated multilamellar liposomes carrier system for cytotoxicity and cellular uptake evaluation. Journal of Liposome Research. 2020:1-35. ISSN: 0898-2104

[2] Popovska O. An overview: Methods for preparation and characterization of liposomes as drug delivery systems. International Journal of Pharmaceutical and Phytopharmacological Research.

**References**

2014;**3**(3):182-189

2012;**2**:78-82

[3] Pandey RS. Formulation of benzocaine loaded PG-liposomes for enhanced skin delivery. Asian Journal of Medical and Pharmaceutical Sciences.

[4] Foteini P, Pippa N, Naziris N, Demetzos C. Physicochemical study of the protein–liposome interactions: Influence of liposome composition and concentration on protein binding.

Journal of Liposome Research.

[5] Sharma SH, Mishra LI, Grover I, Gupta AN, Kaur KI. Liposomes: Vesicular system an overview. International Journal of Pharmacy and Pharmaceutical Sciences.

[6] Dwivedi C, Verma S. Review on preparation and characterization of liposomes with application. Journal of Scientific and Innovative Research.

[7] Sesarman A, Muntean D, Abrudan B, Tefas L, Sylvester B, Licarete E, et al. Improved pharmacokinetics and reduced side effects of doxorubicin therapy by liposomal co-encapsulation with curcumin. Journal of Liposome

2019;**29**(4):313-321

2010;**2**(4):15-21

2013;**2**:486-508

Research. 2019;**8**:1-10

[8] Unga J, Omata D, Kudo N, Ueno S, Munakata L, Shima T, et al. *Liposome-A Comprehensive Approach for Researchers DOI: http://dx.doi.org/10.5772/intechopen.93256*

#### **References**

*Molecular Pharmacology*

**14**

**Author details**

Sandeep Vaidya<sup>2</sup>

Telangana, India

\*, Jyoti Joshi1

provided the original work is properly cited.

and Abhiram Kumar1

\*Address all correspondence to: mninup2015@gmail.com

1 Uttarakhand Technical University [UKTU], Uttarakhand, India

2 National Institute of Pharmaceutical Education and Research [NIPER],

, Neeraj Kumar Chouhan2

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Mamta N. Talati<sup>2</sup>

,

Mani Sharma1

[1] Anishiya Chella ER, Rajendran NK, Jeyaraj M, Ramu A, Rajan M. Retinal photoreceptors targeting SA-g-AA coated multilamellar liposomes carrier system for cytotoxicity and cellular uptake evaluation. Journal of Liposome Research. 2020:1-35. ISSN: 0898-2104

[2] Popovska O. An overview: Methods for preparation and characterization of liposomes as drug delivery systems. International Journal of Pharmaceutical and Phytopharmacological Research. 2014;**3**(3):182-189

[3] Pandey RS. Formulation of benzocaine loaded PG-liposomes for enhanced skin delivery. Asian Journal of Medical and Pharmaceutical Sciences. 2012;**2**:78-82

[4] Foteini P, Pippa N, Naziris N, Demetzos C. Physicochemical study of the protein–liposome interactions: Influence of liposome composition and concentration on protein binding. Journal of Liposome Research. 2019;**29**(4):313-321

[5] Sharma SH, Mishra LI, Grover I, Gupta AN, Kaur KI. Liposomes: Vesicular system an overview. International Journal of Pharmacy and Pharmaceutical Sciences. 2010;**2**(4):15-21

[6] Dwivedi C, Verma S. Review on preparation and characterization of liposomes with application. Journal of Scientific and Innovative Research. 2013;**2**:486-508

[7] Sesarman A, Muntean D, Abrudan B, Tefas L, Sylvester B, Licarete E, et al. Improved pharmacokinetics and reduced side effects of doxorubicin therapy by liposomal co-encapsulation with curcumin. Journal of Liposome Research. 2019;**8**:1-10

[8] Unga J, Omata D, Kudo N, Ueno S, Munakata L, Shima T, et al. Development and evaluation of stability and ultrasound response of DSPC-DPSG-based freeze-dried microbubbles. Journal of Liposome Research. 2019;**29**(4):368-374

[9] Sackmann E. Physical basis of self-organization and function of membranes: Physics of vesicles. In: Handbook of Biological Physics. Vol. 1. 1995. pp. 213-304

[10] Lasic DD, Frederik PM, Stuart MC, Barenholz Y, McIntosh TJ. Gelation of liposome interior A novel method for drug encapsulation. FEBS Letters. 1992;**312**(2-3):255-258

**Chapter 2**

**Abstract**

Capabilities

achieving better therapeutic objective.

dissolution enhancement, floating tablets

**1. Introduction**

**17**

Preparation and *in vitro*

Characterisation of Solid

Dispersion Floating Tablet by

Effervescent Control Release

Technique with Improved Floating

*Peeush Singhal, Rajneesh Dutt Kaushik and Vijay Jyoti Kumar*

In this research, an effort has been done for the development of effervescent

controlled release floating tablet (ECRFT) from solid dispersions (SDs) of diclofenac sodium (DS) for upsurge the solubility and dissolution rate. ECRFT of DS was prepared by using SDs of DS and its SDs prepared with PEG as carrier using thermal method (simple fusion). SDs of DS was formulated in many ratios (1:1, 1:2, 1:3 and 1:4). Prepared SDs were optimised for its solubility, % drug content and % dissolution studies. Tablets were formulated by using optimised SDs products and all formulation was evaluated for various parameters. A clear rise in dissolution rate was detected with entirely SD, amid that the optimised SD (SD4) was considered for ECRFT. Among all the tablet formulations, its F3 formulation was better in all the terms of pre-compression and post-compression parameters. It had all the qualities of a good ECRFT, based on this F3 formulation was selected as the best formulation. Data of *in vitro* release were fitted in several kinetics models to explain release mechanism. The F3 formulation shows zero order release. From this study, we can conclude that ECRFT containing SDs of DS can be successfully used for

**Keywords:** solid dispersion, diclofenac sodium, polyethylene glycol,

Diclofenac sodium (DS) is an effective NSAID with high affinity for both COX-1 and COX-2 receptors and it is one and only maximum frequently recommended drugs in India for the cure of pain, inflammation and joint stiffness caused by arthritis. According to BCS classification system DS belonging from class II means to say having poor solubility and poor dissolution rate [1] hence the focus of this study was on converting BCS class from II to I by increasing its solubility and

#### **Chapter 2**

## Preparation and *in vitro* Characterisation of Solid Dispersion Floating Tablet by Effervescent Control Release Technique with Improved Floating Capabilities

*Peeush Singhal, Rajneesh Dutt Kaushik and Vijay Jyoti Kumar*

### **Abstract**

In this research, an effort has been done for the development of effervescent controlled release floating tablet (ECRFT) from solid dispersions (SDs) of diclofenac sodium (DS) for upsurge the solubility and dissolution rate. ECRFT of DS was prepared by using SDs of DS and its SDs prepared with PEG as carrier using thermal method (simple fusion). SDs of DS was formulated in many ratios (1:1, 1:2, 1:3 and 1:4). Prepared SDs were optimised for its solubility, % drug content and % dissolution studies. Tablets were formulated by using optimised SDs products and all formulation was evaluated for various parameters. A clear rise in dissolution rate was detected with entirely SD, amid that the optimised SD (SD4) was considered for ECRFT. Among all the tablet formulations, its F3 formulation was better in all the terms of pre-compression and post-compression parameters. It had all the qualities of a good ECRFT, based on this F3 formulation was selected as the best formulation. Data of *in vitro* release were fitted in several kinetics models to explain release mechanism. The F3 formulation shows zero order release. From this study, we can conclude that ECRFT containing SDs of DS can be successfully used for achieving better therapeutic objective.

**Keywords:** solid dispersion, diclofenac sodium, polyethylene glycol, dissolution enhancement, floating tablets

#### **1. Introduction**

Diclofenac sodium (DS) is an effective NSAID with high affinity for both COX-1 and COX-2 receptors and it is one and only maximum frequently recommended drugs in India for the cure of pain, inflammation and joint stiffness caused by arthritis. According to BCS classification system DS belonging from class II means to say having poor solubility and poor dissolution rate [1] hence the focus of this study was on converting BCS class from II to I by increasing its solubility and

dissolution rate of DS Which was taken as model drug [2]. The release rate can be improved by increasing surface area of existing drug by using several techniques but among these methods solid dispersion technique is one of the best techniques for increasing the surface area [3]. Hence, an effort was made to increase the dissolution characteristics using the solid dispersion technique [3, 4]. It has absorption site in upper part of gastro intestinal tract. Gastric retention of DS was very short that is why the bioavailability of drug is 54% which is very low because near about 50% portion of orally given drug misses the absorption window. The pharmacokinetic profile of DS showed that the half-life is about 1.2–2 h and hence there is a requirement of frequent dosing (3–4 tablets daily) [5] but this requirement of frequent dose is very dangerous for patients because due to this frequent dosing fluctuation in plasma drug level in body and need constant monitoring of patient for adjustment of dose regimen. That is why this reason may consequently support faster absorption of drug in stomach with higher concentrations for bioavailability improvement. Therefore in order to improve drug dissolution and reduced dosing frequency, it was attempted to formulate solid dispersion of DS [6, 7] and then develop effervescent controlled release floating tablet [8]. The emphasis of the current research was to increase the release rate and bioavailability of DS through preparing ECRFT (effervescent control release floating tablets) with dual approach [9] using solid dispersion product of DS in order to regulate the drug release and make available security from first pass metabolism.

b. A small amount of compound was placed on a clean surface. The compound

(Macro scientific works).The sample was observed continuously, so that the melting point of the sample was not missed. Slow heating was done for most accurate results. The melting range was recorded which beings when the sample first starts to melt and ends when the sample completely melted.

c. The capillary melting point tube was placed in melting point apparatus

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

Solubility may be defined as the spontaneous interaction of two or more substances to form a homogeneous dispersion [11]. The solubility of diclofenac sodium was studied in various aqueous and non-aqueous solvents. About 10 mg of drug was taken in 10 ml of each solvent at room temperature in screw-capped test tubes and shaken for 30 min in a sonicator. The solubility was checked by U-V spectroscopy in

The organic molecule in solution when exposed to light in the ultra-violet region of the spectrum, absorbed light of particular wavelength depending on the type of

The solution (10 μg) of diclofenac sodium was prepared in simulated gastric fluid pH 1.2 and scanned spectrophotometrically (Systronics, Double beam UV-VIS Spectrophotometer: 2201). The scanning range was in between 200 nm to 400 nm. Standard solution of diclofenac sodium was then scanned and graph plotted. The

OBS: 282–283°C

Freely soluble: methanol Soluble: 0.1 N HCl Insoluble: ether, chloroform and toluene

**Parameter Evaluation** API DICLOFENAC SODIUM Description Crystalline Colour White Odour Odourless Bulk Density 0.56 gm/ml True Density 0.64 gm/ml Carr's Index 14.28% Hausner's Ratio 1.14 Melting Point STD: 280°C

Solubility Sparingly soluble: Water

Partition coefficient 1.25

was put in to open end of capillary tube.

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

electronic transition associated with absorption [12].

*2.1.1.3 Solubility studies*

all cases and reported in **Table 1**.

*2.1.1.4 U.V. spectrophotometer*

*2.1.1.4.1 Diclofenac sodium*

**Table 1.**

**19**

*Preformulation characters of diclofenac sodium.*

#### **2. Methodologies**

#### **2.1 Preformulation studies**

Prior to the development of dosage forms, it is essential that certain fundamental, physical and chemical properties of the drug molecule and other derived properties of the drug powder are determined and should be considered in the formulation in relation to the proposed dosage form and route of administration.

These studies should focus on those physiochemical properties of the drug that could affect drug performance and development of an efficacious dosage form.

A typical preformulation program should begin with the description of the organoleptic qualities of the drug substance. The colour, odour and taste are of immense value in developing an aesthetically acceptable formulation.

#### *2.1.1 Identification and characterisation of diclofenac sodium*

#### *2.1.1.1 Physical appearance*

Drug sample has been noted for its organoleptic properties. The drug is white to slightly yellowish crystalline powder, odour: slight and characteristic [1]. Drug was received as gift sample (15 g) from Kwality Pharmaceuticals Ltd., Amritsar.

#### *2.1.1.2 Melting point determination*

The melting point of compound is the temperature at which it changes from a solid to liquid [10]. This is a physical property often used to identify compounds.

#### *2.1.1.2.1 Procedure*

a. A capillary melting tube was taken.

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*


#### *2.1.1.3 Solubility studies*

dissolution rate of DS Which was taken as model drug [2]. The release rate can be improved by increasing surface area of existing drug by using several techniques but among these methods solid dispersion technique is one of the best techniques for increasing the surface area [3]. Hence, an effort was made to increase the dissolution characteristics using the solid dispersion technique [3, 4]. It has absorption site in upper part of gastro intestinal tract. Gastric retention of DS was very short that is why the bioavailability of drug is 54% which is very low because near about 50% portion of orally given drug misses the absorption window. The pharmacokinetic profile of DS showed that the half-life is about 1.2–2 h and hence there is a requirement of frequent dosing (3–4 tablets daily) [5] but this requirement of frequent dose is very dangerous for patients because due to this frequent dosing fluctuation in plasma drug level in body and need constant monitoring of patient for adjustment of dose regimen. That is why this reason may consequently support faster absorption of drug in stomach with higher concentrations for bioavailability improvement. Therefore in order to improve drug dissolution and reduced dosing frequency, it was attempted to formulate solid dispersion of DS [6, 7] and then develop effervescent controlled release floating tablet [8]. The emphasis of the current research was to increase the release rate and bioavailability of DS through preparing ECRFT (effervescent control release floating tablets) with dual approach [9] using solid dispersion product of DS in order to regulate the drug

release and make available security from first pass metabolism.

Prior to the development of dosage forms, it is essential that certain fundamental, physical and chemical properties of the drug molecule and other derived prop-

Drug sample has been noted for its organoleptic properties. The drug is white to slightly yellowish crystalline powder, odour: slight and characteristic [1]. Drug was received as gift sample (15 g) from Kwality Pharmaceuticals Ltd., Amritsar.

The melting point of compound is the temperature at which it changes from a solid to liquid [10]. This is a physical property often used to identify compounds.

erties of the drug powder are determined and should be considered in the formulation in relation to the proposed dosage form and route of administration. These studies should focus on those physiochemical properties of the drug that could affect drug performance and development of an efficacious dosage form. A typical preformulation program should begin with the description of the organoleptic qualities of the drug substance. The colour, odour and taste are of

immense value in developing an aesthetically acceptable formulation.

*2.1.1 Identification and characterisation of diclofenac sodium*

**2. Methodologies**

*Molecular Pharmacology*

**2.1 Preformulation studies**

*2.1.1.1 Physical appearance*

*2.1.1.2.1 Procedure*

**18**

*2.1.1.2 Melting point determination*

a. A capillary melting tube was taken.

Solubility may be defined as the spontaneous interaction of two or more substances to form a homogeneous dispersion [11]. The solubility of diclofenac sodium was studied in various aqueous and non-aqueous solvents. About 10 mg of drug was taken in 10 ml of each solvent at room temperature in screw-capped test tubes and shaken for 30 min in a sonicator. The solubility was checked by U-V spectroscopy in all cases and reported in **Table 1**.

#### *2.1.1.4 U.V. spectrophotometer*

The organic molecule in solution when exposed to light in the ultra-violet region of the spectrum, absorbed light of particular wavelength depending on the type of electronic transition associated with absorption [12].

#### *2.1.1.4.1 Diclofenac sodium*

The solution (10 μg) of diclofenac sodium was prepared in simulated gastric fluid pH 1.2 and scanned spectrophotometrically (Systronics, Double beam UV-VIS Spectrophotometer: 2201). The scanning range was in between 200 nm to 400 nm. Standard solution of diclofenac sodium was then scanned and graph plotted. The


#### **Table 1.**

*Preformulation characters of diclofenac sodium.*

#### **Figure 1.** *U.V. scan of diclofenac sodium in simulated gastric fluid (PH 1.2).*

determined λmax, 276 nm (**Figure 1**) was similar as reported in the literature (276 nm).

#### *2.1.1.5 I.R. spectrophotometry*

About 1 mg of the sample and 100 mg of the potassium bromide (KBr) was taken in a mortar and triturated [13]. A small amount of triturated sample was taken into a pellet maker and compressed at 10 kg/cm<sup>2</sup> . The pellet was kept onto the sample holder and scanned from 4000 to 400 cm<sup>1</sup> .The I-R spectrum of drug sample was obtained using FTIR-8400 s, shimadzu. Important peaks are reported in **Table 2** and graphically represented in **Figures 2**, **3**. This I-R spectrum was found concordant with the IR of diclofenac sodium reported in the official monograph.

#### *2.1.1.6 Quantitative estimation of drug*

For the present study the spectrophotometric method given in the official books was selected for its sensitivity, specificity, simplicity, reproducibility, rapidity and accuracy [14].

this was served as a standard solution [15, 16]. From this solution 10 ml was taken and diluted to 100 ml using simulated gastric fluid pH 1.2 to get a solution of 50 μg/ ml concentration and this solution was served as the standard solution. In to a series of 10 ml volumetric flasks, aliquots of standard solution (i.e. 0.4, 0.8, 1.2, 1.6, 2.0, 3.0, 4.0, 5.0, 6.0 ml) were added and made up the volume up to 10 ml using simulated gastric fluid pH 1.2. The absorbance of these solutions was measured against reagent blank at 276 nm (**Table 3**). A standard curve between concentration and absorbance was plotted (**Figure 4**). A straight line passing through origin is

The partition coefficient directly influences the permeability of drug through various membranes [17–19]. The study has been designed to determine partition coefficient of drug in 1-octanol and pH 1.2 solutions. The partition coefficient between 1-octanol and Simulated gastric fluid (pH 1.2) was determined by shake flask method. About 10 mg of drug was dissolved in one of the phases, and is shaken with the other partitioning solvent for 30 min, allowed to stand for 5 min and then majority of the lower aqueous phase was run off. The drug concentration in both the

obtained.

**21**

**Figure 3.**

**Figure 2.**

*FTIR spectroscopy of pure diclofenac sodium.*

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

*I-R spectra of pure diclofenac sodium from I.P. 1996.*

*2.1.1.8 Partition coefficient*

#### *2.1.1.7 Preparation of calibration curve of diclofenc sodium in simulated gastric fluid (pH 1.2)*

Accurately weighed 50 mg of drug (diclofenac sodium) was dissolved in 100 ml of simulated gastric fluid pH 1.2 to give a solution of 500 μg/ml concentration and


#### **Table 2.** *Characteristic peaks of diclofenac sodium.*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

#### **Figure 2.**

determined λmax, 276 nm (**Figure 1**) was similar as reported in the literature

About 1 mg of the sample and 100 mg of the potassium bromide (KBr) was taken in a mortar and triturated [13]. A small amount of triturated sample was

sample was obtained using FTIR-8400 s, shimadzu. Important peaks are reported in **Table 2** and graphically represented in **Figures 2**, **3**. This I-R spectrum was found concordant with the IR of diclofenac sodium reported in the official monograph.

For the present study the spectrophotometric method given in the official books was selected for its sensitivity, specificity, simplicity, reproducibility, rapidity and

Accurately weighed 50 mg of drug (diclofenac sodium) was dissolved in 100 ml of simulated gastric fluid pH 1.2 to give a solution of 500 μg/ml concentration and

> 1600–1475 1556.61, 1498.74 C]C(aromatic) Stretching 1320–1210 1305.85 CdO stretching Stretching

> > ring

stretching

*2.1.1.7 Preparation of calibration curve of diclofenc sodium in simulated gastric fluid*

**Observed peaks value cm<sup>1</sup>**

1556 1556.61 Dichlorophenyl

1300–1000 1284.63 CdCOdC

. The pellet was kept onto the

.The I-R spectrum of drug

**Groups Stretching/**

**deformation**

Stretching

Stretching

taken into a pellet maker and compressed at 10 kg/cm<sup>2</sup>

sample holder and scanned from 4000 to 400 cm<sup>1</sup>

*U.V. scan of diclofenac sodium in simulated gastric fluid (PH 1.2).*

(276 nm).

**Figure 1.**

accuracy [14].

*(pH 1.2)*

DICLOFENC SODIUM

**Table 2.**

**20**

**IR spectrum Standard peaks**

*Characteristic peaks of diclofenac sodium.*

**value**

*2.1.1.5 I.R. spectrophotometry*

*Molecular Pharmacology*

*2.1.1.6 Quantitative estimation of drug*

*FTIR spectroscopy of pure diclofenac sodium.*

#### **Figure 3.**

this was served as a standard solution [15, 16]. From this solution 10 ml was taken and diluted to 100 ml using simulated gastric fluid pH 1.2 to get a solution of 50 μg/ ml concentration and this solution was served as the standard solution. In to a series of 10 ml volumetric flasks, aliquots of standard solution (i.e. 0.4, 0.8, 1.2, 1.6, 2.0, 3.0, 4.0, 5.0, 6.0 ml) were added and made up the volume up to 10 ml using simulated gastric fluid pH 1.2. The absorbance of these solutions was measured against reagent blank at 276 nm (**Table 3**). A standard curve between concentration and absorbance was plotted (**Figure 4**). A straight line passing through origin is obtained.

#### *2.1.1.8 Partition coefficient*

The partition coefficient directly influences the permeability of drug through various membranes [17–19]. The study has been designed to determine partition coefficient of drug in 1-octanol and pH 1.2 solutions. The partition coefficient between 1-octanol and Simulated gastric fluid (pH 1.2) was determined by shake flask method. About 10 mg of drug was dissolved in one of the phases, and is shaken with the other partitioning solvent for 30 min, allowed to stand for 5 min and then majority of the lower aqueous phase was run off. The drug concentration in both the

*I-R spectra of pure diclofenac sodium from I.P. 1996.*


The drug was identified by IR spectroscopy and the characteristic peaks obtained (**Figure 2**) compared with standard spectra (**Figure 3**) of pure drug reported in official monograph (IP1996). The IR spectra of drug sample are in agreement with the standard IR spectra of pure diclofenac sodium given in official

**S. No Melting ranges Melting point (mean SD)** 1 288–290°C 289.12 0.21

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

Diclofenac Sodium (Batch no. A5/206), hydroxyl propyl methyl cellulose (HPMC) K100M (Batch no. HP121406 MC) and crosspovidone (Batch no.

YPVPP09319040) were obtained from kwality pharmaceutical pvt ltd Amritsar, as gift samples. Sodium bicarbonate (NaHCO3), citric acid, polyvinyl pyrrolidone (PVP K-30), magnesium stearate, lactose and Isopropyl alcohol were purchased from local suppliers. Marketed product, "Voveran SR100 or Voveran 50", (Manufactured by Ranbaxy, India; Batch no.131003 AU or 320,028), used for

Physical mixtures were prepared by mixing the appropriate amounts of the drug

Melt method was used to prepare solid dispersions of diclofenac sodium with PEG 6000 containing different weight ratio (1:1, 1:2, 1:3, 1:4, and 1:5) (**Table 5**). Diclofenac sodium and PEG 6000 were weighed according to their weighed ratios. PEG 6000 was melted at 60°C. In this melted PEG 6000, diclofenac sodium was added. It was mixed well and flashed cooled on an ice bath and then stored overnight in desiccators. The prepared solid dispersion was then grounded by using a mortar and pestle, sieved through a mesh no. 40 and stored over a fused calcium

and carrier (PEG 6000) in the different weight ratios of 1:1, 1:2, 1:3 and 1:4 in mortar [3, 4, 6, 7]. The resulting mixtures were sieved through sieve no. 80, collected and stored in closed container away from light and humidity until use.

In the present study, a reported U-V spectrophometric method was used for the estimation of diclofenac sodium. The calibration curve of diclofenac sodium was prepared in simulated gastric fluid pH 1.2. The data was regressed to obtain straight line. The correlation coefficient was found to be 0.996 in simulated gastric fluid pH 1.2 indicating good linearity. The calibration curve was found to obey Beer-

monograph [1]. Important peaks are reported in **Table 2**.

Lamberts Law in the concentration range studied (0–20 μg/ml).

comparative studies, was purchased from the local retail pharmacy.

**4.1 Preparation of physical mixtures (PM)**

**4.2 Preparation of solid dispersion**

chloride in a desiccators' for further use.

**3. Materials**

**Table 4.**

*Melting point result of diclofenac sodium.*

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

**4. Methods**

**23**

#### **Table 3.**

*Data for calibration curve of diclofenc sodium in simulated gastric fluid pH 1.2 at 276 nm (n = 3).*

**Figure 4.** *Calibration curve of diclofenac sodium in pH 1.2 at 276 nm.*

aqueous and 1-octanol phases was determined spectrophotometrically at 276 nm and calculated the partition coefficient. The partition coefficient was found to be 1.25.

Partition Coefficient ¼ Conc*:*of drug in oil phase*=*Conc*:*of drug in aqueous phase (1)

#### *2.1.2 Result and discussion*

Samples of diclofenac sodium obtained as a gift sample from kwality pharmaceuticals pvt. Ltd., Amritsar was identified and characterised as per the identification test given in official monograph. Physical appearance and melting point of the drug sample under investigation was found to be same as that of the official reports. The results are given in **Table 4**. The solubility of diclofenac sodium was determined in aqueous and non-aqueous solvents. Diclofenac sodium was found to be soluble in 0.1NHCl and ethanol; sparingly soluble in water, practically insoluble in ether, chloroform and toluene. Partition coefficient of the drug was found to be 1.25.The results are given in **Table 1**.

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*


#### **Table 4.**

*Melting point result of diclofenac sodium.*

The drug was identified by IR spectroscopy and the characteristic peaks obtained (**Figure 2**) compared with standard spectra (**Figure 3**) of pure drug reported in official monograph (IP1996). The IR spectra of drug sample are in agreement with the standard IR spectra of pure diclofenac sodium given in official monograph [1]. Important peaks are reported in **Table 2**.

In the present study, a reported U-V spectrophometric method was used for the estimation of diclofenac sodium. The calibration curve of diclofenac sodium was prepared in simulated gastric fluid pH 1.2. The data was regressed to obtain straight line. The correlation coefficient was found to be 0.996 in simulated gastric fluid pH 1.2 indicating good linearity. The calibration curve was found to obey Beer-Lamberts Law in the concentration range studied (0–20 μg/ml).

#### **3. Materials**

Diclofenac Sodium (Batch no. A5/206), hydroxyl propyl methyl cellulose (HPMC) K100M (Batch no. HP121406 MC) and crosspovidone (Batch no. YPVPP09319040) were obtained from kwality pharmaceutical pvt ltd Amritsar, as gift samples. Sodium bicarbonate (NaHCO3), citric acid, polyvinyl pyrrolidone (PVP K-30), magnesium stearate, lactose and Isopropyl alcohol were purchased from local suppliers. Marketed product, "Voveran SR100 or Voveran 50", (Manufactured by Ranbaxy, India; Batch no.131003 AU or 320,028), used for comparative studies, was purchased from the local retail pharmacy.

#### **4. Methods**

aqueous and 1-octanol phases was determined spectrophotometrically at 276 nm and calculated the partition coefficient. The partition coefficient was found

**S. No Concentration (μg/ml) Abs(λmax-276 nm) (mean** � **SD)**

*Data for calibration curve of diclofenc sodium in simulated gastric fluid pH 1.2 at 276 nm (n = 3).*

 0 0.000 � 0.00 2 0.068 � 0.002 4 0.128 � 0.005 6 0.190 � 0.0015 8 0.246 � 0.0021 10 0.315 � 0.0022 12 0.329 � 0.004 14 0.401 � 0.001 16 0.445 � 0.0032 18 0.522 � 0.0051 20 0.589 � 0.0059

Partition Coefficient ¼ Conc*:*of drug in oil phase*=*Conc*:*of drug in aqueous phase

Samples of diclofenac sodium obtained as a gift sample from kwality pharmaceuticals pvt. Ltd., Amritsar was identified and characterised as per the identification test given in official monograph. Physical appearance and melting point of the drug sample under investigation was found to be same as that of the official reports. The results are given in **Table 4**. The solubility of diclofenac sodium was determined in aqueous and non-aqueous solvents. Diclofenac sodium was found to be soluble in 0.1NHCl and ethanol; sparingly soluble in water, practically insoluble in ether, chloroform and toluene. Partition coefficient of the drug was found to be

(1)

to be 1.25.

**22**

**Figure 4.**

**Table 3.**

*Molecular Pharmacology*

*2.1.2 Result and discussion*

1.25.The results are given in **Table 1**.

*Calibration curve of diclofenac sodium in pH 1.2 at 276 nm.*

#### **4.1 Preparation of physical mixtures (PM)**

Physical mixtures were prepared by mixing the appropriate amounts of the drug and carrier (PEG 6000) in the different weight ratios of 1:1, 1:2, 1:3 and 1:4 in mortar [3, 4, 6, 7]. The resulting mixtures were sieved through sieve no. 80, collected and stored in closed container away from light and humidity until use.

#### **4.2 Preparation of solid dispersion**

Melt method was used to prepare solid dispersions of diclofenac sodium with PEG 6000 containing different weight ratio (1:1, 1:2, 1:3, 1:4, and 1:5) (**Table 5**). Diclofenac sodium and PEG 6000 were weighed according to their weighed ratios. PEG 6000 was melted at 60°C. In this melted PEG 6000, diclofenac sodium was added. It was mixed well and flashed cooled on an ice bath and then stored overnight in desiccators. The prepared solid dispersion was then grounded by using a mortar and pestle, sieved through a mesh no. 40 and stored over a fused calcium chloride in a desiccators' for further use.


conical flasks which were then put in orbital shaker at 37°C and at 100 rpm for 72 h. Absorbance of resulting solution was measured on UV/Visible spectrophotometer

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

Shake flask method same as that for saturation solubility [20] was used with

SDs equivalent to 50 mg of diclofenac sodium were weighed accurately and dissolved in 50 ml of ethanol by using mechanical shaker for 30 min. The solutions were filtered using whatman filter paper and drug content was determined by measuring absorbance at 276 nm by UV/visible spectrophotometer [6, 20]. From above evaluation tests, optimised formulation was confirmed (SD4 in **Table 6**)

*In vitro* dissolution studies of prepared SDs were carried out in 900 ml of 0.1 N HCl as a medium using USP type 2 test apparatus with three replicates. The paddle rotation speed was 75 rpm and a temperature of 37°C 0.5 was maintained. In all experiments, 5 ml of dissolution sample was withdrawn at 5 min interval, filtered using a 0.45-mm Whatman filter, and replaced with an equal volume of fresh medium to maintain a constant total volume. Samples were analysed on UV/Visible

IR study was carried out to check the compatibility between the selected Polymers, with the drug. When the spectra were compared it was found that there was no shifting of functional peaks and no overlapping of characteristic peaks and also there was no appearance of new peaks. **Figure 5** shows the IR spectra of various samples. No significant change in the IR spectra of diclofenac sodium complexes was obtained, except for the broadening of the peaks. The broadening of peaks may be probably due to the restriction of bending and stretching vibrations of the

Pure DS 0.3886 0.0044 6.020 0.038 — PM1 (1:1) 0.4481 0.0045 8.328 0.069 82.75 1.54 PM2 (1:2) 0.4603 0.0073 9.765 0.0073 86.68 1.27 PM3 (1:3) 0.5168 0.0034 10.278 0.086 88.01 0.94 **PM4 (1:4) 0.5947 0.0046 11.265 0.101 90.92 1.44** SD1 (1:1) 1.1802 0.0136 11.984 0.064 93.87 1.89 SD2 (1:2) 1.2612 0.0097 12.735 0.028 94.50 2.11 SD3 (1:3) 1.4894 0.0036 13.324 0.071 95.16 1.34 **SD4 (1:4) 1.9261 0.0154 14.291 0.144 96.72 1.53**

*Saturation solubility, pH dependent solubility and percent drug content studies of pure DS, SDs and PMs.*

**pH dependent solubility in 0.1 N HCl (mg/ml)**

**Percent drug content (in 50 mg)**

(UV-1800 Shimadzu, Japan) at 276 nm.

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

0.1 N HCl.

*4.3.4 Percent drug content*

*4.3.5* In vitro *dissolution studies*

spectrophotometer at 276 nm**.**

**4.4 Results and discussions**

**Formulation code**

**Table 6.**

**25**

*4.3.3 Determination of pH dependent solubility*

which was then subjected to *in vitro* dissolution studies.

**Saturation solubility in 0.1 N HCl (mg/ml)**

**Table 5.**

*Composition of solid dispersion and there assign batch code.*

#### **4.3 Characterisation of solid dispersion/ physical mixtures of diclofenac sodium with PEG-6000SDs**

#### *4.3.1 FTIR spectroscopy*

FTIR spectra of drug, PEG 6000 and solid dispersion of DS were obtained. About 1 mg of sample was mixed thoroughly with 100 mg potassium bromide IR powder and compacted under vacuum at a pressure of about 12 psi for 3 min. The resultant disc was mounted in a suitable holder in perkin elmer USA spectrum 65 IR spectrophotometer and the IR spectrum was recorded from 4000 to 400 cm<sup>1</sup> in a scan time of 12 min [20]. The resultant spectra were compared for any spectral changes. **Figure 5** shows the FTIR spectra of the (i) drug, (ii) carrier and (iii) Surface solid dispersion. There was no significant change in the spectrum of solid dispersions, as incorporation of diclofenac into the carrier (PEG6000) did not modify the position of its functional groups.

#### *4.3.2 Determination of saturation solubility*

Saturation solubility was determined by using shake flask method [20]. Excess quantities of pure DS, prepared SDs and PMs were added in 25 ml distilled water in

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

conical flasks which were then put in orbital shaker at 37°C and at 100 rpm for 72 h. Absorbance of resulting solution was measured on UV/Visible spectrophotometer (UV-1800 Shimadzu, Japan) at 276 nm.

#### *4.3.3 Determination of pH dependent solubility*

Shake flask method same as that for saturation solubility [20] was used with 0.1 N HCl.

#### *4.3.4 Percent drug content*

**4.3 Characterisation of solid dispersion/ physical mixtures of diclofenac**

**S. no. Ratio (diclofenac sodium:PEG6000) Batch code** 1 1:1 SD1 2 1:2 SD2 3 1:3 SD3 4 1:4 SD4 5 1:5 SD5

FTIR spectra of drug, PEG 6000 and solid dispersion of DS were obtained. About 1 mg of sample was mixed thoroughly with 100 mg potassium bromide IR powder and compacted under vacuum at a pressure of about 12 psi for 3 min. The resultant disc was mounted in a suitable holder in perkin elmer USA spectrum 65 IR spectrophotometer and the IR spectrum was recorded from 4000 to 400 cm<sup>1</sup> in a scan time of 12 min [20]. The resultant spectra were compared for any spectral changes. **Figure 5** shows the FTIR spectra of the (i) drug, (ii) carrier and (iii) Surface solid dispersion. There was no significant change in the spectrum of solid dispersions, as incorporation of diclofenac into the carrier (PEG6000) did not

Saturation solubility was determined by using shake flask method [20]. Excess quantities of pure DS, prepared SDs and PMs were added in 25 ml distilled water in

*Comparative FTIR spectra of diclofenac, PEG6000, & solid dispersion of diclofenac with PEG6000.*

**sodium with PEG-6000SDs**

*Composition of solid dispersion and there assign batch code.*

modify the position of its functional groups.

*4.3.2 Determination of saturation solubility*

*4.3.1 FTIR spectroscopy*

*Molecular Pharmacology*

**Table 5.**

**Figure 5.**

**24**

SDs equivalent to 50 mg of diclofenac sodium were weighed accurately and dissolved in 50 ml of ethanol by using mechanical shaker for 30 min. The solutions were filtered using whatman filter paper and drug content was determined by measuring absorbance at 276 nm by UV/visible spectrophotometer [6, 20]. From above evaluation tests, optimised formulation was confirmed (SD4 in **Table 6**) which was then subjected to *in vitro* dissolution studies.

#### *4.3.5* In vitro *dissolution studies*

*In vitro* dissolution studies of prepared SDs were carried out in 900 ml of 0.1 N HCl as a medium using USP type 2 test apparatus with three replicates. The paddle rotation speed was 75 rpm and a temperature of 37°C 0.5 was maintained. In all experiments, 5 ml of dissolution sample was withdrawn at 5 min interval, filtered using a 0.45-mm Whatman filter, and replaced with an equal volume of fresh medium to maintain a constant total volume. Samples were analysed on UV/Visible spectrophotometer at 276 nm**.**

#### **4.4 Results and discussions**

IR study was carried out to check the compatibility between the selected Polymers, with the drug. When the spectra were compared it was found that there was no shifting of functional peaks and no overlapping of characteristic peaks and also there was no appearance of new peaks. **Figure 5** shows the IR spectra of various samples. No significant change in the IR spectra of diclofenac sodium complexes was obtained, except for the broadening of the peaks. The broadening of peaks may be probably due to the restriction of bending and stretching vibrations of the


#### **Table 6.**

*Saturation solubility, pH dependent solubility and percent drug content studies of pure DS, SDs and PMs.*

molecule. Various SDs of DS were prepared using PEG-6000, as carriers by thermal method (Simple fusion) technique to increase the solubility as well as dissolution of poorly aqueous soluble drug DS. The prepared SDs and PMs of DS were evaluated for saturation solubility, pH dependent solubility; percent drug content and *in vitro* dissolution studies. The saturation solubility and pH dependent solubility of pure DS, various prepared SDs and PMs of DS in 0.1 N HCl were measured and the results are given in **Table 6**. All PMs showed higher saturation solubility as compared with pure DS. Again, SDs of DS showed higher saturation solubility than their respective PMs of DS and carrier. This might be attributable to an improvement of wetting of drug particles and localised solubilisation by the hydrophilic polymeric carriers.

**4.5 Preparation of floating gastro retentive tablets**

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

ated for their flow and compressibility characteristic**.**

**4.6 Characterisation of granules**

PVP K-30 5% PVP IN IPA

**Table 8.**

**27**

*4.6.1 Drug-polymer interaction studies*

Various ratios of solid dispersions of diclofenac sodium with PEG-6000 were evaluated for percent drug content and out of them the best ratio was selected for preparation of floating tablet of diclofenac sodium. Tablets were prepared by conventional wet granulation method using HPMC K4M, HPMC K15M as a release retardant, carbopol as a swelling agents and NaHCO3 as gas generating agent. Citric acid was also incorporated in the formulation to provide sufficiently acidic medium for NaHCO3 to react and maintain buoyancy. The composition of various formulations is given in **Table 8**. All ingredients (except gas generating agents and magnesium stearate) were passed through sieve no. 60 and mixed in a polybag for 10 min and granulated using PVP K30 (in isopropyl alcohol). The wet mass was passed through sieve number 14 and dried in hot air oven at 50°C for 1.5 h. Dried granules were mixed with magnesium stearate as lubricant, talc as glidant and compressed using 16-station rotary tablet press (Rimek Minipress-I, India) using 13 mm flat punch in order to obtain controlled release floating gastro retentive tablets

containing 50 mg of diclofenac sodium. Prior to compression, granules were evalu-

To study the interaction between drug and polymer, interaction study were performed, drug polymer study were carried out according to the following procedure. Drug and polymer were mixed in 1:1 ratio and put into the glass vials. The glass vials were sealed and placed in the stability chamber at 40°C and 75% RH for 21 days. The sample was analysed for colour change, liquification and bad odours after 7, 15 and 21 days. The IR spectra were taken after 21 days and analysed for any shift in major peaks. No shift was observed in the IR spectrum and no additional

SD4 (solid dispersion of diclofenac sodium) 250 250 250 250 250 250 HPMC K4 70 — 93 — 105 — HPMC K15M — 70 — 93 — 105 Carbopol 934P 70 70 47 47 105 105 Sodium bicarbonate 45 45 45 45 65 65 Citric acid 30 30 30 30 40 40 Avicel PH 102 50 50 50 50 50 50 Magnesium stearate 555555

Total weight 520 520 520 520 620 620

*Composition of different formulations of diclofenac sodium floating tablets.*

**F1 F2 F3 F4 F5 F6**

peak observed indicating no interaction between drug and polymer.

**Ingredient (mg) Formulation code**

Based on the saturation solubility, pH dependent solubility in 0.1 N HCl and drug content among the 8 formulations, PM4 and SD4 were selected to carry out *in vitro* dissolution study and were compared with that of pure DS. The *in vitro* dissolution study of the pure DS, SD4 and PM4 using PEG-6000 as carrier was carried out in 0.1 N HCl at 37°C 1°C for 60 min and it was examined by plotting % drug dissolved against a function of time (**Figure 6**). SD4 and PM4 showed improved dissolution of DS over that of pure DS. Pure DS alone yields the slowest dissolution with only 35.65% drug and the dissolution of PM4 (70.76%) was found to be significantly faster when compared with pure DS. SD4 showed the fastest dissolution (92.99%) than PM4 and pure DS. This observation (**Table 7**) indicated that the increased dissolution of DS from SD4 due to presence of drug in amorphous state as compared PM4 and pure DS. As the proportion of PEG-6000 increased, dissolution rates have also been increased. The improvement of dissolution may be due to its hydrophilic nature of the carrier. Thus it can be concluded that the solubility of the poorly soluble drug, DS can be improved markedly by using solid dispersion technique and the carrier, PEG-6000 has increased the dissolution of the drug.

**Figure 6.** *Dissolution of the pure DS, SD4 and PM4.*


**Table 7.** *Dissolution of the pure DS, SD4 and PM4.*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

#### **4.5 Preparation of floating gastro retentive tablets**

molecule. Various SDs of DS were prepared using PEG-6000, as carriers by thermal method (Simple fusion) technique to increase the solubility as well as dissolution of poorly aqueous soluble drug DS. The prepared SDs and PMs of DS were evaluated for saturation solubility, pH dependent solubility; percent drug content and *in vitro* dissolution studies. The saturation solubility and pH dependent solubility of pure DS, various prepared SDs and PMs of DS in 0.1 N HCl were measured and the results are given in **Table 6**. All PMs showed higher saturation solubility as compared with pure DS. Again, SDs of DS showed higher saturation solubility than their respective PMs of DS and carrier. This might be attributable to an improvement of wetting of drug particles and localised solubilisation by the hydrophilic polymeric

Based on the saturation solubility, pH dependent solubility in 0.1 N HCl and drug content among the 8 formulations, PM4 and SD4 were selected to carry out *in vitro* dissolution study and were compared with that of pure DS. The *in vitro* dissolution study of the pure DS, SD4 and PM4 using PEG-6000 as carrier was carried out in 0.1 N HCl at 37°C 1°C for 60 min and it was examined by plotting % drug dissolved against a function of time (**Figure 6**). SD4 and PM4 showed improved dissolution of DS over that of pure DS. Pure DS alone yields the slowest dissolution with only 35.65% drug and the dissolution of PM4 (70.76%) was found to be significantly faster when compared with pure DS. SD4 showed the fastest dissolution (92.99%) than PM4 and pure DS. This observation (**Table 7**) indicated that the increased dissolution of DS from SD4 due to presence of drug in amorphous state as compared PM4 and pure DS. As the proportion of PEG-6000 increased, dissolution rates have also been increased. The improvement of dissolution may be due to its hydrophilic nature of the carrier. Thus it can be concluded that the solubility of the poorly soluble drug, DS can be improved markedly by using solid dispersion technique and the carrier, PEG-6000 has increased the dissolution

**Percentage of diclofenac sodium dissolved from**

*Time in min/***formulation code 10 15 30 45 60 Pure DS** 3.37 8.50 14.35 18.85 35.65 **PM4** 6.67 21.23 39.44 59.41 70.76 **SD4 9.73 30.51 52.25 74.00 92.99**

carriers.

*Molecular Pharmacology*

of the drug.

**Figure 6.**

**Table 7.**

**26**

*Dissolution of the pure DS, SD4 and PM4.*

*Dissolution of the pure DS, SD4 and PM4.*

Various ratios of solid dispersions of diclofenac sodium with PEG-6000 were evaluated for percent drug content and out of them the best ratio was selected for preparation of floating tablet of diclofenac sodium. Tablets were prepared by conventional wet granulation method using HPMC K4M, HPMC K15M as a release retardant, carbopol as a swelling agents and NaHCO3 as gas generating agent. Citric acid was also incorporated in the formulation to provide sufficiently acidic medium for NaHCO3 to react and maintain buoyancy. The composition of various formulations is given in **Table 8**. All ingredients (except gas generating agents and magnesium stearate) were passed through sieve no. 60 and mixed in a polybag for 10 min and granulated using PVP K30 (in isopropyl alcohol). The wet mass was passed through sieve number 14 and dried in hot air oven at 50°C for 1.5 h. Dried granules were mixed with magnesium stearate as lubricant, talc as glidant and compressed using 16-station rotary tablet press (Rimek Minipress-I, India) using 13 mm flat punch in order to obtain controlled release floating gastro retentive tablets containing 50 mg of diclofenac sodium. Prior to compression, granules were evaluated for their flow and compressibility characteristic**.**

#### **4.6 Characterisation of granules**

#### *4.6.1 Drug-polymer interaction studies*

To study the interaction between drug and polymer, interaction study were performed, drug polymer study were carried out according to the following procedure. Drug and polymer were mixed in 1:1 ratio and put into the glass vials. The glass vials were sealed and placed in the stability chamber at 40°C and 75% RH for 21 days. The sample was analysed for colour change, liquification and bad odours after 7, 15 and 21 days. The IR spectra were taken after 21 days and analysed for any shift in major peaks. No shift was observed in the IR spectrum and no additional peak observed indicating no interaction between drug and polymer.


#### **Table 8.**

*Composition of different formulations of diclofenac sodium floating tablets.*

#### *4.6.2 I-R spectrum of pure drug*

About 1 mg of the sample and 100 mg of the potassium bromide (KBr) was taken in a mortar and triturated. A small amount of triturated sample was taken into a pellet maker and compressed at 10 kg/cm2. The pellet was kept onto the sample holder and scanned from 4000 to 400 cm�<sup>1</sup> .The I-R spectrum of drug sample was obtained using FTIR-8400 s, Shimadzu (**Figure 2**).

*4.6.3 I-R spectra for diclofenac sodium with HPMC K4M + HPMC K15M and carbopol 934P*

Sample mixture of diclofenac sodium with HPMC K4M + HPMC K15M and carbopol 934P were prepared in KBr discs (1 mg sample in 100 mg KBr). A small amount of triturated sample was taken into a pellet maker and was compressed at 10 kg/cm<sup>2</sup> . The scanning range was 4000–400 cm�<sup>1</sup> , and the resolution was 4 cm�<sup>1</sup> (**Figures 7** and **8**).

#### **4.7 Evaluation of granules properties**

#### *4.7.1 Angle of repose*

The angle of repose of was determined by the funnel method. The accurately weighed granules were taken in a funnel. The height of the funnel was adjusted in such a way that the tip of the funnel just touched the apex of the heap of the granules. The granules were allowed to flow through the funnel freely onto the surface [9, 21]. The diameter of the powder cone was measured and angle of repose was calculated using the following equation.

$$\theta = \tan^{-1}(\mathbf{h}/\mathbf{r})\tag{2}$$

agglomerates formed, was introduced into a 10 ml measuring cylinder [9]. After the initial volume was observed, the cylinder was allowed to fall under its own weight on to a hard surface from the height of 2.5 cm at 2 s intervals. The tapping was

BD ¼ Weight of the Powder*=*Volume of the packing*:* (3)

Tap density � 100 (5)

TBD ¼ Weight of the powder*=*Tapped volume of the packing*:* (4)

The flow property was also determined by measuring the compressibility index. It is an important measure that can be obtained from the BD and TBD. According to the theory, the less compressible materials are more flowable. A material having values of less than 20–30% is defined as the free flowing material [9, 21]. Based on the BD and TBD, the percentage compressibility of the bulk drug was determined

Compressibility Index <sup>¼</sup> Tap density–Bulk density

*In vitro* buoyancy studies were performed for all the formulations as per the method described by Rosa *et al* [22]. The randomly selected tablets from each formulation were kept in a 100 ml beaker containing simulated gastric fluid, pH 1.2 as per USP. The time taken for the tablet to rise to the surface and float was taken as floating lag time (FLT). The duration of time the dosage form constantly remained on the surface of medium was determined as the total floating time (TFT) [9, 23–25].

The formulated tablets were assessed for its general appearance.

continued until no further change in volume was noted.

*4.7.3 Compressibility index/carr's index*

*FTIR of diclofenac sodium + carbopol 934P.*

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

**Figure 8.**

by using the following formula.

**4.8 Evaluation of floating tablets**

*4.8.2 General characteristic*

**29**

*4.8.1* In vitro *buoyancy determination studies*

BD and TBD were calculated using the following formulas**.**

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

where, h and r are the height and radius of the powder pile, respectively.

#### *4.7.2 Bulk density*

Both bulk density (BD) and tapped bulk density (TBD) were determined. A quantity of 2 g of powder from each formula, previously lightly shaken to break any

**Figure 7.** *FTIR of diclofenac sodium + HPMC K4M + HPMC K15M.*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

**Figure 8.** *FTIR of diclofenac sodium + carbopol 934P.*

*4.6.2 I-R spectrum of pure drug*

*Molecular Pharmacology*

*934P*

(**Figures 7** and **8**).

*4.7.1 Angle of repose*

*4.7.2 Bulk density*

**Figure 7.**

**28**

10 kg/cm<sup>2</sup>

holder and scanned from 4000 to 400 cm�<sup>1</sup>

**4.7 Evaluation of granules properties**

was calculated using the following equation.

*FTIR of diclofenac sodium + HPMC K4M + HPMC K15M.*

obtained using FTIR-8400 s, Shimadzu (**Figure 2**).

. The scanning range was 4000–400 cm�<sup>1</sup>

About 1 mg of the sample and 100 mg of the potassium bromide (KBr) was taken in a mortar and triturated. A small amount of triturated sample was taken into a pellet maker and compressed at 10 kg/cm2. The pellet was kept onto the sample

*4.6.3 I-R spectra for diclofenac sodium with HPMC K4M + HPMC K15M and carbopol*

Sample mixture of diclofenac sodium with HPMC K4M + HPMC K15M and carbopol 934P were prepared in KBr discs (1 mg sample in 100 mg KBr). A small amount of triturated sample was taken into a pellet maker and was compressed at

The angle of repose of was determined by the funnel method. The accurately weighed granules were taken in a funnel. The height of the funnel was adjusted in such a way that the tip of the funnel just touched the apex of the heap of the granules. The granules were allowed to flow through the funnel freely onto the surface [9, 21]. The diameter of the powder cone was measured and angle of repose

<sup>θ</sup> <sup>¼</sup> tan –<sup>1</sup>

where, h and r are the height and radius of the powder pile, respectively.

Both bulk density (BD) and tapped bulk density (TBD) were determined. A quantity of 2 g of powder from each formula, previously lightly shaken to break any

.The I-R spectrum of drug sample was

, and the resolution was 4 cm�<sup>1</sup>

ð Þ h*=*r (2)

agglomerates formed, was introduced into a 10 ml measuring cylinder [9]. After the initial volume was observed, the cylinder was allowed to fall under its own weight on to a hard surface from the height of 2.5 cm at 2 s intervals. The tapping was continued until no further change in volume was noted.

BD and TBD were calculated using the following formulas**.**

BD ¼ Weight of the Powder*=*Volume of the packing*:* (3)

#### TBD ¼ Weight of the powder*=*Tapped volume of the packing*:* (4)

#### *4.7.3 Compressibility index/carr's index*

The flow property was also determined by measuring the compressibility index. It is an important measure that can be obtained from the BD and TBD. According to the theory, the less compressible materials are more flowable. A material having values of less than 20–30% is defined as the free flowing material [9, 21]. Based on the BD and TBD, the percentage compressibility of the bulk drug was determined by using the following formula.

$$\text{Compressibility Index} = \frac{\text{Tap density-Bulk density}}{\text{Tap density}} \times 100\tag{5}$$

#### **4.8 Evaluation of floating tablets**

#### *4.8.1* In vitro *buoyancy determination studies*

*In vitro* buoyancy studies were performed for all the formulations as per the method described by Rosa *et al* [22]. The randomly selected tablets from each formulation were kept in a 100 ml beaker containing simulated gastric fluid, pH 1.2 as per USP. The time taken for the tablet to rise to the surface and float was taken as floating lag time (FLT). The duration of time the dosage form constantly remained on the surface of medium was determined as the total floating time (TFT) [9, 23–25].

#### *4.8.2 General characteristic*

The formulated tablets were assessed for its general appearance.

#### *4.8.2.1 Thickness and diameter*

Thickness and diameter of tablets was determined using vernier calliper. Three tablets from each batch were used, and average values were calculated.

media, maintained at 37°C � 0.5°C and agitated at 50 rpm. Periodically 5 ml samples were withdrawn and filtered through whatman filter paper and samples were replaced by its equivalent volume of dissolution media. The absorbance of DS was measured UV/Visible spectrophotometrically at 276 nm. The percentage cumulative drug release was calculated and amount of CP released from tablets was deter-

The result of *in vitro* dissolution studies of tablet were fitted with various kinetics models, like zero order (% cumulative drug release vs. time), first order (log % drug remaining vs. time), Higuchi's model (% cumulative drug release vs. square root of time) but these models failed to explain drug release mechanism due to swelling (upon hydration) along with gradual erosion of the matrix. Therefore the dissolution data were also fitted to well-known Korsmeyer and Peppas semi-

Where, M∞ is the amount of drug release after infinite time; k is the release rate constant which considers structural and geometric characteristics of the tablets; and n is the diffusional exponent; indicative of the mechanism of drug release. **Table 9** shows an analysis of diffusional release mechanism obtained by various value of n. The criteria for selecting the most appropriate model were chosen on the basis of goodness of fit test. The data were processed for regression analysis using MS

The biodegradability studies were carried out using USP rotating basket apparatus. A tablet (50 mg) were introduced into the baskets which were rotated at 50 rpm in 900 ml of different pH buffer solution (5.0, 6.8, 8.0) maintained at 37°C � 0.5°C.

To assess the drug and formulation stability, stability studies were done according to ICH guidelines. The promising formulation F4 was tested for accelerated testing for a period of 2 months at 40°C � 2°C/ 75% RH �5% for their drug

**S. No n value Mechanism** 1 n ≤ 0.5 Quasi-fickian diffusion 2 0.5 Fickian diffusion

3 0.5 ≥ n ≤ 1.0 Anomalous (non-fickian) diffusion 4 n ≥ 1.0 Non-fickian super case 11 5 1 Non-fickian case 11

**log** ð Þ¼ Mt*=*M∞ logk þ n logt (8)

mined. The floating tablet is wound with the helical wire sinker.

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

empirical model to ascertain the mechanism of drug release.

*4.8.2.8 Kinetic of drug release*

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

EXCEL statistical function.

*4.8.2.10 Stability studies*

**Table 9.**

**31**

content and other parameters.

*Release mechanism with variation of n values.*

*4.8.2.9 Biodegradability studies of floating tablet*

#### *4.8.2.2 Weight variation*

Formulated floating tablets were tested for weight uniformity, 20 tablets were weighed collectively and individually. From the collective weight, average weight was calculated. Each tablet weight was then compared with average weight to ascertain whether it is within permissible limits or not.

#### *4.8.2.3 Friability test*

The Roche friability test apparatus was used to determine the friability of the tablets. 20 pre-weighed tablets were placed in the apparatus, which was given 100 revolutions, after which the tablets were reweighed. The percentage friability was calculated.

$$\text{\textbulletF} = \{\mathbf{1} - (\text{loss in weight} / \text{initial weight})\} \times \mathbf{100} \tag{6}$$

#### *4.8.2.4 Hardness test*

Hardness of the tablet was determined using the monsanto hardness tester. The lower plunger was placed in contact with the tablet and a zero reading was taken. The plunger was then forced against a spring by tuning a threaded bolt until the tablet fractured. As the spring was compressed a pointer rides along a gauge in the barrel to indicate the force.

#### *4.8.2.5 Percent drug content*

Ten tablets were weighed and powdered. An amount of the powder equivalent to 50 mg of diclofenac sodium was dissolved in 100 ml of 0.1 N HCl, filtered, diluted suitably and analysed for drug content at 276 nm using UV/Visible spectrophotometer.

#### *4.8.2.6 Determination of percent swelling index (percentage water uptake)*

The swelling properties of floating tablet containing drug were determined by placing the tablet matrices in the dissolution test apparatus, in 900 ml of distilled water at 37°C � 0.5°C paddle rotated at 50 rpm. The tablets were removed periodically from dissolution medium. After draining free from water by blotting paper, these were measured for weight gain. Swelling characteristics were expressed in terms of percentage water uptake (%WU) according to the equation shows relationship between swelling index and time**.**

$$\text{WU\%} = \frac{\text{Weight of swollen tablet} - \text{Initial weight of the tablet}}{\text{Initial weight of the tablet}} \times 100 \qquad (7)$$

#### *4.8.2.7 Dissolution studies using USP type II apparatus with wire sinker*

Dissolution test was carried out using USP type II apparatus with wire sinker. The drug release study was carried out for 12 hr. in 900 ml of 0.1 N HCl dissolution media, maintained at 37°C � 0.5°C and agitated at 50 rpm. Periodically 5 ml samples were withdrawn and filtered through whatman filter paper and samples were replaced by its equivalent volume of dissolution media. The absorbance of DS was measured UV/Visible spectrophotometrically at 276 nm. The percentage cumulative drug release was calculated and amount of CP released from tablets was determined. The floating tablet is wound with the helical wire sinker.

#### *4.8.2.8 Kinetic of drug release*

*4.8.2.1 Thickness and diameter*

*4.8.2.2 Weight variation*

*Molecular Pharmacology*

*4.8.2.3 Friability test*

*4.8.2.4 Hardness test*

barrel to indicate the force.

*4.8.2.5 Percent drug content*

tionship between swelling index and time**.**

spectrophotometer.

**30**

calculated.

Thickness and diameter of tablets was determined using vernier calliper. Three

Formulated floating tablets were tested for weight uniformity, 20 tablets were weighed collectively and individually. From the collective weight, average weight was calculated. Each tablet weight was then compared with average weight to

The Roche friability test apparatus was used to determine the friability of the tablets. 20 pre-weighed tablets were placed in the apparatus, which was given 100 revolutions, after which the tablets were reweighed. The percentage friability was

Hardness of the tablet was determined using the monsanto hardness tester. The lower plunger was placed in contact with the tablet and a zero reading was taken. The plunger was then forced against a spring by tuning a threaded bolt until the tablet fractured. As the spring was compressed a pointer rides along a gauge in the

Ten tablets were weighed and powdered. An amount of the powder equivalent

The swelling properties of floating tablet containing drug were determined by placing the tablet matrices in the dissolution test apparatus, in 900 ml of distilled water at 37°C � 0.5°C paddle rotated at 50 rpm. The tablets were removed periodically from dissolution medium. After draining free from water by blotting paper, these were measured for weight gain. Swelling characteristics were expressed in terms of percentage water uptake (%WU) according to the equation shows rela-

Dissolution test was carried out using USP type II apparatus with wire sinker. The drug release study was carried out for 12 hr. in 900 ml of 0.1 N HCl dissolution

Initial weight of the tablet � 100 (7)

to 50 mg of diclofenac sodium was dissolved in 100 ml of 0.1 N HCl, filtered, diluted suitably and analysed for drug content at 276 nm using UV/Visible

*4.8.2.6 Determination of percent swelling index (percentage water uptake)*

WU% <sup>¼</sup> Weight of swollen tablet–Initial weight of the tablet

*4.8.2.7 Dissolution studies using USP type II apparatus with wire sinker*

%F ¼ f g 1 � ð Þ loss in weight*=*initial weight � 100 (6)

tablets from each batch were used, and average values were calculated.

ascertain whether it is within permissible limits or not.

The result of *in vitro* dissolution studies of tablet were fitted with various kinetics models, like zero order (% cumulative drug release vs. time), first order (log % drug remaining vs. time), Higuchi's model (% cumulative drug release vs. square root of time) but these models failed to explain drug release mechanism due to swelling (upon hydration) along with gradual erosion of the matrix. Therefore the dissolution data were also fitted to well-known Korsmeyer and Peppas semiempirical model to ascertain the mechanism of drug release.

$$\log\left(\text{Mt/M}\circ\right) = \log\text{k} + \text{n\log}\tag{8}$$

Where, M∞ is the amount of drug release after infinite time; k is the release rate constant which considers structural and geometric characteristics of the tablets; and n is the diffusional exponent; indicative of the mechanism of drug release. **Table 9** shows an analysis of diffusional release mechanism obtained by various value of n. The criteria for selecting the most appropriate model were chosen on the basis of goodness of fit test. The data were processed for regression analysis using MS EXCEL statistical function.

#### *4.8.2.9 Biodegradability studies of floating tablet*

The biodegradability studies were carried out using USP rotating basket apparatus. A tablet (50 mg) were introduced into the baskets which were rotated at 50 rpm in 900 ml of different pH buffer solution (5.0, 6.8, 8.0) maintained at 37°C � 0.5°C.

#### *4.8.2.10 Stability studies*

To assess the drug and formulation stability, stability studies were done according to ICH guidelines. The promising formulation F4 was tested for accelerated testing for a period of 2 months at 40°C � 2°C/ 75% RH �5% for their drug content and other parameters.


**Table 9.** *Release mechanism with variation of n values.*

#### **5. Result and discussion**

The effervescent floating tablets of SDs of DS were formulated in 6 different batches F1 to F6 by using hydrophilic polymers HPMC K4M, HPMC K15M and hydrophobic polymer carbopol 934P along with effervescing agents, sodium bicarbonate and citric acid (**Table 8**). All the formulations were prepared by wet granulation method. In order to get the longer duration of floating time the high viscosity polymer selected, HPMC K4M was chosen and it was found that, increased viscosity of a polymer prolongs the drug delivery from the dosage form. IR study was carried out to check the compatibility between the selected polymers with the diclofenac sodium drug. This study was performed to assure that there is complete physical entrapment of the drug into the polymer matrix without any mutual interaction. IR spectra were taken for samples like pure drug, and drug-polymer physical mixture at a wavelength of between 4000 and 400 cm<sup>1</sup> . All the spectra were compared for shifting of major functional peaks and also for the loss of functional peaks for identification of incompatibility, if any. When the spectra were compared it was found that there was no shifting of functional peaks and no overlapping of characteristic peaks and also there was no appearance of new peaks. **Figures 2**, **7** and **8** shows the IR spectra of various samples. No significant change in the IR spectra of diclofenac sodium complexes was obtained, except for the broadening of the peaks. The broadening of peaks may be probably due to the restriction of bending and stretching vibrations of the molecule [6]. The preformulation studies such as angle of repose, bulk density, tapped density, and carr's index evaluated were found to be within prescribed limits and indicated good free flowing property (**Table 10**).

spectrophotometrically indicating good content uniformity in the prepared formu-

**Parameter F1 F2 F3 F4 F5 F6** Floating lag time (FLT) (s) 160 182 158 163 221 223 Total Floating time (TFT) (h) 22 21 24 20 24 21

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

The swelling index was calculated with respect to time. As time increase, the swelling index was increased because weight gain by tablet was increased proportionally with rate of hydration. The direct relationship was observed in **Table 13**. The floating formulation F3 was subjected for the dissolution studies using USP type II apparatus with wire sinker in 900 ml of 0.1 N HCl medium. The results are given in **Table 14**. The formulation showed a constant rate of release in a sustained manner similar to zero order kinetics with good buoyancy property. Diclofenac sodium effervescent floating controlled release tablet formulation using solid dis-

persion (F3) showed far better release than marketed products.

**5.1 Effect of sodium bicarbonate concentration on lag time of tablets**

were used. From the results, it was concluded that with the increasing

**Hardness (kg/cm<sup>2</sup> )**

**Thickness (mm)**

The concentration of sodium bicarbonate was found to be critical factor that influenced buoyancy of tablets (**Table 15**). Sodium bicarbonate released CO2 gas that was trapped into the polymeric matrix of HPMC that made the tablets float. Various concentrations of sodium bicarbonate ranging from 5–12% of tablet weight

> **Friability (%)**

F1 4.3 0.016 4.8 0.4 0.24 0.08 542.4 1.9 99.86 0.15 F2 4.4 0.013 5.1 0.3 0.51 0.03 555.8 1.5 99.45 0.08 **F3 4.5 0.015 5.4 0.6** 0.17 **0.04 554.3 1.1 100.01 0.04** F4 4.5 0.013 4.9 0.4 0.46 0.03 545.1 1.8 99.96 0.18 F5 5.5 0.014 4.4 0.1 0.35 0.05 649.1 1.7 98.90 1.05 F6 5.7 0.011 5.8 0.3 0.41 0.04 647.3 0.4 99.02 0.01

**Time (h) Formulation F3** 1 24 2 37 3 48 4 63 5 71 6 88

**Weight variation (mg)**

**Drug content (%)**

lation results were shown in **Table 12**.

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

In vitro *buoyancy determination.*

**Formulation code**

**Table 12.**

**Table 13.**

**33**

*General characteristic of floating tablets.*

**% Swelling index (percentage water uptake)**

*% Swelling index (percentage water uptake) of floating tablets.*

**Table 11.**

*In vitro* Buoyancy of all the prepared tablets formulations were determined using 100 ml beaker containing 0.1NHCl medium shown in (**Table 11**) and the results can be concluded that the batch F3 containing HPMCK4M and carbopol 934P in higher concentration showed good buoyancy lag time is 4.3 min and total floating time is 15 hrs. TFT depends upon the amount of HPMC as the polymer content increased the floating time was increased due to the formation of thick gel which entrapped the gas formed due to NaHCO3 firmly. Among these formulations, the *in vitro* buoyancy was increased in the following order: F3 > F1 > F4 > F2 > F5 > F6. The **Table 9** revealed that FLT minimum for F3 formulation, while its TFT was maximum i.e. 24 h; hence, F3 was selected for further evaluations and *in vitro* drug dissolution studies.

Formulation F3 was evaluated for physical characters like tablet thickness, diameter, hardness, friability, weight variation, percent swelling index, *in vitro* drug release studies. The thickness, diameter and hardness of the formulations satisfied the acceptance criteria. The friability and weight variation was found to be within the limits specified in pharmacopoeia. The drug content was found


#### **Table 10.**

*Pre-compression parameters of granules.*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*


#### **Table 11.**

**5. Result and discussion**

*Molecular Pharmacology*

dissolution studies.

Angle of repose

Bulk density

Tapped density

**Table 10.**

**32**

*Pre-compression parameters of granules.*

at a wavelength of between 4000 and 400 cm<sup>1</sup>

The effervescent floating tablets of SDs of DS were formulated in 6 different batches F1 to F6 by using hydrophilic polymers HPMC K4M, HPMC K15M and hydrophobic polymer carbopol 934P along with effervescing agents, sodium bicarbonate and citric acid (**Table 8**). All the formulations were prepared by wet granulation method. In order to get the longer duration of floating time the high viscosity polymer selected, HPMC K4M was chosen and it was found that, increased viscosity of a polymer prolongs the drug delivery from the dosage form. IR study was carried out to check the compatibility between the selected polymers with the diclofenac sodium drug. This study was performed to assure that there is complete physical entrapment of the drug into the polymer matrix without any mutual interaction. IR spectra were taken for samples like pure drug, and drug-polymer physical mixture

shifting of major functional peaks and also for the loss of functional peaks for identification of incompatibility, if any. When the spectra were compared it was found that there was no shifting of functional peaks and no overlapping of characteristic peaks and also there was no appearance of new peaks. **Figures 2**, **7** and **8** shows the IR spectra of various samples. No significant change in the IR spectra of diclofenac sodium complexes was obtained, except for the broadening of the peaks. The broadening of peaks may be probably due to the restriction of bending and stretching vibrations of the molecule [6]. The preformulation studies such as angle of repose, bulk density, tapped density, and carr's index evaluated were found to be within prescribed limits and indicated good free flowing property (**Table 10**).

*In vitro* Buoyancy of all the prepared tablets formulations were determined using 100 ml beaker containing 0.1NHCl medium shown in (**Table 11**) and the results can be concluded that the batch F3 containing HPMCK4M and carbopol 934P in higher concentration showed good buoyancy lag time is 4.3 min and total floating time is 15 hrs. TFT depends upon the amount of HPMC as the polymer content increased the floating time was increased due to the formation of thick gel which entrapped the gas formed due to NaHCO3 firmly. Among these formulations, the *in vitro* buoyancy was increased in the following order: F3 > F1 > F4 > F2 > F5 > F6. The **Table 9** revealed that FLT minimum for F3 formulation, while its TFT was maximum i.e. 24 h; hence, F3 was selected for further evaluations and *in vitro* drug

Formulation F3 was evaluated for physical characters like tablet thickness, diameter, hardness, friability, weight variation, percent swelling index, *in vitro* drug release studies. The thickness, diameter and hardness of the formulations satisfied the acceptance criteria. The friability and weight variation was found to be within

**Parameter F1 F2 F3 F4 F5 F6**

Carr's index 7.64 0.94 6.66 0.71 5.69 0.56 8.99 0.62 8.68 0.83 7.97 0.49

22.53° 22.17° 23.42° 21.57° 22.87° 23.34°

0.953 0.026 0.948 0.031 0.975 0.0.098 0.881 0.102 0.836 0.057 0.899 0.083

1.05 0.011 1.041 0.019 1.031 0.026 0.978 0.020 0.981 0.017 0.969 0.038

the limits specified in pharmacopoeia. The drug content was found

. All the spectra were compared for

In vitro *buoyancy determination.*

spectrophotometrically indicating good content uniformity in the prepared formulation results were shown in **Table 12**.

The swelling index was calculated with respect to time. As time increase, the swelling index was increased because weight gain by tablet was increased proportionally with rate of hydration. The direct relationship was observed in **Table 13**.

The floating formulation F3 was subjected for the dissolution studies using USP type II apparatus with wire sinker in 900 ml of 0.1 N HCl medium. The results are given in **Table 14**. The formulation showed a constant rate of release in a sustained manner similar to zero order kinetics with good buoyancy property. Diclofenac sodium effervescent floating controlled release tablet formulation using solid dispersion (F3) showed far better release than marketed products.

#### **5.1 Effect of sodium bicarbonate concentration on lag time of tablets**

The concentration of sodium bicarbonate was found to be critical factor that influenced buoyancy of tablets (**Table 15**). Sodium bicarbonate released CO2 gas that was trapped into the polymeric matrix of HPMC that made the tablets float. Various concentrations of sodium bicarbonate ranging from 5–12% of tablet weight were used. From the results, it was concluded that with the increasing


#### **Table 12.**

*General characteristic of floating tablets.*


**Table 13.**

*% Swelling index (percentage water uptake) of floating tablets.*


**5.2 Effect of HPMC grade on lag time of tablets**

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

indicating **Anomalous (non-fickian) diffusion.**

to a major extend along with other plots to some extent.

HPMC (**Table 16**).

**Table 16.**

**Figure 9.**

**35**

*Zero order release kinetics of optimised formulation.*

**5.3 Kinetic of drug release**

It was interesting to note that the grade and quantity of HPMC used in the formulations has impact on floating lag time of the tablet. With the increasing molecular weight/quantity of HPMC, the viscosities of the gel matrix around the tablet also increased which in turn in- creased the floating lag time. The lag time for HPMC K15M tablets was slightly higher compared to HPMC K4M tablet. This may be attributed to the increased density of tablet with increasing molecular weight of

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

The various release kinetic models (**Figures 9**–**12**) were applied to determine the mechanism of drug release from gastro retentive floating tablets and the data is tabulated in **Table 17**. The *in vitro* drug release of optimised formulation (F3) showed the highest regression coefficient values for zero order model, thus indicating absolute correlation between the two variables for the zero order model. Optimised formulations followed Zero order equation proving that the release is by diffusion mechanism. The values of release exponent (n) were calculated from korsmeyer and peppas equation and the 'n' value was determined to be 0.5665

So it can be conclude that the optimised formulation follows the zero order plot

**S. No. Quantity of HPMC (mg) Floating lag time (s)**

 70 (HPMC K4M) 160 93 (HPMC K4M) 158 105 (HPMC K4M) 163 70 (HPMC K15M) 182 93 (HPMC K15M) 221 105 (HPMC K15M) 223

*Comparison of floating lag time prepared from different grade or quantity of HPMC.*

#### **Table 14.**

*Comparative* in vitro *release study of marketed tablets, physical mixture, solid dispersion and floating tablets of diclofenac solid dispersion.*


#### **Table 15.**

*Comparison of floating lag time prepared from concentration of sodium bicarbonate.*

concentration of sodium bicarbonate, the lag time decreased. A concentration of 8.5–9% w/w sodium bicarbonate was found to be optimal that resulted in tablets having lag time < 3 min and floating time of over 12 h. Similar conclusions were also drawn by other researchers working on floating delivery systems. In both the reported works, optimum concentration of sodium bicarbonate was found to be around 10% w/w of the tablet weight [26, 27] which is slightly higher than our optimal concentration.

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

#### **5.2 Effect of HPMC grade on lag time of tablets**

It was interesting to note that the grade and quantity of HPMC used in the formulations has impact on floating lag time of the tablet. With the increasing molecular weight/quantity of HPMC, the viscosities of the gel matrix around the tablet also increased which in turn in- creased the floating lag time. The lag time for HPMC K15M tablets was slightly higher compared to HPMC K4M tablet. This may be attributed to the increased density of tablet with increasing molecular weight of HPMC (**Table 16**).

#### **5.3 Kinetic of drug release**

The various release kinetic models (**Figures 9**–**12**) were applied to determine the mechanism of drug release from gastro retentive floating tablets and the data is tabulated in **Table 17**. The *in vitro* drug release of optimised formulation (F3) showed the highest regression coefficient values for zero order model, thus indicating absolute correlation between the two variables for the zero order model. Optimised formulations followed Zero order equation proving that the release is by diffusion mechanism. The values of release exponent (n) were calculated from korsmeyer and peppas equation and the 'n' value was determined to be 0.5665 indicating **Anomalous (non-fickian) diffusion.**

So it can be conclude that the optimised formulation follows the zero order plot to a major extend along with other plots to some extent.


#### **Table 16.**

*Comparison of floating lag time prepared from different grade or quantity of HPMC.*

**Figure 9.** *Zero order release kinetics of optimised formulation.*

concentration of sodium bicarbonate, the lag time decreased. A concentration of 8.5–9% w/w sodium bicarbonate was found to be optimal that resulted in tablets having lag time < 3 min and floating time of over 12 h. Similar conclusions were also drawn by other researchers working on floating delivery systems. In both the reported works, optimum concentration of sodium bicarbonate was found to be around 10% w/w of the tablet weight [26, 27] which is slightly higher than our

*Comparison of floating lag time prepared from concentration of sodium bicarbonate.*

*Comparative* in vitro *release study of marketed tablets, physical mixture, solid dispersion and floating tablets*

**S. No. Concentration of sodium bicarbonate (%) Floating lag time (s)** 1 5 280 2 6 220 3 7 198 4 8 164 5 9 158 6 10 159 7 11 160 8 12 165

optimal concentration.

**Time (mins)**

*Molecular Pharmacology*

**Table 14.**

**Table 15.**

**34**

*of diclofenac solid dispersion.*

**Marketed tablet (Voveran-50) (% drug release)**

**Physical mixture**

**Diclofenac sodium-solid dispersion (% drug release)**

 0 0.00 0.00 0.000 0.000 15 15.25 0.64 21.23 0.61 30.51 0.54 12.706 0.67 15.266 0.41 30 37.37 0.53 39.44 0.64 52.25 0.49 16.258 1.27 18.365 0.38 60 51.77 0.86 70.76 0.58 92.99 0.78 19.353 0.98 26.548 0.51 24.930 0.79 27.897 0.50 27.966 0.93 31.377 0.43 32.220 0.76 38.323 0.45 38.922 1.22 45.233 0.29 45.875 0.96 54.320 0.27 51.519 1.23 61.522 0.30 60.865 1.31 64.267 0.31 64.037 .55 69.613 0.35 68.561 1.53 73.670 0.51 73.686 0.77 76.568 0.42 77.371 1.16 80.179 0.44 82.957 0.98 85.363 0.47 86.414 0.74 87.573 0.58 89.213 1.78 96.769 1.19

**Marketed tablet (Voveran-SR100) (%drug release)**

**Floating tablet of diclofenac sodium solid dispersion (3) (%drug release)**

**5.4 Biodegradability studies of floating tablet**

*Release kinetic equation values of the optimised formulations.*

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

(**Figure 13**).

**Table 17.**

**5.5 Stability studies**

developmental stages.

and pack profile.

**Figure 13.**

**37**

Biodegradability studies revealed that the gastro retentive floating tablet of diclofenac (F3) was found to disintegrate and dissolve in intestinal pH within 3 h

**S.No Formulation Zero order First order Higuchi Korsmeyer-peppas 1 F3 K R<sup>2</sup> K R2 K R2 N R<sup>2</sup>**

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

10.373 0.9882 0.1373 0.8541 0.3444 0.9837 0.5665 0.9616

but exhibit complete biodegradation in phosphate buffer pH 8.0.

the stability studies. This study pursues two particular aims:

Formulation F3 seemed to completely biodegrade in intestinal fluid, and it is the pH of media, which is responsible for slow dissolution of the tablet in intestinal fluid. This indicates that after gastric emptying the regular shaped tablet, gradually become rough with an irregular surface and thereafter was degraded. Thus the gastro retentive floating tablets of diclofenac proved to be suitable gastro retentive dosage form, as they have a rigid structure that resist biodegradation in gastric pH

Pharmaceutical dosage forms are complex systems composed not only of drug

therapeutic, are intended to contribute desirable, practical properties to the dosage form. These dosage forms may undergo both chemical and physical degradation [28]. Thus, the success of the effective formulation can be evaluated only through

substances but also of various excipients. These excipients, which are non-

• Determination of the optimum formulation and shelf life during

microbiological, therapeutic and toxicological specifications. Ability of a

*Images of complete biodegradation of F3 floating tablet after 3 h.*

• Derivation of the stability of a product, which guarantees the safety and

efficacy of the product up to end of the shelf life at a defined storage condition

So, stability of a pharmaceutical product may be defined as the capability of a particular formulation in a specific container, to remain in its physical, chemical,

**Figure 10.** *First order release kinetics of optimised formulation.*

**Figure 11.** *Higuchi kinetics of optimised formulation.*

**Figure 12.** *Korsmeyer-Peppas kinetics of optimised formulation.*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*


**Table 17.**

**Figure 10.**

*Molecular Pharmacology*

**Figure 11.**

**Figure 12.**

**36**

*Higuchi kinetics of optimised formulation.*

*Korsmeyer-Peppas kinetics of optimised formulation.*

*First order release kinetics of optimised formulation.*

*Release kinetic equation values of the optimised formulations.*

#### **5.4 Biodegradability studies of floating tablet**

Biodegradability studies revealed that the gastro retentive floating tablet of diclofenac (F3) was found to disintegrate and dissolve in intestinal pH within 3 h (**Figure 13**).

Formulation F3 seemed to completely biodegrade in intestinal fluid, and it is the pH of media, which is responsible for slow dissolution of the tablet in intestinal fluid. This indicates that after gastric emptying the regular shaped tablet, gradually become rough with an irregular surface and thereafter was degraded. Thus the gastro retentive floating tablets of diclofenac proved to be suitable gastro retentive dosage form, as they have a rigid structure that resist biodegradation in gastric pH but exhibit complete biodegradation in phosphate buffer pH 8.0.

#### **5.5 Stability studies**

Pharmaceutical dosage forms are complex systems composed not only of drug substances but also of various excipients. These excipients, which are nontherapeutic, are intended to contribute desirable, practical properties to the dosage form. These dosage forms may undergo both chemical and physical degradation [28]. Thus, the success of the effective formulation can be evaluated only through the stability studies. This study pursues two particular aims:


So, stability of a pharmaceutical product may be defined as the capability of a particular formulation in a specific container, to remain in its physical, chemical, microbiological, therapeutic and toxicological specifications. Ability of a

**Figure 13.** *Images of complete biodegradation of F3 floating tablet after 3 h.*

formulation to retain properties in specified limits throughout its shelf-life is referred as stability [28].

The stability of finished pharmaceutical products depends on several factors. On the one hand, it depends on environmental factors such as ambient temperature, humidity and light. On the other hand, it depends on product related factors such as chemical and physical properties of active substance and pharmaceutical excipients, the dosage form and its composition, the manufacturing process, the nature of container closure system and properties of packaging materials.

*5.5.1.2 Physcio-chemical parameters*

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

*5.5.1.4* In vitro *dissolution studies*

*5.5.2 Result and discussion*

(pH 1.2).

**Table 20.**

**39**

Various parameters were evaluated such as Hardness (kg/cm<sup>2</sup>

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

*5.5.1.3 Drug content was assayed by U.V. spectrophotometry*

the drug. The determinations were made in triplicate.

*5.5.2.1 Physical parameters of the optimised tablets formulation*

*5.5.2.2 Physico-chemical parameters of the optimised formulation*

Various parameters were evaluated such as Hardness (kg/cm<sup>2</sup>

**S. No. Sampling interval (days) % Residual drug content**

*Effect of ageing on residual drug content at room temperature &40 2 °C/ 75 5%RH.*

 0 100.01 0.03 100.01 0.03 15 99.82 0.12 99.66 0.09 30 99.56 0.09 99.25 0.18 45 98.75 0.14 98.40 0.15 60 98.56 0.05 97.72 0.9 75 98.07 0.09 97.51 0.10 90 97.69 0.07 96.66 0.06

tions were physically stable at these temperatures.

Uniformity of weight (mg), Drug content (%), Thickness (mm), and *in vitro* release after a period of 15, 30, 45, 60, 75, 90 days. Observations are recorded in **Table 19**.

Gastro retentive floating tablet of diclofenac sodium (50 mg) was dissolved in 100 ml of 0.1 N HCl (pH 1.2) by stirring for 6 h using magnetic stirrer. The resulting solution was then filtered using 0.45 m millipore filter, 1 ml of this solution was taken and added to 100 ml of 0.1 N HCl (pH 1.2). It was then analysed spectrophotometrically at the predetermined λ max (276 nm) to determine concentration of

*In vitro* dissolution studies were carried out using simulated gastric fluid

The Physical parameters after 15th, 30th, 60th, 90th days are as mentioned in **Table 18**. All the Physical parameter are within the acceptable limits which indicated that gastro retentive floating tablet of diclofenac sodium showed no significant change in the physical appearance at room temperature and in stability

chamber at 40°C 2°C and 75 5% relative humidity indicating that the formula-

Uniformity of weight (mg), Drug content (%), Thickness (mm), and *in vitro* release

), Friability (%),

), Friability (%),

**Mean S.D. (n = 3) At room temp. At 40 2°C/75 5% RH**

A study of stability of a pharmaceutical product is essential for safety of the patients, legal requirements concerned with the identity, strength, purity and quality of the drug and to prevent the economic repercussions of marketing an unsuitable product [29, 30].

#### *5.5.1 Experimental*

Optimised formulations were stored in screw capped small glass bottles at room temperature and in stability chamber at 40 1°C and 75% relative humidity. Samples were analysed for physical appearance, Hardness (kg/cm<sup>2</sup> ), Friability (%), Uniformity of weight (mg), Drug content (%), Thickness (mm), Buoyancy lag time (s), Floating time (h) and *in vitro* release after a period of 15, 30, 45, 60, 75, 90 days. Initial drug content was taken as 100% for each formulation. Observations are recorded in **Tables 18** and **19**.

#### *5.5.1.1 Physical characteristics*

Various physical parameters were evaluated such as appearance, Buoyancy lag time (s), floating time. Observations are recorded in **Table 18**.


#### **Table 18.**

*Effect of ageing on physical parameters.*


#### **Table 19.**

*Effect of ageing on physico-chemical parameters of optimised formulation.*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

#### *5.5.1.2 Physcio-chemical parameters*

formulation to retain properties in specified limits throughout its shelf-life is

container closure system and properties of packaging materials.

The stability of finished pharmaceutical products depends on several factors. On the one hand, it depends on environmental factors such as ambient temperature, humidity and light. On the other hand, it depends on product related factors such as chemical and physical properties of active substance and pharmaceutical excipients, the dosage form and its composition, the manufacturing process, the nature of

A study of stability of a pharmaceutical product is essential for safety of the patients, legal requirements concerned with the identity, strength, purity and quality of the drug and to prevent the economic repercussions of marketing an

Optimised formulations were stored in screw capped small glass bottles at room

Uniformity of weight (mg), Drug content (%), Thickness (mm), Buoyancy lag time (s), Floating time (h) and *in vitro* release after a period of 15, 30, 45, 60, 75, 90 days. Initial drug content was taken as 100% for each formulation. Observations are

Various physical parameters were evaluated such as appearance, Buoyancy lag

**S. No. Physical parameters 0 days 15th days 30th days 60th days 90th days** 1 Appearance + + + + + 2 Floating time + + + + + 3 Buoyancy lag time (s) + + + + +

**Parameter Optimised formulation (F3) (n = 3)**

*Effect of ageing on physico-chemical parameters of optimised formulation.*

**At 0 day At 15 days At 30 days At 60 days**

) 5.4 0.08 5.4 0.1 5.4 0.09 5.2 0.07

Friability (%) 0.17 0.02 0.17 0.01 0.19 0.02 0.20 0.01 Uniformity of weight (mg) 554.3 1.1 554.3 1.1 554.3 1.1 554.3 1.1 Drug content (%) 100.01 0.04 100.01 0.04 99.50 0.58 98.89 0.12 Thickness (mm) 4.55 0.12 4.55 0.09 4.55 0.10 4.55 0.11

), Friability (%),

temperature and in stability chamber at 40 1°C and 75% relative humidity.

Samples were analysed for physical appearance, Hardness (kg/cm<sup>2</sup>

time (s), floating time. Observations are recorded in **Table 18**.

referred as stability [28].

*Molecular Pharmacology*

unsuitable product [29, 30].

recorded in **Tables 18** and **19**.

*5.5.1.1 Physical characteristics*

*Effect of ageing on physical parameters.*

Hardness (kg/cm2

*+, no change.*

**Table 18.**

**Table 19.**

**38**

*5.5.1 Experimental*

Various parameters were evaluated such as Hardness (kg/cm<sup>2</sup> ), Friability (%), Uniformity of weight (mg), Drug content (%), Thickness (mm), and *in vitro* release after a period of 15, 30, 45, 60, 75, 90 days. Observations are recorded in **Table 19**.

#### *5.5.1.3 Drug content was assayed by U.V. spectrophotometry*

Gastro retentive floating tablet of diclofenac sodium (50 mg) was dissolved in 100 ml of 0.1 N HCl (pH 1.2) by stirring for 6 h using magnetic stirrer. The resulting solution was then filtered using 0.45 m millipore filter, 1 ml of this solution was taken and added to 100 ml of 0.1 N HCl (pH 1.2). It was then analysed spectrophotometrically at the predetermined λ max (276 nm) to determine concentration of the drug. The determinations were made in triplicate.

#### *5.5.1.4* In vitro *dissolution studies*

*In vitro* dissolution studies were carried out using simulated gastric fluid (pH 1.2).

#### *5.5.2 Result and discussion*

#### *5.5.2.1 Physical parameters of the optimised tablets formulation*

The Physical parameters after 15th, 30th, 60th, 90th days are as mentioned in **Table 18**. All the Physical parameter are within the acceptable limits which indicated that gastro retentive floating tablet of diclofenac sodium showed no significant change in the physical appearance at room temperature and in stability chamber at 40°C 2°C and 75 5% relative humidity indicating that the formulations were physically stable at these temperatures.

#### *5.5.2.2 Physico-chemical parameters of the optimised formulation*

Various parameters were evaluated such as Hardness (kg/cm<sup>2</sup> ), Friability (%), Uniformity of weight (mg), Drug content (%), Thickness (mm), and *in vitro* release


#### **Table 20.**

*Effect of ageing on residual drug content at room temperature &40 2 °C/ 75 5%RH.*

after a period of 15, 30, 45, 60, 75, 90 days. Observations are recorded in **Table 6**. All the physico-chemical parameters are within the acceptable limits which indicated that formulation were stable over the period of 90 days.

#### *5.5.2.3 Residual drug content of stability batch*

Initial drug content of formulations was 100.01 0.04.the drug contents at the end of 15th, 30th, 60th, 90th days were found to be as given in **Table 20**. The drug content was within the permissible limits. The percent residual drug content was determined and the log percent residual content was plotted against time t (**Figures 14**–**17**), which reflected almost linear relationship.

#### *5.5.2.4* In vitro *dissolution studies*

The dissolution results obtained were as given in the **Table 21**.

The dissolution behaviour of samples withdrawn at different interval was similar and the difference in dissolution pattern of samples kept at two different conditions of storage was negligible.

The log % residual drug content vs. time graph was also plotted in order to evaluate shelf-life and half-life of formulations.

**Figure 14.** *Effect of ageing on residual drug content at 40 2°C/75 5%RH.*

Shelf-life was evaluated by the equation:

*Plot of log % residual drug content vs. time at room temp.*

*Effect of ageing on residual drug content at room temperature.*

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

of 10% drug was calculated as T10%.

**Figure 17.**

**Table 21.**

**41**

**Figure 16.**

T10% ¼ 0*:*104*=*K (9)

**Room temperature 40** � **2°C/75** � **5% RH**

Degradation rate constant K was calculated from the slope of straight line between log of % residual drug and time interval. The time required for degradation

**Time interval (days) % Cumulative drug release in 8 h** � **SD (n = 3)**

 96.769 � 1.19 96.769 � 1.19 95.78 � 0.84 94.34 � 1.52 94.81 � 1.64 93.05 � 0.81 94.45 � 0.56 92.89 � 0.69 93.97 � 0.93 92.45 � 1.21

*Effect of ageing on % cumulative drug release at room temperature & 40* � *2°C/75* � *5%RH.*

**Figure 15.** *Plot of log % residual drug content Vs time at 40 2°C/75 5%RH.*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

**Figure 16.** *Effect of ageing on residual drug content at room temperature.*

**Figure 17.** *Plot of log % residual drug content vs. time at room temp.*


**Table 21.**

after a period of 15, 30, 45, 60, 75, 90 days. Observations are recorded in **Table 6**. All the physico-chemical parameters are within the acceptable limits which

Initial drug content of formulations was 100.01 0.04.the drug contents at the

The dissolution behaviour of samples withdrawn at different interval was similar and the difference in dissolution pattern of samples kept at two different conditions

The log % residual drug content vs. time graph was also plotted in order to

end of 15th, 30th, 60th, 90th days were found to be as given in **Table 20**. The drug content was within the permissible limits. The percent residual drug content was determined and the log percent residual content was plotted against time t

indicated that formulation were stable over the period of 90 days.

(**Figures 14**–**17**), which reflected almost linear relationship.

The dissolution results obtained were as given in the **Table 21**.

*5.5.2.3 Residual drug content of stability batch*

evaluate shelf-life and half-life of formulations.

*Effect of ageing on residual drug content at 40 2°C/75 5%RH.*

*Plot of log % residual drug content Vs time at 40 2°C/75 5%RH.*

*5.5.2.4* In vitro *dissolution studies*

of storage was negligible.

*Molecular Pharmacology*

**Figure 14.**

**Figure 15.**

**40**

*Effect of ageing on % cumulative drug release at room temperature & 40* � *2°C/75* � *5%RH.*

Shelf-life was evaluated by the equation:

$$\mathbf{T}\_{10\%} = \mathbf{0}.\mathbf{104}/\mathbf{K} \tag{9}$$

Degradation rate constant K was calculated from the slope of straight line between log of % residual drug and time interval. The time required for degradation of 10% drug was calculated as T10%.


**Table 22.**

*Shelf life of optimised formulation.*

Half-life was evaluated by the equation:

$$\mathbf{T}\_{1/2} = \mathbf{0}.\mathbf{693/K} \tag{10}$$

**Acronyms and abbreviations**

PM physical mixtures DT disintegration time

SDs solid dispersions DS diclofenac sodium BD bulk density

FLT floating lag time

**Author details**

Peeush Singhal<sup>1</sup>

**43**

Vishwavidyalaya, Haridwar, India

provided the original work is properly cited.

Srinagar, Uttarakhand, India

TBD tapped bulk density TFT total floating time %WU percentage water uptake

SEM scanning electron microscopy

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

ECRFT effervescent controlled release floating tablet

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

\*, Rajneesh Dutt Kaushik<sup>1</sup> and Vijay Jyoti Kumar<sup>2</sup>

2 Department of Pharmaceutical Sciences, H.N.B. Garhwal Central University,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Department of Pharmaceutical Sciences (FMSH), Gurukul Kangri

\*Address all correspondence to: peeushpharma@gmail.com

Gastro retentive floating tablet of diclofenac sodium stored at 40 � 2°C/75 � 5% RH showed K value as 3.822 � <sup>10</sup>�<sup>4</sup> and t10% value as 272.039 days, while those stored at room temperature showed K value as 2.303 � <sup>10</sup>�**<sup>4</sup>** and t10% value as 451.58 days (**Table 22**).

The T10% obtained in case of formulation stored at 40°C � 2°C/75 � 5%RH was found to be lower in comparison with the formulation stored at room temperature which indicated that the formulations tend to degrade faster at higher temperatures and humidity.

The results of stability studies suggest that for adequate shelf life of optimised gastro retentive floating tablet of diclofenac sodium, it should be stored in cool and dry place.

#### **6. Conclusions**

In the above research work, ECRFT has been developed by using dual approach; one is solid dispersion (for solubility enhancement) and other is effervescent floating technique (for achieving extended retention in upper G.I.T.), which was prepared from previously optimised solid dispersion of diclofenac sodium. Formulated tablets showed outstanding physicochemical properties, biodegradation studies, stabilities studies, and prolong gastric retention with control release. When compared with marketed tablets of immediate release (Voveran-50) and control release (Voveran-100SR), the optimised formulation F3 was found to be favourable for improving bioavailability of drug, enhancing its therapeutics efficacy and improving patient compliance due to less frequent dosing requirement. Hence, it can be concluded that the prepared formulation can be used positively as a particular oral controlled release-floating tablet for once a day administration.

#### **Acknowledgements**

The authors would like to express their hearty gratitude to Kwality Pharmaceutical Pvt Ltd, Amritsar for supplying diclofenac sodium raw material and excipients. We are also thankful to Dr. Abhishek Bansal, Department of Pharmaceutical sciences, Gurukul Kangri University, Haridwar for the help provided during research.

#### **Conflict of interest**

There is no conflict of interest.

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

#### **Acronyms and abbreviations**

Half-life was evaluated by the equation:

**S. No. Storage condition K (day**�**<sup>1</sup>**

451.58 days (**Table 22**).

*Shelf life of optimised formulation.*

*Molecular Pharmacology*

and humidity.

**6. Conclusions**

**Acknowledgements**

**Conflict of interest**

**42**

There is no conflict of interest.

dry place.

**Table 22.**

T1*<sup>=</sup>*<sup>2</sup> ¼ 0*:*693*=*K (10)

**) T10% (days) t1/2 (days)**

Gastro retentive floating tablet of diclofenac sodium stored at 40 � 2°C/75 � 5% RH showed K value as 3.822 � <sup>10</sup>�<sup>4</sup> and t10% value as 272.039 days, while those stored at room temperature showed K value as 2.303 � <sup>10</sup>�**<sup>4</sup>** and t10% value as

<sup>1</sup> <sup>40</sup> � 2 °C/75 � 5%RH 3.822 � <sup>10</sup>�<sup>4</sup> 272.039 1812.72 <sup>2</sup> Room temperature 2.303 � <sup>10</sup>�<sup>4</sup> 451.58 3009.11

The T10% obtained in case of formulation stored at 40°C � 2°C/75 � 5%RH was found to be lower in comparison with the formulation stored at room temperature which indicated that the formulations tend to degrade faster at higher temperatures

The results of stability studies suggest that for adequate shelf life of optimised gastro retentive floating tablet of diclofenac sodium, it should be stored in cool and

In the above research work, ECRFT has been developed by using dual approach; one is solid dispersion (for solubility enhancement) and other is effervescent floating technique (for achieving extended retention in upper G.I.T.), which was prepared from previously optimised solid dispersion of diclofenac sodium. Formulated tablets showed outstanding physicochemical properties, biodegradation studies, stabilities studies, and prolong gastric retention with control release. When compared with marketed tablets of immediate release (Voveran-50) and control release (Voveran-100SR), the optimised formulation F3 was found to be favourable for improving bioavailability of drug, enhancing its therapeutics efficacy and improving patient compliance due to less frequent dosing requirement. Hence, it can be concluded that the prepared formulation can be used positively as a particular oral

The authors would like to express their hearty gratitude to Kwality Pharmaceutical Pvt Ltd, Amritsar for supplying diclofenac sodium raw material and excipients. We are also thankful to Dr. Abhishek Bansal, Department of Pharmaceutical sciences, Gurukul Kangri University, Haridwar for the help provided during research.

controlled release-floating tablet for once a day administration.


#### **Author details**

Peeush Singhal<sup>1</sup> \*, Rajneesh Dutt Kaushik<sup>1</sup> and Vijay Jyoti Kumar<sup>2</sup>

1 Department of Pharmaceutical Sciences (FMSH), Gurukul Kangri Vishwavidyalaya, Haridwar, India

2 Department of Pharmaceutical Sciences, H.N.B. Garhwal Central University, Srinagar, Uttarakhand, India

\*Address all correspondence to: peeushpharma@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Available from: http://www.drugs. com/diclofenac.html

[2] Rao GK, Mandapalli PK, Manthri R, Reddy VP. Development and in vivo evaluation of gastro retentive delivery systems for cefuroxime axetil. Saudi Pharmaceutical Journal. 2013;**21**(1): 53-59. DOI: 10.1016/j.jsps.2012.01.003

[3] Chau Le-Ngoc VO, Chulhun P, Beom-Jin L. Current trends and future perspectives of solid dispersions containing poorly water-soluble drugs review article. European Journal of Pharmaceutics and Biopharmaceutics. 2013;**85**(3) Part B:799-813

[4] Giri TK, Kumar K, Alexander A, Ajazuddin, Badwaik H, Tripathi DK. A novel and alternative approach to controlled release drug delivery system based on solid dispersion technique review article. Bulletin of Faculty Pharmacy, Cairo University. 2012;**50**(2): 147-159

[5] Willis JV, Kendall MJ, Flinn RM, Thornill DP, Welling PG. The pharmacokinetics of diclofenac sodium following intravenous and oral administration. European Journal of Clinical Pharmacology. 1979;**16**(6): 405-410

[6] Sharma N, Jain N, Sudhakar CK, Jain S. Formulation and evaluation of gastro retentive floating tablets containing cefpodoxim proxetil solid dispersions. International Journal of Current Pharmaceutical Research. 2012; **4**(4):82-87

[7] Shivakumar HN, Desai BG, Deshmukh G. Design and optimization of diclofenac sodium controlled release solid dispersions by response surface methodology. Indian Journal of Pharmaceutical Sciences. 2008;**70**(1):22-30

[8] Baumgartner S, Kristl J, Vrecer F, Vodopivec P, Zorko B. Optimization of floating matrix tablets and evaluation of their gastric residence time. International Journal of Pharmaceutics. 2000;**195**:125-135

[19] Sharma BK. Instrumental Methods of Chemical Analysis. 23rd ed. Goel Publishing House; 2004. pp. 292-302

*DOI: http://dx.doi.org/10.5772/intechopen.92187*

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet…*

of cefuroxime axetil: In vitro evaluation. AAPS PharmSciTech. 2006;**7**:E1-E7

[28] Yoshika S, Stell VJ. Stability of Drugs and Dosage Forms. 2nd ed. New Delhi: Springer Private Ltd; 2006.

[29] Garret NM. Stability studies of floating drug delivery dosage form. Journal of Pharmaceutical Sciences.

[30] Gennaro AR. Remington:

Pharmaceutical Sciences and Practice of Pharmacy. 20th ed. Vol. 1. Philadelphia: Lippincott Williams & Wilkins; 1990.

pp. 151-186

1965;**54**:1557

p. 986

[20] Barzegar-Jalali M, Alaei-Beirami M,

Hamidi A, Andalib S, et al. Comparison of physicochemical characteristics and drug release of diclofenac sodiumeudragit® RS100 nanoparticles and solid dispersions. Powder Technology.

[21] Davis SS, Stockwel AF, Taylor MJ, Hardy JG, Whalley DR, Wilson CG. The effect of density on the gastric emptying of single and multiple unit dosage forms. Pharmaceutical Research. 1986;**3**:

[22] Rosa M, Zia H, Rhodes T. Design and testing in vitro of a bioadhesive and floating drug delivery system for oral application. International Journal of Pharmaceutics. 1994;**105**:65-70

Mirchandani HL, Chien YW. Effect of HPMC and carbopol on the release and floating properties of gastric floating drug delivery system using factorial design. International Journal of Pharmaceutics. 2003;**253**:13-22

[24] Machida Y, Inouy K, Tokumura T, Iwata M, Nagai T. Preparation and evaluation of intragastric buoyant preparations. Drug Design and Delivery.

[25] Moes AJ. Gastro retentive dosage forms. Critical Reviews in Therapeutic Drug Carrier Systems. 1993;**10**:143-159

Intragastricoating drug delivery system

[26] Dhumal RS, Rajmane ST, Dhumal ST, Pawar AP. Design and evaluation of bi-layered floating tablets of cefuroxime axetil for bimodal release. Journal of Scientific and Industrial

Research. 2006;**65**:812-816

[27] Patel VF, Patel NM.

**45**

[23] Li S, Lin S, Daggy BP,

1989;**4**:155-161

Javadzadeh Y, Mohammadi G,

2012;**219**:211-216

208-213

[9] Chanvanpatil M, Jain P, Chaudhari S, Shear R, Vavia P. Novel sustained release, swellable and bio adhesive gastro retentive drug delivery system for ofloxacin. International Journal of Pharmaceutics. 2006;**316**:86-92

[10] United State Pharmacopoeia 31. The National Formulary. Asian Edition. 26 (I). North Bethesda, Maryland, United States: The Board of Trustees; 2008. pp. 297-298

[11] Seedher N, Bhatia S. Solubility enhancement of cox-2 inhibitors using various solvent systems. AAPS PharmSciTech. 2003;**4**(3):33

[12] Instruction manual pharmaspec. UV-1700 Series. User's System Guide. Kyoto Japan: Shimadzu Corporation;

[13] Pavia LK. Introduction to Spectroscopy. 3rd ed. United States: Thomson Brooks/Cole; 2001. p. 358

[14] ICH Harmonized Tripartite Guideline, Text on Validation of Analytical Procedure (Q2A). London, UK: EMEA; 1994

[15] Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry. 4th ed. Vol. 2. London, UK; 2007. pp. 282-288

[16] Garatt DC. The Quantitative Analysis of Drugs. 3rd ed. CBS publishers and Distributors; 2005. p. 876

[17] British Pharmacopoeia. The British pharmacopoeia commission offices, London. 2008;**1&2**:685-686

[18] Indian Pharmacopoeia. The Controlled Publication, New Delhi. Vol. 2. 2007. pp. 402-403

*Preparation and* in vitro *Characterisation of Solid Dispersion Floating Tablet… DOI: http://dx.doi.org/10.5772/intechopen.92187*

[19] Sharma BK. Instrumental Methods of Chemical Analysis. 23rd ed. Goel Publishing House; 2004. pp. 292-302

**References**

com/diclofenac.html

*Molecular Pharmacology*

[1] Available from: http://www.drugs.

floating matrix tablets and evaluation of

International Journal of Pharmaceutics.

[9] Chanvanpatil M, Jain P, Chaudhari S, Shear R, Vavia P. Novel sustained release, swellable and bio adhesive gastro retentive drug delivery system for ofloxacin. International Journal of Pharmaceutics. 2006;**316**:86-92

[10] United State Pharmacopoeia 31. The National Formulary. Asian Edition. 26 (I). North Bethesda, Maryland, United States: The Board of Trustees; 2008.

[11] Seedher N, Bhatia S. Solubility enhancement of cox-2 inhibitors using

[12] Instruction manual pharmaspec. UV-1700 Series. User's System Guide. Kyoto Japan: Shimadzu Corporation;

various solvent systems. AAPS PharmSciTech. 2003;**4**(3):33

[13] Pavia LK. Introduction to Spectroscopy. 3rd ed. United States: Thomson Brooks/Cole; 2001. p. 358

[14] ICH Harmonized Tripartite Guideline, Text on Validation of Analytical Procedure (Q2A). London,

[15] Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry. 4th ed. Vol. 2. London, UK; 2007. pp. 282-288

publishers and Distributors; 2005. p. 876

[17] British Pharmacopoeia. The British pharmacopoeia commission offices,

[16] Garatt DC. The Quantitative Analysis of Drugs. 3rd ed. CBS

London. 2008;**1&2**:685-686

Vol. 2. 2007. pp. 402-403

[18] Indian Pharmacopoeia. The Controlled Publication, New Delhi.

UK: EMEA; 1994

their gastric residence time.

2000;**195**:125-135

pp. 297-298

[2] Rao GK, Mandapalli PK, Manthri R, Reddy VP. Development and in vivo evaluation of gastro retentive delivery systems for cefuroxime axetil. Saudi Pharmaceutical Journal. 2013;**21**(1): 53-59. DOI: 10.1016/j.jsps.2012.01.003

[3] Chau Le-Ngoc VO, Chulhun P, Beom-Jin L. Current trends and future perspectives of solid dispersions containing poorly water-soluble drugs review article. European Journal of Pharmaceutics and Biopharmaceutics.

[4] Giri TK, Kumar K, Alexander A, Ajazuddin, Badwaik H, Tripathi DK. A novel and alternative approach to controlled release drug delivery system based on solid dispersion technique review article. Bulletin of Faculty Pharmacy, Cairo University. 2012;**50**(2):

[5] Willis JV, Kendall MJ, Flinn RM, Thornill DP, Welling PG. The

[6] Sharma N, Jain N, Sudhakar CK, Jain S. Formulation and evaluation of gastro retentive floating tablets containing cefpodoxim proxetil solid dispersions. International Journal of Current Pharmaceutical Research. 2012;

[7] Shivakumar HN, Desai BG,

dispersions by response surface

Deshmukh G. Design and optimization of diclofenac sodium controlled release solid

methodology. Indian Journal of Pharmaceutical Sciences. 2008;**70**(1):22-30

[8] Baumgartner S, Kristl J, Vrecer F, Vodopivec P, Zorko B. Optimization of

following intravenous and oral administration. European Journal of Clinical Pharmacology. 1979;**16**(6):

pharmacokinetics of diclofenac sodium

2013;**85**(3) Part B:799-813

147-159

405-410

**4**(4):82-87

**44**

[20] Barzegar-Jalali M, Alaei-Beirami M, Javadzadeh Y, Mohammadi G, Hamidi A, Andalib S, et al. Comparison of physicochemical characteristics and drug release of diclofenac sodiumeudragit® RS100 nanoparticles and solid dispersions. Powder Technology. 2012;**219**:211-216

[21] Davis SS, Stockwel AF, Taylor MJ, Hardy JG, Whalley DR, Wilson CG. The effect of density on the gastric emptying of single and multiple unit dosage forms. Pharmaceutical Research. 1986;**3**: 208-213

[22] Rosa M, Zia H, Rhodes T. Design and testing in vitro of a bioadhesive and floating drug delivery system for oral application. International Journal of Pharmaceutics. 1994;**105**:65-70

[23] Li S, Lin S, Daggy BP, Mirchandani HL, Chien YW. Effect of HPMC and carbopol on the release and floating properties of gastric floating drug delivery system using factorial design. International Journal of Pharmaceutics. 2003;**253**:13-22

[24] Machida Y, Inouy K, Tokumura T, Iwata M, Nagai T. Preparation and evaluation of intragastric buoyant preparations. Drug Design and Delivery. 1989;**4**:155-161

[25] Moes AJ. Gastro retentive dosage forms. Critical Reviews in Therapeutic Drug Carrier Systems. 1993;**10**:143-159

[26] Dhumal RS, Rajmane ST, Dhumal ST, Pawar AP. Design and evaluation of bi-layered floating tablets of cefuroxime axetil for bimodal release. Journal of Scientific and Industrial Research. 2006;**65**:812-816

[27] Patel VF, Patel NM. Intragastricoating drug delivery system of cefuroxime axetil: In vitro evaluation. AAPS PharmSciTech. 2006;**7**:E1-E7

[28] Yoshika S, Stell VJ. Stability of Drugs and Dosage Forms. 2nd ed. New Delhi: Springer Private Ltd; 2006. pp. 151-186

[29] Garret NM. Stability studies of floating drug delivery dosage form. Journal of Pharmaceutical Sciences. 1965;**54**:1557

[30] Gennaro AR. Remington: Pharmaceutical Sciences and Practice of Pharmacy. 20th ed. Vol. 1. Philadelphia: Lippincott Williams & Wilkins; 1990. p. 986

Section 2

Pharmaceutical Dosage

Forms

**47**

Section 2

## Pharmaceutical Dosage Forms

**Chapter 3**

**Abstract**

**1. Introduction**

healthcare system [4].

**2. History**

**49**

Advances

*and Marilena Vlachou*

3D-Printed Modified-Release

Tablets: A Review of the Recent

*Angeliki Siamidi, Eleni Tsintavi, Dimitrios M. Rekkas*

The broad spectrum of applications of three-dimensional printing (3D printing,

3D printing is an object fabrication technique based on the sequential deposition of layers of materials. Using a computer-aided design (CAD) software, structures of various sizes and shapes can be produced. This method has found application in many sectors, from industrial engineering to personalized biomaterials and devices in medicine [1, 2]. Within the pharmaceutical field, 3D printing can produce small batches of medicinal products, with tailored dosages, shapes, sizes, and release characteristics [3]. These advantages of 3D printing facilitate the efforts towards personalized therapies. The need for the modification of a dose that will fit better a patient's individual needs arises from differences in the patient's age, weight, and severity of disease(s) [4]. Even though there are great advancements in drug administration methods, the orally administered drugs remain the most preferred choice by patients due to the fact that it is relatively safe, very convenient and costeffective. The preference on oral solid pharmaceutical forms, especially tablets, has rendered the personalization of oral solid dosage forms a step forward in the

The increasing applications of 3D printing have made it a well-accepted concept

at present times. Charles Hull is considered the pioneer of 3D printing, as he

pharmaceutics, the main idea behind 3D printing products is to design and develop delivery systems that are suited to an individual's needs. In this way, the size, appearance, shape, and rate of delivery of a wide array of medicines could be easily adjusted. The aim of this chapter is to provide a compilation of the 3D printing techniques, used for the fabrication of oral drug delivery systems, and review the relevant scientific

3DP) has attracted the attention of researchers working in diverse fields. In

developments in particular those with modified-release characteristics.

**Keywords:** 3D printing, modified release, oral drug delivery, tablets

#### **Chapter 3**

## 3D-Printed Modified-Release Tablets: A Review of the Recent Advances

*Angeliki Siamidi, Eleni Tsintavi, Dimitrios M. Rekkas and Marilena Vlachou*

#### **Abstract**

The broad spectrum of applications of three-dimensional printing (3D printing, 3DP) has attracted the attention of researchers working in diverse fields. In pharmaceutics, the main idea behind 3D printing products is to design and develop delivery systems that are suited to an individual's needs. In this way, the size, appearance, shape, and rate of delivery of a wide array of medicines could be easily adjusted. The aim of this chapter is to provide a compilation of the 3D printing techniques, used for the fabrication of oral drug delivery systems, and review the relevant scientific developments in particular those with modified-release characteristics.

**Keywords:** 3D printing, modified release, oral drug delivery, tablets

#### **1. Introduction**

3D printing is an object fabrication technique based on the sequential deposition of layers of materials. Using a computer-aided design (CAD) software, structures of various sizes and shapes can be produced. This method has found application in many sectors, from industrial engineering to personalized biomaterials and devices in medicine [1, 2]. Within the pharmaceutical field, 3D printing can produce small batches of medicinal products, with tailored dosages, shapes, sizes, and release characteristics [3]. These advantages of 3D printing facilitate the efforts towards personalized therapies. The need for the modification of a dose that will fit better a patient's individual needs arises from differences in the patient's age, weight, and severity of disease(s) [4]. Even though there are great advancements in drug administration methods, the orally administered drugs remain the most preferred choice by patients due to the fact that it is relatively safe, very convenient and costeffective. The preference on oral solid pharmaceutical forms, especially tablets, has rendered the personalization of oral solid dosage forms a step forward in the healthcare system [4].

#### **2. History**

The increasing applications of 3D printing have made it a well-accepted concept at present times. Charles Hull is considered the pioneer of 3D printing, as he

developed, patented, and commercialized the first equipment for the 3D printing of objects in 1983. Hull's 3D printing technique was based on stereolithography. It consisted of a laser that moved across the surface of a liquid resin, curing it. This process was repeated layer by layer many times until the desired shape was formed. In 1988, Charles Deckard filed a patent for selective laser sintering. In this process, a laser beam is scanned over a powder bed to sinter or fuse the powder that is placed on a powder bed. The powder bed is then lowered, fresh powder is spread, and the process is repeated to produce a solid object. The un-bonded is then removed, and the structure can be further treated, for example, with heat, to enforce the bonding. In 1989, Scott Crump filed a patent on fused deposition modeling. Using this technique, the object is formed by depositing layers of solidifying materials (selfhardening waxes, thermoplastic resins, and molten metals) until the desired shape is formed [5, 6]. In 2015, the FDA approved Spritam®, the first 3D-printed prescription drug product to treat partial onset seizures, myoclonic seizures, and primary generalized tonic-clonic seizures. Since then, many innovations have been evolved using the 3DP technology.

#### **3. Advantages and limitations**

The oral dosage form production by the 3DP processes has many advantages specially for customizing drug delivery. The active ingredient can be included in the dosage form as per each patient requirements to achieve a personalized dose and release pattern. 3D printing aids also in achieving multidrug combinations with complex release profiles [7]. On-demand production and tailor-made products with specific geometries, designs, and shape forms can be achieved which otherwise would be difficult with the conventional tableting. Even though there has been intense research to circumvent the 3D printing flaws, this new technology has still some limitations. Few 3D printing techniques may produce relatively porous structures and uneven shapes of dosage forms [6]. When fused deposition modeling technique is utilized, the use of only thermostable drugs and the few available compatible excipients is a limiting step. Also, with stereolithography, the challenge lies on the potential drug degradation due to the exposure to UV light that induces polymerization reaction [5].

printing techniques categorized by the raw materials employed. Among them, stereolithography, selective laser sintering, binder jetting, and fused deposition modeling are the most used techniques in the literature for the production of

*3D-Printed Modified-Release Tablets: A Review of the Recent Advances*

*DOI: http://dx.doi.org/10.5772/intechopen.90868*

Stereolithography employs raw materials in the liquid form, such as photosensitive/photopolymerizable liquid resins. A high-energy light source like ultraviolet irradiation solidifies the liquid resins, creating the 3D object [6]. Among the advantages of this technique are the high accuracy and good surface quality of the object. This method has been widely used for implant design and manufacture as well as for creating accurate 3D models acquired from various anatomical scans

Selective laser sintering (SLS) technique utilizes raw materials in a powder form, and the laser used melts and bonds the layers of material powders together. SLS

On the other hand, binder jetting technique, also called drop-on-demand inkjet

printing or 3D printing, is able to create 3D objects from powder materials by depositing liquid binder droplets onto a powder substrate and sticking the particles together [9, 10]. This technique along with the continuous inkjet printing belongs to the printing-based inkjet systems and has been utilized for the fabrication of implants and solid dosage forms, such as the first commercially printed tablet

Finally, the most widely spread technique is the fused deposition modeling (FDM), and it belongs to the nozzle-based printing techniques. FDM is characterized by the use of thermoplastic polymers that pass through a pre-heated printing head and is melted and extruded through a precise nozzle with a specific diameter. In contact with the cold printing surface, the polymers solidify and thus create the 3D object. A variation of this type of 3D printing technique is the semisolid extrusion system, in which semiconductors (gels, ointments) are printed through a syringe-shaped extruder [10]. In the recent years, these techniques have been extensively used for the research and development of various pharmaceutical forms

**Figure 3** depicts the main additive manufacturing technologies which either experimentally or industrially have been used for the manufacturing of pharma-

pharmaceutical dosage forms [10].

*3D printing technique categorization.*

has been used for the manufacturing of artificial tissue.

such as hydrogels or coated solid dosage forms [11].

of a patient.

**Figure 2.**

Spritam®.

ceutical dosage forms.

**51**

#### **4. Various techniques used in 3D printing**

Irrespective of the 3D printing technique employed, the process follows three basic steps: the creation of a computer-aided design file; followed by its transformation to a rapid prototyping stereolithography file (.stl), which describes the surface geometry of the 3D object; and finally, its conversion to a machine specific code (.gcode) which is recognized by the 3D printer machine and creates the final object [8] (**Figure 1**).

There are various ways to classify the 3D techniques, according to the additive process followed, the form of the raw materials used, the mechanism of layering, or even the kind of printing heads utilized [9]. **Figure 2** illustrates the different 3D

**Figure 1.** *The basic steps of 3D printing process.*

*3D-Printed Modified-Release Tablets: A Review of the Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.90868*

#### **Figure 2.** *3D printing technique categorization.*

developed, patented, and commercialized the first equipment for the 3D printing of objects in 1983. Hull's 3D printing technique was based on stereolithography. It consisted of a laser that moved across the surface of a liquid resin, curing it. This process was repeated layer by layer many times until the desired shape was formed. In 1988, Charles Deckard filed a patent for selective laser sintering. In this process, a laser beam is scanned over a powder bed to sinter or fuse the powder that is placed on a powder bed. The powder bed is then lowered, fresh powder is spread, and the process is repeated to produce a solid object. The un-bonded is then removed, and the structure can be further treated, for example, with heat, to enforce the bonding. In 1989, Scott Crump filed a patent on fused deposition modeling. Using this technique, the object is formed by depositing layers of solidifying materials (selfhardening waxes, thermoplastic resins, and molten metals) until the desired shape is formed [5, 6]. In 2015, the FDA approved Spritam®, the first 3D-printed prescription drug product to treat partial onset seizures, myoclonic seizures, and primary generalized tonic-clonic seizures. Since then, many innovations have been

The oral dosage form production by the 3DP processes has many advantages specially for customizing drug delivery. The active ingredient can be included in the dosage form as per each patient requirements to achieve a personalized dose and release pattern. 3D printing aids also in achieving multidrug combinations with complex release profiles [7]. On-demand production and tailor-made products with specific geometries, designs, and shape forms can be achieved which otherwise would be difficult with the conventional tableting. Even though there has been intense research to circumvent the 3D printing flaws, this new technology has still some limitations. Few 3D printing techniques may produce relatively porous structures and uneven shapes of dosage forms [6]. When fused deposition modeling technique is utilized, the use of only thermostable drugs and the few available compatible excipients is a limiting step. Also, with stereolithography, the challenge lies on the potential drug degradation due to the exposure to UV light that induces

Irrespective of the 3D printing technique employed, the process follows three basic steps: the creation of a computer-aided design file; followed by its transformation to a rapid prototyping stereolithography file (.stl), which describes the surface geometry of the 3D object; and finally, its conversion to a machine specific code (.gcode) which is recognized by the 3D printer machine and creates the final

There are various ways to classify the 3D techniques, according to the additive process followed, the form of the raw materials used, the mechanism of layering, or even the kind of printing heads utilized [9]. **Figure 2** illustrates the different 3D

evolved using the 3DP technology.

*Molecular Pharmacology*

**3. Advantages and limitations**

polymerization reaction [5].

object [8] (**Figure 1**).

*The basic steps of 3D printing process.*

**Figure 1.**

**50**

**4. Various techniques used in 3D printing**

printing techniques categorized by the raw materials employed. Among them, stereolithography, selective laser sintering, binder jetting, and fused deposition modeling are the most used techniques in the literature for the production of pharmaceutical dosage forms [10].

Stereolithography employs raw materials in the liquid form, such as photosensitive/photopolymerizable liquid resins. A high-energy light source like ultraviolet irradiation solidifies the liquid resins, creating the 3D object [6]. Among the advantages of this technique are the high accuracy and good surface quality of the object. This method has been widely used for implant design and manufacture as well as for creating accurate 3D models acquired from various anatomical scans of a patient.

Selective laser sintering (SLS) technique utilizes raw materials in a powder form, and the laser used melts and bonds the layers of material powders together. SLS has been used for the manufacturing of artificial tissue.

On the other hand, binder jetting technique, also called drop-on-demand inkjet printing or 3D printing, is able to create 3D objects from powder materials by depositing liquid binder droplets onto a powder substrate and sticking the particles together [9, 10]. This technique along with the continuous inkjet printing belongs to the printing-based inkjet systems and has been utilized for the fabrication of implants and solid dosage forms, such as the first commercially printed tablet Spritam®.

Finally, the most widely spread technique is the fused deposition modeling (FDM), and it belongs to the nozzle-based printing techniques. FDM is characterized by the use of thermoplastic polymers that pass through a pre-heated printing head and is melted and extruded through a precise nozzle with a specific diameter. In contact with the cold printing surface, the polymers solidify and thus create the 3D object. A variation of this type of 3D printing technique is the semisolid extrusion system, in which semiconductors (gels, ointments) are printed through a syringe-shaped extruder [10]. In the recent years, these techniques have been extensively used for the research and development of various pharmaceutical forms such as hydrogels or coated solid dosage forms [11].

**Figure 3** depicts the main additive manufacturing technologies which either experimentally or industrially have been used for the manufacturing of pharmaceutical dosage forms.

**Release behavior\***

Modified Dual-

compartment tablet

*DOI: http://dx.doi.org/10.5772/intechopen.90868*

Threecompartment tablet

Tablets of various shapes

Rifampicin and isoniazid

*3D-Printed Modified-Release Tablets: A Review of the Recent Advances*

Caplets Budesonide Polyvinyl alcohol,

and 4-aminosalicylic

and paracetamol

Extended Tablets Acetaminophen Benecel™ HPMC E5

nifedipine, and glipizide

Sustained Polypill Captopril,

**53**

Tablets Theophylline Eudragit RL 100, RS

Tablets Acetaminophen Methocel™ E50,

Tablets 5-Aminosalicylic acid

acid

Tablets 4-Aminosalicylic acid

Caplets Paracetamol or caffeine

**Dosage form API(s) Excipient(s) Technique Ref.**

mannitol

Hydrochlorothiazide Partially hydrolyzed

Polyethylene oxide, polylactic acid (PLA), polyvinyl alcohol (PVA)

PVA (Mowiol®4–88),

Eudragit® L 100, triethyl citrate, talc, isopropanol-water solution

Polyethylene glycol diacrylate, diphenyl

trimethylbenzoyl) phosphine oxide, and (PEG 300)

polyvinylpyrrolidone (Povidone K30), ethyl cellulose, Eudragit® RS 100, stearic acid, sodium lauryl sulfate, fluorescein, colloidal silicon dioxide

and Aqualon™ EC N14 with either Klucel™ HPC EF and LF, Soluplus®, or Eudragit® L 100

100, and E and hydroxypropyl cellulose (SSL grade), triethyl citrate

Tablets Prednisolone Polyvinyl alcohol FDM [26] Tablets Fluorescein Polyvinyl alcohol FDM [27]

> Hydroxypropyl methylcellulose (HPMC 2208), polyethylene glycol (PEG 6000),

Controlled Tablets Fenofibrate White beeswax Inkjet printing [28]

(2,4,6-

Paracetamol Polyvinyl alcohol FDM/HME [18]

FDM/HME [16]

FDM/HME [17]

FDM/HME and fluid bed coating

Stereolithography [22]

Inkjet printing [23]

FDM/HME [24]

FDM/HME [25]

Extrusion [29]

Polyvinyl alcohol FDM [20]

Polyvinyl alcohol FDM [21]

[19]

**Figure 3.** *Basic 3D printing techniques deployed for the manufacturing of pharmaceutical dosage forms.*

#### **5. Recent accomplishments in modified-release 3D-printed tablets**

Orodispersible, sublingual, fast-dissolving drug delivery formulations that rapidly disintegrate in the oral cavity or immediate-release tablets by 3D printing have been produced [12–15].

Multipurpose therapeutic systems offering tailored combinations of drugs, drug doses, and the desired release kinetic properties have attracted increasing attention, due to the advantages that these personalized pharmaceutical products could offer. In this respect, many scientists have designed modified-release oral dosage medicines, using 3D printing. The drug release from modified-release formulations is changed on purpose from that of an immediate-release formulation to achieve a preferred therapeutic goal. The applications of 3DP on modified *per oral* drug delivery are summarized in **Table 1**.

Genina et al. [16] have shown that coupling fused deposition modeling 3D printing with the hot-melt extrusion offers a new method for manufacturing tailored-dosage medicines, with modified-release properties. In detail an oral dualcompartmental dosage unit (dcDU) has been designed, and the in vitro and in vivo release profiles of an antitubercular drug combination of rifampicin and isoniazid have been evaluated. These two active ingredients are considered as first-line therapy for tuberculosis but interact negatively with each other upon simultaneous release in acidic environment. This was circumvented by the compartmentalization of rifampicin and isoniazid into sealable compartments of 3D-printed dualcompartmental dosage units. This novel delivery system was characterized with focus on microscopic verification of the designed attributes, the modulation of drug release from dcDUs, and the pharmacokinetic profile of dcDUs in rats. In another study [17] an oral solid dosage form was developed by employing the fused deposition modeling, using a custom-built filament consisted of polyvinyl alcohol, mannitol, and hydrochlorothiazide, as a model drug, co-formulated via hot-melt extrusion. The dissolution studies performed demonstrated zero-order release kinetics. In another study [18], hot-melt extrusion and fused deposition modeling were used to produce different shaped tablets (cube, pyramid, cylinder, sphere, and torus) containing acetaminophen. It was found that drug's release was not


*3D-Printed Modified-Release Tablets: A Review of the Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.90868*

**5. Recent accomplishments in modified-release 3D-printed tablets**

*Basic 3D printing techniques deployed for the manufacturing of pharmaceutical dosage forms.*

been produced [12–15].

*Molecular Pharmacology*

**Figure 3.**

**52**

delivery are summarized in **Table 1**.

Orodispersible, sublingual, fast-dissolving drug delivery formulations that rapidly disintegrate in the oral cavity or immediate-release tablets by 3D printing have

Multipurpose therapeutic systems offering tailored combinations of drugs, drug doses, and the desired release kinetic properties have attracted increasing attention, due to the advantages that these personalized pharmaceutical products could offer. In this respect, many scientists have designed modified-release oral dosage medicines, using 3D printing. The drug release from modified-release formulations is changed on purpose from that of an immediate-release formulation to achieve a preferred therapeutic goal. The applications of 3DP on modified *per oral* drug

Genina et al. [16] have shown that coupling fused deposition modeling 3D printing with the hot-melt extrusion offers a new method for manufacturing tailored-dosage medicines, with modified-release properties. In detail an oral dualcompartmental dosage unit (dcDU) has been designed, and the in vitro and in vivo release profiles of an antitubercular drug combination of rifampicin and isoniazid have been evaluated. These two active ingredients are considered as first-line therapy for tuberculosis but interact negatively with each other upon simultaneous release in acidic environment. This was circumvented by the compartmentalization

of rifampicin and isoniazid into sealable compartments of 3D-printed dualcompartmental dosage units. This novel delivery system was characterized with focus on microscopic verification of the designed attributes, the modulation of drug release from dcDUs, and the pharmacokinetic profile of dcDUs in rats. In another study [17] an oral solid dosage form was developed by employing the fused deposition modeling, using a custom-built filament consisted of polyvinyl alcohol, mannitol, and hydrochlorothiazide, as a model drug, co-formulated via hot-melt extrusion. The dissolution studies performed demonstrated zero-order release kinetics. In another study [18], hot-melt extrusion and fused deposition modeling were used to produce different shaped tablets (cube, pyramid, cylinder, sphere, and

torus) containing acetaminophen. It was found that drug's release was not


dependent on the tablet surface area, but on the surface-area-to-volume ratio, indicating the effect of the shape on the release profile. The results showed that the tablets of similar mass showed little difference in dissolution profiles that could be explained by the erosion-mediated process that controlled the drug release. Tablets of various shapes may alter the drug dissolution profiles and can aid in the design of new dosage forms with specific pharmacokinetic characteristic targeted to different sites in the gastrointestinal track. Fused deposition 3D printing technology alongside with hot-melt extrusion and fluid bed coating was used to fabricate modifiedrelease budesonide dosage forms. The drug was loaded into polyvinyl alcohol filaments which were then engineered into capsule-shaped tablets and coated with a layer of enteric polymer. The dissolution studies showed that the drug release from the caplet formulation started at the small intestine and continued in a sustained manner throughout the large intestine and colon [19]. The same group of researchers has also produced tablets containing as model drugs the two

*An overview of the 3DP technique applications in modified per oral drug delivery (FDM, fused deposition*

Tablets Diclofenac Avicel PH301, lactose,

Tablets Diclofenac Eudragit® E 100 and L

**Dosage form API(s) Excipient(s) Technique Ref.**

(Povidon K30), hydroxypropyl methylcellulose (Methocel™ K100MCR), lactose

Microcrystalline cellulose powder, Eudragit® E 100,

Eudragit® L 100,

Inkjet [36]

Inkjet [36]

Inkjet [36]

RLPO

100

**Release behavior\***

Immediate/ extended

Enteric dual pulsatory

Dual pulsatory

*\**

**55**

**Table 1.**

Tablets Chlorpheniramine maleate

*3D-Printed Modified-Release Tablets: A Review of the Recent Advances*

*The release behavior reported as defined by the author.*

*DOI: http://dx.doi.org/10.5772/intechopen.90868*

*modeling; HME, hot-melt extrusion).*

aminosalicylic acid isomers, 5-aminosalicylic acid and 4-aminosalicylic acid, using fused deposition modeling. The results indicated that the release profiles obtained could be easily modified by the proper selection of the printing parameters [20]. Furthermore, fused deposition modeling was used to produce acetaminophen or caffeine caplets from polyvinyl alcohol filaments. The dissolution tests performed in biorelevant bicarbonate media revealed distinctive modified-release profiles, which were dependent on drug solubility and drug loading. The results indicated that the drug release can be faster from formulations incorporating the drug with higher solubility and higher loading [21]. Additionally, Wang et al. [22] managed to formulate modified-release tablets of paracetamol and 4-aminosalicylic acid using polyethylene glycol diacrylate as monomer and diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide as a photoinitiator in stereolithographic 3D printing. Also, in another study [23], researchers employed the powder bed/jetting method to construct a methacrylic or ethylcellulose matrix tablet to achieve a modified release of acetaminophen. Erosion and in vitro dissolution studies in ethylcellulose-containing tablets indicated that the drug was released via a two-dimensional surface erosion mechanism and 98% of the drug could be released linearly in 12 h. Tablets with other release-retardation materials, such as sodium lauryl sulfate, stearic acid, and


#### *3D-Printed Modified-Release Tablets: A Review of the Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.90868*

#### **Table 1.**

*\**

pulsatory

*The release behavior reported as defined by the author.*

**Release behavior\***

*Molecular Pharmacology*

Delayed DuoCaplet Paracetamol and

Shell-core tablets

compartment capsular device

Pulsatile Two-

Immediate /sustained

**54**

caffeine

Theophylline, budesonide, and diclofenac sodium

Tablets Paracetamol Hypromellose acetate

**Dosage form API(s) Excipient(s) Technique Ref.**

acetate

MG, HG), methylparaben NF grade, magnesium stearate

Core:

and talc

Blue and

Bilayer tablet Guaifenesin Hydroxypropyl

hydrochlorothiazide, ramipril, pravastatin sodium, atenolol

Polypill Aspirin,

Kollicoat®IRyellow)

Cellulose acetate, Dmannitol, polyethylene glycol (PEG 6000) sodium starch glycolate,

Polyvinylpyrrolidone

methylcellulose (HPMC 2910 & 2208), poly(acrylic acid), microcrystalline cellulose, sodium starch glycolate

polyvinyl alcohol, hydroxypropyl methylcellulose (HPMC), HPMC acetate succinate, polyvinyl alcoholpolyethylene glycol graft copolymer, glycerol, polyethylene glycol (PEG 400, PEG8000), blue and yellow dye-containing formulations (Kollicoat®IR Brilliant

Acetaminophen Polylactic acid,

tromethamine, lactose, sodium chloride, D-mannitol,

croscarmellose sodium, microcrystalline cellulose, sodium starch glycolate, hydroxypropyl methylcellulose (Methocel™), cellulose

succinate (HPMC LG,

Polyvinylpyrrolidone, triethyl citrate, talc or tribasic phosphate sodium, and API Shell: Eudragit® L 100–55, triethyl citrate,

Polyvinyl alcohol FDM/HME [30]

FDM/HME [31]

Dual FDM/HME [32]

Extrusion [34]

Extrusion [35]

[33]

FDM/HME injection molding *An overview of the 3DP technique applications in modified per oral drug delivery (FDM, fused deposition modeling; HME, hot-melt extrusion).*

100

dependent on the tablet surface area, but on the surface-area-to-volume ratio, indicating the effect of the shape on the release profile. The results showed that the tablets of similar mass showed little difference in dissolution profiles that could be explained by the erosion-mediated process that controlled the drug release. Tablets of various shapes may alter the drug dissolution profiles and can aid in the design of new dosage forms with specific pharmacokinetic characteristic targeted to different sites in the gastrointestinal track. Fused deposition 3D printing technology alongside with hot-melt extrusion and fluid bed coating was used to fabricate modifiedrelease budesonide dosage forms. The drug was loaded into polyvinyl alcohol filaments which were then engineered into capsule-shaped tablets and coated with a layer of enteric polymer. The dissolution studies showed that the drug release from the caplet formulation started at the small intestine and continued in a sustained manner throughout the large intestine and colon [19]. The same group of researchers has also produced tablets containing as model drugs the two aminosalicylic acid isomers, 5-aminosalicylic acid and 4-aminosalicylic acid, using fused deposition modeling. The results indicated that the release profiles obtained could be easily modified by the proper selection of the printing parameters [20]. Furthermore, fused deposition modeling was used to produce acetaminophen or caffeine caplets from polyvinyl alcohol filaments. The dissolution tests performed in biorelevant bicarbonate media revealed distinctive modified-release profiles, which were dependent on drug solubility and drug loading. The results indicated that the drug release can be faster from formulations incorporating the drug with higher solubility and higher loading [21]. Additionally, Wang et al. [22] managed to formulate modified-release tablets of paracetamol and 4-aminosalicylic acid using polyethylene glycol diacrylate as monomer and diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide as a photoinitiator in stereolithographic 3D printing. Also, in another study [23], researchers employed the powder bed/jetting method to construct a methacrylic or ethylcellulose matrix tablet to achieve a modified release of acetaminophen. Erosion and in vitro dissolution studies in ethylcellulose-containing tablets indicated that the drug was released via a two-dimensional surface erosion mechanism and 98% of the drug could be released linearly in 12 h. Tablets with other release-retardation materials, such as sodium lauryl sulfate, stearic acid, and

Eudragit RS-100, showed similar release-retardation effects by different release mechanisms.

HPMCAS-based tablets showed initial delayed release in the gastric medium and in the intestinal conditions, and the drug release was faster from the tablets prepared using polymers with a lower pH threshold. These results confirm that the fused deposition modeling 3DP makes possible the production of delayed-release printlets, without the need of enteric coating. Okwuosa et al. [32] managed to fabricate shell-core delayed-release tablets of theophylline, budesonide, and diclofenac sodium with dual fused deposition modeling 3D printing and hot-melt extrusion. For the core structure, filaments consisting of the polymer (PVP), plasticizer (triethyl citrate), filler (talc) or tribasic phosphate sodium, and the active ingredient were created with hot-melt extrusion. While for the shell, Eudragit L100–55, triethyl citrate, and talc were used. The created filaments were then used for the printing of caplets containing the active ingredient in the core, while the shell serves as an enteric coating. This study demonstrated the potential of fabricating patient-specific pH-responsive tablets in one step. In another article, Maroni et al. [33] have reported on the manufacture of a two-compartment capsular device conveying incompatible drugs or differing drug formulations using the fused deposition modeling and injection molding. Through the assembly of compartments that had different wall thickness and/or composition, the drug release could be charac-

*3D-Printed Modified-Release Tablets: A Review of the Recent Advances*

*DOI: http://dx.doi.org/10.5772/intechopen.90868*

Khaled et al. [34] used the extrusion-based 3DP for the preparation of guaifenesin bilayer tablets with an immediate-release and a sustained-release layer. Drug release kinetics indicated Fickian diffusion drug release through the hydrated HPMC gel layer. The same group of researchers [35] used the same technique for the production of a novel complex geometry "five-in-one" polypill. The drugs, aspirin, hydrochlorothiazide, ramipril, and pravastatin atenolol, were physically separated in the polypill to avoid incompatibility issues and allow maximum flexibility. Release studies revealed immediate and sustained drug release mechanisms. A research group formulated immediate/extended-release tablets, which were composed of two drug-containing sections of different pH-based release mechanisms. The pulsed release of the model drug, chlorpheniramine maleate, took place after a lag time of 10 min followed by extended release of the compound over a period of 7 h. Furthermore, enteric dual pulsed-release tablets were constructed and the dissolution profiles showed that two pulses of diclofenac sodium, released, one immediately at t = 1 h and the second pulse began after a lag time of 4 h. The same group of researchers [36] also fabricated dual pulsed-release tablets, where one section eroded immediately in the acidic environment stage releasing diclofenac during the first 30 min, while the second section eroded 5 h later, at higher pH values.

The present chapter offers a review of the 3D-printed modified-release oral solid pharmaceutical formulations that have been created up to date. It aims at demonstrating the potential role of this technology in the tailored manufacture of pharmaceutical products. Even though 3DP has been introduced since the 1980s, there is still a lot of exploration in this field, especially for the creation of materials suitable for pharmaceutical and medical applications. One of the ongoing researches in the area is the 3DP of new, versatile materials that have the ability to change their properties under the influence of external factors or over time. The structural modification over time or otherwise called the fourth dimension, created a new term called "4D printing" [37]. In oral dosage forms, this technology allows the modification of drug delivery, since the timely release profile can be triggered by

stimuli, such as pH, temperature, enzymes action, and time [38].

terized as pulsatile.

**6. Conclusions**

**57**

Zhang et al. fabricated solid-dispersion filaments with acetaminophen dissolved or dispersed in a polymer matrix by hot-melt extrusion technology, which was suitable for fused deposition modeling-based 3D printing. The 3D printed tablets showed more extended drug release rates than the directly compressed tablets [24]. In another study [25], using the same methods, scientists presented a flexible dose tablet system, suitable for both immediate and extended-release tablets. As excipients three methacrylic polymers (Eudragit RL, RS, and E) as well as a cellulosebased material (hydroxypropyl cellulose, HPC SSL) were used, while theophylline was used as a model drug substance. Moreover, in another report [26], the feasibility of using a fused deposition modeling-based 3D printer to fabricate extendedrelease tablets, using prednisolone loaded poly(vinyl alcohol) filaments, and to control its release was investigated. The results indicated that the in vitro drug release was extended up to 24 h, showing that the fused deposition modeling is a promising method to control the dose of extended-release tablets. Moreover in another work, polyvinyl alcohol filaments have been loaded with fluorescein as a model drug, by swelling of the polymer in ethanolic drug solution, and 10-mmdiameter tablets of polyvinyl alcohol/fluorescein using fused deposition modeling 3DP were printed. The dissolution tests that were conducted in modified Hank's buffer indicated controlled-release profiles [27].

Kyobula et al. [28] have prepared drug-loaded solid dosage forms with complex geometries such as honeycomb based, using hot-melt 3D inkjet printing. The model drug used was fenofibrate, and the relevant studies indicated controlled release. This study verified an alternative production approach for solid dosage forms with different geometry, which could achieve various release profiles for personalized drug products.

Khaled et al. [29] have employed 3D extrusion-based printing as a technique for the production of multi-active tablets with well-defined and separate controlledrelease profiles for three different drugs, namely, captopril, nifedipine, and glipizide. This "polypill" incorporated an osmotic pump for captopril and sustained release compartments for nifedipine and glipizide. The dissolution testing showed that the captopril portion exhibited the expected zero-order drug release from an osmotic pump, while the others showed either first-order release or Korsmeyer-Peppas release kinetics dependent on the active/excipient ratio used.

Goyanes et al. [30] used the fused deposition modeling 3D printing to fabricate novel oral drug delivery systems with paracetamol and caffeine filaments of poly (vinyl alcohol), with the intent of applying this process to the production of personalized products, tailored at the point of dispensing or use. The design included a multilayer device, with each layer containing drug, whose identity was different to the drug in the adjacent layers, and a two-compartment device, comprising of a caplet in caplet (DuoCaplet), with each compartment containing a different drug. The drug release tests in biorelevant bicarbonate media showed unique drug release profiles depending on the macrostructure of the devices. In the multilayer device incorporating two drugs, the drug release rate was similar for both drugs but faster when the drug loading was higher. In DuoCaplets the drug incorporated in the external layer was released first, and there was a lag time until the release of the drug contained in the core, depending on the characteristics of the external layer. Moreover, the same group [31] used the fused deposition modeling and the hotmelt extrusion to generate paracetamol-loaded tablets from filaments produced from three different grades of hypromellose acetate succinate. The dissolution tests showed that the drug release from the tablets depended on the polymer composition, the drug loading, and the internal structure of the formulations. Especially, all

#### *3D-Printed Modified-Release Tablets: A Review of the Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.90868*

HPMCAS-based tablets showed initial delayed release in the gastric medium and in the intestinal conditions, and the drug release was faster from the tablets prepared using polymers with a lower pH threshold. These results confirm that the fused deposition modeling 3DP makes possible the production of delayed-release printlets, without the need of enteric coating. Okwuosa et al. [32] managed to fabricate shell-core delayed-release tablets of theophylline, budesonide, and diclofenac sodium with dual fused deposition modeling 3D printing and hot-melt extrusion. For the core structure, filaments consisting of the polymer (PVP), plasticizer (triethyl citrate), filler (talc) or tribasic phosphate sodium, and the active ingredient were created with hot-melt extrusion. While for the shell, Eudragit L100–55, triethyl citrate, and talc were used. The created filaments were then used for the printing of caplets containing the active ingredient in the core, while the shell serves as an enteric coating. This study demonstrated the potential of fabricating patient-specific pH-responsive tablets in one step. In another article, Maroni et al. [33] have reported on the manufacture of a two-compartment capsular device conveying incompatible drugs or differing drug formulations using the fused deposition modeling and injection molding. Through the assembly of compartments that had different wall thickness and/or composition, the drug release could be characterized as pulsatile.

Khaled et al. [34] used the extrusion-based 3DP for the preparation of guaifenesin bilayer tablets with an immediate-release and a sustained-release layer. Drug release kinetics indicated Fickian diffusion drug release through the hydrated HPMC gel layer. The same group of researchers [35] used the same technique for the production of a novel complex geometry "five-in-one" polypill. The drugs, aspirin, hydrochlorothiazide, ramipril, and pravastatin atenolol, were physically separated in the polypill to avoid incompatibility issues and allow maximum flexibility. Release studies revealed immediate and sustained drug release mechanisms.

A research group formulated immediate/extended-release tablets, which were composed of two drug-containing sections of different pH-based release mechanisms. The pulsed release of the model drug, chlorpheniramine maleate, took place after a lag time of 10 min followed by extended release of the compound over a period of 7 h. Furthermore, enteric dual pulsed-release tablets were constructed and the dissolution profiles showed that two pulses of diclofenac sodium, released, one immediately at t = 1 h and the second pulse began after a lag time of 4 h. The same group of researchers [36] also fabricated dual pulsed-release tablets, where one section eroded immediately in the acidic environment stage releasing diclofenac during the first 30 min, while the second section eroded 5 h later, at higher pH values.

#### **6. Conclusions**

Eudragit RS-100, showed similar release-retardation effects by different release

or dispersed in a polymer matrix by hot-melt extrusion technology, which was suitable for fused deposition modeling-based 3D printing. The 3D printed tablets showed more extended drug release rates than the directly compressed tablets [24]. In another study [25], using the same methods, scientists presented a flexible dose tablet system, suitable for both immediate and extended-release tablets. As excipients three methacrylic polymers (Eudragit RL, RS, and E) as well as a cellulosebased material (hydroxypropyl cellulose, HPC SSL) were used, while theophylline was used as a model drug substance. Moreover, in another report [26], the feasibility of using a fused deposition modeling-based 3D printer to fabricate extendedrelease tablets, using prednisolone loaded poly(vinyl alcohol) filaments, and to control its release was investigated. The results indicated that the in vitro drug release was extended up to 24 h, showing that the fused deposition modeling is a promising method to control the dose of extended-release tablets. Moreover in another work, polyvinyl alcohol filaments have been loaded with fluorescein as a model drug, by swelling of the polymer in ethanolic drug solution, and 10-mmdiameter tablets of polyvinyl alcohol/fluorescein using fused deposition modeling 3DP were printed. The dissolution tests that were conducted in modified Hank's

buffer indicated controlled-release profiles [27].

Zhang et al. fabricated solid-dispersion filaments with acetaminophen dissolved

Kyobula et al. [28] have prepared drug-loaded solid dosage forms with complex geometries such as honeycomb based, using hot-melt 3D inkjet printing. The model drug used was fenofibrate, and the relevant studies indicated controlled release. This study verified an alternative production approach for solid dosage forms with different geometry, which could achieve various release profiles for personalized

Khaled et al. [29] have employed 3D extrusion-based printing as a technique for the production of multi-active tablets with well-defined and separate controlledrelease profiles for three different drugs, namely, captopril, nifedipine, and

glipizide. This "polypill" incorporated an osmotic pump for captopril and sustained release compartments for nifedipine and glipizide. The dissolution testing showed that the captopril portion exhibited the expected zero-order drug release from an osmotic pump, while the others showed either first-order release or Korsmeyer-

Goyanes et al. [30] used the fused deposition modeling 3D printing to fabricate novel oral drug delivery systems with paracetamol and caffeine filaments of poly (vinyl alcohol), with the intent of applying this process to the production of personalized products, tailored at the point of dispensing or use. The design included a multilayer device, with each layer containing drug, whose identity was different to the drug in the adjacent layers, and a two-compartment device, comprising of a caplet in caplet (DuoCaplet), with each compartment containing a different drug. The drug release tests in biorelevant bicarbonate media showed unique drug release profiles depending on the macrostructure of the devices. In the multilayer device incorporating two drugs, the drug release rate was similar for both drugs but faster when the drug loading was higher. In DuoCaplets the drug incorporated in the external layer was released first, and there was a lag time until the release of the drug contained in the core, depending on the characteristics of the external layer. Moreover, the same group [31] used the fused deposition modeling and the hotmelt extrusion to generate paracetamol-loaded tablets from filaments produced from three different grades of hypromellose acetate succinate. The dissolution tests showed that the drug release from the tablets depended on the polymer composition, the drug loading, and the internal structure of the formulations. Especially, all

Peppas release kinetics dependent on the active/excipient ratio used.

mechanisms.

*Molecular Pharmacology*

drug products.

**56**

The present chapter offers a review of the 3D-printed modified-release oral solid pharmaceutical formulations that have been created up to date. It aims at demonstrating the potential role of this technology in the tailored manufacture of pharmaceutical products. Even though 3DP has been introduced since the 1980s, there is still a lot of exploration in this field, especially for the creation of materials suitable for pharmaceutical and medical applications. One of the ongoing researches in the area is the 3DP of new, versatile materials that have the ability to change their properties under the influence of external factors or over time. The structural modification over time or otherwise called the fourth dimension, created a new term called "4D printing" [37]. In oral dosage forms, this technology allows the modification of drug delivery, since the timely release profile can be triggered by stimuli, such as pH, temperature, enzymes action, and time [38].

*Molecular Pharmacology*

#### **Conflict of interest**

The authors declare no conflict of interest.

**References**

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*DOI: http://dx.doi.org/10.5772/intechopen.90868*

*3D-Printed Modified-Release Tablets: A Review of the Recent Advances*

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[6] Pravin S, Sudhir A. Integration of 3D printing with dosage forms: A new perspective for modern healthcare. Biomedicine and Pharmacotherapy. 2018;**107**:146-154. DOI: 10.1016/j.

[7] Sandler N, Preis M. Printed drugdelivery systems for improved patient treatment. Trends in Pharmacological Sciences. 2016;**37**(12):1070-1080. DOI:

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tips.2018.02.006

#### **Author details**

Angeliki Siamidi, Eleni Tsintavi, Dimitrios M. Rekkas and Marilena Vlachou\* Sector of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece

\*Address all correspondence to: vlachou@pharm.uoa.gr

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*3D-Printed Modified-Release Tablets: A Review of the Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.90868*

#### **References**

**Conflict of interest**

*Molecular Pharmacology*

**Author details**

**58**

Angeliki Siamidi, Eleni Tsintavi, Dimitrios M. Rekkas and Marilena Vlachou\* Sector of Pharmaceutical Technology, Department of Pharmacy, School of Health

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Sciences, National and Kapodistrian University of Athens, Athens, Greece

\*Address all correspondence to: vlachou@pharm.uoa.gr

provided the original work is properly cited.

The authors declare no conflict of interest.

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[3] Norman J, Madurawe RD, Moore CMV, Khan MA, Khairuzzaman A. A new chapter in pharmaceutical manufacturing: 3Dprinted drug products. Advanced Drug Delivery Reviews. 2017;**108**:39-50. DOI: 10.1016/j.addr.2016.03.001

[4] Alhnan MA, Okwuosa TC, Sadia M, Wan KW, Ahmed W, Arafat B. Emergence of 3D printed dosage forms: Opportunities and challenges. Pharmaceutical Research. 2016;**33**(8): 1817-1832. DOI: 10.1007/s11095-016- 1933-1

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[26] Skowyra J, Pietrzak K, Alhnan MA. Fabrication of extended-release patienttailored prednisolone tablets via fused deposition modelling (FDM) 3D printing. European Journal of Pharmaceutical Sciences. 2015;**68**:11-17. DOI: 10.1016/j.ejps.2014.11.009

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*3D-Printed Modified-Release Tablets: A Review of the Recent Advances DOI: http://dx.doi.org/10.5772/intechopen.90868*

[28] Kyobula M, Adedeji A, Alexander MR, Saleh E, Wildman R, Ashcroft I, et al. 3D inkjet printing of tablets exploiting bespoke complex geometries for controlled and tuneable drug release. Journal of Controlled Release. 2017;**261**:207-215. DOI: 10.1016/j.jconrel.2017.06.025

[15] Solanki NG, Tahsin M, Shah AV, Serajuddin ATM. Formulation of 3D printed tablet for rapid drug release by fused deposition modeling: Screening polymers for drug release, drugpolymer miscibility and printability. Journal of Pharmaceutical Sciences. 2018;**107**(1):390-401. DOI: 10.1016/j.

[21] Goyanes A, Kobayashi M, Martínez-Pacheco R, Gaisford S, Basit AW. Fusedfilament 3D printing of drug products: Microstructure analysis and drug release characteristics of PVA-based caplets. International Journal of Pharmaceutics. 2016;**514**(1):290-295. DOI: 10.1016/j.

[22] Wang J, Goyanes A, Gaisford S, Basit AW. Stereolithographic (SLA) 3D printing of oral modified-release dosage

Pharmaceutics. 2016;**503**(1–2):207-212. DOI: 10.1016/j.ijpharm.2016.03.016

[23] Yu DG, Yang XL, Huang WD, Liu J, Wang YG, Xu H. Tablets with material

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controlled-release tablets. International Journal of Pharmaceutics. 2017;**519**(1–

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[26] Skowyra J, Pietrzak K, Alhnan MA. Fabrication of extended-release patienttailored prednisolone tablets via fused deposition modelling (FDM) 3D printing. European Journal of

Pharmaceutical Sciences. 2015;**68**:11-17.

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International Journal of Pharmaceutics. 2014;**476**(1–2):88-92. DOI: 10.1016/j.

DOI: 10.1016/j.ejps.2014.11.009

ijpharm.2014.09.044

forms. International Journal of

gradients fabricated by threedimensional printing. Journal of Pharmaceutical Sciences. 2007;**96**(9): 2446-2456. DOI: 10.1002/jps.20864

hot-melt extrusion to produce

2):186-197. DOI: 10.1016/j. ijpharm.2016.12.049

ijpharm.2016.06.021

[16] Genina N, Boetker JP, Colombo S, Harmankaya N, Rantanen J, Bohr A. Anti-tuberculosis drug combination for controlled oral delivery using 3D printed compartmental dosage forms: From drug product design to in vivo testing. Journal of Controlled Release. 2017;**268**:40-48. DOI: 10.1016/j.

[17] Gioumouxouzis CI, Katsamenis OL, Bouropoulos N, Fatouros DG. 3D printed oral solid dosage forms containing hydrochlorothiazide for controlled drug delivery. Journal of Drug Delivery Science and Technology. 2017;**40**:164-171. DOI: 10.1016/j.

[18] Goyanes A, Robles Martinez P, Buanz A, Basit AW, Gaisford S. Effect of geometry on drug release from 3D printed tablets. International Journal of Pharmaceutics. 2015;**494**(2):657-663. DOI: 10.1016/j.ijpharm.2015.04.069

[19] Goyanes A, Chang H, Sedough D, Hatton GB, Wang J, Buanz A, et al. Fabrication of controlled-release budesonide tablets via desktop (FDM) 3D printing. International Journal of Pharmaceutics. 2015;**496**(2):414-420. DOI: 10.1016/j.ijpharm.2015.10.039

[20] Goyanes A, Buanz ABM,

printing of modified-release

tablets. European Journal of

ejpb.2014.12.003

**60**

Hatton GB, Gaisford S, Basit AW. 3D

aminosalicylate (4-ASA and 5-ASA)

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xphs.2017.10.021

*Molecular Pharmacology*

jconrel.2017.10.003

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[29] Khaled SA, Burley JC, Alexander MR, Yang J, Roberts CJ. 3D printing of tablets containing multiple drugs with defined release profiles. International Journal of Pharmaceutics. 2015;**494**(2):643-650. DOI: 10.1016/j. ijpharm.2015.07.067

[30] Goyanes A, Wang J, Buanz A, Martínez-Pacheco R, Telford R, Gaisford S, et al. 3D printing of medicines: Engineering novel oral devices with unique design and drug release characteristics. Molecular Pharmaceutics. 2015;**12**(11):4077-4084. DOI: 10.1021/acs.molpharmaceut. 5b00510

[31] Goyanes A, Fina F, Martorana A, Sedough D, Gaisford S, Basit AW. Development of modified release 3D printed tablets (printlets) with pharmaceutical excipients using additive manufacturing. International Journal of Pharmaceutics. 2017;**527**(1– 2):21-30. DOI: 10.1016/j. ijpharm.2017.05.021

[32] Okwuosa TC, Pereira BC, Arafat B, Cieszynska M, Isre A, Alhnan MA. Fabricating a shell-core delayed release tablet using dual FDM3D printing for patient-centred therapy. Pharmaceutical Research. 2017;**34**:427. DOI: 10.1007/ s11095-016-2073-3

[33] Maroni A, Melocchi A, Parietti F, Foppoli A, Zema L, Gazzaniga A. 3D printed multi-compartment capsular devices for two-pulse oral drug delivery. Journal of Controlled Release. 2017;**268**: 10-18. DOI: 10.1016/j. jconrel.2017.10.008

[34] Khaled SA, Burley JC, Alexander MR, Roberts CJ. Desktop 3D printing of controlled release pharmaceutical bilayer tablets. International Journal of Pharmaceutics. 2014;**461**(1–2):105-111. DOI: 10.1016/j. ijpharm.2013.11.021

[35] Khaled SA, Burley JC, Alexander MR, Yang J, Roberts CJ. 3D printing of five-in-one dose combination polypill with defined immediate and sustained release profiles. Journal of Controlled Release. 2015;**217**:308-314. DOI: 10.1016/j. jconrel.2015.09.028

[36] Rowe C, Katstra W, Palazzolo R, Giritlioglu B, Teung P, Cima M. Multimechanism oral dosage forms fabricated by three dimensional printing™. Journal of Controlled Release. 2000;**66**(1):11-17. DOI: 10.1016/s0168-3659(99)00224-2

[37] Khoo ZX, Teoh JEM, Liu Y, Chua CK, Yang S, An J, et al. 3D printing of smart materials: A review on recent progresses in 4D printing. Virtual and Physical Prototyping. 2015;**10**(3): 103-122. DOI: 10.1080/ 17452759.2015.1097054

[38] Lui YS, Sow WT, Tan LP, Wu Y, Lai Y, Li H. 4D printing and stimuliresponsive materials in biomedical aspects. Acta Biomaterialia. 2019;**92**: 19-36. DOI: 10.1016/j.actbio.2019. 05.005

**63**

Section 3

Structure of

Drug-Receptor Complex

Section 3

## Structure of Drug-Receptor Complex

**65**

**Chapter 4**

**Abstract**

cology in the cancer field.

resistance

**1. Introduction**

Integrated Molecular Profiling

as an Approach to Identify PI3K

Inhibitor Resistance Mechanisms

*Nicole L. Michmerhuizen, Jiayu Wang and J. Chad Brenner*

The identification of drug resistance pathways and approaches to target these pathways remains a significant and important challenge in cancer biology. Here, we address this challenge in the context of ongoing efforts to advance phosphatidylinositol 3-kinase (PI3K) inhibitors for the treatment of PI3K-aberrant cancers. While PI3K inhibitors have had tremendous success in some diseases, such as breast cancer, early clinical trials in other malignancies, such as head and neck squamous cell carcinoma (HNSCC), have not had the same level of success. Since HNSCC and other cancers display relatively high PI3K pathway alteration rates (>45%), these underwhelming results suggest that additional or unexpected factors may contribute to the lower response rates. Here, we highlight some of the emerging functional genomic and sequencing approaches being used to identify predictive biomarkers of PI3K inhibitor response using both cancer cell lines and clinical trial specimens. Importantly, these approaches have uncovered both innate genetic and adaptive mechanisms driving PI3K inhibitor resistance. In this chapter, we describe recent technological advances that have revolutionized our understanding of PI3K inhibitor resistance pathways in HNSCC and highlight how these and other approaches lay the groundwork to make significant strides in our understanding of molecular pharma-

**Keywords:** PI3K, targeted therapy, drug combination, drug screening, drug

patients with HNSCC survive for 5 years following diagnosis [5].

Head and neck squamous cell carcinomas (HNSCCs) are malignant neoplasms that can occur in regions including the oral cavity, oropharynx, and larynx. HNSCC is the sixth most common cancer, by incidence, worldwide and constitutes approximately 4% of all cancers globally [1–3]. Tobacco use, alcohol consumption, and/or infection with oncogenic high-risk types of HPV, primarily HPV16, are regarded as the major risk factors for HNSCC [4]. Although traditional treatments for HNSCC include surgery, radiotherapy, and cytotoxic chemotherapy [1], these approaches have only modestly reduced the mortality of HNSCC. In fact, only 40–50% of

#### **Chapter 4**

## Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms

*Nicole L. Michmerhuizen, Jiayu Wang and J. Chad Brenner*

#### **Abstract**

The identification of drug resistance pathways and approaches to target these pathways remains a significant and important challenge in cancer biology. Here, we address this challenge in the context of ongoing efforts to advance phosphatidylinositol 3-kinase (PI3K) inhibitors for the treatment of PI3K-aberrant cancers. While PI3K inhibitors have had tremendous success in some diseases, such as breast cancer, early clinical trials in other malignancies, such as head and neck squamous cell carcinoma (HNSCC), have not had the same level of success. Since HNSCC and other cancers display relatively high PI3K pathway alteration rates (>45%), these underwhelming results suggest that additional or unexpected factors may contribute to the lower response rates. Here, we highlight some of the emerging functional genomic and sequencing approaches being used to identify predictive biomarkers of PI3K inhibitor response using both cancer cell lines and clinical trial specimens. Importantly, these approaches have uncovered both innate genetic and adaptive mechanisms driving PI3K inhibitor resistance. In this chapter, we describe recent technological advances that have revolutionized our understanding of PI3K inhibitor resistance pathways in HNSCC and highlight how these and other approaches lay the groundwork to make significant strides in our understanding of molecular pharmacology in the cancer field.

**Keywords:** PI3K, targeted therapy, drug combination, drug screening, drug resistance

#### **1. Introduction**

Head and neck squamous cell carcinomas (HNSCCs) are malignant neoplasms that can occur in regions including the oral cavity, oropharynx, and larynx. HNSCC is the sixth most common cancer, by incidence, worldwide and constitutes approximately 4% of all cancers globally [1–3]. Tobacco use, alcohol consumption, and/or infection with oncogenic high-risk types of HPV, primarily HPV16, are regarded as the major risk factors for HNSCC [4]. Although traditional treatments for HNSCC include surgery, radiotherapy, and cytotoxic chemotherapy [1], these approaches have only modestly reduced the mortality of HNSCC. In fact, only 40–50% of patients with HNSCC survive for 5 years following diagnosis [5].

### **2. The phosphatidylinositol 3-kinase (PI3K) pathway in HNSCC**

HNSCC sequencing studies have identified highly prevalent PI3K pathway alterations that activate PI3K signaling. Following activation by receptor tyrosine kinases (RTKs) and/or G-protein coupled receptors (GPCRs), PI3K phosphorylates phosphatidylinositol (4,5)-bisphosphate (PIP2) into an essential second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) [6]. PIP3 then recruits and activates proteins like PDK1 and AKT to mediate PI3K's pro-survival functions (**Figure 1A**). As a tumor suppressor, PTEN dephosphorylates PIP3 into PIP2 to prevent downstream signal propagation; the deregulation of PTEN is also related to multiple cancers including HNSCC [7].

There are three classes of PI3K (Class I, Class II, and Class III), and Class I PI3K is further divided into Class IA and Class IB [8]. Among the Class IA PI3Ks is p110alpha encoded by the gene *PIK3CA*, which represents the catalytic subunit and alpha isoform of PI3K. Importantly, *PIK3CA* is the most frequently altered gene in the PI3K pathway across all tumors and in HNSCC [9–11]. Alterations of other Class I PI3Ks isoforms, like p110β, and of some regulatory isoforms have been detected in various cancers, albeit with a relatively low frequency [12] (**Figure 2**). Although studies have also examined Class II and Class III PI3Ks, more research is needed to understand their role in human disease [16].

Functionally, the PI3K signaling pathway has a wide range of intracellular effects, including participation in cell cycle, survival, metabolism, motility, and genomic instability [17]. Mutations or other genetic aberrations can lead to hyperactivation of PI3K signaling, and in turn increase cell growth and viability. Angiogenesis and inflammatory cell recruitment, which are thought to be cancer-promoting, may also drive tumor progression and are common in advanced-stage tumors [9, 18].

#### **Figure 1.**

*Rationale for PI3K inhibitor combination therapy. (A) The PI3K signaling pathway has diverse cellular functions. (B) Alterations in PI3K pathway genes may lead to increased signaling. (C) Resistance to PI3K inhibitor monotherapy prevents cell death. (D) Co-targeting PI3K and resistance mechanisms leads to cell death.*

**67**

(**Figure 1B**) [21].

**Figure 2.**

*[13–15].*

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms*

The first manuscript detailing *PIK3CA* mutations in HNSCC was published in 2006; this study described "hotspot," activating mutations (E542K, E545K, and H1047R) among other *PIK3CA* alterations that are less frequent and have not been as well characterized [19]. Since then, evidence has continued to support the significant role of PI3K signaling in HNSCC. The Cancer Genome Atlas (TCGA) dataset, one of the largest HNSCC sequencing studies performed to date, shows that the PI3K signaling pathway is the most frequently mutated oncogenic and targetable signaling pathway in this cancer type [13, 15, 20]. Additionally, Chung and co-authors independently found that almost 32% of HNSCC patients have *PIK3CA* mutation or copy number alteration after analyzing 252 HNSCC patient samples. This study also indicated that 11% of HPV-positive and 5% of HPV-negative HNSCC patients have loss-of-function mutations or copy number alterations in *PTEN*, the gene responsible for restraining PI3K pathway activation

*PI3K pathway gene alteration rates in HNSCC tumors from the cancer genome atlas (TCGA). Amplifications, deletions, mutations and/or multiple alterations (e.g., amplification and mutation) are shown as indicated* 

The frequency of PI3K pathway alteration suggests that inhibitors targeting this pathway may be of clinical use, and several teams have advanced PI3K inhibitors to test their effectiveness for HNSCC treatment. Early clinical trials demonstrated that PI3K inhibitors were safe for use in patients with solid tumors [22, 23], and studies evaluated the potential benefits of using pan-PI3K-targeting agents in recurrent and metastatic HNSCC specifically. However, PI3K inhibitors have more recently shown limitations in efficacy as well as safety (**Figure 1C**). Jimeno et al. reported in 2015 that pan-PI3K inhibitor PX-866 did not improve outcomes when added to cytotoxic chemotherapy (docetaxel) in unselected recurrent or metastatic (R/M) HNSCC patients [24]. Later, Soulieres and colleagues achieved improvements in overall and progression-free survival when administering another pan-PI3K inhibitor, BKM120, with cytotoxic agent paclitaxel (as compared to paclitaxel alone); this study, the BERIL-1 trial, is discussed further below [25]. Unfortunately, BKM120 has not been evaluated further due to undesired side effects. Current trials are evaluating the clinical effects of other PI3K-targeting drugs (NCT03740100), including those with

isoform selectivity (NCT02145312, NCT02822482), in HNSCC patients.

The lack of patient selection is one potential contributor to the underwhelming efficacy of PI3K inhibitor treatment in HNSCCs to date. Although the majority of TCGA HNSCC patients display aberration in one or more PI3K pathway genes [14],

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.92875*

#### **Figure 2.**

*Molecular Pharmacology*

multiple cancers including HNSCC [7].

understand their role in human disease [16].

**2. The phosphatidylinositol 3-kinase (PI3K) pathway in HNSCC**

HNSCC sequencing studies have identified highly prevalent PI3K pathway alterations that activate PI3K signaling. Following activation by receptor tyrosine kinases (RTKs) and/or G-protein coupled receptors (GPCRs), PI3K phosphorylates phosphatidylinositol (4,5)-bisphosphate (PIP2) into an essential second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) [6]. PIP3 then recruits and activates proteins like PDK1 and AKT to mediate PI3K's pro-survival functions (**Figure 1A**). As a tumor suppressor, PTEN dephosphorylates PIP3 into PIP2 to prevent downstream signal propagation; the deregulation of PTEN is also related to

There are three classes of PI3K (Class I, Class II, and Class III), and Class I PI3K is further divided into Class IA and Class IB [8]. Among the Class IA PI3Ks is p110alpha encoded by the gene *PIK3CA*, which represents the catalytic subunit and alpha isoform of PI3K. Importantly, *PIK3CA* is the most frequently altered gene in the PI3K pathway across all tumors and in HNSCC [9–11]. Alterations of other Class I PI3Ks isoforms, like p110β, and of some regulatory isoforms have been detected in various cancers, albeit with a relatively low frequency [12] (**Figure 2**). Although studies have also examined Class II and Class III PI3Ks, more research is needed to

Functionally, the PI3K signaling pathway has a wide range of intracellular effects, including participation in cell cycle, survival, metabolism, motility, and genomic instability [17]. Mutations or other genetic aberrations can lead to hyperactivation of PI3K signaling, and in turn increase cell growth and viability. Angiogenesis and inflammatory cell recruitment, which are thought to be cancer-promoting, may also

drive tumor progression and are common in advanced-stage tumors [9, 18].

*Rationale for PI3K inhibitor combination therapy. (A) The PI3K signaling pathway has diverse cellular functions. (B) Alterations in PI3K pathway genes may lead to increased signaling. (C) Resistance to PI3K inhibitor monotherapy prevents cell death. (D) Co-targeting PI3K and resistance mechanisms leads to cell* 

**66**

*death.*

**Figure 1.**

*PI3K pathway gene alteration rates in HNSCC tumors from the cancer genome atlas (TCGA). Amplifications, deletions, mutations and/or multiple alterations (e.g., amplification and mutation) are shown as indicated [13–15].*

The first manuscript detailing *PIK3CA* mutations in HNSCC was published in 2006; this study described "hotspot," activating mutations (E542K, E545K, and H1047R) among other *PIK3CA* alterations that are less frequent and have not been as well characterized [19]. Since then, evidence has continued to support the significant role of PI3K signaling in HNSCC. The Cancer Genome Atlas (TCGA) dataset, one of the largest HNSCC sequencing studies performed to date, shows that the PI3K signaling pathway is the most frequently mutated oncogenic and targetable signaling pathway in this cancer type [13, 15, 20]. Additionally, Chung and co-authors independently found that almost 32% of HNSCC patients have *PIK3CA* mutation or copy number alteration after analyzing 252 HNSCC patient samples. This study also indicated that 11% of HPV-positive and 5% of HPV-negative HNSCC patients have loss-of-function mutations or copy number alterations in *PTEN*, the gene responsible for restraining PI3K pathway activation (**Figure 1B**) [21].

The frequency of PI3K pathway alteration suggests that inhibitors targeting this pathway may be of clinical use, and several teams have advanced PI3K inhibitors to test their effectiveness for HNSCC treatment. Early clinical trials demonstrated that PI3K inhibitors were safe for use in patients with solid tumors [22, 23], and studies evaluated the potential benefits of using pan-PI3K-targeting agents in recurrent and metastatic HNSCC specifically. However, PI3K inhibitors have more recently shown limitations in efficacy as well as safety (**Figure 1C**). Jimeno et al. reported in 2015 that pan-PI3K inhibitor PX-866 did not improve outcomes when added to cytotoxic chemotherapy (docetaxel) in unselected recurrent or metastatic (R/M) HNSCC patients [24]. Later, Soulieres and colleagues achieved improvements in overall and progression-free survival when administering another pan-PI3K inhibitor, BKM120, with cytotoxic agent paclitaxel (as compared to paclitaxel alone); this study, the BERIL-1 trial, is discussed further below [25]. Unfortunately, BKM120 has not been evaluated further due to undesired side effects. Current trials are evaluating the clinical effects of other PI3K-targeting drugs (NCT03740100), including those with isoform selectivity (NCT02145312, NCT02822482), in HNSCC patients.

The lack of patient selection is one potential contributor to the underwhelming efficacy of PI3K inhibitor treatment in HNSCCs to date. Although the majority of TCGA HNSCC patients display aberration in one or more PI3K pathway genes [14], the status of any single gene or any group of genes has not been validated as a critical biomarker of response to PI3K inhibition. This is in contrast to recent data for hormone receptor-positive breast cancer, where PI3K inhibitor alpelisib is used in combination with fulvestrant to prolong survival for patients with *PIK3CA* mutant tumors [26]. While precision medicine trials across a variety of cancer types have also shown a trend supporting *PIK3CA* alterations as a marker for response to PI3K inhibitors [27], *PIK3CA* mutation has not yet been associated with sensitivity to PI3K inhibition in HNSCC trials [24, 28]. Studies evaluating the role of *PIK3CA* mutations in response to PI3K inhibition in HNSCC often do not reach statistical significance or have a very limited number of patients enrolling. For example, Janku et al. conducted an analysis of responses to PI3K/AKT/mTOR inhibitors in tumors with H1047R *PIK3CA* mutations including four HNSCC patients; after inhibitor treatment, two of these patients experienced progressive disease, one had little change in tumor burden, and another had an incomplete response to therapy [29]. It is possible that the difference in outcomes between tumors with and without *PIK3CA* mutations has not been noted due to an insufficient number of *PIK3CA* mutant tumors in any single clinical trial. Ongoing studies of PI3K inhibitor copanlisib in HNSCC patients with *PIK3CA* mutation or amplification or *PTEN* loss will better elucidate any potential differences in response attributable to PI3K activation (NCT02822482). It is also feasible that other features predict responses to PI3K inhibitor. In the recent BERIL-1 study, which compared outcomes in R/M HNSCC patients (n = 79 per group) treated with paclitaxel with or without pan-PI3K inhibitor BKM120, follow-up analysis revealed that *TP53* alteration, low tumor mutation burden, HPV negativity, and high infiltration of tumor infiltrating lymphocytes (TILs) or CD8+ T-cells was associated with improved response to BKM120. Recent preclinical work has proposed that loss-of-function mutations in *NOTCH1* may predict response to PI3K inhibition [30]. Together these data suggest that a more nuanced understanding of tissue typespecific factors and PI3K inhibitor response mechanisms may be required to develop clinically effective companion diagnostics for this class of inhibitors in HNSCC.

Our recent report indicates that responses to PI3K inhibition, either as monotherapy or in combination with other targeted inhibitors, are complex and cannot be predicted solely based on genetic mutation, copy number alteration, or RNA expression of a single gene (submitted). In this study, a diverse set of HNSCC cell lines were treated with PI3K inhibitors of varying isoform selectivity as monotherapies. Alpha isoform-targeting agents were clearly more effective than other PI3K inhibitors, but the sensitivity profiles for any individual pan- or alpha-isoform PI3K inhibitor were more difficult to stratify. This was increasingly true when PI3K inhibitors were used in drug combinations: *PIK3CA* mutation, copy number, RNA expression, and HPV status did not prove to be meaningful biomarkers for either PI3K and epidermal growth factor receptor (EGFR) inhibitor dual-therapies [31] or for other synergistic drug pairs. In the case of PI3Kalpha inhibitor HS-173 and FAK inhibitor TAE226, greater synergy was observed in *PIK3CA* mutant cell lines as compared to *PIK3CA* wildtype cell lines in initial validation experiments. However, when this association was tested more rigorously with other PI3K and FAK inhibitors, dual-therapy was beneficial also in many *PIK3CA* wildtype models; this could be due to differences in selectivity or mechanism of action for individual small molecule drugs [32]. Thus, PI3K pathway activation, measured at the DNA level via mutation or copy number status or at the protein level via relative downstream phosphorylation, appears to be an insufficient biomarker for sensitivity in HNSCC cell lines; other cellular features, including additional alteration in PI3K pathway members (such as downstream mutations in *AKT1*) or activation of receptor tyrosine kinases (perhaps via upstream overexpression of EGFR), may contribute to signaling through the PI3K pathway and thereby affect inhibitor responses. Indeed,

**69**

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms*

multifaceted analyses, such as those considering gene sets rather than individual genetic changes, may be needed to predict sensitivity. For example, responses to EGFR or FAK inhibitor may be better stratified using gene sets incorporating activation of PI3K, mTOR, or other related signaling nodes instead of *PIK3CA* mutation status alone. Alternatively, an additional pathway that is changed as a result of PI3K activation (e.g., epithelial to mesenchymal transition, cell cycle, or apoptosis)

More broadly, activation or blockade of PI3K signaling may impact response to other forms of cancer treatment. Clinical trial data have not demonstrated that mutation, amplification, or loss of PI3K pathway genes is linked to sensitivity or resistance to PI3K inhibitors. However, recent trials for EGFR-targeting agents have noted poorer outcomes following EGFR inhibition in patients with PI3K activation. In the phase III E2303 trial, which compared cisplatin with or without cetuximab in without cetuximab in R/M HNSCC patients [33], *PIK3CA* mutation or *PTEN* loss was associated with poor response to EGFR-targeting therapy [34]. This finding was also noted in the LUX-H&N1 trial, which compared second-line treatments with afatinib and methotrexate in R/M HNSCCs [35]; here, tumors lacking *PTEN* received inferior benefit from afatinib as compared to those with high levels of *PTEN* [36]. Preclinical studies have also noted that PI3K activation or *PTEN* loss may serve as a biomarker for resistance to cetuximab [37, 38] and that *PIK3CA* mutant HNSCC models may be more resistant to cyclin-dependent kinase (CDK) inhibitor palbociclib [39]. Further

studies are warranted to validate these results in larger, prospective trials.

**3. Combination drug strategies may overcome compensatory PI3K** 

One of the most widely studied mechanisms of resistance to PI3K inhibition is signaling through members of the ERBB family, primarily EGFR, and the downstream Ras-MEK-ERK effector pathway. The human ERBB gene family encodes four members of the ERBB family of receptors including EGFR/ERBB1/HER1, NEU/ ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4. As a transmembrane receptor, EGFR can be activated by ligands including epidermal growth factor (EGF). EGFR signaling subsequently activates downstream pathways, including RAS-RAF-MEK-ERK. MAPK and PI3K-AKT-mTOR signaling pathways, to promote cell proliferation and inhibit apoptosis in many head and neck models [40, 41]. EGFR overexpression has been reported in the vast majority of HNSCCs [42]. Further genetic dysregulations of EGFR signaling caused by *EGFR* gene mutation (although rare in head and neck cancer) and *EGFR* gene copy number amplification (approximately 10% of

Activation of EGFR signaling as a mechanism of PI3K inhibitor resistance has been extensively characterized using a wide variety of cell lines (displaying a diverse array of genetic alterations) and a large set of ERBB family-targeting drugs [1, 31, 44–47]. Early work focused on *PIK3CA-*amplified HNSCCs demonstrated that two-thirds (67%) of cell lines with additional copies of wildtype *PIK3CA* maintained RAS-RAF-MEK-ERK MAPK pathway activity following PI3K inhibitor treatment and that two of these models were also sensitive to dual inhibition of PI3K and EGFR or MEK [1]. A more recent publication extended this observation to a larger panel of ERBB inhibitors and cell lines, including several with *PIK3CA* mutations [31]. Overall, findings from the latter study mirrored those of previous publications showing that dual-therapy with PI3K and EGFR inhibitors was often

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

**inhibitor resistance**

**3.1 Epidermal growth factor receptor (EGFR)**

cases) also contribute to activated EGFR signaling [41, 43].

may be even more effective in predicting response.

#### *Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.92875*

multifaceted analyses, such as those considering gene sets rather than individual genetic changes, may be needed to predict sensitivity. For example, responses to EGFR or FAK inhibitor may be better stratified using gene sets incorporating activation of PI3K, mTOR, or other related signaling nodes instead of *PIK3CA* mutation status alone. Alternatively, an additional pathway that is changed as a result of PI3K activation (e.g., epithelial to mesenchymal transition, cell cycle, or apoptosis) may be even more effective in predicting response.

More broadly, activation or blockade of PI3K signaling may impact response to other forms of cancer treatment. Clinical trial data have not demonstrated that mutation, amplification, or loss of PI3K pathway genes is linked to sensitivity or resistance to PI3K inhibitors. However, recent trials for EGFR-targeting agents have noted poorer outcomes following EGFR inhibition in patients with PI3K activation. In the phase III E2303 trial, which compared cisplatin with or without cetuximab in without cetuximab in R/M HNSCC patients [33], *PIK3CA* mutation or *PTEN* loss was associated with poor response to EGFR-targeting therapy [34]. This finding was also noted in the LUX-H&N1 trial, which compared second-line treatments with afatinib and methotrexate in R/M HNSCCs [35]; here, tumors lacking *PTEN* received inferior benefit from afatinib as compared to those with high levels of *PTEN* [36]. Preclinical studies have also noted that PI3K activation or *PTEN* loss may serve as a biomarker for resistance to cetuximab [37, 38] and that *PIK3CA* mutant HNSCC models may be more resistant to cyclin-dependent kinase (CDK) inhibitor palbociclib [39]. Further studies are warranted to validate these results in larger, prospective trials.

#### **3. Combination drug strategies may overcome compensatory PI3K inhibitor resistance**

#### **3.1 Epidermal growth factor receptor (EGFR)**

One of the most widely studied mechanisms of resistance to PI3K inhibition is signaling through members of the ERBB family, primarily EGFR, and the downstream Ras-MEK-ERK effector pathway. The human ERBB gene family encodes four members of the ERBB family of receptors including EGFR/ERBB1/HER1, NEU/ ERBB2/HER2, ERBB3/HER3 and ERBB4/HER4. As a transmembrane receptor, EGFR can be activated by ligands including epidermal growth factor (EGF). EGFR signaling subsequently activates downstream pathways, including RAS-RAF-MEK-ERK. MAPK and PI3K-AKT-mTOR signaling pathways, to promote cell proliferation and inhibit apoptosis in many head and neck models [40, 41]. EGFR overexpression has been reported in the vast majority of HNSCCs [42]. Further genetic dysregulations of EGFR signaling caused by *EGFR* gene mutation (although rare in head and neck cancer) and *EGFR* gene copy number amplification (approximately 10% of cases) also contribute to activated EGFR signaling [41, 43].

Activation of EGFR signaling as a mechanism of PI3K inhibitor resistance has been extensively characterized using a wide variety of cell lines (displaying a diverse array of genetic alterations) and a large set of ERBB family-targeting drugs [1, 31, 44–47]. Early work focused on *PIK3CA-*amplified HNSCCs demonstrated that two-thirds (67%) of cell lines with additional copies of wildtype *PIK3CA* maintained RAS-RAF-MEK-ERK MAPK pathway activity following PI3K inhibitor treatment and that two of these models were also sensitive to dual inhibition of PI3K and EGFR or MEK [1]. A more recent publication extended this observation to a larger panel of ERBB inhibitors and cell lines, including several with *PIK3CA* mutations [31]. Overall, findings from the latter study mirrored those of previous publications showing that dual-therapy with PI3K and EGFR inhibitors was often

*Molecular Pharmacology*

the status of any single gene or any group of genes has not been validated as a critical biomarker of response to PI3K inhibition. This is in contrast to recent data for hormone receptor-positive breast cancer, where PI3K inhibitor alpelisib is used in combination with fulvestrant to prolong survival for patients with *PIK3CA* mutant tumors [26]. While precision medicine trials across a variety of cancer types have also shown a trend supporting *PIK3CA* alterations as a marker for response to PI3K inhibitors [27], *PIK3CA* mutation has not yet been associated with sensitivity to PI3K inhibition in HNSCC trials [24, 28]. Studies evaluating the role of *PIK3CA* mutations in response to PI3K inhibition in HNSCC often do not reach statistical significance or have a very limited number of patients enrolling. For example, Janku et al. conducted an analysis of responses to PI3K/AKT/mTOR inhibitors in tumors with H1047R *PIK3CA* mutations including four HNSCC patients; after inhibitor treatment, two of these patients experienced progressive disease, one had little change in tumor burden, and another had an incomplete response to therapy [29]. It is possible that the difference in outcomes between tumors with and without *PIK3CA* mutations has not been noted due to an insufficient number of *PIK3CA* mutant tumors in any single clinical trial. Ongoing studies of PI3K inhibitor copanlisib in HNSCC patients with *PIK3CA* mutation or amplification or *PTEN* loss will better elucidate any potential differences in response attributable to PI3K activation (NCT02822482). It is also feasible that other features predict responses to PI3K inhibitor. In the recent BERIL-1 study, which compared outcomes in R/M HNSCC patients (n = 79 per group) treated with paclitaxel with or without pan-PI3K inhibitor BKM120, follow-up analysis revealed that *TP53* alteration, low tumor mutation burden, HPV negativity, and high infiltration of tumor infiltrating lymphocytes (TILs) or CD8+ T-cells was associated with improved response to BKM120. Recent preclinical work has proposed that loss-of-function mutations in *NOTCH1* may predict response to PI3K inhibition [30]. Together these data suggest that a more nuanced understanding of tissue typespecific factors and PI3K inhibitor response mechanisms may be required to develop clinically effective companion diagnostics for this class of inhibitors in HNSCC. Our recent report indicates that responses to PI3K inhibition, either as monotherapy or in combination with other targeted inhibitors, are complex and cannot be predicted solely based on genetic mutation, copy number alteration, or RNA expression of a single gene (submitted). In this study, a diverse set of HNSCC cell lines were treated with PI3K inhibitors of varying isoform selectivity as monotherapies. Alpha isoform-targeting agents were clearly more effective than other PI3K inhibitors, but the sensitivity profiles for any individual pan- or alpha-isoform PI3K inhibitor were more difficult to stratify. This was increasingly true when PI3K inhibitors were used in drug combinations: *PIK3CA* mutation, copy number, RNA expression, and HPV status did not prove to be meaningful biomarkers for either PI3K and epidermal growth factor receptor (EGFR) inhibitor dual-therapies [31] or for other synergistic drug pairs. In the case of PI3Kalpha inhibitor HS-173 and FAK inhibitor TAE226, greater synergy was observed in *PIK3CA* mutant cell lines as compared to *PIK3CA* wildtype cell lines in initial validation experiments. However, when this association was tested more rigorously with other PI3K and FAK inhibitors, dual-therapy was beneficial also in many *PIK3CA* wildtype models; this could be due to differences in selectivity or mechanism of action for individual small molecule drugs [32]. Thus, PI3K pathway activation, measured at the DNA level via mutation or copy number status or at the protein level via relative downstream phosphorylation, appears to be an insufficient biomarker for sensitivity in HNSCC cell lines; other cellular features, including additional alteration in PI3K pathway members (such as downstream mutations in *AKT1*) or activation of receptor

tyrosine kinases (perhaps via upstream overexpression of EGFR), may contribute to signaling through the PI3K pathway and thereby affect inhibitor responses. Indeed,

**68**

more effective than either monotherapy [44–47], but also extended this observation to consider individual classes of ERBB-targeting agents that might result in heightened responses when used as part of combination treatments. Results in HNSCC cell lines showed that *irreversible* inhibitors of EGFR were more effective in combination with PI3K inhibitors than reversible ERBB-targeting agents. As previous work had primarily considered dual-therapies that included either reversible EGFR inhibitors or EGFR-targeting antibodies such as cetuximab, this work was the first direct comparison of PI3K inhibitors in combination with distinct pharmacologies against EGFR.

Beyond direct inhibition of the receptor tyrosine kinases PI3K and EGFR themselves, previous work has also examined drug combinations targeting PI3K and EGFR via inhibition of downstream effectors including mTOR and MEK, respectively. Several papers have described synergy with mTOR inhibitors and EGFR agents [48–50]. In one of these studies, Jimeno et al. used H1047R *PIK3CA* mutant Detroit 562 cells in a xenograft model and noted improved response to mTOR inhibitor temsirolimus and erlotinib. This response co-occurred with changes in MAPK and p70 S6 kinase phosphorylation (downstream of EGFR and mTOR, respectively) and in Ki67, effects that were not evidenced in less responsive xenograft models or after single-agent treatment. Other work with Detroit 562 in vitro showed minimal responses to PI3K inhibitor HS-173 and reversible EGFR inhibitors, including erlotinib, that could be enhanced to synergistic levels with multiple irreversible EGFR inhibitors [31]. The combination of erlotinib with mTOR inhibitors (temsirolimus or otherwise) has not been reported in this model. Nevertheless, since additional data show that ineffective reversible EGFR inhibitor combinations block MAPK phosphorylation [31] (p70 S6 kinase phosphorylation was not tested), it is possible that one or more additional effectors, perhaps further downstream of MAPK/p70 S6 kinase or as part of a second escape pathway, may be responsible for synergistic effects. Alternatively, in vitro and in vivo responses to mTOR and EGFR agents in Detroit 562 and potentially other HNSCCs could be dependent on distinct mechanisms. PI3K and irreversible EGFR inhibitor combinations have not yet been tested in xenografts, but these experiments would enhance our understanding of the potential for such dual-therapies to translate clinically.

In light of the synergy observed following treatment with agents targeting the PI3K and EGFR pathways in preclinical models, phase I and II trials have been performed to examine these dual-therapies in HNSCC patients. Of these trials, three have been completed, all in patients receiving second-line treatment due to chemotherapy resistance, recurrence, and/or metastasis. The first of these trials examined temsirolimus with cetuximab and resulted in dose-limited toxicities in one-third of patients [51]. The second considered another mTOR inhibitor, everolimus, with erlotinib. This combination had a reasonable toxicity profile and stopped or decreased tumor growth in several patients, but it did not result in clinical benefit as compared to previous trials considering erlotinib as a monotherapy [52]. The third trial, which considered cetuximab with or without PI3K inhibitor PX-866, also did not provide evidence of improvement with the addition of PI3K inhibitor [53]. Several other trials using PI3K- and EGFR-targeting agents, sometimes alongside cytotoxic chemotherapy or radiotherapy, have been initiated and are in various stages of completion. Toxicity seems to be a major concern in many of these studies and may limit the use of such combinations in patients. As a result, the development of more specific combinations is warranted.

Nevertheless, previous work also suggests that the use of currently available PI3K and EGFR therapies may be optimized in other ways. For example, the sequence of combination treatments may be an important consideration. Lattanzio

**71**

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms*

et al. showed that Cal-33 cells responded to treatment with EGFR antibody followed by PI3K inhibitor [54], while other data show minimal responses in this model when EGFR- and PI3K-targeting small molecules were co-administered [31]. The type of EGFR-targeting agent that was used may also explain this conflicting data: small molecules (e.g., gefitinib, erlotinib, afatinib) and biologics (e.g., cetuximab) could

Additional studies have successfully implicated other means of upstream inhibition in combination with PI3K-targeting drugs to improve responses in HNSCC, including other members of the ERBB family. While the structure of these members is similar to that of EGFR, each ERBB receptor has a different ligand binding specificity and physiological role [55]. Abnormalities of ERBB2, ERBB3, and ERBB4 have been reported in various malignancies such as breast cancer and occur in smaller subsets of head and neck cancer; therefore, they also became the targets for cancer

For example, Brand and co-authors blocked ERBB3 signaling as a means of reversing PI3K inhibitor resistance mediated by HPV oncoproteins E6 and E7 in HPV-positive HNSCCs [57]. These studies support the role of RTKs in HNSCC resistance mechanisms and validate that combination PI3K and RTK blockade may result in improved PI3K inhibitor responses. Likewise, Meister et al. also reported that PI3K inhibition induces ERBB3 upregulation and activation while the combination of PI3K and ERBB3 inhibition has synergistic effect, suppressing HNSCC growth both in vitro and in vivo [58]. Furthermore, in breast cancer, researchers also found that amplification of *ERBB2* can activate PI3K-AKT signaling directly and indirectly [59], and ERBB2 inhibition potentiates the antitumor effect of PI3K

PI3K inhibitor resistance mechanisms, of course, span far beyond the ERBB family of RTKs alone. For instance, the AXL receptor tyrosine kinase is highly expressed in various cancers including esophageal squamous cell carcinoma and HNSCC [61]. Based on work by Elkabets et al., AXL is upregulated particularly in PI3K inhibitor-resistant HNSCCs [62]. These researchers also showed that the dimerization of AXL and EGFR promotes signaling through PI3K and mTOR by activating the PLCγ-PKC signaling pathway, thereby limiting the efficacy of PI3K inhibitors. This PI3K-independent activation of mTOR counteracts the growth inhibitory effect of PI3K inhibition and mediates drug resistance in some head and neck models [62]. Further, Badarni et al. chose to identify and target the transcription factors that were responsible for the increased expression of AXL in HNSCCs. In doing so, they discovered that a c-JUN, a member of the AP-1 transcription factor (TF) complex, was likely responsible for AXL upregulation and that blocking

this TF improved the response to PI3K inhibitor BYL719 [63].

have very different response profiles in combination with PI3K inhibitors. Overall, although responses to PI3K and EGFR inhibitor combinations in HNSCC have been studied more extensively than many other dual-therapies for this cancer type, current results highlight the diversity of responses to agents targeting these two signaling pathways. Additional factors, including but not limited to timing, patient selection, and other co-treatments, require further consideration before compensation through the PI3K and EGFR pathways will be most effectively

therapies and have been considered in drug combinations [55, 56].

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

exploited in a population of HNSCC patients.

inhibitor BEZ235 in this cancer type [60].

**3.3 AXL receptor tyrosine kinase**

**3.2 Other ERBB family members**

#### *Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.92875*

et al. showed that Cal-33 cells responded to treatment with EGFR antibody followed by PI3K inhibitor [54], while other data show minimal responses in this model when EGFR- and PI3K-targeting small molecules were co-administered [31]. The type of EGFR-targeting agent that was used may also explain this conflicting data: small molecules (e.g., gefitinib, erlotinib, afatinib) and biologics (e.g., cetuximab) could have very different response profiles in combination with PI3K inhibitors.

Overall, although responses to PI3K and EGFR inhibitor combinations in HNSCC have been studied more extensively than many other dual-therapies for this cancer type, current results highlight the diversity of responses to agents targeting these two signaling pathways. Additional factors, including but not limited to timing, patient selection, and other co-treatments, require further consideration before compensation through the PI3K and EGFR pathways will be most effectively exploited in a population of HNSCC patients.

#### **3.2 Other ERBB family members**

*Molecular Pharmacology*

against EGFR.

more effective than either monotherapy [44–47], but also extended this observation to consider individual classes of ERBB-targeting agents that might result in heightened responses when used as part of combination treatments. Results in HNSCC cell lines showed that *irreversible* inhibitors of EGFR were more effective in combination with PI3K inhibitors than reversible ERBB-targeting agents. As previous work had primarily considered dual-therapies that included either reversible EGFR inhibitors or EGFR-targeting antibodies such as cetuximab, this work was the first direct comparison of PI3K inhibitors in combination with distinct pharmacologies

Beyond direct inhibition of the receptor tyrosine kinases PI3K and EGFR themselves, previous work has also examined drug combinations targeting PI3K and EGFR via inhibition of downstream effectors including mTOR and MEK, respectively. Several papers have described synergy with mTOR inhibitors and EGFR agents [48–50]. In one of these studies, Jimeno et al. used H1047R *PIK3CA* mutant Detroit 562 cells in a xenograft model and noted improved response to mTOR inhibitor temsirolimus and erlotinib. This response co-occurred with changes in MAPK and p70 S6 kinase phosphorylation (downstream of EGFR and mTOR, respectively) and in Ki67, effects that were not evidenced in less responsive xenograft models or after single-agent treatment. Other work with Detroit 562 in vitro showed minimal responses to PI3K inhibitor HS-173 and reversible EGFR inhibitors, including erlotinib, that could be enhanced to synergistic levels with multiple irreversible EGFR inhibitors [31]. The combination of erlotinib with mTOR inhibitors (temsirolimus or otherwise) has not been reported in this model. Nevertheless, since additional data show that ineffective reversible EGFR inhibitor combinations block MAPK phosphorylation [31] (p70 S6 kinase phosphorylation was not tested), it is possible that one or more additional effectors, perhaps further downstream of MAPK/p70 S6 kinase or as part of a second escape pathway, may be responsible for synergistic effects. Alternatively, in vitro and in vivo responses to mTOR and EGFR agents in Detroit 562 and potentially other HNSCCs could be dependent on distinct mechanisms. PI3K and irreversible EGFR inhibitor combinations have not yet been tested in xenografts, but these experiments would enhance our understanding of

In light of the synergy observed following treatment with agents targeting the PI3K and EGFR pathways in preclinical models, phase I and II trials have been performed to examine these dual-therapies in HNSCC patients. Of these trials, three have been completed, all in patients receiving second-line treatment due to chemotherapy resistance, recurrence, and/or metastasis. The first of these trials examined temsirolimus with cetuximab and resulted in dose-limited toxicities in one-third of patients [51]. The second considered another mTOR inhibitor, everolimus, with erlotinib. This combination had a reasonable toxicity profile and stopped or decreased tumor growth in several patients, but it did not result in clinical benefit as compared to previous trials considering erlotinib as a monotherapy [52]. The third trial, which considered cetuximab with or without PI3K inhibitor PX-866, also did not provide evidence of improvement with the addition of PI3K inhibitor [53]. Several other trials using PI3K- and EGFR-targeting agents, sometimes alongside cytotoxic chemotherapy or radiotherapy, have been initiated and are in various stages of completion. Toxicity seems to be a major concern in many of these studies and may limit the use of such combinations in patients. As a result, the development

Nevertheless, previous work also suggests that the use of currently available PI3K and EGFR therapies may be optimized in other ways. For example, the sequence of combination treatments may be an important consideration. Lattanzio

the potential for such dual-therapies to translate clinically.

of more specific combinations is warranted.

**70**

Additional studies have successfully implicated other means of upstream inhibition in combination with PI3K-targeting drugs to improve responses in HNSCC, including other members of the ERBB family. While the structure of these members is similar to that of EGFR, each ERBB receptor has a different ligand binding specificity and physiological role [55]. Abnormalities of ERBB2, ERBB3, and ERBB4 have been reported in various malignancies such as breast cancer and occur in smaller subsets of head and neck cancer; therefore, they also became the targets for cancer therapies and have been considered in drug combinations [55, 56].

For example, Brand and co-authors blocked ERBB3 signaling as a means of reversing PI3K inhibitor resistance mediated by HPV oncoproteins E6 and E7 in HPV-positive HNSCCs [57]. These studies support the role of RTKs in HNSCC resistance mechanisms and validate that combination PI3K and RTK blockade may result in improved PI3K inhibitor responses. Likewise, Meister et al. also reported that PI3K inhibition induces ERBB3 upregulation and activation while the combination of PI3K and ERBB3 inhibition has synergistic effect, suppressing HNSCC growth both in vitro and in vivo [58]. Furthermore, in breast cancer, researchers also found that amplification of *ERBB2* can activate PI3K-AKT signaling directly and indirectly [59], and ERBB2 inhibition potentiates the antitumor effect of PI3K inhibitor BEZ235 in this cancer type [60].

#### **3.3 AXL receptor tyrosine kinase**

PI3K inhibitor resistance mechanisms, of course, span far beyond the ERBB family of RTKs alone. For instance, the AXL receptor tyrosine kinase is highly expressed in various cancers including esophageal squamous cell carcinoma and HNSCC [61]. Based on work by Elkabets et al., AXL is upregulated particularly in PI3K inhibitor-resistant HNSCCs [62]. These researchers also showed that the dimerization of AXL and EGFR promotes signaling through PI3K and mTOR by activating the PLCγ-PKC signaling pathway, thereby limiting the efficacy of PI3K inhibitors. This PI3K-independent activation of mTOR counteracts the growth inhibitory effect of PI3K inhibition and mediates drug resistance in some head and neck models [62]. Further, Badarni et al. chose to identify and target the transcription factors that were responsible for the increased expression of AXL in HNSCCs. In doing so, they discovered that a c-JUN, a member of the AP-1 transcription factor (TF) complex, was likely responsible for AXL upregulation and that blocking this TF improved the response to PI3K inhibitor BYL719 [63].

#### **3.4 Insulin-like growth factor-1 receptor (IGF-1R)**

IGFR signaling is vital in the development of tissues [64] and is aberrantly implicated in several types of cancer including adenomas, breast cancer, lung cancer, ovarian cancer, and HNSCC [65]. *IGF1R* is amplified in 4% of HNSCC tumors from TCGA, placing it among the most commonly amplified genes in this tumor type [13–15]. Both IGF-1R and IGF binding protein-3 (IGFBP-3) have overt impact on HNSCC prognoses, as clinical data reveal that high expression of IGFBP-3 as well as the co-expression of IGFBP-3 with IGF-1R may predict poor prognosis in this cancer type [66]. Preclinical models also demonstrate the role of the IGF-1R signaling pathway in HNSCC: small molecule IGF-1R inhibitor BMS-536924 is effective in both cell lines derived from transgenic mice that spontaneously developed salivary gland tumors and in xenograft mouse models [67].

The tyrosine kinase activity of IGF-1R suggests that the activation of IGF-1R could induce activation of PI3K signaling [68, 69]. Interestingly, studies suggest that the activation of mTOR signaling could be part of a negative feedback loop to reduce PI3K signaling by the phosphorylation of insulin receptor substrate-1 (IRS-1) [70]. This complex relationship between the PI3K and IGFR signaling pathways indicates that IGFR signaling could be a compensatory mechanism contributing to PI3K inhibitor resistance, a phenomenon that has been previously observed in multiple other cancer types [71, 72]. Recent data (submitted) also indicate the potential efficacy of PI3K and IGF-1R inhibitor combinations in HNSCC models, but such dual-therapies have yet to be evaluated in patients.

#### **3.5 Anaplastic lymphoma kinase (ALK)**

Aberration in the *ALK* gene is associated with several cancer types; gene fusions have been noted in anaplastic large cell lymphoma and one subset of non-small cell lung cancer [73], while mutations are present in nearly 15% of neuroblastomas [74]. ALK has also been shown to have a pleiotropic role in the aggressive growth and invasiveness of oral squamous cell carcinoma [75]. As a downstream member of the ALK signaling pathway, PI3K/AKT signaling may be activated followed by ALK activation [76]. Collectively, these data motivate consideration of ALK signaling as a compensatory signaling pathway responsible for resistance to PI3K inhibitor monotherapies.

ALK signaling has not historically been evaluated as a resistance mechanism in HNSCC, since fusion events are rare and ALK expression is often quite low in this cancer type. Nevertheless, recent work has shown that EGFR inhibitor treatments may increase the expression of ALK and display greater efficacy with ALK inhibitors [77, 78]. Gonzales et al. showed that the combination of ALK inhibitor and EGFR inhibitor could decrease HNSCC cell proliferation in vitro as well as reduce the volumes of xenotransplantation tumors in vivo [77]. These studies demonstrate that ALK signaling is indeed an important mediator of drug resistance in some head and neck cancers. Additionally, *ALK* was also identified in a CRISPR/Cas9 knockout screen as one of the kinases responsible for resistance to PI3K inhibitor treatment (submitted). PI3K and ALK dual-therapy, however, has not been considered in HNSCC. The only previous studies of PI3K and ALK inhibitors examine such combinations in the context of other tumor types driven by ALK signaling due to fusion with *EML4* or other genes [79–82]. Our recent work shows that co-treatment with ALK inhibitor brigatinib and PI3K inhibitor pictilisib is synergistic in HNSCC models (submitted); this may represent the first evidence of interaction between PI3K and ALK signaling in the absence of *ALK* gene fusion.

**73**

gistic in a subset of patients.

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms*

However, brigatinib is not a perfectly selective inhibitor of ALK—it also displays activity at IGF-1R, EGFR, and other RTKs, especially at higher concentrations [83, 84]. While ALK inhibition may be an important component of the response to combinations of brigatinib and PI3K inhibitor, we cannot exclude the possibility that the blockade of IGF-1R and/or EGFR contribute at least partially to this response. Both small molecule profiling studies and subsequent validation analyses show that IGF-1R or EGFR inhibition can improve responses to PI3Ktargeting therapy, as noted above. Genetic knockouts of *ALK* and *IGF1R* that were generated in combination-responsive HSC-4 cells offer insight into the effect of blocking an individual RTK, mimicking a perfectly selective pharmacologic treatment. *ALK* knockout HSC-4 cells are more sensitive to monotherapy with AKT inhibitor GDC-0068 than wildtype HSC-4 cells. Responses to PI3K inhibition are not markedly different in the knockout model, suggesting that ALK alone may not be responsible for the synergy of ALK- and PI3K-targeting agents in HNSCC. In spite of this, *ALK* and *IGF1R* knockout cell lines display increased levels of p110alpha, the protein encoded by the *PIK3CA* gene (submitted). This indicates an important molecular relationship between the ALK, IGF-1R, and PI3K pathways and may explain the lack of response to PI3K inhibition in knock-

While the mechanistic basis for responses to pictilisib and brigatinib has not been fully elucidated, co-treatment with these agents three times per week inhibited tumor growth in a cell line-derived xenograft mouse model (submitted). However, despite the significant result observed after a 3-week course with these inhibitors, tumors did progress as treatment continued for extended lengths of time. This observation is in support of the development of additional compensatory mechanisms mediating treatment resistance. Although the combination of PI3K and ALK inhibitor extended survival for weeks past what would have been observed in mice with vehicle- and brigatinib-treated tumors, further exploration of compensatory signaling in HNSCC and the development of improved treatment paradigms

FGFRs also have been nominated as a critical candidate for compensatory signaling pathway in HNSCC. After the binding of the fibroblast growth factor (FGF) ligands, the FGFR signaling pathway will modify downstream phosphorylation and gene expression. Importantly, this pathway also has a well-described role in HNSCC pathogenesis due to recurrent of genetic alterations that occur in the disease [85]. According to previous studies, recurrent FGFR1 amplification is present in 17% of OSCC cases [86]. A high level of FGFR2 expression, FGFR3 expression and FGFR2/FGFR3 co-expression are also observed in HNSCC cell lines [87], and such alterations have been suggested to contribute to the early stages of tumor initiation and disease progression [88]. In fact, previous in vitro and in vivo studies show that inhibition of the FGFR signaling pathway could decrease the proliferation of HNSCC supporting cells (fibroblast and endothelial cells) and ultimately results in a decline of tumor cell proliferation through increased cell apoptosis [89]. Combinations of EGFR and FGFR inhibitors have been evaluated in both HNSCC and other cancers, most notably lung cancer [90, 91], but have not translated to the clinic due to toxic effects in patients [92]. Based on these and other data demonstrating the important role of FGFR signaling in HNSCC [93], combinations of PI3K and FGFR inhibitors might be syner-

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

out cell lines.

is needed.

**3.6 Fibroblast growth factor receptor (FGFR)**

#### *Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.92875*

However, brigatinib is not a perfectly selective inhibitor of ALK—it also displays activity at IGF-1R, EGFR, and other RTKs, especially at higher concentrations [83, 84]. While ALK inhibition may be an important component of the response to combinations of brigatinib and PI3K inhibitor, we cannot exclude the possibility that the blockade of IGF-1R and/or EGFR contribute at least partially to this response. Both small molecule profiling studies and subsequent validation analyses show that IGF-1R or EGFR inhibition can improve responses to PI3Ktargeting therapy, as noted above. Genetic knockouts of *ALK* and *IGF1R* that were generated in combination-responsive HSC-4 cells offer insight into the effect of blocking an individual RTK, mimicking a perfectly selective pharmacologic treatment. *ALK* knockout HSC-4 cells are more sensitive to monotherapy with AKT inhibitor GDC-0068 than wildtype HSC-4 cells. Responses to PI3K inhibition are not markedly different in the knockout model, suggesting that ALK alone may not be responsible for the synergy of ALK- and PI3K-targeting agents in HNSCC. In spite of this, *ALK* and *IGF1R* knockout cell lines display increased levels of p110alpha, the protein encoded by the *PIK3CA* gene (submitted). This indicates an important molecular relationship between the ALK, IGF-1R, and PI3K pathways and may explain the lack of response to PI3K inhibition in knockout cell lines.

While the mechanistic basis for responses to pictilisib and brigatinib has not been fully elucidated, co-treatment with these agents three times per week inhibited tumor growth in a cell line-derived xenograft mouse model (submitted). However, despite the significant result observed after a 3-week course with these inhibitors, tumors did progress as treatment continued for extended lengths of time. This observation is in support of the development of additional compensatory mechanisms mediating treatment resistance. Although the combination of PI3K and ALK inhibitor extended survival for weeks past what would have been observed in mice with vehicle- and brigatinib-treated tumors, further exploration of compensatory signaling in HNSCC and the development of improved treatment paradigms is needed.

#### **3.6 Fibroblast growth factor receptor (FGFR)**

FGFRs also have been nominated as a critical candidate for compensatory signaling pathway in HNSCC. After the binding of the fibroblast growth factor (FGF) ligands, the FGFR signaling pathway will modify downstream phosphorylation and gene expression. Importantly, this pathway also has a well-described role in HNSCC pathogenesis due to recurrent of genetic alterations that occur in the disease [85]. According to previous studies, recurrent FGFR1 amplification is present in 17% of OSCC cases [86]. A high level of FGFR2 expression, FGFR3 expression and FGFR2/FGFR3 co-expression are also observed in HNSCC cell lines [87], and such alterations have been suggested to contribute to the early stages of tumor initiation and disease progression [88]. In fact, previous in vitro and in vivo studies show that inhibition of the FGFR signaling pathway could decrease the proliferation of HNSCC supporting cells (fibroblast and endothelial cells) and ultimately results in a decline of tumor cell proliferation through increased cell apoptosis [89]. Combinations of EGFR and FGFR inhibitors have been evaluated in both HNSCC and other cancers, most notably lung cancer [90, 91], but have not translated to the clinic due to toxic effects in patients [92]. Based on these and other data demonstrating the important role of FGFR signaling in HNSCC [93], combinations of PI3K and FGFR inhibitors might be synergistic in a subset of patients.

*Molecular Pharmacology*

**3.4 Insulin-like growth factor-1 receptor (IGF-1R)**

gland tumors and in xenograft mouse models [67].

dual-therapies have yet to be evaluated in patients.

PI3K and ALK signaling in the absence of *ALK* gene fusion.

**3.5 Anaplastic lymphoma kinase (ALK)**

IGFR signaling is vital in the development of tissues [64] and is aberrantly implicated in several types of cancer including adenomas, breast cancer, lung cancer, ovarian cancer, and HNSCC [65]. *IGF1R* is amplified in 4% of HNSCC tumors from TCGA, placing it among the most commonly amplified genes in this tumor type [13–15]. Both IGF-1R and IGF binding protein-3 (IGFBP-3) have overt impact on HNSCC prognoses, as clinical data reveal that high expression of IGFBP-3 as well as the co-expression of IGFBP-3 with IGF-1R may predict poor prognosis in this cancer type [66]. Preclinical models also demonstrate the role of the IGF-1R signaling pathway in HNSCC: small molecule IGF-1R inhibitor BMS-536924 is effective in both cell lines derived from transgenic mice that spontaneously developed salivary

The tyrosine kinase activity of IGF-1R suggests that the activation of IGF-1R could induce activation of PI3K signaling [68, 69]. Interestingly, studies suggest that the activation of mTOR signaling could be part of a negative feedback loop to reduce PI3K signaling by the phosphorylation of insulin receptor substrate-1 (IRS-1) [70]. This complex relationship between the PI3K and IGFR signaling pathways indicates that IGFR signaling could be a compensatory mechanism contributing to PI3K inhibitor resistance, a phenomenon that has been previously observed in multiple other cancer types [71, 72]. Recent data (submitted) also indicate the potential efficacy of PI3K and IGF-1R inhibitor combinations in HNSCC models, but such

Aberration in the *ALK* gene is associated with several cancer types; gene fusions have been noted in anaplastic large cell lymphoma and one subset of non-small cell lung cancer [73], while mutations are present in nearly 15% of neuroblastomas [74]. ALK has also been shown to have a pleiotropic role in the aggressive growth and invasiveness of oral squamous cell carcinoma [75]. As a downstream member of the ALK signaling pathway, PI3K/AKT signaling may be activated followed by ALK activation [76]. Collectively, these data motivate consideration of ALK signaling as a compensatory signaling pathway responsible for resistance to PI3K inhibitor

ALK signaling has not historically been evaluated as a resistance mechanism in HNSCC, since fusion events are rare and ALK expression is often quite low in this cancer type. Nevertheless, recent work has shown that EGFR inhibitor treatments may increase the expression of ALK and display greater efficacy with ALK inhibitors [77, 78]. Gonzales et al. showed that the combination of ALK inhibitor and EGFR inhibitor could decrease HNSCC cell proliferation in vitro as well as reduce the volumes of xenotransplantation tumors in vivo [77]. These studies demonstrate that ALK signaling is indeed an important mediator of drug resistance in some head and neck cancers. Additionally, *ALK* was also identified in a CRISPR/Cas9 knockout screen as one of the kinases responsible for resistance to PI3K inhibitor treatment (submitted). PI3K and ALK dual-therapy, however, has not been considered in HNSCC. The only previous studies of PI3K and ALK inhibitors examine such combinations in the context of other tumor types driven by ALK signaling due to fusion with *EML4* or other genes [79–82]. Our recent work shows that co-treatment with ALK inhibitor brigatinib and PI3K inhibitor pictilisib is synergistic in HNSCC models (submitted); this may represent the first evidence of interaction between

**72**

monotherapies.

#### **3.7 Focal adhesion kinase (FAK)**

Although most pathways nominated as drivers of PI3K inhibitor resistance are RTKs, a potential relationship between PI3K and the cytoplasmic FAK receptor has also been described. FAK, which is encoded by the Protein Tyrosine Kinase 2 (*PTK2*) gene [94], has a wide range of intracellular functions, involving regulation of cell adhesion, cell proliferation, migration, and cell apoptosis, all of which are commonly altered in head and neck cancer [95]. One previous study assessed 147 HNSCC clinical tumor samples and reported FAK protein overexpression and *PTK2* gene amplification in 62 and 39% of these samples, respectively, supporting a molecular role for this kinase in HNSCC [96]. FAK inhibitors serve to arrest the cell cycle and decrease tumor cell viability via the induction of apoptosis [97]. One such inhibitor, TAE226, is able to induce the tumor cell apoptosis and in turn suppress HNSCC growth in vitro; this inhibitor also blocked tumor growth, metastasis, and angiogenesis in vivo [98]. The mechanisms by which the FAK signaling pathway is involved in tumor cell metastases and growth may be kinase-dependent; in this case, FAK is upstream of PI3K signaling pathway. One hypothesis is that activation of FAK could induce the PI3K and subsequent downstream signaling including remodeling of the cytoskeletal, matrix metalloproteinases (MMPs) as well as formation or turnover of focal adhesion. Such effects might contribute to the metastases of tumor cells. Activation of the FAK-PI3K signaling pathway has also been shown to inhibit tumor cell apoptosis [99–102]. Interestingly, loss of *PTEN*, has been shown to stimulate FAK and downstream targets [103]. However, a role for FAK inhibition has not been well described in cases of *PIK3CA* amplification or mutation. One clinical trial (NCT02372227) sought to evaluate the safety of combining PI3K and FAK inhibitors VS-5584 and VS-6063 in malignant mesothelioma but was terminated prior to completion. A recent report (submitted) shows that FAK/IGF-1R inhibitor TAE226 may be synergistic with PI3K inhibitors in HNSCC cell lines and xenografts, but the contribution of FAK signaling and the mechanism for this effect have yet to be fully determined. Taken together, however, this collection of data nominates FAK signaling as a means of resistance to PI3K inhibitor monotherapy in HNSCC.

#### **3.8 Aurora kinase A (AURKA)**

Aurora kinase A (AURKA) is another player identified as having a potentially indispensable role in PI3K inhibitor response in some HNSCCs. AURKA is one of the aurora serine-threonine kinase family members, which play an important role in the regulation of cell cycle and cell division by controlling mitosis and meiosis [104]. In normal cells, AURKA regulates mitosis by contributing to maturation of the centrosome, synthesizing of bipolar spindle, and controlling cytokinesis. Researchers also showed that the upregulation of AURKA is associated with worse prognosis and decreased survival of patients with HNSCCs [105–107]. These poor outcomes are postulated to result from associated centrosome abnormalities and chromosomal aneusomy as well as activation of the spindle assembly checkpoint [106, 108]. AURKA is also involved in both AKT and FAK signaling pathways, and this AURKA/AKT/FAK signaling axis can be responsible for the migration and invasion of HNSCCs [109]. Previous research shows that suppression of AURKA via shRNA could inhibit the ability of laryngeal carcinoma cells to grow and invade both in vitro and in vivo, suggesting that AURKA inhibitor monotherapy may have a substantial effect on oncogenic phenotypes in some models [110]. Furthermore, recent data (submitted) indicate that the regulation of cell cycle gene expression and protein levels, including AURKA, is reduced by synergistic PI3K inhibitor

**75**

for combination effects.

sible to ascertain from this study alone.

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms*

dual-therapies. The altered function of AURKA shows that aurora kinases could

Overactivation of PI3K/AKT pathway can inhibit tumor cell apoptosis and promote cell proliferation. The family of forkhead transcription factors (FOXOs) is among the specific downstream PI3K/AKT effectors involved in the regulation of cell apoptosis. The PI3K/AKT pathway mediates serine/threonine phosphorylation of FOXO transcription factors, which reverses the pro-apoptotic effect of FOXOs by downregulating the pro-apoptotic protein BIM, a Bcl-2 family member. Conversely, the use of PI3K and AKT inhibitors may induce cell apoptosis in various tumor types, a phenomenon already detailed in breast cancer models [111]. PI3K/AKT inhibition reduces FOXO phosphorylation leading to the upregulation of BIM [112], an effect that may contribute to apoptosis. The role of specific FOXO family members and their transcriptional targets as well as how these functions are altered by modulation of PI3K signaling has yet to be fully determined in

Much of the previous work on compensatory resistance to PI3K inhibition has noted contributions from pathways downstream of PI3K or other codependent RTKs, including PDK1 [30], AKT [113], mTOR [114–116], and MEK [117, 118]. Previous publications [117, 118] show that inhibiting PI3K and MEK is functionally similar to inhibiting PI3K and EGFR (see above). Prior studies have also highlighted the importance of PI3K effector AKT in combination responses; these publications describe similar evidence of synergy when replacing PI3K inhibitors with AKT inhibition or siRNA [46] and report reduced AKT phosphorylation following combination PI3K and EGFR inhibitor treatments [44, 46, 47, 82, 119]. Similarly, the work of Sambandam et al. demonstrates the importance of PDK1 inhibition in PI3K inhibitor responses by showing that: (1) reductions in the level of phosphorylated and total PDK1 were present in cell lines that were more sensitive to PI3K inhibition and (2) AKT inhibitor MK-2206 and PDK1 inhibitor GSK2334470 were synergistic when used together, recapitulating or exceeding the effects of PI3K inhibitor monotherapy [30]. In contrast, other analyses (submitted) demonstrate that AKT phosphorylation was similarly reduced following treatment with PI3K monotherapy and ineffective combinations as compared to treatment with synergistic drug pairs. This may suggest that AKT inhibition is necessary, but not sufficient, for response to PI3K inhibitor therapy. PDK1 phosphorylation was also unchanged with PI3K mono- and dual-therapy in this recent dataset (submitted), and little benefit was noted with PDK1, AKT, or mTOR inhibitors to PI3K inhibitors. Based on these findings, it seems that although PDK1, AKT, and mTOR are downstream effectors common to PI3K and the other RTKs involved in synergistic drug pairs, additional mechanisms are responsible

Clinical data evaluating the contribution of targeting both PI3K and its downstream PI3K remain immature. Trials have been performed to determine the safe dose of PI3K inhibitor BEZ235 in combination with mTOR inhibitors RAD001 (NCT01482156) or everolimus (NCT01508104), but results have yet to be published. While there is a trial of PI3K/mTOR inhibitor bimiralisib (NCT03740100) currently ongoing, the contribution of PI3K and mTOR individually will be impos-

play a pivotal compensatory signaling role in PI3K inhibitor resistance.

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

**3.9 Cell apoptosis**

HNSCC.

**3.10 PDK1, AKT, and mTOR**

dual-therapies. The altered function of AURKA shows that aurora kinases could play a pivotal compensatory signaling role in PI3K inhibitor resistance.

#### **3.9 Cell apoptosis**

*Molecular Pharmacology*

monotherapy in HNSCC.

**3.8 Aurora kinase A (AURKA)**

**3.7 Focal adhesion kinase (FAK)**

Although most pathways nominated as drivers of PI3K inhibitor resistance are RTKs, a potential relationship between PI3K and the cytoplasmic FAK receptor has also been described. FAK, which is encoded by the Protein Tyrosine Kinase 2 (*PTK2*) gene [94], has a wide range of intracellular functions, involving regulation of cell adhesion, cell proliferation, migration, and cell apoptosis, all of which are commonly altered in head and neck cancer [95]. One previous study assessed 147 HNSCC clinical tumor samples and reported FAK protein overexpression and *PTK2* gene amplification in 62 and 39% of these samples, respectively, supporting a molecular role for this kinase in HNSCC [96]. FAK inhibitors serve to arrest the cell cycle and decrease tumor cell viability via the induction of apoptosis [97]. One such inhibitor, TAE226, is able to induce the tumor cell apoptosis and in turn suppress HNSCC growth in vitro; this inhibitor also blocked tumor growth, metastasis, and angiogenesis in vivo [98]. The mechanisms by which the FAK signaling pathway is involved in tumor cell metastases and growth may be kinase-dependent; in this case, FAK is upstream of PI3K signaling pathway. One hypothesis is that activation of FAK could induce the PI3K and subsequent downstream signaling including remodeling of the cytoskeletal, matrix metalloproteinases (MMPs) as well as formation or turnover of focal adhesion. Such effects might contribute to the metastases of tumor cells. Activation of the FAK-PI3K signaling pathway has also been shown to inhibit tumor cell apoptosis [99–102]. Interestingly, loss of *PTEN*, has been shown to stimulate FAK and downstream targets [103]. However, a role for FAK inhibition has not been well described in cases of *PIK3CA* amplification or mutation. One clinical trial (NCT02372227) sought to evaluate the safety of combining PI3K and FAK inhibitors VS-5584 and VS-6063 in malignant mesothelioma but was terminated prior to completion. A recent report (submitted) shows that FAK/IGF-1R inhibitor TAE226 may be synergistic with PI3K inhibitors in HNSCC cell lines and xenografts, but the contribution of FAK signaling and the mechanism for this effect have yet to be fully determined. Taken together, however, this collection of data nominates FAK signaling as a means of resistance to PI3K inhibitor

Aurora kinase A (AURKA) is another player identified as having a potentially indispensable role in PI3K inhibitor response in some HNSCCs. AURKA is one of the aurora serine-threonine kinase family members, which play an important role in the regulation of cell cycle and cell division by controlling mitosis and meiosis [104]. In normal cells, AURKA regulates mitosis by contributing to maturation of the centrosome, synthesizing of bipolar spindle, and controlling cytokinesis. Researchers also showed that the upregulation of AURKA is associated with worse prognosis and decreased survival of patients with HNSCCs [105–107]. These poor outcomes are postulated to result from associated centrosome abnormalities and chromosomal aneusomy as well as activation of the spindle assembly checkpoint [106, 108]. AURKA is also involved in both AKT and FAK signaling pathways, and this AURKA/AKT/FAK signaling axis can be responsible for the migration and invasion of HNSCCs [109]. Previous research shows that suppression of AURKA via shRNA could inhibit the ability of laryngeal carcinoma cells to grow and invade both in vitro and in vivo, suggesting that AURKA inhibitor monotherapy may have a substantial effect on oncogenic phenotypes in some models [110]. Furthermore, recent data (submitted) indicate that the regulation of cell cycle gene expression and protein levels, including AURKA, is reduced by synergistic PI3K inhibitor

**74**

Overactivation of PI3K/AKT pathway can inhibit tumor cell apoptosis and promote cell proliferation. The family of forkhead transcription factors (FOXOs) is among the specific downstream PI3K/AKT effectors involved in the regulation of cell apoptosis. The PI3K/AKT pathway mediates serine/threonine phosphorylation of FOXO transcription factors, which reverses the pro-apoptotic effect of FOXOs by downregulating the pro-apoptotic protein BIM, a Bcl-2 family member. Conversely, the use of PI3K and AKT inhibitors may induce cell apoptosis in various tumor types, a phenomenon already detailed in breast cancer models [111]. PI3K/AKT inhibition reduces FOXO phosphorylation leading to the upregulation of BIM [112], an effect that may contribute to apoptosis. The role of specific FOXO family members and their transcriptional targets as well as how these functions are altered by modulation of PI3K signaling has yet to be fully determined in HNSCC.

#### **3.10 PDK1, AKT, and mTOR**

Much of the previous work on compensatory resistance to PI3K inhibition has noted contributions from pathways downstream of PI3K or other codependent RTKs, including PDK1 [30], AKT [113], mTOR [114–116], and MEK [117, 118]. Previous publications [117, 118] show that inhibiting PI3K and MEK is functionally similar to inhibiting PI3K and EGFR (see above). Prior studies have also highlighted the importance of PI3K effector AKT in combination responses; these publications describe similar evidence of synergy when replacing PI3K inhibitors with AKT inhibition or siRNA [46] and report reduced AKT phosphorylation following combination PI3K and EGFR inhibitor treatments [44, 46, 47, 82, 119]. Similarly, the work of Sambandam et al. demonstrates the importance of PDK1 inhibition in PI3K inhibitor responses by showing that: (1) reductions in the level of phosphorylated and total PDK1 were present in cell lines that were more sensitive to PI3K inhibition and (2) AKT inhibitor MK-2206 and PDK1 inhibitor GSK2334470 were synergistic when used together, recapitulating or exceeding the effects of PI3K inhibitor monotherapy [30]. In contrast, other analyses (submitted) demonstrate that AKT phosphorylation was similarly reduced following treatment with PI3K monotherapy and ineffective combinations as compared to treatment with synergistic drug pairs. This may suggest that AKT inhibition is necessary, but not sufficient, for response to PI3K inhibitor therapy. PDK1 phosphorylation was also unchanged with PI3K mono- and dual-therapy in this recent dataset (submitted), and little benefit was noted with PDK1, AKT, or mTOR inhibitors to PI3K inhibitors. Based on these findings, it seems that although PDK1, AKT, and mTOR are downstream effectors common to PI3K and the other RTKs involved in synergistic drug pairs, additional mechanisms are responsible for combination effects.

Clinical data evaluating the contribution of targeting both PI3K and its downstream PI3K remain immature. Trials have been performed to determine the safe dose of PI3K inhibitor BEZ235 in combination with mTOR inhibitors RAD001 (NCT01482156) or everolimus (NCT01508104), but results have yet to be published. While there is a trial of PI3K/mTOR inhibitor bimiralisib (NCT03740100) currently ongoing, the contribution of PI3K and mTOR individually will be impossible to ascertain from this study alone.

#### **3.11 Immunotherapy**

Emerging data suggest that the PI3K pathway may interact with immune responses and improve the efficacy of immune checkpoint receptor (ICR) blockade. One indication of the involvement of the PI3K pathway in responses to immunotherapies is based on data using models with loss of tumor suppressor *PTEN*. In multiple cancer types, increased expression of programmed death ligand-1 (PD-L1) has been observed in models with loss of *PTEN* [120–123], and there is some evidence that this may also be the case in HNSCC [124].

PI3K signaling also may interface with immune responses through its important role in cellular metabolism. In cancerous cells, the metabolic balance shifts from oxidative phosphorylation to aerobic glycolysis as part of a paradigm known as the Warburg effect [17]. A notable consequence of this effect is the inefficient use of glucose, which limits the maximum possible amount of ATP or energy. Since PI3K pathway activity contributes to glucose uptake and glycolysis [125], cancer cells displaying PI3K pathway activation (through *PIK3CA* mutation, *PTEN* loss or other genetic or functional changes) may utilize additional glucose from the tumor microenvironment. Nearby cytotoxic T-lymphocytes (CTLs) and other immunomodulatory cells, which require vast amounts of glucose to launch and maintain effective immune attack on tumor spread, are therefore in heated competition with PI3K pathway-activated cells for the limited glucose supply. As such, CTL activation, including migration to the tumor site, production of cytokines, and other immune functions, is limited and CTL exhaustion occurs more rapidly [126–130]. PI3K inhibition, however, alters cellular metabolism to prevent or delay CTL exhaustion and thereby allows for a faster and/or more durable CTL response.

Additionally, PI3K inhibition may enhance the effects of ICR blockade by preventing adaptive resistance. One mechanism that may blunt responses to ICR blockade involves the compensatory upregulation of other ICRs. Shayan and coworkers showed that in HNSCC tumor samples, two ICRs, programmed cell death receptor-1 (PD-1) and T-cell Ig and mucin domain-3 protein (TIM-3), were co-expressed on CTLs displaying high levels of exhaustion [131]. PD-1 blockade resulted in further upregulation of TIM-3; this upregulation was reversible using PI3K inhibition [131]. Thus, through multiple means, PI3K inhibition may augment responses to immunotherapy in HNSCC. Clinical trials to evaluate therapies that combine PI3K inhibitor and ICR blockade are ongoing (including NCT04317105 to evaluate PD-1 inhibitor nivolumab and PI3K inhibitor copanlisib in solid tumors), and the results of these studies will offer critical insights for the future of targeted and immunotherapies in HNSCC and other cancers.

#### **4. Conclusions**

Due to advances in diverse profiling strategies ranging from next generation sequencing to combinatorial high throughput small molecule profiling to pooled CRISPR and shRNA screens, recent technological advances have led to significant scientific advances in understanding the mechanisms that drive response to PI3K inhibitors in HNSCC and other cancers. Indeed, the future is bright for the advancement of PI3K inhibitors, especially as combination therapies, in HNSCC and other cancer types. Nevertheless, multiple questions remain regarding the role of specific signaling pathways in PI3K inhibitor resistance, and additional studies will be required to further our understanding of this important intersection of pharmacology and cancer biology. More work is needed to develop safer, more effective drugs, to establish biomarkers for response, and to target critical

**77**

**Author details**

United States

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms*

resistance mechanisms. The studies described in this chapter serve as a contribution in these efforts. With the combined efforts of the community, PI3K inhibitors

may, in time, have a place among standard-of-care treatments for HNSCC.

Nicole L. Michmerhuizen1,2, Jiayu Wang1,2 and J. Chad Brenner1,2,3,4\*

2 Department of Pharmacology, University of Michigan Medical School,

3 Program in Cellular and Molecular Biology, University of Michigan Medical

4 Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Medical School, Ann Arbor, MI, United States

\*Address all correspondence to: chadbren@umich.edu

Ann Arbor, MI, United States

School, Ann Arbor, MI, United States

provided the original work is properly cited.

1 Department of Otolaryngology – Head and Neck Surgery, University of Michigan

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

The authors declare no conflict of interest.

**Conflict of interest**

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.92875*

resistance mechanisms. The studies described in this chapter serve as a contribution in these efforts. With the combined efforts of the community, PI3K inhibitors may, in time, have a place among standard-of-care treatments for HNSCC.

### **Conflict of interest**

*Molecular Pharmacology*

**3.11 Immunotherapy**

Emerging data suggest that the PI3K pathway may interact with immune responses and improve the efficacy of immune checkpoint receptor (ICR) blockade. One indication of the involvement of the PI3K pathway in responses to immunotherapies is based on data using models with loss of tumor suppressor *PTEN*. In multiple cancer types, increased expression of programmed death ligand-1 (PD-L1) has been observed in models with loss of *PTEN* [120–123], and there is some

PI3K signaling also may interface with immune responses through its important role in cellular metabolism. In cancerous cells, the metabolic balance shifts from oxidative phosphorylation to aerobic glycolysis as part of a paradigm known as the Warburg effect [17]. A notable consequence of this effect is the inefficient use of glucose, which limits the maximum possible amount of ATP or energy. Since PI3K pathway activity contributes to glucose uptake and glycolysis [125], cancer cells displaying PI3K pathway activation (through *PIK3CA* mutation, *PTEN* loss or other genetic or functional changes) may utilize additional glucose from the tumor microenvironment. Nearby cytotoxic T-lymphocytes (CTLs) and other immunomodulatory cells, which require vast amounts of glucose to launch and maintain effective immune attack on tumor spread, are therefore in heated competition with PI3K pathway-activated cells for the limited glucose supply. As such, CTL activation, including migration to the tumor site, production of cytokines, and other immune functions, is limited and CTL exhaustion occurs more rapidly [126–130]. PI3K inhibition, however, alters cellular metabolism to prevent or delay CTL exhaustion and thereby allows for a faster and/or more durable CTL response. Additionally, PI3K inhibition may enhance the effects of ICR blockade by preventing adaptive resistance. One mechanism that may blunt responses to ICR blockade involves the compensatory upregulation of other ICRs. Shayan and coworkers showed that in HNSCC tumor samples, two ICRs, programmed cell death receptor-1 (PD-1) and T-cell Ig and mucin domain-3 protein (TIM-3), were co-expressed on CTLs displaying high levels of exhaustion [131]. PD-1 blockade resulted in further upregulation of TIM-3; this upregulation was reversible using PI3K inhibition [131]. Thus, through multiple means, PI3K inhibition may augment responses to immunotherapy in HNSCC. Clinical trials to evaluate therapies that combine PI3K inhibitor and ICR blockade are ongoing (including NCT04317105 to evaluate PD-1 inhibitor nivolumab and PI3K inhibitor copanlisib in solid tumors), and the results of these studies will offer critical insights for the future of targeted

evidence that this may also be the case in HNSCC [124].

and immunotherapies in HNSCC and other cancers.

Due to advances in diverse profiling strategies ranging from next generation sequencing to combinatorial high throughput small molecule profiling to pooled CRISPR and shRNA screens, recent technological advances have led to significant scientific advances in understanding the mechanisms that drive response to PI3K inhibitors in HNSCC and other cancers. Indeed, the future is bright for the advancement of PI3K inhibitors, especially as combination therapies, in HNSCC and other cancer types. Nevertheless, multiple questions remain regarding the role of specific signaling pathways in PI3K inhibitor resistance, and additional studies will be required to further our understanding of this important intersection of pharmacology and cancer biology. More work is needed to develop safer, more effective drugs, to establish biomarkers for response, and to target critical

**76**

**4. Conclusions**

The authors declare no conflict of interest.

### **Author details**

Nicole L. Michmerhuizen1,2, Jiayu Wang1,2 and J. Chad Brenner1,2,3,4\*

1 Department of Otolaryngology – Head and Neck Surgery, University of Michigan Medical School, Ann Arbor, MI, United States

2 Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, United States

3 Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, United States

4 Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, United States

\*Address all correspondence to: chadbren@umich.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[14] Cancer Genome Atlas

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2013;**6**(269):pl1

[12] Thorpe LM, Yuzugullu H,

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*Molecular Pharmacology*

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[39] Zainal NS, Lee BKB, Wong ZW, Chin IS, Yee PS, Gan CP, et al. Effects of palbociclib on oral squamous cell carcinoma and the role of PIK3CA in conferring resistance. Cancer Biology &

[40] Singh B, Carpenter G, Coffey RJ. EGF receptor ligands: Recent advances. F1000Research. 2016;**5**(F1000 Faculty

[41] Wee P, Wang Z. Epidermal growth factor receptor cell proliferation signaling pathways. Cancers (Basel).

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of Pathology. 1986;**149**(1):9-14

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Medicine. 2019;**16**(2):264-275

Oral Oncology. 2019;**91**:69-78

[38] Keysar SB, Astling DP,

2013;**7**(4):776-790

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2017;**9**(5):52

S24-S31

2017;**28**(10):2526-2532

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kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo. Molecular Cancer Therapeutics.

[33] Burtness B, Goldwasser MA, Flood W, Mattar B, Forastiere AA. Phase III randomized trial of cisplatin plus placebo compared with cisplatin

plus cetuximab in metastatic/ recurrent head and neck cancer: An Eastern Cooperative Oncology Group study. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology.

2005;**23**(34):8646-8654

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2014;**20**(11):3023-3032

2015;**16**(5):583-594

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squamous-cell carcinoma of the head and neck progressing on or after platinum-based therapy (LUX-Head & Neck 1): An open-label, randomised phase 3 trial. The Lancet Oncology.

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model of head and neck cancer. The Laryngoscope. 2016;**126**(4):E156-E163

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[88] Vairaktaris E, Ragos V, Yapijakis C, Derka S, Vassiliou S, Nkenke E, et al. FGFR-2 and-3 play an important role in initial stages of oral oncogenesis. Anticancer Research. 2006;**26**(6B): 4217-4221

[89] Sweeny L, Liu Z, Lancaster W, Hart J, Hartman YE, Rosenthal EL. Inhibition of fibroblasts reduced head and neck cancer growth by targeting fibroblast growth factor receptor. The Laryngoscope. 2012;**122**(7):1539-1544

[90] Koole K, Brunen D, van Kempen PM, Noorlag R, de Bree R, Lieftink C, et al. FGFR1 is a potential prognostic biomarker and therapeutic target in head and neck squamous cell carcinoma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2016;**22**(15):3884-3893

[91] Quintanal-Villalonga A, Molina-Pinelo S, Cirauqui C, Ojeda-Marquez L, Marrugal A, Suarez R, et al. FGFR1 cooperates with EGFR in lung cancer oncogenesis, and their combined inhibition shows improved efficacy. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer. 2019;**14**(4):641-655

[92] Das M, Padda SK, Frymoyer A, Zhou L, Riess JW, Neal JW, et al. Dovitinib and erlotinib in patients with metastatic non-small cell lung cancer: A drug-drug interaction. Lung Cancer (Amsterdam, Netherlands). 2015;**89**(3):280-286

[93] Chae YK, Pai SG, Sun P, Costa R, Matsangou M, Agulnik M, et al. Fibroblast growth factor receptor (FGFR) as a therapeutic target in lung and head and neck cancer. American Journal of Hematology/Oncology®. 2016;**12**(3):13-19

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[99] Chen J-S, Huang X-h, Wang Q, Huang J-Q, L-j Z, Chen X-L, et al. Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer. Carcinogenesis. 2012;**34**(1):10-19

[100] Zhao J, Guan J-L. Signal transduction by focal adhesion kinase in cancer. Cancer Metastasis Reviews. 2009;**28**(1-2):35-49

[101] Reif S, Lang A, Lindquist JN, Yata Y, Gäbele E, Scanga A, et al. The role of focal adhesion kinasephosphatidylinositol 3-kinase-akt

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signaling pathway. Oncology Letters.

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[112] Urbich C, Knau A, Fichtlscherer S, Walter DH, Brühl T, Potente M, et al. FOXO-dependent expression of the proapoptotic protein Bim: Pivotal role for apoptosis signaling in endothelial progenitor cells. The FASEB Journal.

Rickwood D, Endo-Munoz L, Cameron S, Dahler A, et al. Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma. British Journal of Cancer. 2012;**106**(1):107-115

2016;**11**(3):1889-1894

2011;**357**(1-2):95-106

2014;**4**(3):334-347

2005;**19**(8):974-976

[113] Erlich RB, Kherrouche Z,

[114] Tonlaar N, Galoforo S,

Thibodeau BJ, Ahmed S, Wilson TG, Yumpo Cardenas P, et al. Antitumor activity of the dual PI3K/MTOR

inhibitor, PF-04691502, in combination

with radiation in head and neck cancer. Radiotherapy and Oncology.

[115] Elkabets M, Vora S, Juric D, Morse N, Mino-Kenudson M,

Muranen T, et al. mTORC1 inhibition is required for sensitivity to PI3K p110alpha inhibitors in PIK3CA-mutant breast cancer. Science Translational Medicine. 2013;**5**(196):196ra99

2017;**124**(3):504-512

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

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[104] Crane R, Gadea B, Littlepage L, Wu H, Ruderman JV. Aurora A, meiosis

[105] Mehra R, Serebriiskii IG, Burtness B, Astsaturov I, Golemis EA. Aurora kinases in head and neck cancer. The Lancet Oncology. 2013;**14**(10):e425-ee35

[106] Marumoto T, Zhang D, Saya H. Aurora-A—A guardian of poles. Nature

Reviews. Cancer. 2005;**5**(1):42

[107] Reiter R, Gais P, Jütting U, Steuer-Vogt MK, Pickhard A, Bink K, et al. Aurora kinase A messenger RNA overexpression is correlated with tumor progression and shortened survival in head and neck squamous cell carcinoma. Clinical Cancer Research.

[108] Anand S, Penrhyn-Lowe S, Venkitaraman AR. AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol. Cancer Cell.

[109] Wu J, Yang L, Shan Y, Cai C, Wang S, Zhang H. AURKA promotes cell migration and invasion of head and neck squamous cell carcinoma through regulation of the AURKA/Akt/FAK

2006;**12**(17):5136-5141

2003;**3**(1):51-62

and mitosis. Biology of the Cell.

2004;**96**(3):215-229

signaling in hepatic stellate cell proliferation and type I collagen expression. The Journal of Biological Chemistry. 2003;**278**(10):8083-8090

Cancer. 2014;**14**(9):598

2017;**7**:170

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*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.92875*

signaling in hepatic stellate cell proliferation and type I collagen expression. The Journal of Biological Chemistry. 2003;**278**(10):8083-8090

*Molecular Pharmacology*

4217-4221

cell carcinoma cells. Clinical Cancer Research. 2011;**17**(15):5016-5025

[94] André E, Beckerandre M.

form of human focal adhesion kinase in brain. Biochemical and Biophysical Research Communications.

1993;**190**(1):140-147

2010;**10**(10):735-741

2006;**12**(11):3272-3279

Expression of an N-terminally truncated

[95] Golubovskaya VM. Focal adhesion kinase as a cancer therapy target. Anti-Cancer Agents in Medicinal

Chemistry (Formerly Current Medicinal

Chemistry-Anti-Cancer Agents).

[96] Canel M, Secades P, Rodrigo J-P, Cabanillas R, Herrero A, Suarez C, et al. Overexpression of focal adhesion kinase in head and neck squamous cell carcinoma is independent of FAK gene copy number. Clinical Cancer Research.

[97] Tai Y-L, Chen L-C, Shen T-L. Emerging roles of focal adhesion kinase in cancer. BioMed Research International. 2015;**2015**:1-13

[98] Kurio N, Shimo T, Fukazawa T, Okui T, Hassan NMM, Honami T, et al. Anti-tumor effect of a novel FAK inhibitor TAE226 against human oral squamous cell carcinoma. Oral Oncology. 2012;**48**(11):1159-1170

[99] Chen J-S, Huang X-h, Wang Q, Huang J-Q, L-j Z, Chen X-L, et al. Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer. Carcinogenesis.

2012;**34**(1):10-19

2009;**28**(1-2):35-49

[100] Zhao J, Guan J-L. Signal

transduction by focal adhesion kinase in cancer. Cancer Metastasis Reviews.

[101] Reif S, Lang A, Lindquist JN, Yata Y, Gäbele E, Scanga A, et al. The role of focal adhesion kinasephosphatidylinositol 3-kinase-akt

[88] Vairaktaris E, Ragos V, Yapijakis C, Derka S, Vassiliou S, Nkenke E, et al. FGFR-2 and-3 play an important role in initial stages of oral oncogenesis. Anticancer Research. 2006;**26**(6B):

[89] Sweeny L, Liu Z, Lancaster W, Hart J, Hartman YE, Rosenthal EL. Inhibition of fibroblasts reduced head and neck cancer growth by targeting fibroblast growth factor receptor. The Laryngoscope. 2012;**122**(7):1539-1544

[90] Koole K, Brunen D, van Kempen PM, Noorlag R, de Bree R, Lieftink C, et al. FGFR1 is a potential prognostic biomarker and therapeutic target in head and neck squamous cell carcinoma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research.

2016;**22**(15):3884-3893

[91] Quintanal-Villalonga A, Molina-Pinelo S, Cirauqui C,

Ojeda-Marquez L, Marrugal A, Suarez R, et al. FGFR1 cooperates with EGFR in lung cancer oncogenesis, and their combined inhibition shows improved efficacy. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer. 2019;**14**(4):641-655

[92] Das M, Padda SK, Frymoyer A, Zhou L, Riess JW, Neal JW, et al. Dovitinib and erlotinib in patients with metastatic non-small cell lung cancer: A drug-drug interaction. Lung Cancer (Amsterdam, Netherlands).

[93] Chae YK, Pai SG, Sun P, Costa R, Matsangou M, Agulnik M, et al. Fibroblast growth factor receptor (FGFR) as a therapeutic target in lung and head and neck cancer. American Journal of Hematology/Oncology®.

2015;**89**(3):280-286

2016;**12**(3):13-19

**84**

[102] Sulzmaier FJ, Jean C, Schlaepfer DD. FAK in cancer: Mechanistic findings and clinical applications. Nature Reviews. Cancer. 2014;**14**(9):598

[103] Alfieri R, Giovannetti E, Bonelli M, Cavazzoni A. New treatment opportunities in phosphatase and Tensin homolog (PTEN)-deficient tumors: Focus on PTEN/focal adhesion kinase pathway. Frontiers in Oncology. 2017;**7**:170

[104] Crane R, Gadea B, Littlepage L, Wu H, Ruderman JV. Aurora A, meiosis and mitosis. Biology of the Cell. 2004;**96**(3):215-229

[105] Mehra R, Serebriiskii IG, Burtness B, Astsaturov I, Golemis EA. Aurora kinases in head and neck cancer. The Lancet Oncology. 2013;**14**(10):e425-ee35

[106] Marumoto T, Zhang D, Saya H. Aurora-A—A guardian of poles. Nature Reviews. Cancer. 2005;**5**(1):42

[107] Reiter R, Gais P, Jütting U, Steuer-Vogt MK, Pickhard A, Bink K, et al. Aurora kinase A messenger RNA overexpression is correlated with tumor progression and shortened survival in head and neck squamous cell carcinoma. Clinical Cancer Research. 2006;**12**(17):5136-5141

[108] Anand S, Penrhyn-Lowe S, Venkitaraman AR. AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol. Cancer Cell. 2003;**3**(1):51-62

[109] Wu J, Yang L, Shan Y, Cai C, Wang S, Zhang H. AURKA promotes cell migration and invasion of head and neck squamous cell carcinoma through regulation of the AURKA/Akt/FAK

signaling pathway. Oncology Letters. 2016;**11**(3):1889-1894

[110] Zhang H, Chen X, Liu B, Zhou L. Effects of stable knockdown of Aurora kinase A on proliferation, migration, chromosomal instability, and expression of focal adhesion kinase and matrix metalloproteinase-2 in HEp-2 cells. Molecular and Cellular Biochemistry. 2011;**357**(1-2):95-106

[111] Will M, Qin ACR, Toy W, Yao Z, Rodrik-Outmezguine V, Schneider C, et al. Rapid induction of apoptosis by PI3K inhibitors is dependent upon their transient inhibition of RAS-ERK signaling. Cancer Discovery. 2014;**4**(3):334-347

[112] Urbich C, Knau A, Fichtlscherer S, Walter DH, Brühl T, Potente M, et al. FOXO-dependent expression of the proapoptotic protein Bim: Pivotal role for apoptosis signaling in endothelial progenitor cells. The FASEB Journal. 2005;**19**(8):974-976

[113] Erlich RB, Kherrouche Z, Rickwood D, Endo-Munoz L, Cameron S, Dahler A, et al. Preclinical evaluation of dual PI3K-mTOR inhibitors and histone deacetylase inhibitors in head and neck squamous cell carcinoma. British Journal of Cancer. 2012;**106**(1):107-115

[114] Tonlaar N, Galoforo S, Thibodeau BJ, Ahmed S, Wilson TG, Yumpo Cardenas P, et al. Antitumor activity of the dual PI3K/MTOR inhibitor, PF-04691502, in combination with radiation in head and neck cancer. Radiotherapy and Oncology. 2017;**124**(3):504-512

[115] Elkabets M, Vora S, Juric D, Morse N, Mino-Kenudson M, Muranen T, et al. mTORC1 inhibition is required for sensitivity to PI3K p110alpha inhibitors in PIK3CA-mutant breast cancer. Science Translational Medicine. 2013;**5**(196):196ra99

[116] Ruicci KM, Pinto N, Khan MI, Yoo J, Fung K, MacNeil D, et al. ERK-TSC2 signalling in constitutively-active HRAS mutant HNSCC cells promotes resistance to PI3K inhibition. Oral Oncology. 2018;**84**:95-103

[117] Mohan S, Vander Broek R, Shah S, Eytan DF, Pierce ML, Carlson SG, et al. MEK inhibitor PD-0325901 overcomes resistance to PI3K/mTOR inhibitor PF-5212384 and potentiates antitumor effects in human head and neck squamous cell carcinoma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2015;**21**(17):3946-3956

[118] Mazumdar T, Byers LA, Ng PK, Mills GB, Peng S, Diao L, et al. A comprehensive evaluation of biomarkers predictive of response to PI3K inhibitors and of resistance mechanisms in head and neck squamous cell carcinoma. Molecular Cancer Therapeutics. 2014;**13**(11):2738-2750

[119] Benavente S, Huang S, Armstrong EA, Chi A, Hsu KT, Wheeler DL, et al. Establishment and characterization of a model of acquired resistance to epidermal growth factor receptor targeting agents in human cancer cells. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2009;**15**(5):1585-1592

[120] Xu C, Fillmore CM, Koyama S, Wu H, Zhao Y, Chen Z, et al. Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression. Cancer Cell. 2014;**25**(5):590-604

[121] Zhu J, Chen L, Zou L, Yang P, Wu R, Mao Y, et al. MiR-20b, -21, and -130b inhibit PTEN expression resulting in B7-H1 over-expression in advanced colorectal cancer. Human Immunology. 2014;**75**(4):348-353

[122] Zhang Y, Zhang J, Xu K, Xiao Z, Sun J, Xu J, et al. PTEN/PI3K/ mTOR/B7-H1 signaling pathway regulates cell progression and immuno-resistance in pancreatic cancer. Hepato-Gastroenterology. 2013;**60**(127):1766-1772

[123] Parsa AT, Waldron JS, Panner A, Crane CA, Parney IF, Barry JJ, et al. Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nature Medicine. 2007;**13**(1):84-88

[124] Lyford-Pike S, Peng S, Young GD, Taube JM, Westra WH, Akpeng B, et al. Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPVassociated head and neck squamous cell carcinoma. Cancer Research. 2013;**73**(6):1733-1741

[125] Courtnay R, Ngo DC, Malik N, Ververis K, Tortorella SM, Karagiannis TC. Cancer metabolism and the Warburg effect: The role of HIF-1 and PI3K. Molecular Biology Reports. 2015;**42**(4):841-851

[126] Sugiura A, Rathmell JC. Metabolic barriers to T cell function in tumors. Journal of Immunology. 2018;**200**(2):400-407

[127] Siska PJ, Rathmell JC. T cell metabolic fitness in antitumor immunity. Trends in Immunology. 2015;**36**(4):257-264

[128] Delgoffe GM, Powell JD. Feeding an army: The metabolism of T cells in activation, anergy, and exhaustion. Molecular Immunology. 2015;**68**(2 Pt C):492-496

[129] Topalian SL, Taube JM, Anders RA, Pardoll DM. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nature Reviews. Cancer. 2016;**16**(5):275-287

**87**

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms*

*DOI: http://dx.doi.org/10.5772/intechopen.92875*

[130] Palucka AK, Coussens LM. The basis of oncoimmunology. Cell.

[131] Shayan G, Srivastava R, Li J, Schmitt N, Kane LP, Ferris RL. Adaptive resistance to anti-PD1 therapy by Tim-3 upregulation is mediated by the PI3K-Akt pathway in head and neck cancer. Oncoimmunology. 2017;**6**(1):e1261779

2016;**164**(6):1233-1247

*Integrated Molecular Profiling as an Approach to Identify PI3K Inhibitor Resistance Mechanisms DOI: http://dx.doi.org/10.5772/intechopen.92875*

[130] Palucka AK, Coussens LM. The basis of oncoimmunology. Cell. 2016;**164**(6):1233-1247

*Molecular Pharmacology*

Oncology. 2018;**84**:95-103

[116] Ruicci KM, Pinto N, Khan MI, Yoo J, Fung K, MacNeil D, et al. ERK-TSC2 signalling in constitutively-active HRAS mutant HNSCC cells promotes resistance to PI3K inhibition. Oral

[122] Zhang Y, Zhang J, Xu K, Xiao Z,

[123] Parsa AT, Waldron JS, Panner A, Crane CA, Parney IF, Barry JJ, et al. Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nature

[124] Lyford-Pike S, Peng S, Young GD, Taube JM, Westra WH, Akpeng B, et al. Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPVassociated head and neck squamous cell carcinoma. Cancer Research.

[125] Courtnay R, Ngo DC, Malik N,

Karagiannis TC. Cancer metabolism and the Warburg effect: The role of HIF-1 and PI3K. Molecular Biology Reports.

Sun J, Xu J, et al. PTEN/PI3K/ mTOR/B7-H1 signaling pathway regulates cell progression and immuno-resistance in pancreatic cancer. Hepato-Gastroenterology.

2013;**60**(127):1766-1772

Medicine. 2007;**13**(1):84-88

2013;**73**(6):1733-1741

2015;**42**(4):841-851

2018;**200**(2):400-407

2015;**36**(4):257-264

C):492-496

Ververis K, Tortorella SM,

[126] Sugiura A, Rathmell JC. Metabolic barriers to T cell function in tumors. Journal of Immunology.

[127] Siska PJ, Rathmell JC. T cell metabolic fitness in antitumor immunity. Trends in Immunology.

[128] Delgoffe GM, Powell JD. Feeding an army: The metabolism of T cells in activation, anergy, and exhaustion. Molecular Immunology. 2015;**68**(2 Pt

[129] Topalian SL, Taube JM, Anders RA,

biomarkers to guide immune checkpoint blockade in cancer therapy. Nature Reviews. Cancer. 2016;**16**(5):275-287

Pardoll DM. Mechanism-driven

[117] Mohan S, Vander Broek R, Shah S, Eytan DF, Pierce ML, Carlson SG, et al. MEK inhibitor PD-0325901 overcomes resistance to PI3K/mTOR inhibitor PF-5212384 and potentiates antitumor effects in human head and neck squamous cell carcinoma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 2015;**21**(17):3946-3956

[118] Mazumdar T, Byers LA, Ng PK, Mills GB, Peng S, Diao L, et al. A comprehensive evaluation of biomarkers predictive of response to PI3K inhibitors and of resistance mechanisms in head and neck squamous cell carcinoma. Molecular Cancer Therapeutics. 2014;**13**(11):2738-2750

[119] Benavente S, Huang S, Armstrong EA, Chi A, Hsu KT, Wheeler DL, et al. Establishment and characterization of a model of acquired resistance to epidermal growth factor receptor targeting agents in human cancer cells. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research.

2009;**15**(5):1585-1592

2014;**25**(5):590-604

2014;**75**(4):348-353

[120] Xu C, Fillmore CM, Koyama S, Wu H, Zhao Y, Chen Z, et al. Loss of Lkb1 and Pten leads to lung squamous

[121] Zhu J, Chen L, Zou L, Yang P, Wu R, Mao Y, et al. MiR-20b, -21, and -130b inhibit PTEN expression resulting in B7-H1 over-expression in advanced colorectal cancer. Human Immunology.

cell carcinoma with elevated PD-L1 expression. Cancer Cell.

**86**

[131] Shayan G, Srivastava R, Li J, Schmitt N, Kane LP, Ferris RL. Adaptive resistance to anti-PD1 therapy by Tim-3 upregulation is mediated by the PI3K-Akt pathway in head and neck cancer. Oncoimmunology. 2017;**6**(1):e1261779

**89**

**Chapter 5**

**Abstract**

Allosteric Modulators for GPCRs

as a Therapeutic Alternative with

High Potential in Drug Discovery

The superfamily of G protein-coupled receptors (GPCRs) consists of biological microprocessors that can activate multiple signaling pathways. Most GPCRs have an orthosteric pocket where the endogenous ligand(s) typically binds. Conversely, allosteric ligands bind to GPCRs at sites that are distinct from the orthosteric binding region and they modulate the response elicited by the endogenous ligand. Allosteric ligands can also switch the response of a GPCR after ligand binding to a unique signaling pathway, these ligands are termed biased allosteric modulators. Thus, the development of allosteric ligands opens new and multiple ways in which the signaling pathways of GPCRs can be manipulated for potential therapeutic benefit. Furthermore, the mechanisms by which allosteric ligands modulate the effects of endogenous ligands have provided new insights into the interactions between allosteric ligands and GPCRs. These new findings have a high potential to improve drug discovery and development and, therefore, creating the need for better screening methods for allosteric drugs to increase the chances of success in

*Arfaxad Reyes Alcaraz, Emilio Y. Lucero Garcia-Rojas,* 

the development of allosteric modulators as lead clinical compounds.

orthosteric site, endogenous agonist

agonist response to a single signaling pathway [1, 2].

**1. Introduction**

**Keywords:** GPCRs, allosteric modulators, biased signaling, β-Arrestin, G-protein,

Allosteric modulators are small molecules or peptides that by specifically interacting with the receptor can alter the affinity, and/or efficacy of the endogenous hormone or other orthosteric ligands including antagonists, and possibly even constitutive signaling by GPCRs. By modifying these pharmacological parameters, allosteric modulators can exert multiple effects on the signaling of GPCRs. Positive Allosteric Modulators (PAMs) potentiate the signaling of the receptor by increasing the affinity and/or efficacy of the endogenous ligand or other administered agonists. On the contrary, Negative Allosteric Modulators (NAMs) decrease the affinity and/or efficacy of the agonists. Biased Allosteric Modulators (BAMs) will direct the

Before going deeper into pharmacological concepts, it is necessary to define fundamental parameters used to describe the activity of a ligand. Affinity refers to the

*Richard A. Bond and Bradley K. McConnell*

#### **Chapter 5**

## Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug Discovery

*Arfaxad Reyes Alcaraz, Emilio Y. Lucero Garcia-Rojas, Richard A. Bond and Bradley K. McConnell*

#### **Abstract**

The superfamily of G protein-coupled receptors (GPCRs) consists of biological microprocessors that can activate multiple signaling pathways. Most GPCRs have an orthosteric pocket where the endogenous ligand(s) typically binds. Conversely, allosteric ligands bind to GPCRs at sites that are distinct from the orthosteric binding region and they modulate the response elicited by the endogenous ligand. Allosteric ligands can also switch the response of a GPCR after ligand binding to a unique signaling pathway, these ligands are termed biased allosteric modulators. Thus, the development of allosteric ligands opens new and multiple ways in which the signaling pathways of GPCRs can be manipulated for potential therapeutic benefit. Furthermore, the mechanisms by which allosteric ligands modulate the effects of endogenous ligands have provided new insights into the interactions between allosteric ligands and GPCRs. These new findings have a high potential to improve drug discovery and development and, therefore, creating the need for better screening methods for allosteric drugs to increase the chances of success in the development of allosteric modulators as lead clinical compounds.

**Keywords:** GPCRs, allosteric modulators, biased signaling, β-Arrestin, G-protein, orthosteric site, endogenous agonist

#### **1. Introduction**

Allosteric modulators are small molecules or peptides that by specifically interacting with the receptor can alter the affinity, and/or efficacy of the endogenous hormone or other orthosteric ligands including antagonists, and possibly even constitutive signaling by GPCRs. By modifying these pharmacological parameters, allosteric modulators can exert multiple effects on the signaling of GPCRs. Positive Allosteric Modulators (PAMs) potentiate the signaling of the receptor by increasing the affinity and/or efficacy of the endogenous ligand or other administered agonists. On the contrary, Negative Allosteric Modulators (NAMs) decrease the affinity and/or efficacy of the agonists. Biased Allosteric Modulators (BAMs) will direct the agonist response to a single signaling pathway [1, 2].

Before going deeper into pharmacological concepts, it is necessary to define fundamental parameters used to describe the activity of a ligand. Affinity refers to the

capacity of a ligand to bind to a receptor. The efficacy of a ligand, is the ability of a ligand to activate or amplify a response after binding to a receptor. Then Furchgott defined intrinsic efficacy as efficacy divided by the total receptor number, in hopes of defining a unique ligand-receptor value much like a ligand's affinity [3]. Earlier, Ariens had introduced the term 'intrinsic activity' of a ligand to explain the behavior of partial agonists [4]. Ariens proposed assigning the maximum response of the endogenous ligand a value of 1 or 100% and partial agonists were expressed as a fraction of this response. The discovery of inverse agonists, ligands that can shut down constitutive signaling by a GPCR expanded the scale from −1 to 1 (or − 100– 100%). A ligand that does not produce a cellular response (i.e., zero efficacy) when bound to the orthosteric site of the receptor is termed an antagonist [5].

The site to which endogenous agonists bind to is defined as the orthosteric site. Allosteric modulators do not bind to this site. They bind to other sites and are thus termed allosteric sites [6]. Upon binding, modulators generally stabilize a preexisting conformation or change the structural conformation of the receptor. This will often modulate the orthosteric site and can modify the effects of the agonists, or in theory, inverse agonists [1]. Allosteric modulators can also stabilize one of the multiple conformational states of the receptor [7].

Experimentally, allosteric modulation can be challenging because the allosteric modulators may affect the affinity and/or efficacy differently for each agonist (see agonist or probe dependence discussed below). For instance, different agonists that induce the same cellular response, after binding to the same receptor, can be differentially modulated by the same allosteric modulator [1]. Furthermore, they are usually difficult to screen for because they do not produce an effect by themselves and may not displace radiolabeled ligands used in binding assays.

Drugs that target the orthosteric site of G protein-coupled receptors (GPCRs) are currently the most common therapeutic tools. Allosteric binding sites (e.g., sites elsewhere on the receptors) are less well-defined and, therefore, less exploited clinically. Diversity in location, mechanism, and specificity of allosteric ligands are characteristics giving them a great potential to extend the range of the ways that drugs can modulate GPCR signaling.

#### **2. Advantages of allosteric modulators**

Allosteric modulators with no intrinsic efficacy will usually only exert their effects in the presence of an endogenous agonist. Thus, they can selectively tune cellular responses in tissues where the endogenous agonist exerts its physiological or pathophysiological functions. As a result, temporal and spatial aspects of the endogenous agonist signaling can be chronically maintained or even corrected in pathological states. Also, the saturation of allosteric binding sites limits itself the action of the allosteric modulator and the effect on the function of the agonist. This excellent property of the agonist overcomes the overdosing of a drug, making the allosteric modulators much safer than classical drugs [8].

A great benefit of using allosteric ligands for therapeutic applications is their huge potential to achieve greater selectivity at subtypes of GPCRs [8]. This could be due to greater diversity in the amino acid sequence of the allosteric binding sites compared to the orthosteric binding pocket. Another possibility is via selective cooperativity between the allosteric and orthosteric binding sites at a given receptor subtype. In addition, in some GPCRs, where the orthosteric binding site is not clear or its structure is poorly defined, the allosteric binding site might be a good alternative to target with small molecules; this has been observed in receptors with long

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target validation.

*Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug…*

peptidic ligands and is frequently found with the class B GPCRs (which are GPCRs characterized of having a long N-terminal extracellular domain). In general, the advantages of allosteric modulators apply regardless of the specific therapeutic area

Radioligand binding and functional assays have particular advantages when they are used to screen for allosteric modulators of GPCRs. Nevertheless, the extent and direction (that is positive or negative) of the interaction between the allosteric and orthosteric ligand for receptors with more than one endogenous ligand will depend on the orthosteric agonist interacting with the receptor; this phenomenon is known as probe or agonist dependence (**Figure 1**). Agonist dependence makes more complex the identification and pharmacological characterization of allosteric modulators. Particularly, the case of aplaviroc (an allosteric modulator for CC-chemokine receptor 5) showed no effect in the binding of CCL5 to the receptor but totally

Agonist dependence, or better known as probe dependence, can seriously affect potential therapeutics and also the development of allosteric ligands. In the ideal situation, the endogenous ligand would be used as a prototypic ligand in the high throughput screening process during drug development. However, in a real situation, the endogenous agonists, most of the time are unsuitable and susceptible to rapid degradation along the drug screening process and even more during *in vivo* studies. For this reason, highly stable agonists are preferentially used. Therefore, the allosteric ligand is first tested with the natural ligand on an early stage of the process in order to conclude if the achieved effects are equivalent to those registered

In order to better illustrate this concept a good example would be when a PAM has the ability to increase the cholinergic neuronal transmission in Alzheimer's disease [10], but the endogenous ligand acetylcholine is of rapid degradation making it unsuitable for drug screening. Cholinergic substitute ligands such as carbachol or pilocarpine are used for screening, and the stable analog oxotremorine is used to validate the effects of muscarinic acetylcholine receptor (mAChR) allosteric modulators *in vivo* [11]. However, the effects of the PAM LY2033298 are dependent on the orthosteric agonist (carbachol or pilocarpine) being used [12, 13] and these effects could lead to unexpected profiles of the allosteric ligand in later stages of the

The characteristic of some allosteric ligands to have differential probe dependence on multiple receptor subtypes is an additional consideration that can have significant implications for the drug screening process. For instance, LY2033298 was reported to be a specific positive allosteric modulator of the M4 mAChR. Accordingly, PAM LY2033298 increased acetylcholine-mediated calcium responses at the M4 mAChR but not for the rest of the four receptor subtypes [14]. This allosteric modulator has also been shown to have high positive cooperativity with the surrogate orthosteric ligands oxotremorine and tetramethylammonium at the M2 mAChR [13]; this positive cooperative effect is similar to that observed with LY2033298 and oxotremorine at the M4 mAChR. This is an example of how the probe-dependent interaction of oxotremorine at both the M2 and M4 receptor subtypes can confound experimental interpretation of the effect of the allosteric ligand *in vivo* [11]. This highlights the need to understand the probe dependence of allosteric ligands at related receptors to ensure a robust

*DOI: http://dx.doi.org/10.5772/intechopen.91838*

**2.1 The challenge of agonist dependence**

prevented the binding of CCL3 [9].

with the prototypical agonist.

drug discovery process.

or the tissue where the receptor is being targeted.

peptidic ligands and is frequently found with the class B GPCRs (which are GPCRs characterized of having a long N-terminal extracellular domain). In general, the advantages of allosteric modulators apply regardless of the specific therapeutic area or the tissue where the receptor is being targeted.

#### **2.1 The challenge of agonist dependence**

*Molecular Pharmacology*

capacity of a ligand to bind to a receptor. The efficacy of a ligand, is the ability of a ligand to activate or amplify a response after binding to a receptor. Then Furchgott defined intrinsic efficacy as efficacy divided by the total receptor number, in hopes of defining a unique ligand-receptor value much like a ligand's affinity [3]. Earlier, Ariens had introduced the term 'intrinsic activity' of a ligand to explain the behavior of partial agonists [4]. Ariens proposed assigning the maximum response of the endogenous ligand a value of 1 or 100% and partial agonists were expressed as a fraction of this response. The discovery of inverse agonists, ligands that can shut down constitutive signaling by a GPCR expanded the scale from −1 to 1 (or − 100– 100%). A ligand that does not produce a cellular response (i.e., zero efficacy) when

bound to the orthosteric site of the receptor is termed an antagonist [5].

and may not displace radiolabeled ligands used in binding assays.

multiple conformational states of the receptor [7].

drugs can modulate GPCR signaling.

**2. Advantages of allosteric modulators**

allosteric modulators much safer than classical drugs [8].

The site to which endogenous agonists bind to is defined as the orthosteric site. Allosteric modulators do not bind to this site. They bind to other sites and are thus termed allosteric sites [6]. Upon binding, modulators generally stabilize a preexisting conformation or change the structural conformation of the receptor. This will often modulate the orthosteric site and can modify the effects of the agonists, or in theory, inverse agonists [1]. Allosteric modulators can also stabilize one of the

Experimentally, allosteric modulation can be challenging because the allosteric modulators may affect the affinity and/or efficacy differently for each agonist (see agonist or probe dependence discussed below). For instance, different agonists that induce the same cellular response, after binding to the same receptor, can be differentially modulated by the same allosteric modulator [1]. Furthermore, they are usually difficult to screen for because they do not produce an effect by themselves

Drugs that target the orthosteric site of G protein-coupled receptors (GPCRs) are currently the most common therapeutic tools. Allosteric binding sites (e.g., sites elsewhere on the receptors) are less well-defined and, therefore, less exploited clinically. Diversity in location, mechanism, and specificity of allosteric ligands are characteristics giving them a great potential to extend the range of the ways that

Allosteric modulators with no intrinsic efficacy will usually only exert their effects in the presence of an endogenous agonist. Thus, they can selectively tune cellular responses in tissues where the endogenous agonist exerts its physiological or pathophysiological functions. As a result, temporal and spatial aspects of the endogenous agonist signaling can be chronically maintained or even corrected in pathological states. Also, the saturation of allosteric binding sites limits itself the action of the allosteric modulator and the effect on the function of the agonist. This excellent property of the agonist overcomes the overdosing of a drug, making the

A great benefit of using allosteric ligands for therapeutic applications is their huge potential to achieve greater selectivity at subtypes of GPCRs [8]. This could be due to greater diversity in the amino acid sequence of the allosteric binding sites compared to the orthosteric binding pocket. Another possibility is via selective cooperativity between the allosteric and orthosteric binding sites at a given receptor subtype. In addition, in some GPCRs, where the orthosteric binding site is not clear or its structure is poorly defined, the allosteric binding site might be a good alternative to target with small molecules; this has been observed in receptors with long

**90**

Radioligand binding and functional assays have particular advantages when they are used to screen for allosteric modulators of GPCRs. Nevertheless, the extent and direction (that is positive or negative) of the interaction between the allosteric and orthosteric ligand for receptors with more than one endogenous ligand will depend on the orthosteric agonist interacting with the receptor; this phenomenon is known as probe or agonist dependence (**Figure 1**). Agonist dependence makes more complex the identification and pharmacological characterization of allosteric modulators. Particularly, the case of aplaviroc (an allosteric modulator for CC-chemokine receptor 5) showed no effect in the binding of CCL5 to the receptor but totally prevented the binding of CCL3 [9].

Agonist dependence, or better known as probe dependence, can seriously affect potential therapeutics and also the development of allosteric ligands. In the ideal situation, the endogenous ligand would be used as a prototypic ligand in the high throughput screening process during drug development. However, in a real situation, the endogenous agonists, most of the time are unsuitable and susceptible to rapid degradation along the drug screening process and even more during *in vivo* studies. For this reason, highly stable agonists are preferentially used. Therefore, the allosteric ligand is first tested with the natural ligand on an early stage of the process in order to conclude if the achieved effects are equivalent to those registered with the prototypical agonist.

In order to better illustrate this concept a good example would be when a PAM has the ability to increase the cholinergic neuronal transmission in Alzheimer's disease [10], but the endogenous ligand acetylcholine is of rapid degradation making it unsuitable for drug screening. Cholinergic substitute ligands such as carbachol or pilocarpine are used for screening, and the stable analog oxotremorine is used to validate the effects of muscarinic acetylcholine receptor (mAChR) allosteric modulators *in vivo* [11]. However, the effects of the PAM LY2033298 are dependent on the orthosteric agonist (carbachol or pilocarpine) being used [12, 13] and these effects could lead to unexpected profiles of the allosteric ligand in later stages of the drug discovery process.

The characteristic of some allosteric ligands to have differential probe dependence on multiple receptor subtypes is an additional consideration that can have significant implications for the drug screening process. For instance, LY2033298 was reported to be a specific positive allosteric modulator of the M4 mAChR. Accordingly, PAM LY2033298 increased acetylcholine-mediated calcium responses at the M4 mAChR but not for the rest of the four receptor subtypes [14]. This allosteric modulator has also been shown to have high positive cooperativity with the surrogate orthosteric ligands oxotremorine and tetramethylammonium at the M2 mAChR [13]; this positive cooperative effect is similar to that observed with LY2033298 and oxotremorine at the M4 mAChR. This is an example of how the probe-dependent interaction of oxotremorine at both the M2 and M4 receptor subtypes can confound experimental interpretation of the effect of the allosteric ligand *in vivo* [11]. This highlights the need to understand the probe dependence of allosteric ligands at related receptors to ensure a robust target validation.

#### **Figure 1.**

*Agonist dependence and biased agonism. (A) Agonist dependence of an allosteric ligand is shown with several endogenous agonists. (Aa) An allosteric modulator potentiates the ability of endogenous Agonist-1 to inhibit the binding of an antagonist to a human GPCR. (Ab) An allosteric modulator displaying weak or almost neutral cooperativity with endogenous Agonist-2. (Ac) An allosteric modulator displaying neutral cooperativity with endogenous Agonist-3. (B) Biased agonism is the capacity of different agonists to differentially activate the same GPCR, producing specific sets of signaling pathways. Changes in efficacy or potency by different agonists are indicators of potential biased agonism for a given GPCR. (Ba) Allosteric modulator-A showing stronger potency for signaling-A than for signaling-B, and not showing effect at signaling-C. (Bb) Allosteric modulator-B displaying higher potency for signaling-B than for signaling-A. (Bc) A biased agonist in complex with a GPCR can by itself preferentially activate to a unique set of signaling pathways and the interaction of the ligand-GPCR complex with an allosteric ligand will affect the signaling bias of the GPCR. Ligand-C is co-bound with an allosteric modulator-A and it potentiates the stimulus towards pathway-B and generates activity on signaling-C, however it down regulates signaling-A. This can be observed by a change in potency and efficacy between signaling-B and signaling-C in comparison with signaling-A.*

Probe dependence is irrelevant in many physiological systems because the therapeutically targeted GPCRs have only one endogenous ligand. However, as we mentioned above, receptors can also respond to several endogenous agonists, under physiological conditions and disease. It is important to mention few more examples like the case of the chemokine receptors [15], melanocortin receptors [16], parathyroid hormone receptor 1 [17], relaxin receptors [18], calcium-sensing receptors [19], calcitonin and calcitonin-like receptors [20], as well as glucagon and glucagonlike peptide 1 (GLP1) receptors [21] and galanin receptors. In these examples, agonist dependence represents a huge challenge in the development of allosteric ligands as well as their therapeutic application.

**93**

**Figure 2.**

*Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug…*

Distinct ligands can show different capacities to differentially activate signaling pathways from a GPCR by inducing different structural conformations [8, 22]; this effect is termed as biased signaling (also known as biased agonism see **Figure 2**). Examples of ligands that produce biased signaling include classical orthosteric adrenoceptor antagonists and inverse agonists (also known as beta-blockers) that antagonize receptor-mediated cyclic AMP production but promote cAMP response

Research of carvedilol, an adrenoceptor antagonist, has shown to be a superior

At the present time, the terms biased agonism and allosteric modulation are usually considered to be different pharmacological phenomena. However, both events share in common that they are due to ligand-specific conformational changes in the GPCR that implicates a change in the three-dimensional structure of the GPCR. Having as a result that some specific signaling pathways can be either

For instance, the allosteric modulators of parturition (PDC113.824) induce biased signaling when an orthosteric ligand is co-bound to the prostaglandin F2α receptor. In mouse models, this compound acts as a negative allosteric modulator of prostaglandin F2α receptor-mediated cytosolic calcium oscillations and myometrial contraction. Specifically, PDC113.824 uncouples the receptor from the Gα12–RHO– ROCK (RHO-associated protein kinase) signaling pathway, but still significantly increases the phosphorylation of ERK1 and ERK2 Gαq dependent [26]. An autoantibody for the calcium-sensing receptor that produces acquired hypocalciuric hypercalcemia by selectively increasing Gαq-dependent signaling and inhibiting

*Biased agonism is the ability of a receptor-ligand complex to selectively activate its downstream signaling pathways. (a) A balanced signal that stimulates both signaling pathways, G-protein dependent or β-arrestin dependent, equally in the same manner. (b) A biased ligand acting on the receptor as an agonist of one pathway (e.g., β-arrestins) while acting as an antagonist of another pathway (e.g., through heterotrimeric G proteins). (c) A biased ligand acting on the receptor as an agonist of one pathway (e.g., through heterotrimeric* 

*G proteins) while acting as an antagonist of another pathway (e.g., beta-arrestins).*

therapeutic, as compared to other adrenoceptor antagonists, for heart failure therapy [24] Despite that the drug was shown to be an inverse agonist for Gαs dependent signaling, it was also observed that carvedilol exerts partial agonism inβ -arrestin-dependent extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation [25]. Based on these observations we can hypothesize that a different set of efficacies in different signaling pathways determines the final therapeutic outcome of GPCR ligands. Biased signaling has been widely studied for orthosteric ligands, however, there is also the possibility that many, if not all allosteric ligands, will exert biased signaling properties when the receptor is co-

*DOI: http://dx.doi.org/10.5772/intechopen.91838*

**2.2 Biased agonism in allosteric modulation**

element-mediated gene transcription [23].

bound to the agonist.

positively or negatively regulated.

*Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug… DOI: http://dx.doi.org/10.5772/intechopen.91838*

#### **2.2 Biased agonism in allosteric modulation**

*Molecular Pharmacology*

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**Figure 1.**

Probe dependence is irrelevant in many physiological systems because the therapeutically targeted GPCRs have only one endogenous ligand. However, as we mentioned above, receptors can also respond to several endogenous agonists, under physiological conditions and disease. It is important to mention few more examples like the case of the chemokine receptors [15], melanocortin receptors [16], parathyroid hormone receptor 1 [17], relaxin receptors [18], calcium-sensing receptors [19], calcitonin and calcitonin-like receptors [20], as well as glucagon and glucagonlike peptide 1 (GLP1) receptors [21] and galanin receptors. In these examples, agonist dependence represents a huge challenge in the development of allosteric

*and efficacy between signaling-B and signaling-C in comparison with signaling-A.*

*Agonist dependence and biased agonism. (A) Agonist dependence of an allosteric ligand is shown with several endogenous agonists. (Aa) An allosteric modulator potentiates the ability of endogenous Agonist-1 to inhibit the binding of an antagonist to a human GPCR. (Ab) An allosteric modulator displaying weak or almost neutral cooperativity with endogenous Agonist-2. (Ac) An allosteric modulator displaying neutral cooperativity with endogenous Agonist-3. (B) Biased agonism is the capacity of different agonists to differentially activate the same GPCR, producing specific sets of signaling pathways. Changes in efficacy or potency by different agonists are indicators of potential biased agonism for a given GPCR. (Ba) Allosteric modulator-A showing stronger potency for signaling-A than for signaling-B, and not showing effect at signaling-C. (Bb) Allosteric modulator-B displaying higher potency for signaling-B than for signaling-A. (Bc) A biased agonist in complex with a GPCR can by itself preferentially activate to a unique set of signaling pathways and the interaction of the ligand-GPCR complex with an allosteric ligand will affect the signaling bias of the GPCR. Ligand-C is co-bound with an allosteric modulator-A and it potentiates the stimulus towards pathway-B and generates activity on signaling-C, however it down regulates signaling-A. This can be observed by a change in potency* 

ligands as well as their therapeutic application.

Distinct ligands can show different capacities to differentially activate signaling pathways from a GPCR by inducing different structural conformations [8, 22]; this effect is termed as biased signaling (also known as biased agonism see **Figure 2**). Examples of ligands that produce biased signaling include classical orthosteric adrenoceptor antagonists and inverse agonists (also known as beta-blockers) that antagonize receptor-mediated cyclic AMP production but promote cAMP response element-mediated gene transcription [23].

Research of carvedilol, an adrenoceptor antagonist, has shown to be a superior therapeutic, as compared to other adrenoceptor antagonists, for heart failure therapy [24] Despite that the drug was shown to be an inverse agonist for Gαs dependent signaling, it was also observed that carvedilol exerts partial agonism inβ -arrestin-dependent extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation [25]. Based on these observations we can hypothesize that a different set of efficacies in different signaling pathways determines the final therapeutic outcome of GPCR ligands. Biased signaling has been widely studied for orthosteric ligands, however, there is also the possibility that many, if not all allosteric ligands, will exert biased signaling properties when the receptor is cobound to the agonist.

At the present time, the terms biased agonism and allosteric modulation are usually considered to be different pharmacological phenomena. However, both events share in common that they are due to ligand-specific conformational changes in the GPCR that implicates a change in the three-dimensional structure of the GPCR. Having as a result that some specific signaling pathways can be either positively or negatively regulated.

For instance, the allosteric modulators of parturition (PDC113.824) induce biased signaling when an orthosteric ligand is co-bound to the prostaglandin F2α receptor. In mouse models, this compound acts as a negative allosteric modulator of prostaglandin F2α receptor-mediated cytosolic calcium oscillations and myometrial contraction. Specifically, PDC113.824 uncouples the receptor from the Gα12–RHO– ROCK (RHO-associated protein kinase) signaling pathway, but still significantly increases the phosphorylation of ERK1 and ERK2 Gαq dependent [26]. An autoantibody for the calcium-sensing receptor that produces acquired hypocalciuric hypercalcemia by selectively increasing Gαq-dependent signaling and inhibiting

#### **Figure 2.**

*Biased agonism is the ability of a receptor-ligand complex to selectively activate its downstream signaling pathways. (a) A balanced signal that stimulates both signaling pathways, G-protein dependent or β-arrestin dependent, equally in the same manner. (b) A biased ligand acting on the receptor as an agonist of one pathway (e.g., β-arrestins) while acting as an antagonist of another pathway (e.g., through heterotrimeric G proteins). (c) A biased ligand acting on the receptor as an agonist of one pathway (e.g., through heterotrimeric G proteins) while acting as an antagonist of another pathway (e.g., beta-arrestins).*

Gαi-dependent signaling [27]. This is only one example of how an allosteric modulator can induce biased signaling that results in disease.

Based on these examples is that we can see the need to deeply understand the effect of allosteric modulation at different signaling pathways, as positive and negative regulation of every pathway from the receptor, does not always generate a beneficial therapeutic effect.

In theory, the operational model describes that biased agonism via allosteric modulation is a pathway-dependent variation in the signaling produced by the agonist in such a way that is not correlated with the intrinsic efficacy of the agonist or allosteric modulators [28]. However, in real conditions, biased signaling by an allosteric modulator is when the allosteric ligand potentiates one pathway but decreases the other one, as we previously described.

For a better understanding of this pharmacological phenomenon, we can continue describing more examples in this regard. Another very good example is the case of the allosteric modulation of the muscarinic receptor M4 subtype (M4 mAChR). Increasing signaling at M4 mAChR by the allosteric modulator LY2033298 generated significant changes in the grade of positive cooperativity in various signaling cascades of this receptor [29]. An additional case to exemplify this is the biased allosteric modulation of the GLP1 receptor. Some allosteric ligands can potentiate cAMP production and having a smaller effect on β-arrestin dependent signaling [30, 31].

Currently, most allosteric modulators have been discovered following screening assays; such as those used to identify agonists and antagonists, instead of a thoughtful rational process. The development of novel allosteric modulators that can induce biased agonism has the potential of specifically targeting therapeutic signaling pathways and excluding off-target pathways providing in this way a novel mechanism of biased agonism and produce new drugs with fewer side effects. However, we also face the problem that poses a big challenge in drug discovery and development of allosteric ligands and is that for the vast majority of GPCRs, it is still not well understood which set of signaling pathways efficacies will produce the desired therapeutic effect. The most important issue in the development of biased allosteric ligands is required a full understanding of the molecular determinants and also structural signatures that will lead to biased signaling on a receptor.

#### **2.3 Agonist dependence in allosteric modulation and biased agonism**

Many GPCRs have more than one endogenous agonist in such a way that their action is differentially regulated by the same allosteric modulator, having, as a result, a phenomenon called probe dependence. For instance, in the case of GLP1 receptor, there are two PAMs (compound 2 and BETP (4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl) pyrimidine)) that show agonist dependence; where they increased the affinity of the oxyntomodulin (an endogenous agonist) by 18–25 times respectively. In contrast, none of them had no effect on four more ligands of GLP1 receptor [30, 32]. Interestingly, these allosteric modulators induced biased signaling during GLP-1 activation by oxyntomodulin. Observing an increase in Gαs activation, β-arrestin1/2 recruitment and insulin secretion, but they did not show any allosteric effect in ERK1/2 activation. The capacity of a modulator to regulate all or only some endogenous ligands in a pathway-dependent manner could not only seriously affect the development of novel allosteric modulators but also their therapeutics.

#### **2.4 Implications in drug screening**

Currently, allosteric screening routinely relies on seeking compounds that regulate the actions of the main endogenous ligand. Conversely, screening against

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targeted.

**2.6 Therapeutic relevance**

*Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug…*

alternative endogenous ligands, even if they have lower affinity or efficacy, may yield new leads that might not be possible to identify if screening for cooperative

The possibility that an allosteric modulator will antagonize or potentiate the effects of an endogenous agonist without affecting other endogenous agonists would be seen as a therapeutic advantage, only in the case that each agonist exerts a different physiological effect. For example, according to previous studies some CCR5 allosteric modulators prevent the interaction of HIV-1 to CCR5, inhibiting in this way the infection [33]. However, it has not been well understood, whether inhibiting the chemokine physiological function of CCR5, would be desirable from a therapeutic point of view. During AIDS treatment, it is highly desirable the availability of allosteric modulators that prevent the HIV-1 entry without affecting CCR5 internalization by chemokines, since CCR5 plays a key role in favorable protection

Probe dependent effects and the capacity of allosteric ligands to induce allosteric bias could be used to regulate GPCR physiological function in such a way that the signaling pathways that lead to favorable physiological outputs can be selectively

All GPCRs are involved in nearly all physiological functions in humans and are the target of intense drug discovery efforts [35, 36]. Recent structures of GPCRs bound to allosteric modulators have revealed that the receptor surface is characterized by diverse cavities and crevices that may serve as binding sites for allosteric modulators [37]. This supports the notion that GPCRs are structurally flexible and they can be regulated by different allosteric ligands through a wide variety of mechanisms [38–43]. The vast majority of these structures have been solved with NAMs, which stabilize receptors in their inactive states [37]. Currently, only a single structure of an active GPCR bound to a small-molecule PAM has been described, the M2 muscarinic acetylcholine receptor with LY2119620 [44]. Thus, mechanisms

Receptor subtypes have orthosteric sites that are similar in its tridimensional structure and sometimes even in their amino acid sequence since mutations within this site, may especially decrease receptor function with detrimental consequences for the system. This can be harmful in complex systems and thus, evolution does not frequently favor such changes. In contrast, allosteric binding sites are less critical for receptor function and this is why they often have great structural variation between receptor subtypes. Moreover, in contrast to orthosteric ligands, allosteric drugs have the potential of being highly specific by only targeting a very specific set of receptor subtypes. But also, it is worth to highlight that the same allosteric site might be structurally different across species, having as a consequence differential

Allosteric ligands cannot activate or inactivate receptors. Specifically, allosteric action will depend on endogenous ligands like neurotrophins, hormones, nucleotides or lipid moieties whose levels in the organism are tightly regulated. This can lower overdose risk relative to similarly acting orthosteric drugs. It may also allow a strategy where large enough doses that saturate all the receptors of the target tissue can be administrated to prolong the drug effect [5]. These characteristics enable receptors to be activated at specific times (i.e., in response

effects is only performed using the main endogenous ligand.

in the progression of AIDs after HIV-1 infection [34].

**2.5 GPCR structure and allosteric modulation**

of PAMs and their potential binding sites remain unexplored.

effects of the same allosteric ligand between species [45].

*DOI: http://dx.doi.org/10.5772/intechopen.91838*

#### *Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug… DOI: http://dx.doi.org/10.5772/intechopen.91838*

alternative endogenous ligands, even if they have lower affinity or efficacy, may yield new leads that might not be possible to identify if screening for cooperative effects is only performed using the main endogenous ligand.

The possibility that an allosteric modulator will antagonize or potentiate the effects of an endogenous agonist without affecting other endogenous agonists would be seen as a therapeutic advantage, only in the case that each agonist exerts a different physiological effect. For example, according to previous studies some CCR5 allosteric modulators prevent the interaction of HIV-1 to CCR5, inhibiting in this way the infection [33]. However, it has not been well understood, whether inhibiting the chemokine physiological function of CCR5, would be desirable from a therapeutic point of view. During AIDS treatment, it is highly desirable the availability of allosteric modulators that prevent the HIV-1 entry without affecting CCR5 internalization by chemokines, since CCR5 plays a key role in favorable protection in the progression of AIDs after HIV-1 infection [34].

Probe dependent effects and the capacity of allosteric ligands to induce allosteric bias could be used to regulate GPCR physiological function in such a way that the signaling pathways that lead to favorable physiological outputs can be selectively targeted.

#### **2.5 GPCR structure and allosteric modulation**

All GPCRs are involved in nearly all physiological functions in humans and are the target of intense drug discovery efforts [35, 36]. Recent structures of GPCRs bound to allosteric modulators have revealed that the receptor surface is characterized by diverse cavities and crevices that may serve as binding sites for allosteric modulators [37]. This supports the notion that GPCRs are structurally flexible and they can be regulated by different allosteric ligands through a wide variety of mechanisms [38–43]. The vast majority of these structures have been solved with NAMs, which stabilize receptors in their inactive states [37]. Currently, only a single structure of an active GPCR bound to a small-molecule PAM has been described, the M2 muscarinic acetylcholine receptor with LY2119620 [44]. Thus, mechanisms of PAMs and their potential binding sites remain unexplored.

#### **2.6 Therapeutic relevance**

Receptor subtypes have orthosteric sites that are similar in its tridimensional structure and sometimes even in their amino acid sequence since mutations within this site, may especially decrease receptor function with detrimental consequences for the system. This can be harmful in complex systems and thus, evolution does not frequently favor such changes. In contrast, allosteric binding sites are less critical for receptor function and this is why they often have great structural variation between receptor subtypes. Moreover, in contrast to orthosteric ligands, allosteric drugs have the potential of being highly specific by only targeting a very specific set of receptor subtypes. But also, it is worth to highlight that the same allosteric site might be structurally different across species, having as a consequence differential effects of the same allosteric ligand between species [45].

Allosteric ligands cannot activate or inactivate receptors. Specifically, allosteric action will depend on endogenous ligands like neurotrophins, hormones, nucleotides or lipid moieties whose levels in the organism are tightly regulated. This can lower overdose risk relative to similarly acting orthosteric drugs. It may also allow a strategy where large enough doses that saturate all the receptors of the target tissue can be administrated to prolong the drug effect [5]. These characteristics enable receptors to be activated at specific times (i.e., in response

*Molecular Pharmacology*

beneficial therapeutic effect.

Gαi-dependent signaling [27]. This is only one example of how an allosteric modu-

Based on these examples is that we can see the need to deeply understand the effect of allosteric modulation at different signaling pathways, as positive and negative regulation of every pathway from the receptor, does not always generate a

In theory, the operational model describes that biased agonism via allosteric modulation is a pathway-dependent variation in the signaling produced by the agonist in such a way that is not correlated with the intrinsic efficacy of the agonist or allosteric modulators [28]. However, in real conditions, biased signaling by an allosteric modulator is when the allosteric ligand potentiates one pathway but

For a better understanding of this pharmacological phenomenon, we can continue describing more examples in this regard. Another very good example is the case of the allosteric modulation of the muscarinic receptor M4 subtype (M4 mAChR). Increasing signaling at M4 mAChR by the allosteric modulator LY2033298 generated significant changes in the grade of positive cooperativity in various signaling cascades of this receptor [29]. An additional case to exemplify this is the biased allosteric modulation of the GLP1 receptor. Some allosteric ligands can potentiate cAMP production and having a smaller effect on β-arrestin dependent signaling [30, 31]. Currently, most allosteric modulators have been discovered following screening assays; such as those used to identify agonists and antagonists, instead of a thoughtful rational process. The development of novel allosteric modulators that can induce biased agonism has the potential of specifically targeting therapeutic signaling pathways and excluding off-target pathways providing in this way a novel mechanism of biased agonism and produce new drugs with fewer side effects. However, we also face the problem that poses a big challenge in drug discovery and development of allosteric ligands and is that for the vast majority of GPCRs, it is still not well understood which set of signaling pathways efficacies will produce the desired therapeutic effect. The most important issue in the development of biased allosteric ligands is required a full understanding of the molecular determinants and also

structural signatures that will lead to biased signaling on a receptor.

**2.3 Agonist dependence in allosteric modulation and biased agonism**

Many GPCRs have more than one endogenous agonist in such a way that their action is differentially regulated by the same allosteric modulator, having, as a result, a phenomenon called probe dependence. For instance, in the case of GLP1 receptor, there are two PAMs (compound 2 and BETP (4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl) pyrimidine)) that show agonist dependence; where they increased the affinity of the oxyntomodulin (an endogenous agonist) by 18–25 times respectively. In contrast, none of them had no effect on four more ligands of GLP1 receptor [30, 32]. Interestingly, these allosteric modulators induced biased signaling during GLP-1 activation by oxyntomodulin. Observing an increase in Gαs activation, β-arrestin1/2 recruitment and insulin secretion, but they did not show any allosteric effect in ERK1/2 activation. The capacity of a modulator to regulate all or only some endogenous ligands in a pathway-dependent manner could not only seriously affect the development of novel allosteric modulators but also their therapeutics.

Currently, allosteric screening routinely relies on seeking compounds that regulate the actions of the main endogenous ligand. Conversely, screening against

lator can induce biased signaling that results in disease.

decreases the other one, as we previously described.

**94**

**2.4 Implications in drug screening**

to a physiological stimulus) with the difference of the constant activation by an orthosteric agonist [45].

Allosteric ligands regulate the responses already existing within tissues and making possible the drug response on specific tissue. Contrary to orthosteric ligands where they produce a less targeted effect within the organism since they bind to every receptor they can, affecting multiple tissues expressing the target receptor [1].

Some allosteric modulators have also been shown to lack the desensitizing effect that some agonists. Nicotinic acetylcholine receptors, for example, quickly desensitize in the presence of agonist drugs but maintain normal function in the presence of PAMs [46].

#### **3. Conclusions**

A huge number of drugs with fewer side effects are being developed using allosteric targets. Only two types of screening strategies are the principal approach in drug discovery; phage display and high-throughput screening. It is foreseen that complex computations will be conducted in years to come in order to gain better insights about the binding pockets within the receptors for which allosteric modulators can potentially be designed [47–49]. Information from crystal structures of receptors bound to different ligands would provide structural insights about the conformational changes that occur upon ligand binding. These studies would be fundamental during a rational drug design of these kind of ligands. In the last few years, we have seen a great advance in the design of novel allosteric modulators and it will possibly intensify even more in the near future. Progress in drug delivery will help to obtain further spatial specificity of therapeutic drugs, and as a result, we expect will translate into identifying significantly increased number of new drug possibilities that are more effective and with fewer side effects [50, 51]. However, no matter how selective such drugs can be designed, they cannot equalize the spatiotemporal basis of specificity that occurs naturally in our systems. Therefore, every single advance in allosteric drug discovery that promotes the homeostasis of our biological systems, will significantly contribute to the goals of developing more effective drugs with fewer side effects.

#### **Acknowledgements**

This work was supported by grants from the National Institute of Health, the American Heart Association, and Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation.

**97**

**Author details**

and Bradley K. McConnell

*Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug…*

Arfaxad Reyes-Alcaraz\*, Emilio Y. Lucero Garcia-Rojas, Richard A. Bond

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

College of Pharmacy, University of Houston, Houston, TX, USA

\*Address all correspondence to: areyesa2@central.uh.edu

provided the original work is properly cited.

*DOI: http://dx.doi.org/10.5772/intechopen.91838*

#### **Conflict of interest**

The authors declare no conflict of interest.

*Allosteric Modulators for GPCRs as a Therapeutic Alternative with High Potential in Drug… DOI: http://dx.doi.org/10.5772/intechopen.91838*

#### **Author details**

*Molecular Pharmacology*

orthosteric agonist [45].

receptor [1].

of PAMs [46].

**3. Conclusions**

effective drugs with fewer side effects.

The authors declare no conflict of interest.

**Acknowledgements**

**Conflict of interest**

Foundation.

to a physiological stimulus) with the difference of the constant activation by an

Allosteric ligands regulate the responses already existing within tissues and making possible the drug response on specific tissue. Contrary to orthosteric ligands where they produce a less targeted effect within the organism since they bind to every receptor they can, affecting multiple tissues expressing the target

Some allosteric modulators have also been shown to lack the desensitizing effect that some agonists. Nicotinic acetylcholine receptors, for example, quickly desensitize in the presence of agonist drugs but maintain normal function in the presence

A huge number of drugs with fewer side effects are being developed using allosteric targets. Only two types of screening strategies are the principal approach in drug discovery; phage display and high-throughput screening. It is foreseen that complex computations will be conducted in years to come in order to gain better insights about the binding pockets within the receptors for which allosteric modulators can potentially be designed [47–49]. Information from crystal structures of receptors bound to different ligands would provide structural insights about the conformational changes that occur upon ligand binding. These studies would be fundamental during a rational drug design of these kind of ligands. In the last few years, we have seen a great advance in the design of novel allosteric modulators and it will possibly intensify even more in the near future. Progress in drug delivery will help to obtain further spatial specificity of therapeutic drugs, and as a result, we expect will translate into identifying significantly increased number of new drug possibilities that are more effective and with fewer side effects [50, 51]. However, no matter how selective such drugs can be designed, they cannot equalize the spatiotemporal basis of specificity that occurs naturally in our systems. Therefore, every single advance in allosteric drug discovery that promotes the homeostasis of our biological systems, will significantly contribute to the goals of developing more

This work was supported by grants from the National Institute of Health, the American Heart Association, and Robert J. Kleberg, Jr. and Helen C. Kleberg

**96**

Arfaxad Reyes-Alcaraz\*, Emilio Y. Lucero Garcia-Rojas, Richard A. Bond and Bradley K. McConnell College of Pharmacy, University of Houston, Houston, TX, USA

\*Address all correspondence to: areyesa2@central.uh.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[39] Staus DP, Strachan RT, Manglik A, Pani B, Kahsai AW, Kim TH, et al. Allosteric nanobodies reveal the

dynamic range and diverse mechanisms

[40] Manglik A, Kim TH, Masureel M, Altenbach C, Yang Z, Hilger D, et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell;**161**:1001-1011

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[42] Ned Van Eps LNC, Morizumi T, Kusnetzow AK, Szczepek M, Hofmann KP, Bayburt TH, et al. Conformational equilibria of lightactivated rhodopsin in nanodiscs. Proceedings of the National Academy of

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**103**

**Chapter 6**

**Abstract**

**1. Introduction**

as muscular contraction [2].

of selective drug candidates.

Potassium Channels as a Potential

Aberrant function or expression of potassium channels can be underlying in pathologies such as cardiac arrhythmia, diabetes mellitus, hypertension, preterm birth, and various types of cancer. The expression of potassium channels is altered in many types of diseases. Also, we have previously shown that natural polyphenols, such as resveratrol, and selective synthetic modulators of potassium channels, like pinacidil, can alter their function and lead to the desired outcome. Therefore, targeting potassium channels with substance, which has an influence on their function, is promising access to cancer, diabetes mellitus, preterm birth, or hypertension therapy. In this chapter, we could discuss strategies for targeting different types of potassium channels as potential targets for synthetic and natural molecules therapy.

**Keywords:** potassium channels, K channels, modulators of K channels, activators,

Ion channels are protein molecules that form pores in the cell membrane and membranes of cellular organelles and allow passive flow of ions in the direction of their electrochemical gradient and result in electrical currents. Ion channels play key roles in membrane potential generation and many cellular activities such as signal transduction, neurotransmitter release, muscle contraction, hormone secre-

It is widely known that potassium channels (K channels) are transmembrane proteins that allow the flow of potassium across the membrane to regulate ion homeostasis, cell proliferation, migration, cell volume, and specific processes such

K channels are the most diverse ion channel type, and each subtype has a specific physiological role. K channels are highly attractive as targets for the development of novel therapeutics. However, the lack of detailed structural and functional insight of K channels and their diversity and ubiquity pose challenges for the development

For many years the structure and types of K channels were unknown due to the lack of specific ligands for their activation or blocking. A progressive shift in the study of these structures has emerged since the discovery of animal toxins that are highly specific to individual types but also with the introduction of electrophysiological methods—voltage clamp and patch clamp, which have made it possible to test individual channel. In recent years, many genes detected participate in the encoding of these ion channels. Some subtypes are cloned and their biophysical properties determined,

inhibitors, natural polyphenols, resveratrol, K channels antibody

tion, volume regulation, growth, motility, and apoptosis [1].

Target Spot for Drugs

*Vladimir Djokic and Radmila Novakovic*

#### **Chapter 6**

### Potassium Channels as a Potential Target Spot for Drugs

*Vladimir Djokic and Radmila Novakovic*

#### **Abstract**

Aberrant function or expression of potassium channels can be underlying in pathologies such as cardiac arrhythmia, diabetes mellitus, hypertension, preterm birth, and various types of cancer. The expression of potassium channels is altered in many types of diseases. Also, we have previously shown that natural polyphenols, such as resveratrol, and selective synthetic modulators of potassium channels, like pinacidil, can alter their function and lead to the desired outcome. Therefore, targeting potassium channels with substance, which has an influence on their function, is promising access to cancer, diabetes mellitus, preterm birth, or hypertension therapy. In this chapter, we could discuss strategies for targeting different types of potassium channels as potential targets for synthetic and natural molecules therapy.

**Keywords:** potassium channels, K channels, modulators of K channels, activators, inhibitors, natural polyphenols, resveratrol, K channels antibody

#### **1. Introduction**

Ion channels are protein molecules that form pores in the cell membrane and membranes of cellular organelles and allow passive flow of ions in the direction of their electrochemical gradient and result in electrical currents. Ion channels play key roles in membrane potential generation and many cellular activities such as signal transduction, neurotransmitter release, muscle contraction, hormone secretion, volume regulation, growth, motility, and apoptosis [1].

It is widely known that potassium channels (K channels) are transmembrane proteins that allow the flow of potassium across the membrane to regulate ion homeostasis, cell proliferation, migration, cell volume, and specific processes such as muscular contraction [2].

K channels are the most diverse ion channel type, and each subtype has a specific physiological role. K channels are highly attractive as targets for the development of novel therapeutics. However, the lack of detailed structural and functional insight of K channels and their diversity and ubiquity pose challenges for the development of selective drug candidates.

For many years the structure and types of K channels were unknown due to the lack of specific ligands for their activation or blocking. A progressive shift in the study of these structures has emerged since the discovery of animal toxins that are highly specific to individual types but also with the introduction of electrophysiological methods—voltage clamp and patch clamp, which have made it possible to test individual channel. In recent years, many genes detected participate in the encoding of these ion channels. Some subtypes are cloned and their biophysical properties determined,

but this has not led to a complete elucidation of their function [3]. Especially in recent years, studies on this membrane protein family in different tissue types dramatically increased. Parallel with this remarkable progress in our understanding of molecular diversity, structure, and function, a growing number of discoveries have linked K channel gene mutations with various diseases. Such diseases of the heart, kidney, pancreas, and central nervous system involve either mutation(s) in the K channel gene(s) and/or altered regulation of K channel function. The enhanced understanding of these diseases, facilitated by a combination of genomic and biophysical approaches, has helped our understanding of how various mutations affect channel function, contribute to disease etiology, and rationalize novel treatment strategies.

This review will survey the K channels crucial role in the physiologic and pathophysiological function and discuss the emerging understanding of their clinical influence. Once taken into account these strategies, K channels may represent suitable and easily accessible different disease biomarkers and targets for therapy.

#### **2. Classification of K channels**

Ion channels classified according to the type of ions they conduct, their structure, their expression pattern and mode of activation [4]. Potassium channels are composed of complexes of several protein subunits, each encoded by a different gene.

The general model of the K channel is a complex of four α subunits grouped in the form of a pore through the membrane. Two transmembrane helices and a short loop between them (called the P-loop) are trademarks of these channels. The P-loop contains the amino acid sequence of threonine-valine-glycine-tyrosine-glycine, which is the selective filter most responsible for the selective passage of K+ ions through the channel. This architecture with two transmembrane helices and a loop is an essential and universal feature of the K channel, but further, different features characterize each channel subfamily. In addition to α subunits, a variety of accessory-regulatory subunits such as SUR, β, MinK, and KChiP and others enter the K channel composition [5].

Different types of K channels have been shown to comprise more than 100 different protein subunits that are tissue-specific and species-specific [6]. About 75 genes coding different types of K channels have detected in human genomes. Molecular studies of K channels have allowed their classification based on the primary amino acid sequence of the pore-containing subunit. This sequence motif, conserved across all K channels, was proposed to correspond to the selectivity filter of the poreforming region of the channel protein. There are many (sub)types and isoforms of K channels divided into three groups based on transmembrane domains that make up the α subunit [3]. These channels are classified into three groups, based on the number of transmembrane domains (TMDs) (**Figure 1**). Within each family, ion channels with 65% identical amino acid sequences are further grouped into subfamilies [7]. The standard nomenclature for K channels is proposed by the *International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification* (NC-IUPHAR) [8], presented in **Figure 1** and **Tables 1**–**4**.

Orange is a Kv type with six transmembrane domains, with red main subtypes KCa channels; green is a Kir subtype with four transmembrane domains, and blue is a subtype with two transmembrane domains K2P.

Understanding the role of K channels and detecting their subunits/proteins in different physiological and pathophysiological condition are essential, along with linking dysfunction of these channels to specific diseases and disorders. These facts speak to the importance of these channels, as a possible therapeutic site for the action of drugs that should prevent or stop unwanted conditions/states. Therefore, the study of their function and the expression of their proteins are of great importance.

**105**

**Figure 1.**

and others [12, 13].

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

**2.1 Voltage-dependent K channels (Kv) and their modulators**

*Classification of main types and subtypes of K channels.*

Kv channels are the largest superfamily of the K channel, coding with about 40 genes and containing 12 subfamilies, each with several representatives [9]. Representatives of Kv1–4, 7, and 10–12 subfamilies can form functional channels as homomers, while Kv5, 6, 8, and 9 must associate with Kv2 or three subunits to form a functional channel. The official nomenclature for Kv channels is Kva.b, where a and b denote the subfamily and ordinal number of channel discovery (**Figure 1**). There are six transmembrane domains in each subunit, designated as S1–S6. Four β-subunits are associated with α-subunits and located on the cytoplasmic side of the membrane [10]. There is a peptide loop between the S5 and S6 domains. Loops of α-subunits face the lumen of the pores and contribute to the formation of K+ conductive pores [3]. Domain S4 is the central part of the voltage sensor necessary to activate the Kv channel. The opening/closing mechanism also contributes to the

electrostatic interaction of negative charges on the S2 and S3 domains [11]. The biophysical properties, physiological regulation, and pharmacological properties of Kv channels are dependent on the combination of α subunits. The combination on four α subunits may be homo- or heteromultimers. Even more complex to these heteromultimers is their interaction with smaller accessory proteins including β subunits, KChIP, KchAP, and minK proteins, miRP peptide,

The isoforms of Kv channels dominant for smooth muscle are mainly Kv1, Kv2, Kv3, and Kv4. For example, in vascular smooth muscle cells, the most important subtypes of Kv channels are Kv1 (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6), Kv2 (Kv2.1), Kv3 (Kv3.1). Kv4 (Kv4 .2–3), and Kv7 (Kv7.1–5); in the smooth muscle of the uterus is Kv4 (Kv4.2, Kv4.3). *Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

*Molecular Pharmacology*

**2. Classification of K channels**

but this has not led to a complete elucidation of their function [3]. Especially in recent years, studies on this membrane protein family in different tissue types dramatically increased. Parallel with this remarkable progress in our understanding of molecular diversity, structure, and function, a growing number of discoveries have linked K channel gene mutations with various diseases. Such diseases of the heart, kidney, pancreas, and central nervous system involve either mutation(s) in the K channel gene(s) and/or altered regulation of K channel function. The enhanced understanding of these diseases, facilitated by a combination of genomic and biophysical approaches, has helped our understanding of how various mutations affect channel function,

contribute to disease etiology, and rationalize novel treatment strategies.

is the selective filter most responsible for the selective passage of K+

This review will survey the K channels crucial role in the physiologic and pathophysiological function and discuss the emerging understanding of their clinical influence. Once taken into account these strategies, K channels may represent suitable and easily accessible different disease biomarkers and targets for therapy.

Ion channels classified according to the type of ions they conduct, their structure, their expression pattern and mode of activation [4]. Potassium channels are composed of complexes of several protein subunits, each encoded by a different gene. The general model of the K channel is a complex of four α subunits grouped in the form of a pore through the membrane. Two transmembrane helices and a short loop between them (called the P-loop) are trademarks of these channels. The P-loop contains the amino acid sequence of threonine-valine-glycine-tyrosine-glycine, which

channel. This architecture with two transmembrane helices and a loop is an essential and universal feature of the K channel, but further, different features characterize each channel subfamily. In addition to α subunits, a variety of accessory-regulatory subunits such as SUR, β, MinK, and KChiP and others enter the K channel composition [5].

Different types of K channels have been shown to comprise more than 100 different protein subunits that are tissue-specific and species-specific [6]. About 75 genes coding different types of K channels have detected in human genomes. Molecular studies of K channels have allowed their classification based on the primary amino acid sequence of the pore-containing subunit. This sequence motif, conserved across all K channels, was proposed to correspond to the selectivity filter of the poreforming region of the channel protein. There are many (sub)types and isoforms of K channels divided into three groups based on transmembrane domains that make up the α subunit [3]. These channels are classified into three groups, based on the number of transmembrane domains (TMDs) (**Figure 1**). Within each family, ion channels with 65% identical amino acid sequences are further grouped into subfamilies [7]. The standard nomenclature for K channels is proposed by the *International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification* (NC-IUPHAR) [8], presented in **Figure 1** and **Tables 1**–**4**.

Orange is a Kv type with six transmembrane domains, with red main subtypes KCa channels; green is a Kir subtype with four transmembrane domains, and blue is

Understanding the role of K channels and detecting their subunits/proteins in different physiological and pathophysiological condition are essential, along with linking dysfunction of these channels to specific diseases and disorders. These facts speak to the importance of these channels, as a possible therapeutic site for the action of drugs that should prevent or stop unwanted conditions/states. Therefore, the study of their function and the expression of their proteins are of great importance.

a subtype with two transmembrane domains K2P.

ions through the

**104**

**Figure 1.** *Classification of main types and subtypes of K channels.*

#### **2.1 Voltage-dependent K channels (Kv) and their modulators**

Kv channels are the largest superfamily of the K channel, coding with about 40 genes and containing 12 subfamilies, each with several representatives [9]. Representatives of Kv1–4, 7, and 10–12 subfamilies can form functional channels as homomers, while Kv5, 6, 8, and 9 must associate with Kv2 or three subunits to form a functional channel. The official nomenclature for Kv channels is Kva.b, where a and b denote the subfamily and ordinal number of channel discovery (**Figure 1**). There are six transmembrane domains in each subunit, designated as S1–S6. Four β-subunits are associated with α-subunits and located on the cytoplasmic side of the membrane [10]. There is a peptide loop between the S5 and S6 domains. Loops of α-subunits face the lumen of the pores and contribute to the formation of K+ conductive pores [3]. Domain S4 is the central part of the voltage sensor necessary to activate the Kv channel. The opening/closing mechanism also contributes to the electrostatic interaction of negative charges on the S2 and S3 domains [11].

The biophysical properties, physiological regulation, and pharmacological properties of Kv channels are dependent on the combination of α subunits. The combination on four α subunits may be homo- or heteromultimers. Even more complex to these heteromultimers is their interaction with smaller accessory proteins including β subunits, KChIP, KchAP, and minK proteins, miRP peptide, and others [12, 13].

The isoforms of Kv channels dominant for smooth muscle are mainly Kv1, Kv2, Kv3, and Kv4. For example, in vascular smooth muscle cells, the most important subtypes of Kv channels are Kv1 (Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6), Kv2 (Kv2.1), Kv3 (Kv3.1). Kv4 (Kv4 .2–3), and Kv7 (Kv7.1–5); in the smooth muscle of the uterus is Kv4 (Kv4.2, Kv4.3).


**107**

**Table 1.**

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

gabapentin,

retigabine,

**-** 

RPR260243

*Selective modulators of voltage-sensitive channels.*




retigabine XE991, linopiridine, TEA Kv7.2 -

retigabine linopiridine Kv7.3 -

retigabine XE991, linopiridine, TEA Kv7.4 -

gabapentin linopiridine, XE991 Kv7.5 -






**-** LY97241, hinidin Kv10.2 **-** 

**-** E4031 Kv11.2 minK

**-** E4031 Kv11.3 minK

**-** Ba2+ Kv12.1 minK


Kv10.1 **-** 

Kv11.1 minK, MiRP1

LY97241, terfenadine, dofetilide, kalmodulin, astemizole

astemizole, terfenadin, disopyramide, E4031, dofetilide, ibutilide

The expression pattern depends on the compartment and/or conditions like gravid or not in the uterus and part of lent blood vessels. This heteromultimerization seems to modulate Kv current expression, sensitivity to various substances, as well as biophysical properties of channels such as inactivation [14–18]. Further, research shows that the most important isoforms for the proliferation, activation, migration, and apoptosis of tumor cells are Kv1.3, Kv1.5, Kv2.1, Kv41, Kv9.3, Kv10.1, and Kv11.1 [19]. A recognizable feature of Kv channels is their sensitivity to pharmacological modulators. The compounds most commonly used to inhibit Kv channels are 4-aminopyridine (4-AP) and tetraethylammonium (TEA). In general, Kv channels of vascular

#### *Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

*Molecular Pharmacology*




**Activators Inhibitors Subtypes Heteromultimers** 














Kv1.1

Kv1.2

Kv1.2, Kv1.4,

Kv 1 i Kv 2

Kv1.1, Kv1.4,

Kv 1 i Kv 2

Kv 1 i Kv 2

Kv1.1, Kv1.2,

Kv 1 i Kv 2

Kv5.1, Kv6.1‐6.4, Kv8.1‐ 8.2, Kv9.1‐9.3

Kv5.1, Kv6.1‐6.4, Kv8.1‐ 8.2, Kv9.1‐9.3

KChIP 1‐4, DPP6, DPP10, Kv 1, NCS‐1, Nav 1

DPP10, MinK, MiRPs

Kv1.3 Kv1.1, Kv1.2, Kv1.4, Kv1.6,


margatoxin,


margatoxin, noxiustoxin, TEA, maurotoxin, correolide





**106**


#### **Table 1.**

*Selective modulators of voltage-sensitive channels.*

The expression pattern depends on the compartment and/or conditions like gravid or not in the uterus and part of lent blood vessels. This heteromultimerization seems to modulate Kv current expression, sensitivity to various substances, as well as biophysical properties of channels such as inactivation [14–18]. Further, research shows that the most important isoforms for the proliferation, activation, migration, and apoptosis of tumor cells are Kv1.3, Kv1.5, Kv2.1, Kv41, Kv9.3, Kv10.1, and Kv11.1 [19].

A recognizable feature of Kv channels is their sensitivity to pharmacological modulators. The compounds most commonly used to inhibit Kv channels are 4-aminopyridine (4-AP) and tetraethylammonium (TEA). In general, Kv channels of vascular

smooth muscle cells exhibit higher sensitivity to 4-AP, which nonspecifically inhibits members of the Kv1–4 subfamilies of these channels [20]. Electrophysiological imaging showed that the channels encoded by Kv1.2 and Kv1.5 genes were relatively sensitive to 4-AP, while Kv2 channels inhibited TEA more effectively. The 4-AP concentrations required for the half-maximal inhibition of Kv channel function varied between 0.3 mM and 1.1 mM. These differences in channel sensitivity attribute to the different expressions of Kv channel subtypes, the use of different animal species in studies, differences in sex, cell isolation techniques, and imaging conditions [12, 20].

#### *2.1.1 Ca2+- and voltage-dependent K channels (KCa) channels and their modulators*

Ca2+-sensitive Kv channels other than voltage-dependent depolarizations, for opening, also require an increased concentration of Ca2+ ions in the cytoplasm [21]. They are divided into channels with high (KCa1-BKCa, maxi K, 100–300 pS), intermediate (KCa3-IKCa, 25–100 pS), and low conductivity (KCa2-SKCa, 2–25 pS) [22]. KCa1 channels are activated by membrane depolarization and/or Ca2+ binding to the channel; KCa2 and KCa3 channels are voltage-independent and activated by Ca2+ binding to calmodulin that constitutively binds to the channels [23].

*KCa1.1 channels* form of α pore-forming subunits and auxiliary β or γ subunits. The four α subunits can build a functional channel on their own. Associated accessory subunits act as potent regulators of most channel characteristics, including voltage and Ca2+ sensitivity, as well as sensitivity to pharmacological modulators.

The differences in KCa1.1 channels and Kv channels are the presence of an additional transmembrane (S0) segment with the extracellular N-terminus, as well as the presence of a long intracellular C-terminus, forming the so-called "channel tail." Furthermore, unlike Kv channels, where the voltage sensor is localizing in the S4 domain, the positively charged residues responsible for the voltage dependence of the KCa1.1 channels are less centralized and present in the S2, S3, and S4 domains [21]. There are different intracellular partners of these channels. Also, KCa1.1 channels can be the targets of a number of posttranslational modifications such as oxidation, glycosylation, and phosphorylation reactions. Finally, the association of α subunits with different regulatory subunits further contributes to increasing the diversity of these channels [23, 24].

Furthermore, four types of β subunits (β1–4) and four types of γ subunits (γ1–4) modulate almost all the physiological and pharmacological properties of


**109**

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

by PKC-independent mechanisms [29].

KCa1.1 channels. β subunits contain two, while γ subunits are constructed from a single transmembrane domain. The mechanism by which helper subunits interact with α subunits and regulate KCa1.1 channel activity is extremely complicated, but it is critical for the study and understanding of the vascular disease. In vascular smooth muscle cells, the β1 subunit is the predominant isoform, and its dysfunction is associated with diabetes, hypertension, and other vascular diseases. Deletion of the gene for the β1 subunit causes a significant decrease in Ca2+-sensitivity of the channel. As auxiliary subunits of KCa1.1 channels, γ subunits also affect their activity by modulating voltage and Ca2+-dependence. They play a role in the regulation of smooth muscle tone, and change in the gene encoding them leads to a decrease in the activity of functional KCa1.1 channels lead to constrictions [25–27]. Expression of β1 subunit can be selectively upstream or downstream-regulated in smooth muscle cells, without reflecting to α subunit expression. Occurs under the influence of various physiological and pathophysiological conditions, as well as during hormonal stimulation and that increase or decrease the channel activity [26, 28]. KCa1.1 channels also represent important targets in the mechanism of action of various activators or inhibitors. Adenosine and β-adrenergic agonists activate KCa1.1 channels via cAMP-dependent and cGMP-dependent pathways. Especially in vascular smooth muscle cells, elevated levels of cGMP and activation of PKG with NO result in the phosphorylation of BKCa and their subsequent activation. On the other hand, angiotensin II and endothelin-1 inhibit KCa1.1 channels in coronary arteries

The pharmacology of KCa1.1 channels spread from nonspecific blockade with TEA and alkaloids, like paxillin, to more effective specific inhibitors scorpion toxins, such as iberiotoxin and charybdotoxin. Although these compounds do not have pure therapeutic potential, they are very useful tools for studying the function of these channels. Several small-molecule KCa1.1 channel openers have been detected for both native and cloned channels. For example, benzimidazole NS-1619 activates KCa1.1 channels, but its functional effects also include inhibition of Ca2+ currents and Kv channels. And many other substances can modulate the activity of KCa1.1 channels, such as estrogen, reactive oxygen species and ethanol [22, 30, 31]. *KCa2.1, KCa2.2, and KCa3.1 channels* are mostly present in neurons, endothelium of the blood vessels, epithelium, and in several types of smooth muscle, e.g., in the urinary tract. Thus, the opening of these channels is independent of the membrane potential but strictly dependent on Ca2+. Complex Ca2+ calmodulin induces a

Pharmacologically, KCa2 channels are highly sensitive to bee venom and apamin with different affinity for all channel isoforms: KCa2 channels are the most sensitive and KCa1 the least. Scilatoxin, biculculin, dequalinium, and its more potent derivative UCL1684 are also potent inhibitors of these channels group. The compounds, such as clotrimazole and TRAM-34, are more specific inhibitors of KCa3.1 channel. KCa3.1 current can be reduced by the scorpion toxin charybdotoxin, like KCa1.1 channels. Both types of channels are activated by chlorzoxazone, zoxazolamine, riluzole, 1-EBIO, its more potent DC-EBIO derivative, and NS-309. However, these compounds are not successful, such as Ca2+ for channel activation. Moreover, an increase

ions into the cell at a mem-

current is limited and barely detectable [33]. The

, while at more

currents.

in channel sensitivity for Ca2+ is an assumed mechanism of their action [31].

explanation for the phenomenon of inward rectification is in the fact that intracel-

**2.2 Inwardly rectifying K channels (Kir) and their modulators**

brane potential more negative than the equilibrium potential for K+

lular Mg2+ and polyamines (spermine and spermidine) block output K+

The inwardly rectifying (Kir) channels conduct K<sup>+</sup>

positive potentials, the output K+

conformational change leading to the opening of the pore [32].

**Table 2.** *Selective modulators of Ca2+- and voltage-sensitive channels (KCa).*

#### *Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

*Molecular Pharmacology*

*modulators*

diversity of these channels [23, 24].

*Selective modulators of Ca2+- and voltage-sensitive channels (KCa).*

smooth muscle cells exhibit higher sensitivity to 4-AP, which nonspecifically inhibits members of the Kv1–4 subfamilies of these channels [20]. Electrophysiological imaging showed that the channels encoded by Kv1.2 and Kv1.5 genes were relatively sensitive to 4-AP, while Kv2 channels inhibited TEA more effectively. The 4-AP concentrations required for the half-maximal inhibition of Kv channel function varied between 0.3 mM and 1.1 mM. These differences in channel sensitivity attribute to the different expressions of Kv channel subtypes, the use of different animal species in studies, differences in sex, cell isolation techniques, and imaging conditions [12, 20].

Ca2+-sensitive Kv channels other than voltage-dependent depolarizations, for opening, also require an increased concentration of Ca2+ ions in the cytoplasm [21]. They are divided into channels with high (KCa1-BKCa, maxi K, 100–300 pS), intermediate (KCa3-IKCa, 25–100 pS), and low conductivity (KCa2-SKCa, 2–25 pS) [22]. KCa1 channels are activated by membrane depolarization and/or Ca2+ binding to the channel; KCa2 and KCa3 channels are voltage-independent and activated by

*KCa1.1 channels* form of α pore-forming subunits and auxiliary β or γ subunits. The four α subunits can build a functional channel on their own. Associated accessory subunits act as potent regulators of most channel characteristics, including voltage and Ca2+ sensitivity, as well as sensitivity to pharmacological modulators. The differences in KCa1.1 channels and Kv channels are the presence of an additional transmembrane (S0) segment with the extracellular N-terminus, as well as the presence of a long intracellular C-terminus, forming the so-called "channel tail." Furthermore, unlike Kv channels, where the voltage sensor is localizing in the S4 domain, the positively charged residues responsible for the voltage dependence of the KCa1.1 channels are less centralized and present in the S2, S3, and S4 domains [21]. There are different intracellular partners of these channels. Also, KCa1.1 channels can be the targets of a number of posttranslational modifications such as oxidation, glycosylation, and phosphorylation reactions. Finally, the association of α subunits with different regulatory subunits further contributes to increasing the

Furthermore, four types of β subunits (β1–4) and four types of γ subunits (γ1–4) modulate almost all the physiological and pharmacological properties of

*2.1.1 Ca2+- and voltage-dependent K channels (KCa) channels and their* 

Ca2+ binding to calmodulin that constitutively binds to the channels [23].

**108**

**Table 2.**

KCa1.1 channels. β subunits contain two, while γ subunits are constructed from a single transmembrane domain. The mechanism by which helper subunits interact with α subunits and regulate KCa1.1 channel activity is extremely complicated, but it is critical for the study and understanding of the vascular disease. In vascular smooth muscle cells, the β1 subunit is the predominant isoform, and its dysfunction is associated with diabetes, hypertension, and other vascular diseases. Deletion of the gene for the β1 subunit causes a significant decrease in Ca2+-sensitivity of the channel. As auxiliary subunits of KCa1.1 channels, γ subunits also affect their activity by modulating voltage and Ca2+-dependence. They play a role in the regulation of smooth muscle tone, and change in the gene encoding them leads to a decrease in the activity of functional KCa1.1 channels lead to constrictions [25–27]. Expression of β1 subunit can be selectively upstream or downstream-regulated in smooth muscle cells, without reflecting to α subunit expression. Occurs under the influence of various physiological and pathophysiological conditions, as well as during hormonal stimulation and that increase or decrease the channel activity [26, 28].

KCa1.1 channels also represent important targets in the mechanism of action of various activators or inhibitors. Adenosine and β-adrenergic agonists activate KCa1.1 channels via cAMP-dependent and cGMP-dependent pathways. Especially in vascular smooth muscle cells, elevated levels of cGMP and activation of PKG with NO result in the phosphorylation of BKCa and their subsequent activation. On the other hand, angiotensin II and endothelin-1 inhibit KCa1.1 channels in coronary arteries by PKC-independent mechanisms [29].

The pharmacology of KCa1.1 channels spread from nonspecific blockade with TEA and alkaloids, like paxillin, to more effective specific inhibitors scorpion toxins, such as iberiotoxin and charybdotoxin. Although these compounds do not have pure therapeutic potential, they are very useful tools for studying the function of these channels. Several small-molecule KCa1.1 channel openers have been detected for both native and cloned channels. For example, benzimidazole NS-1619 activates KCa1.1 channels, but its functional effects also include inhibition of Ca2+ currents and Kv channels. And many other substances can modulate the activity of KCa1.1 channels, such as estrogen, reactive oxygen species and ethanol [22, 30, 31].

*KCa2.1, KCa2.2, and KCa3.1 channels* are mostly present in neurons, endothelium of the blood vessels, epithelium, and in several types of smooth muscle, e.g., in the urinary tract. Thus, the opening of these channels is independent of the membrane potential but strictly dependent on Ca2+. Complex Ca2+ calmodulin induces a conformational change leading to the opening of the pore [32].

Pharmacologically, KCa2 channels are highly sensitive to bee venom and apamin with different affinity for all channel isoforms: KCa2 channels are the most sensitive and KCa1 the least. Scilatoxin, biculculin, dequalinium, and its more potent derivative UCL1684 are also potent inhibitors of these channels group. The compounds, such as clotrimazole and TRAM-34, are more specific inhibitors of KCa3.1 channel. KCa3.1 current can be reduced by the scorpion toxin charybdotoxin, like KCa1.1 channels. Both types of channels are activated by chlorzoxazone, zoxazolamine, riluzole, 1-EBIO, its more potent DC-EBIO derivative, and NS-309. However, these compounds are not successful, such as Ca2+ for channel activation. Moreover, an increase in channel sensitivity for Ca2+ is an assumed mechanism of their action [31].

#### **2.2 Inwardly rectifying K channels (Kir) and their modulators**

The inwardly rectifying (Kir) channels conduct K<sup>+</sup> ions into the cell at a membrane potential more negative than the equilibrium potential for K+ , while at more positive potentials, the output K+ current is limited and barely detectable [33]. The explanation for the phenomenon of inward rectification is in the fact that intracellular Mg2+ and polyamines (spermine and spermidine) block output K+ currents.

As carriers of positive charge, polyamines and Mg2+ interact with the negatively charged amino acids present in the transmembrane M1 domain and terminal C-terminus of the Kir channel, thereby inhibiting the passage of K<sup>+</sup> ions through the pore [34]. Kir channels have been identified in many types of cells. Seven subfamilies are identified so far (**Figure 1** and **Table 3**).

Structure of Kir channel is consisting of four α subunit-forming subunits, each containing two transmembrane domains (M1 and M2) and a P-loop between them. The properties and functions of Kir channels vary between different tissues and species. For example, Kir channel expression is most pronounced in the smooth muscle of the autoregulatory vascular compartments, such as the coronary and cerebral circulation. Kir duct expression increases with decreased blood vessel diameter. The difference in expressive Kir channels can be explained by the fact that the conduction artery shows a very small response to a smaller version of extracellular K, whereas resistant arteries show a positive response [35].


**111**

K+

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

myometrial smooth muscle cells [17, 25, 35, 36].

and/or potentiate NO activation [37].

KATP channel [38–40].

*ATP-sensitive Kir channels (KATP)* are the exceptions, which have a more complex heterooctamer structure with multiple types of accessory subunits [3, 5]. Structurally, KATP channels form of four pore-forming Kir6.x subunits and four regulatory subunits known as sulfonylurea receptors, SURx. Kir6.x subunits are responsible for ATP inhibition and SURx for nucleoside-diphosphate activation. Functional expression of the KATP channel requires coexpression of Kir6.x and SURx subunits in a 1:1 ratio. SUR1 is predominantly present in pancreatic β cells. SUR2 has two variants, SUR2A and SUR2B, which are generated by alternative excision of exon 38 in the ABCC9 gene. SUR2A is mainly present in the myocardium and skeletal muscle, while SUR2B generally is distributed in the vascular and

Numerous studies have highlighted that KATP channels in vascular smooth muscle cells play an important role in achieving systemic vasodilation during hypoxia, increasing blood flow to the heart, kidney, and muscle. This vasodilation is attributed to the release of NO or adenosine due to hypoxia but also to the direct effect of hypoxia itself. During hypoxia, KATP channels may be activated by a decrease in ATP, a decrease in pH and partial pressure of oxygen, or an increase in intracellular lactates and ADP. These factors can activate KATP channels directly

KATP channels play their roles in the mechanism of the action of relaxation and contraction by interacting with various protein kinases. Thus, KATP channels activated by protein kinase A (PKA) and cGMP-dependent protein kinase participate in the mechanism of action of endogenous vasodilators such as adenosine and prostacyclin. On the other hand, activation of protein kinase C (PKC) and vasoconstrictor induced increases in intracellular Ca2+ caused by noradrenaline, vasopressin, endothelin, and angiotensin II were accompanied by inhibition of the

KATP channels in the smooth muscle are inhibited by antidiabetics from a group of sulfonylurea derivatives, such as glibenclamide and tolbutamide. Glibenclamide is the most commonly used KATP channel inhibitor in vascular smooth muscle stud-

In the study of potential drugs, the core interest is directed towards KATP channels, since they exhibit activity under basic conditions and significantly contribute

K2P channel subunit consists of two regions that participate in pore formation (P1 and P2, hence their name) and four transmembrane domains (M1–M4). Functional channels form as dimers of these subunits that form a single pore selectively permeable to potassium [43]. Activity of these channels is voltageindependent, and they under physiological conditions (high concentration of

extracellular space [44], leading to stability of the resting membrane potential. K2P channel activity is regulated by a wide variety of factors such as pH, stretching of membrane, temperature, and endogenic compounds like arachidonic acid. K2P channels play important roles in many physiological processes: neuroprotection, cerebrovascular vasodilation, regulation of aldosterone production and secretion,

The neuroprotective agent riluzole, currently in use for the treatment of amyotrophic lateral sclerosis, has been shown to be an activator of TREK-1 and TRAAK channels. Volatile general anesthetics such as chloroform and isoflurane have also

depression, chemoreception, and pulmonary vasoconstriction [45].

ions from the cell into the

ies, whereas tolbutamide shows much lower potency [13, 41].

**2.3 K channels with two pores (K2P) and their modulators**

in the cytoplasm and low extracellular) conduct K+

been shown to target TREK-1 channels [3].

to the control of the resting membrane potential [42].

**Table 3.**

*Selective modulators of inwardly rectifying K channels (Kir).*

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

*Molecular Pharmacology*

As carriers of positive charge, polyamines and Mg2+ interact with the negatively charged amino acids present in the transmembrane M1 domain and terminal

the pore [34]. Kir channels have been identified in many types of cells. Seven

extracellular K, whereas resistant arteries show a positive response [35].

Structure of Kir channel is consisting of four α subunit-forming subunits, each containing two transmembrane domains (M1 and M2) and a P-loop between them. The properties and functions of Kir channels vary between different tissues and species. For example, Kir channel expression is most pronounced in the smooth muscle of the autoregulatory vascular compartments, such as the coronary and cerebral circulation. Kir duct expression increases with decreased blood vessel diameter. The difference in expressive Kir channels can be explained by the fact that the conduction artery shows a very small response to a smaller version of

ions through

C-terminus of the Kir channel, thereby inhibiting the passage of K<sup>+</sup>

subfamilies are identified so far (**Figure 1** and **Table 3**).

**110**

**Table 3.**

*Selective modulators of inwardly rectifying K channels (Kir).*

*ATP-sensitive Kir channels (KATP)* are the exceptions, which have a more complex heterooctamer structure with multiple types of accessory subunits [3, 5]. Structurally, KATP channels form of four pore-forming Kir6.x subunits and four regulatory subunits known as sulfonylurea receptors, SURx. Kir6.x subunits are responsible for ATP inhibition and SURx for nucleoside-diphosphate activation.

Functional expression of the KATP channel requires coexpression of Kir6.x and SURx subunits in a 1:1 ratio. SUR1 is predominantly present in pancreatic β cells. SUR2 has two variants, SUR2A and SUR2B, which are generated by alternative excision of exon 38 in the ABCC9 gene. SUR2A is mainly present in the myocardium and skeletal muscle, while SUR2B generally is distributed in the vascular and myometrial smooth muscle cells [17, 25, 35, 36].

Numerous studies have highlighted that KATP channels in vascular smooth muscle cells play an important role in achieving systemic vasodilation during hypoxia, increasing blood flow to the heart, kidney, and muscle. This vasodilation is attributed to the release of NO or adenosine due to hypoxia but also to the direct effect of hypoxia itself. During hypoxia, KATP channels may be activated by a decrease in ATP, a decrease in pH and partial pressure of oxygen, or an increase in intracellular lactates and ADP. These factors can activate KATP channels directly and/or potentiate NO activation [37].

KATP channels play their roles in the mechanism of the action of relaxation and contraction by interacting with various protein kinases. Thus, KATP channels activated by protein kinase A (PKA) and cGMP-dependent protein kinase participate in the mechanism of action of endogenous vasodilators such as adenosine and prostacyclin. On the other hand, activation of protein kinase C (PKC) and vasoconstrictor induced increases in intracellular Ca2+ caused by noradrenaline, vasopressin, endothelin, and angiotensin II were accompanied by inhibition of the KATP channel [38–40].

KATP channels in the smooth muscle are inhibited by antidiabetics from a group of sulfonylurea derivatives, such as glibenclamide and tolbutamide. Glibenclamide is the most commonly used KATP channel inhibitor in vascular smooth muscle studies, whereas tolbutamide shows much lower potency [13, 41].

In the study of potential drugs, the core interest is directed towards KATP channels, since they exhibit activity under basic conditions and significantly contribute to the control of the resting membrane potential [42].

#### **2.3 K channels with two pores (K2P) and their modulators**

K2P channel subunit consists of two regions that participate in pore formation (P1 and P2, hence their name) and four transmembrane domains (M1–M4). Functional channels form as dimers of these subunits that form a single pore selectively permeable to potassium [43]. Activity of these channels is voltageindependent, and they under physiological conditions (high concentration of K+ in the cytoplasm and low extracellular) conduct K+ ions from the cell into the extracellular space [44], leading to stability of the resting membrane potential. K2P channel activity is regulated by a wide variety of factors such as pH, stretching of membrane, temperature, and endogenic compounds like arachidonic acid. K2P channels play important roles in many physiological processes: neuroprotection, cerebrovascular vasodilation, regulation of aldosterone production and secretion, depression, chemoreception, and pulmonary vasoconstriction [45].

The neuroprotective agent riluzole, currently in use for the treatment of amyotrophic lateral sclerosis, has been shown to be an activator of TREK-1 and TRAAK channels. Volatile general anesthetics such as chloroform and isoflurane have also been shown to target TREK-1 channels [3].


**Table 4.**

*Selective modulators of K channels with two pores K2P.*

#### **3. Natural polyphenols and antibody for K channels a promise for future cancer treatment**

#### **3.1 Natural polyphenols**

Natural polyphenols are secondary metabolism of plants that have multiple activities in determining plant properties such as color, aroma, taste, solution, pathogen resistance, etc. Natural polyphenols have been expanding in the interest of

**113**

hearts [51].

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

sis, and angiogenesis [50].

of K channel function.

both the scientific community and the public over the past decade when they have shown to have a significant function in the prevention of cancer, cardiovascular disease, diabetes and neurodegenerative disorders, etc. [46–49]. Polyphenols are the most common antioxidant constituents, and their source is various foods of plant origin: fruits, vegetables, seeds, especially nuts, chocolate, wine, tea, and coffee. Research carried out during the last decade provided evidence that natural, biologically active polyphenols, such as resveratrol, genistein, quercetin, and catechin-gallate, and curcumin have a wide spectrum of pharmacologic properties such as anti-inflammatory, antioxidant, anticarcinogenic, antiaging, neuroprotective, and cardioprotective effect. Resveratrol, stilbene from grape and red wine, genistein, isoflavone from soy-based food, and catechin-gallate from tea influence cancer initiation, promotion, and progression through diverse signal-transduction pathways that control cell growth and division, inflammation, apoptosis, metasta-

Many studies suggested that Kv channels could be the targets of polyphenols, directly and indirectly [17, 47, 51]. Furthermore, there is evidence that modulation of Kv channels via the PI3K/Akt/mTOR pathway may be a possible indirect mode of action of polyphenols [52, 53]. The PI3K/Akt/mTOR pathway is known to play an important role in cell survival (inhibition of apoptosis), proliferation, and cell metabolism, and PI3K activity has been linked to a variety of human cancers [54]. As described above, the effect on the kinase pathway would result in the modulation

Polyphenols are thought to have several different mechanisms that prevent cardiovascular disease. The following effects are reported in the literature: antioxidant, anti-aggregation, beneficial effects on plasma HDL-cholesterol levels (raising HDL-cholesterol levels), inhibition of LDL-cholesterol oxidation, improvement of endothelial function, and stabilization of atherosclerotic plaque [55]. The mechanism of the vasodilatory action of polyphenols is not fully understood. Still, there are many results that polyphenols included K channels in their mechanism of action [46–48]. It is shown that these molecules can cause endothelium-dependent and endothelium-independent relaxation of the blood vessel. Polyphenols are known to modulate many intracellular signaling pathways as well as the expression of individual genes. Thus, plant polyphenols have been described to activate endothelial NO synthase (eNOS), increase nitric oxide production, and thus induce endothelium-dependent vasodilation. Activation of eNOS occurs due to an increase

in the intracellular level of Ca2+ ions and phosphorylation of PI3 kinase.

addressing the need for innovative tocolytic therapeutics.

Previous reports indicated that resveratrol inhibits vasocontractile response and relaxes different arteries and vein by the activation of smooth muscle Kv and KCa1.1 channels [47, 48]. But, data from electrophysiological studies suggest that resveratrol inhibits L-type Ca2+ channels and enhances activity of the KATP channels in rat

Additionally, it has been shown that the K channel family affects cell function and plays a significant role in regulating myometrium contractility [5, 19]. Changes in the expression or activity of K channels can translate into inadequate repolarization leading to aberrant uterine activity. Thus, K channel alterations may contribute to certain pathophysiological conditions such as preterm labor. Many studies have shown that opening of different types of K channels leads to the relaxation of nonpregnant as well as pregnant myometrium [5, 17, 19, 26, 56]. It seems that function and molecular expression of K channels are dependent of stages of pregnancy, the age, and of hormones influences [17, 57]. K channels, as a novel target to prevent preterm delivery with nontoxic natural polyphenols, are the important work in

#### *Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

*Molecular Pharmacology*

**112**

**Table 4.**

**cancer treatment**

*Selective modulators of K channels with two pores K2P.*

**3.1 Natural polyphenols**

**3. Natural polyphenols and antibody for K channels a promise for future** 

Natural polyphenols are secondary metabolism of plants that have multiple activities in determining plant properties such as color, aroma, taste, solution, pathogen resistance, etc. Natural polyphenols have been expanding in the interest of both the scientific community and the public over the past decade when they have shown to have a significant function in the prevention of cancer, cardiovascular disease, diabetes and neurodegenerative disorders, etc. [46–49]. Polyphenols are the most common antioxidant constituents, and their source is various foods of plant origin: fruits, vegetables, seeds, especially nuts, chocolate, wine, tea, and coffee.

Research carried out during the last decade provided evidence that natural, biologically active polyphenols, such as resveratrol, genistein, quercetin, and catechin-gallate, and curcumin have a wide spectrum of pharmacologic properties such as anti-inflammatory, antioxidant, anticarcinogenic, antiaging, neuroprotective, and cardioprotective effect. Resveratrol, stilbene from grape and red wine, genistein, isoflavone from soy-based food, and catechin-gallate from tea influence cancer initiation, promotion, and progression through diverse signal-transduction pathways that control cell growth and division, inflammation, apoptosis, metastasis, and angiogenesis [50].

Many studies suggested that Kv channels could be the targets of polyphenols, directly and indirectly [17, 47, 51]. Furthermore, there is evidence that modulation of Kv channels via the PI3K/Akt/mTOR pathway may be a possible indirect mode of action of polyphenols [52, 53]. The PI3K/Akt/mTOR pathway is known to play an important role in cell survival (inhibition of apoptosis), proliferation, and cell metabolism, and PI3K activity has been linked to a variety of human cancers [54]. As described above, the effect on the kinase pathway would result in the modulation of K channel function.

Polyphenols are thought to have several different mechanisms that prevent cardiovascular disease. The following effects are reported in the literature: antioxidant, anti-aggregation, beneficial effects on plasma HDL-cholesterol levels (raising HDL-cholesterol levels), inhibition of LDL-cholesterol oxidation, improvement of endothelial function, and stabilization of atherosclerotic plaque [55]. The mechanism of the vasodilatory action of polyphenols is not fully understood. Still, there are many results that polyphenols included K channels in their mechanism of action [46–48]. It is shown that these molecules can cause endothelium-dependent and endothelium-independent relaxation of the blood vessel. Polyphenols are known to modulate many intracellular signaling pathways as well as the expression of individual genes. Thus, plant polyphenols have been described to activate endothelial NO synthase (eNOS), increase nitric oxide production, and thus induce endothelium-dependent vasodilation. Activation of eNOS occurs due to an increase in the intracellular level of Ca2+ ions and phosphorylation of PI3 kinase.

Previous reports indicated that resveratrol inhibits vasocontractile response and relaxes different arteries and vein by the activation of smooth muscle Kv and KCa1.1 channels [47, 48]. But, data from electrophysiological studies suggest that resveratrol inhibits L-type Ca2+ channels and enhances activity of the KATP channels in rat hearts [51].

Additionally, it has been shown that the K channel family affects cell function and plays a significant role in regulating myometrium contractility [5, 19]. Changes in the expression or activity of K channels can translate into inadequate repolarization leading to aberrant uterine activity. Thus, K channel alterations may contribute to certain pathophysiological conditions such as preterm labor. Many studies have shown that opening of different types of K channels leads to the relaxation of nonpregnant as well as pregnant myometrium [5, 17, 19, 26, 56]. It seems that function and molecular expression of K channels are dependent of stages of pregnancy, the age, and of hormones influences [17, 57]. K channels, as a novel target to prevent preterm delivery with nontoxic natural polyphenols, are the important work in addressing the need for innovative tocolytic therapeutics.

In such a scenario, combination treatment with K channel modulators and natural polyphenols could be beneficial for cardiovascular, renal, or gynecologic disorders.

#### **3.2 Antibodies**

The increasing knowledge on the expression of K channels in tumors, together with the information on the structure and function of these molecules and the possibility of detailed in vitro and in vivo studies, makes this family of channels an attractive candidate for the design of personalized therapies for oncological diseases.

As already stated, in addition to regulating many physiological functions, K channels are aberrantly expressed in different types of tumors. In cancer cells, K channel activity regulates cell proliferation, resistance to apoptotic cell death, tumor angiogenesis, invasiveness, and metastatic spread. Moreover, being expressed in cells of the tumor microenvironment, K channels can also modulate the immune/inflammatory response, which contributes to the drive of cancer establishment and progression [18, 58]. After many years of studies, some K channels are emerging as novel cancer biomarkers, to be employed to stratify patients for either prognostic or predictive purposes [59].

Although the attempts to generate blocking monoclonal antibodies using conventional approaches have shown limited success, the insight that structural studies have provided in the last few years makes it possible to design alternative strategies with higher chances of success. This opens doors for a new approach to combine the advantageous features of K channels-specific antibodies and their modulators and will undoubtedly result in improved therapy alternatives in the near future.

It anticipates that a detailed understanding of structural aspects would revolutionize and refine approaches targeting K channels for therapeutic purposes.

#### **4. Conclusion**

K channels are crucial for all aspects of life by regulating the excitability of neurons and the heart, contracting muscles, secreting hormones, moving fluid, and activating the immune cell. K channel modulation accordingly offers tremendous opportunities for drug development. However, with 7% of clinically used drugs targeting ligand-ion channels and only 5% of voltage-gated channels targeted, ion channels are currently "underrepresented" drugs in clinical practice [60]. The reason for this discrepancy is the fact that K channels belonging to a single subtype can be found in the different tissues, e.g., the heart and brain, where they play different roles in the nervous excitability and contractility of the heart muscle. It was mentioned above that even within the same tissue there are subtypes of channels that potentially play different roles in disease and physiology, thus making sub-selective modulators of each subtype of K channels desirable as candidates for drug development. The ubiquity of K channels makes it important to develop highly selective agents. Furthermore, numerous studies have shown that different diseases as diabetes mellitus and hypertension cause changes in K channel expression and function that further complicate solution innovative sub-selective therapeutics or antibodies. However, it is not remote the time in which it will be possible to target specific K channels for therapeutic purposes.

**115**

**Author details**

Vladimir Djokic and Radmila Novakovic\*

provided the original work is properly cited.

Faculty of Medicine, Institute of Pharmacology, Clinical Pharmacology

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: radmila.novakovic@med.bg.ac.rs

and Toxicology, University of Belgrade, Belgrade, Serbia

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

of Serbia [No TR31020, OI175064].

This work was supported by the Scientific Research Grant from Ministry of Education, Science, and Technological Development Government of the Republic

**Funding**

#### **Conflict of interest**

The author declares no conflict of interest.

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

#### **Funding**

*Molecular Pharmacology*

**3.2 Antibodies**

**4. Conclusion**

prognostic or predictive purposes [59].

specific K channels for therapeutic purposes.

The author declares no conflict of interest.

In such a scenario, combination treatment with K channel modulators and natural polyphenols could be beneficial for cardiovascular, renal, or gynecologic disorders.

The increasing knowledge on the expression of K channels in tumors, together with the information on the structure and function of these molecules and the possibility of detailed in vitro and in vivo studies, makes this family of channels an attractive candidate for the design of personalized therapies for oncological diseases. As already stated, in addition to regulating many physiological functions, K channels are aberrantly expressed in different types of tumors. In cancer cells, K channel activity regulates cell proliferation, resistance to apoptotic cell death, tumor angiogenesis, invasiveness, and metastatic spread. Moreover, being expressed in cells of the tumor microenvironment, K channels can also modulate the immune/inflammatory response, which contributes to the drive of cancer establishment and progression [18, 58]. After many years of studies, some K channels are emerging as novel cancer biomarkers, to be employed to stratify patients for either

Although the attempts to generate blocking monoclonal antibodies using conventional approaches have shown limited success, the insight that structural studies have provided in the last few years makes it possible to design alternative strategies with higher chances of success. This opens doors for a new approach to combine the advantageous features of K channels-specific antibodies and their modulators and will undoubtedly result in improved therapy alternatives in the near future.

It anticipates that a detailed understanding of structural aspects would revolu-

K channels are crucial for all aspects of life by regulating the excitability of neurons and the heart, contracting muscles, secreting hormones, moving fluid, and activating the immune cell. K channel modulation accordingly offers tremendous opportunities for drug development. However, with 7% of clinically used drugs targeting ligand-ion channels and only 5% of voltage-gated channels targeted, ion channels are currently "underrepresented" drugs in clinical practice [60]. The reason for this discrepancy is the fact that K channels belonging to a single subtype can be found in the different tissues, e.g., the heart and brain, where they play different roles in the nervous excitability and contractility of the heart muscle. It was mentioned above that even within the same tissue there are subtypes of channels that potentially play different roles in disease and physiology, thus making sub-selective modulators of each subtype of K channels desirable as candidates for drug development. The ubiquity of K channels makes it important to develop highly selective agents. Furthermore, numerous studies have shown that different diseases as diabetes mellitus and hypertension cause changes in K channel expression and function that further complicate solution innovative sub-selective therapeutics or antibodies. However, it is not remote the time in which it will be possible to target

tionize and refine approaches targeting K channels for therapeutic purposes.

**114**

**Conflict of interest**

This work was supported by the Scientific Research Grant from Ministry of Education, Science, and Technological Development Government of the Republic of Serbia [No TR31020, OI175064].

### **Author details**

Vladimir Djokic and Radmila Novakovic\* Faculty of Medicine, Institute of Pharmacology, Clinical Pharmacology and Toxicology, University of Belgrade, Belgrade, Serbia

\*Address all correspondence to: radmila.novakovic@med.bg.ac.rs

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**116**

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[60] Gerlach AC, Antonio BM. Validation

of ion channel targets. Channels (Austin, Tex.). 2015;**9**(6):376-379. DOI:

10.1080/19336950.2015.1081725

10.1139/bcb-2017-0062

secretion by blocking K (ATP) and K (V) channels of beta cells. European Journal of Pharmacology.

[52] Kim YA, Kim GY, Park KY, Choi YH. Resveratrol inhibits nitric oxide and prostaglandin E2 production by lipopolysaccharide-activated C6 microglia. Journal of Medicinal Food.

[53] Ko EA, Park WS, Son YK, Kim Do H, Kim N, Kim HK, et al. The effect of tyrosine kinase inhibitor genistein on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells. Vascular Pharmacology. 2009;**50**:51-56

[54] Chamcheu JC, Roy T, Uddin MB, Banang-Mbeumi S, Chamcheu RN, Walker AL, Liu YY, Huang S. Role and therapeutic targeting of the PI3K/Akt/ mTOR signaling pathway in skin cancer: A review of current status and future trends on natural and synthetic agents therapy. Cells. 2019;**8**(8). pii: E803. DOI:

2007;**568**:269-277

2007;**10**(2):218-224

10.3390/cells8080803

2009;**2**:270-278

2007;**101**:181-186

rep-10-0492

[55] Pandey KB, Rizvi SI. Plant

[56] Novakovic R, Milovanovic S, Protic D, Djokic J, Heinle H, Gojkovic-Bukarica L. The effect of potassium channel opener pinacidil on the non-pregnant rat uterus. Basic & Clinical Pharmacology & Toxicology.

[57] Lovasz N, Ducza E, Gaspar R, Falkay G. Ontogeny of sulfonylureabinding regulatory subunits of KATP channels in the pregnant rat myometrium. Reproduction. 2011;**142**(1):175-181. DOI: 10.1530/

[58] Pardo LA, Stuhmer W. The roles of K(+) channels in cancer. Nature Reviews. Cancer. 2013;**14**(1):39-48

polyphenols as dietary antioxidants in human health and disease. Oxidative Medicine and Cellular Longevity.

*Potassium Channels as a Potential Target Spot for Drugs DOI: http://dx.doi.org/10.5772/intechopen.92176*

secretion by blocking K (ATP) and K (V) channels of beta cells. European Journal of Pharmacology. 2007;**568**:269-277

*Molecular Pharmacology*

[35] Hibino H, Inanobe A, Furutani K, Murakami S, Findlay I, Kurachi Y. Inwardly rectifying potassium channels: channels. The Journal of Physiology.

[44] Enyedi P, Czirjak G. Molecular background of leak K+ currents: Two-pore domain potassium channels. Physiological Reviews.

[45] Gurney A, Manoury B. Twopore potassium channels in the cardiovascular system. European Biophysics Journal. 2009;**38**(3):305-318

[46] Protić D, Beleslin-Čokić B, Novaković R, Kanjuh V, Heinle H, Sćepanović R, et al. Effect of wine polyphenol resveratrol on the contractions elicited electrically or by norepinephrine in the rat portal vein. Phytotherapy Research.

[47] Gojkovic-Bukarica L, Markovic-Lipkovski J, Heinle H, Cirovic S, Rajkovic J, Djokic V, et al. The red wine

polyphenol resveratrol induced relaxation of the isolated renal artery of diabetic rats: The role of potassium channels. Journal of Functional Foods.

[48] Gojkovic Bukarica LJ, Protic D, Kanjuh V, Heinle H, Novakovic R, Šcepanovic R. Cardiovascular effects of resveratrol. Vojnosanitetski Pregled.

[49] Scalbert A, Manach C, Morand C,

[50] Afaq F, Katiyar SK. Polyphenols: Skin photoprotection and inhibition of photocarcinogenesis. Mini Reviews in Medicinal Chemistry.

[51] Chen WP, Chi TC, Chuang LM, Su MJ. Resveratrol enhances insulin

Rémésy C, Jiménez L. Dietary polyphenols and the prevention of diseases. Critical Reviews in Food Science and Nutrition.

2010;**588**(17):3149-3156

2010;**90**(2):559-605

2013;**27**:1685-1693

2019;**52**:266-275

2013;**70**(12):1145-1150

2005;**45**(4):287-306

2011;**11**(14):1200-1215

[36] Chrissobolis S, Sobey CG. Inwardly rectifying potassium channels in the regulation of vascular tone. Current Drug Targets. 2003;**4**(4):281-289

[37] Djokic V, Jankovic-Raznatovic S, Novakovic R, Kostic M, Rajkovic J, Labudovic-Borovic M, et al. Effect of gestational diabetes mellitus and pregnancy-induced hypertension on human umbilical vein smooth muscle KATP channels. Experimental and Molecular Pathology. 2019;**111**:104323. DOI: 10.1016/j.yexmp.2019.104323

[38] Landry DW, Oliver JA. The ATP-sensitive K+ channel mediates hypotension in endotoxemia and hypoxic lactic acidosis in dog. The Journal of Clinical Investigation.

[39] Jackson WF. Potassium channels in the peripheral microcirculation. Microcirculation. 2005;**12**:113-127

[40] Ko EA, Han J, Jung ID, Park WS. Physiological roles of K+ channels in vascular smooth muscle cells. Journal of Smooth Muscle Research. 2008;**44**:65-

81. DOI: 10.1540/jsmr.44.65

2006;**572**(3):617-624

[41] Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement JP 4th, Boyd AE 3rd, González G, et al. Cloning of the beta cell high-affinity sulfonylurea receptor: A regulator of insulin secretion. Science. 1995;**268**(5209):423-426

[42] Teramoto N. Physiological roles of ATP-sensitive K+ channels in smooth muscle. The Journal of Physiology.

[43] Mathie A, Al-Moubarak E, Veale EL. Gating of two pore domain potassium

1992;**89**:2071-2074

Their structure, function, and physiological roles. Physiological Reviews. 2010;**90**(1):291-366

**118**

[52] Kim YA, Kim GY, Park KY, Choi YH. Resveratrol inhibits nitric oxide and prostaglandin E2 production by lipopolysaccharide-activated C6 microglia. Journal of Medicinal Food. 2007;**10**(2):218-224

[53] Ko EA, Park WS, Son YK, Kim Do H, Kim N, Kim HK, et al. The effect of tyrosine kinase inhibitor genistein on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells. Vascular Pharmacology. 2009;**50**:51-56

[54] Chamcheu JC, Roy T, Uddin MB, Banang-Mbeumi S, Chamcheu RN, Walker AL, Liu YY, Huang S. Role and therapeutic targeting of the PI3K/Akt/ mTOR signaling pathway in skin cancer: A review of current status and future trends on natural and synthetic agents therapy. Cells. 2019;**8**(8). pii: E803. DOI: 10.3390/cells8080803

[55] Pandey KB, Rizvi SI. Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Medicine and Cellular Longevity. 2009;**2**:270-278

[56] Novakovic R, Milovanovic S, Protic D, Djokic J, Heinle H, Gojkovic-Bukarica L. The effect of potassium channel opener pinacidil on the non-pregnant rat uterus. Basic & Clinical Pharmacology & Toxicology. 2007;**101**:181-186

[57] Lovasz N, Ducza E, Gaspar R, Falkay G. Ontogeny of sulfonylureabinding regulatory subunits of KATP channels in the pregnant rat myometrium. Reproduction. 2011;**142**(1):175-181. DOI: 10.1530/ rep-10-0492

[58] Pardo LA, Stuhmer W. The roles of K(+) channels in cancer. Nature Reviews. Cancer. 2013;**14**(1):39-48

[59] Cázares-Ordoñez V, Pardo LA. Kv10.1 potassium channel: From the brain to the tumors. Biochemistry and Cell Biology. 2017;**95**(5):531-536. DOI: 10.1139/bcb-2017-0062

[60] Gerlach AC, Antonio BM. Validation of ion channel targets. Channels (Austin, Tex.). 2015;**9**(6):376-379. DOI: 10.1080/19336950.2015.1081725

**Chapter 7**

*Akira Naito*

**Abstract**

Environments

bilayers to form small particles.

lipid bilayer, solid-state NMR

**1. Introduction**

polyglutamine [11].

**121**

Fibril Formation by Glucagon

in Solution and in Membrane

Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic because they activate apoptotic signaling pathways. First, fibril formation by glucagon in acidic solution is discussed in light of morphological and structural changes during elapsed time. Second, we provide kinetic analyses using a two-step autocatalytic reaction mechanism; the first step is a homogeneous nuclear formation process, and the second step is an autocatalytic heterogeneous fibril elongation process. Third, the processes of fibril formation by glucagon in a membrane environment are discussed based on the structural changes in the fibrils. In the presence of bicelles in acidic solution, glucagon interacts with the bicelles and forms fibril intermediates on the bicelle surface and grows into elongated fibrils. Glucagon-dimyristoylphosphatidylcholine (DMPC) bilayers in neutral solution mimic the environment for fibril formation by glucagon under near-physiological condition. Under these conditions, glucagon forms fibril intermediates that grow into elongated fibrils inside the lipid bilayer. Many days after preparing the glucagon-DMPC bilayer sample, the fibrils form networks inside and outside the bilayer. Furthermore, fibril intermediates strongly interact with lipid

**Keywords:** glucagon fibril, fibrillation mechanism, two-step autocatalytic reaction,

Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs, where it activates the glycogenolysis and gluconeogenic pathways, resulting in raised blood glucose levels [1–3]. Glucagon tends to form gel-like fibrillar aggregates in acidic condition [4]. These aggregates are cytotoxic due to the activation of apoptotic signaling pathways [5]. These fibrils are similar to those of other therapeutic peptides and proteins such as human calcitonin (hCT) [6] and insulin [7] and pathologically related fibrils such as prion [8], amylin (type 2 diabetes) [9], β-amyloid (Alzheimer's disease) [10], and

Some non-fibrillar proteins and peptides have been observed by electron

microscopy to form amyloid fibrils with similar morphologies [12].

#### **Chapter 7**

## Fibril Formation by Glucagon in Solution and in Membrane Environments

*Akira Naito*

#### **Abstract**

Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic because they activate apoptotic signaling pathways. First, fibril formation by glucagon in acidic solution is discussed in light of morphological and structural changes during elapsed time. Second, we provide kinetic analyses using a two-step autocatalytic reaction mechanism; the first step is a homogeneous nuclear formation process, and the second step is an autocatalytic heterogeneous fibril elongation process. Third, the processes of fibril formation by glucagon in a membrane environment are discussed based on the structural changes in the fibrils. In the presence of bicelles in acidic solution, glucagon interacts with the bicelles and forms fibril intermediates on the bicelle surface and grows into elongated fibrils. Glucagon-dimyristoylphosphatidylcholine (DMPC) bilayers in neutral solution mimic the environment for fibril formation by glucagon under near-physiological condition. Under these conditions, glucagon forms fibril intermediates that grow into elongated fibrils inside the lipid bilayer. Many days after preparing the glucagon-DMPC bilayer sample, the fibrils form networks inside and outside the bilayer. Furthermore, fibril intermediates strongly interact with lipid bilayers to form small particles.

**Keywords:** glucagon fibril, fibrillation mechanism, two-step autocatalytic reaction, lipid bilayer, solid-state NMR

#### **1. Introduction**

Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs, where it activates the glycogenolysis and gluconeogenic pathways, resulting in raised blood glucose levels [1–3]. Glucagon tends to form gel-like fibrillar aggregates in acidic condition [4]. These aggregates are cytotoxic due to the activation of apoptotic signaling pathways [5]. These fibrils are similar to those of other therapeutic peptides and proteins such as human calcitonin (hCT) [6] and insulin [7] and pathologically related fibrils such as prion [8], amylin (type 2 diabetes) [9], β-amyloid (Alzheimer's disease) [10], and polyglutamine [11].

Some non-fibrillar proteins and peptides have been observed by electron microscopy to form amyloid fibrils with similar morphologies [12].

Several characteristics of these fibrils are related to the misfolding of proteins, leading to severe conditions such as fibril deposits in the brains of Alzheimer's disease patients [13] and in the pancreas of patients with type 2 diabetes [9].

presence of bicelles. The nucleation rate is slower, and the fibril elongation rate is

In neutral conditions, glucagon molecules are incorporated into lipid bilayers above the phase transition temperature, and the properties of the lipids appear to remain unperturbed. Below the phase transition temperature, glucagon forms discoidal particle with DMPC [37, 38] and induces closer packing of the phospholipid bilayers [39]. Similar peptide-lipid interaction to form discoidal particles below the phase transition temperature is seen in the melittin-DMPC bilayer system [40, 41]. The time course behavior of glucagon fibril formation inside a DMPC bilayer under neutral conditions, which approximates the physiological condition, and the kinetic behavior of glucagon under these conditions have been investigated to understand the fibrillation process under near-physiological conditions [42].

Gel formation by glucagon in the β-sheet conformation in acid solutions is a relatively slow process at room temperature [4] and can be followed by observing the change in viscosity as shown in **Figure 1**. The most marked feature of the change in viscosity in these time profiles is the presence of a substantial lag phase during which oligomeric nuclei are likely formed and function as initiation sites. These phenomena were confirmed by adding a small seed of preformed glucagon gel on the end of a wire into a viscometer containing a fresh acidic glucagon solution. The viscosity increased immediately with essentially no lag (**Figure 1a**). After a prolonged reaction time, the viscosity began to decrease and fibrils sometimes precipitated. These fibrils represent a variant of the β-structure of glucagon. Fibril formation strongly depends on the peptide concentration, proceeding very slowly at less than about 1.5 mg/ml and occurring more readily at higher concentration (**Figure 1b**). Increasing the ionic strength of the solution results in both an increasing aggregation rate and more rapid production of fibrils. The change in viscosity with time in even 0.01 M sodium chloride occurs much more rapidly than

The effect of temperature on the polymerization rate is also very marked. Both the aggregation rate and the size of the aggregates as reflected in the maximal values

Transmission electron microscopy (TEM) time-elapsed pictures were obtained

The α-helical content of aged glucagon at 5.0 mg/ml decreased significantly to approximately 1%. The CD spectrum of a β-sheet structure typically shows an intense positive band at 198 nm and a negative band at 218 nm [5]. The CD spectral pattern of aged glucagon was that of a β-sheet structure, indicating a conformational transition from α-helical to β-sheet structure under these conditions.

The FTIR spectrum of glucagon immediately after dissolution showed a low-

, and aging resulted in progressively

of the reduced viscosity show a strong temperature maximum around 30°C (**Figure 1c**). An addition of 5% (v/v) of the nonaqueous solvent dioxin completely

during fibril formation by glucagon dissolved in 0.015 M acetic acid solution (18 mg/ml) at pH 3.3 [36]. TEM pictures were measured approximately 2 hrs, then 1 week, and 6 months after the dissolution of glucagon (**Figure 2**). A small number of spherical-shaped fibril intermediates appeared after approximately 2 hrs, as shown in **Figure 2a**. After 1 week, the number of spherical fibril intermediates had increased, and elongated fibrils had appeared (**Figure 2b**). After 6 months, long mature fibrils about 10 nm in diameter were observed, and the spherical fibril

faster in acidic solution than in the presence of bicelles.

*DOI: http://dx.doi.org/10.5772/intechopen.91681*

*Fibril Formation by Glucagon in Solution and in Membrane Environments*

**2. Fibril formation by glucagon in acidic solution**

in the absence of salt, and viscosity decreases quickly.

intermediates had completely disappeared (**Figure 2c**).

intensity β-sheet band at 1620–1630 cm<sup>1</sup>

**123**

inhibits the aggregation (**Figure 1a**).

Kinetic analyses of fibril formation by the therapeutic peptide human calcitonin indicate that hCT molecules associate to form fibril intermediates via a two-step autocatalytic reaction mechanism. The first step of kinetic reaction (rate constant, k1) is a homogeneous reaction from micelle-like oligomers to fibril intermediates. These intermediates react with monomeric molecules to elongate into longer fibrils via a heterogeneous fibril elongation process (rate constant, k2) [14–17]. Elucidating the molecular structure of amyloid fibrils is important for understanding the mechanism of self-aggregation, but it is difficult to determine high-resolution molecular structures using typical spectroscopic methods because fibrils are heterogeneous solids. Solid-state NMR spectroscopy has demonstrated advantages for the conformational determination of Alzheimer's amyloid β-peptides (Aβ), which mainly comprise 40 or 42 amino acid residues and are the main component of the amyloid plaques found in Alzheimer's disease patients [12, 18]. Both the intra-chain conformation of the Aβ molecule in fibrils and their intermolecular alignment have been analyzed to explore the mechanism of molecular association underlying the formation of Aβ (1–40) [19–21] and the more toxic Aβ (1–42) [22–24] fibrils.

The primary structure of the glucagon peptide is His<sup>1</sup> -Ser-Gln-Gly-Thr<sup>5</sup> -Phe-Thr-Ser-Asp-Tyr10-Ser-Lys-Tyr-Leu-Asp15-Ser-Arg-Arg-Ala-Gln20-Asp-Phe-Val-Gln-Trp25-Leu-Met-Asn-Thr-OH.

An X-ray crystallographic study showed that glucagon adopts a trimeric α-helix structure stabilized by hydrophobic interactions between molecules related by threefold symmetry [25], whereas a solution NMR study showed that glucagon in dilute aqueous solution may not form a specific structure, with the exception of the 22–25 region [26]. The secondary structure of glucagon in the presence of dodecylphosphocholine micelles comprises three turns of an irregular α-helix formed by residues 17 to 29 near the C-terminus, a stretch of extended polypeptide chain from residues 14 to 17, an α-helix-like turn formed by residue 10 to 14, and another extended region from residue 5 to 10 [27].

Fibril formation by glucagon molecules was observed by Beaven et al. in undisturbed aqueous solution at pH 2 [4]. The viscosity initially increased and a birefringent gel is formed. With time, a precipitate appeared comprising long fibrils, as determined using electron microscopy. Infrared spectra of the gel, solid film, and precipitate showed that in all these states, glucagon is in the form of antiparallel β-sheet chains [5, 28]. Kinetic analysis of fibril formation by glucagon under acidic conditions demonstrated a complex fibrillation mechanism in which suitable changes in the fibrillation condition can alter the type of fibril formed or result in the formation of a mixture of several types of fibrils [29, 30]. Furthermore, the fibrils come in two forms: one composed entirely of glucagon monomers and the other entirely of glucagon trimers [31]. Studies of fibril formation typically use acidic pH solutions because of the low solubility of glucagon in neutral solution.

Understanding the cytotoxicity of amyloid-forming peptides requires investigating the interaction of these peptides with membranes because lipid bilayer components dramatically alter most aspects of amyloid aggregation [32–35]. We previously reported glucagon fibrillation in the presence of dimyristoylphosphatidylcholine/1,2-dihexanoyl-sn-glycero-3-phophocholine (DMPC-DHPC) bicelles in acidic solution. The glucagon structure in the fibril in the presence of these bicelles is different from that in their absence [36]: the N- and C-termini both change from α-helix to β-sheet in acidic solution, while the N-terminus remains in an α-helical conformation, whereas the C-terminus changes from α-helix to β-sheet in the

#### *Fibril Formation by Glucagon in Solution and in Membrane Environments DOI: http://dx.doi.org/10.5772/intechopen.91681*

presence of bicelles. The nucleation rate is slower, and the fibril elongation rate is faster in acidic solution than in the presence of bicelles.

In neutral conditions, glucagon molecules are incorporated into lipid bilayers above the phase transition temperature, and the properties of the lipids appear to remain unperturbed. Below the phase transition temperature, glucagon forms discoidal particle with DMPC [37, 38] and induces closer packing of the phospholipid bilayers [39]. Similar peptide-lipid interaction to form discoidal particles below the phase transition temperature is seen in the melittin-DMPC bilayer system [40, 41].

The time course behavior of glucagon fibril formation inside a DMPC bilayer under neutral conditions, which approximates the physiological condition, and the kinetic behavior of glucagon under these conditions have been investigated to understand the fibrillation process under near-physiological conditions [42].

#### **2. Fibril formation by glucagon in acidic solution**

Gel formation by glucagon in the β-sheet conformation in acid solutions is a relatively slow process at room temperature [4] and can be followed by observing the change in viscosity as shown in **Figure 1**. The most marked feature of the change in viscosity in these time profiles is the presence of a substantial lag phase during which oligomeric nuclei are likely formed and function as initiation sites. These phenomena were confirmed by adding a small seed of preformed glucagon gel on the end of a wire into a viscometer containing a fresh acidic glucagon solution. The viscosity increased immediately with essentially no lag (**Figure 1a**). After a prolonged reaction time, the viscosity began to decrease and fibrils sometimes precipitated. These fibrils represent a variant of the β-structure of glucagon.

Fibril formation strongly depends on the peptide concentration, proceeding very slowly at less than about 1.5 mg/ml and occurring more readily at higher concentration (**Figure 1b**). Increasing the ionic strength of the solution results in both an increasing aggregation rate and more rapid production of fibrils. The change in viscosity with time in even 0.01 M sodium chloride occurs much more rapidly than in the absence of salt, and viscosity decreases quickly.

The effect of temperature on the polymerization rate is also very marked. Both the aggregation rate and the size of the aggregates as reflected in the maximal values of the reduced viscosity show a strong temperature maximum around 30°C (**Figure 1c**). An addition of 5% (v/v) of the nonaqueous solvent dioxin completely inhibits the aggregation (**Figure 1a**).

Transmission electron microscopy (TEM) time-elapsed pictures were obtained during fibril formation by glucagon dissolved in 0.015 M acetic acid solution (18 mg/ml) at pH 3.3 [36]. TEM pictures were measured approximately 2 hrs, then 1 week, and 6 months after the dissolution of glucagon (**Figure 2**). A small number of spherical-shaped fibril intermediates appeared after approximately 2 hrs, as shown in **Figure 2a**. After 1 week, the number of spherical fibril intermediates had increased, and elongated fibrils had appeared (**Figure 2b**). After 6 months, long mature fibrils about 10 nm in diameter were observed, and the spherical fibril intermediates had completely disappeared (**Figure 2c**).

The α-helical content of aged glucagon at 5.0 mg/ml decreased significantly to approximately 1%. The CD spectrum of a β-sheet structure typically shows an intense positive band at 198 nm and a negative band at 218 nm [5]. The CD spectral pattern of aged glucagon was that of a β-sheet structure, indicating a conformational transition from α-helical to β-sheet structure under these conditions.

The FTIR spectrum of glucagon immediately after dissolution showed a lowintensity β-sheet band at 1620–1630 cm<sup>1</sup> , and aging resulted in progressively

Several characteristics of these fibrils are related to the misfolding of proteins, leading to severe conditions such as fibril deposits in the brains of Alzheimer's disease patients [13] and in the pancreas of patients with type 2 diabetes [9].

tion of Aβ (1–40) [19–21] and the more toxic Aβ (1–42) [22–24] fibrils.

Thr-Ser-Asp-Tyr10-Ser-Lys-Tyr-Leu-Asp15-Ser-Arg-Arg-Ala-Gln20-Asp-Phe-Val-

structure stabilized by hydrophobic interactions between molecules related by threefold symmetry [25], whereas a solution NMR study showed that glucagon in dilute aqueous solution may not form a specific structure, with the exception of the

Fibril formation by glucagon molecules was observed by Beaven et al. in undisturbed aqueous solution at pH 2 [4]. The viscosity initially increased and a birefringent gel is formed. With time, a precipitate appeared comprising long fibrils, as determined using electron microscopy. Infrared spectra of the gel, solid film, and precipitate showed that in all these states, glucagon is in the form of antiparallel β-sheet chains [5, 28]. Kinetic analysis of fibril formation by glucagon under acidic conditions demonstrated a complex fibrillation mechanism in which suitable changes in the fibrillation condition can alter the type of fibril formed or result in the formation of a mixture of several types of fibrils [29, 30]. Furthermore, the fibrils come in two forms: one composed entirely of glucagon monomers and the other entirely of glucagon trimers [31]. Studies of fibril formation typically use acidic pH solutions because of the low solubility of glucagon in

Understanding the cytotoxicity of amyloid-forming peptides requires investigating the interaction of these peptides with membranes because lipid bilayer components dramatically alter most aspects of amyloid aggregation [32–35]. We previously reported glucagon fibrillation in the presence of dimyristoylphosphatidylcholine/1,2-dihexanoyl-sn-glycero-3-phophocholine (DMPC-DHPC) bicelles in acidic solution. The glucagon structure in the fibril in the presence of these bicelles is different from that in their absence [36]: the N- and C-termini both change from α-helix to β-sheet in acidic solution, while the N-terminus remains in an α-helical conformation, whereas the C-terminus changes from α-helix to β-sheet in the

22–25 region [26]. The secondary structure of glucagon in the presence of dodecylphosphocholine micelles comprises three turns of an irregular α-helix formed by residues 17 to 29 near the C-terminus, a stretch of extended polypeptide chain from residues 14 to 17, an α-helix-like turn formed by residue 10 to 14, and

An X-ray crystallographic study showed that glucagon adopts a trimeric α-helix



The primary structure of the glucagon peptide is His<sup>1</sup>

another extended region from residue 5 to 10 [27].

Gln-Trp25-Leu-Met-Asn-Thr-OH.

*Molecular Pharmacology*

neutral solution.

**122**

Kinetic analyses of fibril formation by the therapeutic peptide human calcitonin indicate that hCT molecules associate to form fibril intermediates via a two-step autocatalytic reaction mechanism. The first step of kinetic reaction (rate constant, k1) is a homogeneous reaction from micelle-like oligomers to fibril intermediates. These intermediates react with monomeric molecules to elongate into longer fibrils via a heterogeneous fibril elongation process (rate constant, k2) [14–17]. Elucidating the molecular structure of amyloid fibrils is important for understanding the mechanism of self-aggregation, but it is difficult to determine high-resolution molecular structures using typical spectroscopic methods because fibrils are heterogeneous solids. Solid-state NMR spectroscopy has demonstrated advantages for the conformational determination of Alzheimer's amyloid β-peptides (Aβ), which mainly comprise 40 or 42 amino acid residues and are the main component of the amyloid plaques found in Alzheimer's disease patients [12, 18]. Both the intra-chain conformation of the Aβ molecule in fibrils and their intermolecular alignment have been analyzed to explore the mechanism of molecular association underlying the forma-

nucleation-dependent aggregation pathways influenced by the solution conditions and can be self-propagated by seeding. Type A fibrils that form at high glucagon concentration (>5 mg/ml, pH 2.5) represent the least stable fibril type with a low melting midpoint (Tmapp < 32°C) and single protofilament fibrils observable by TEM (**Figure 3a**). Type B (Bunagitated and Bagitated) fibrils form under low glucagon concentration (<0.5 mg/ml, pH 2.5). Type Bunagitated fibrils grow by branching in the absence of agitation and appear as branched twisted fibrils by TEM (**Figure 3b**). Type Bagitated glucagon fibrils form when the solution is agitated, suggesting that agitation breaks the fibril creating more free fibril ends that align as parallel pairs (**Figure 3c**). Type D fibrils grow under low glucagon concentration (<0.5 mg/ml, pH 2.5) in the presence of 150–250 mM Cl and are twisted and tightly packed

*Fibril Formation by Glucagon in Solution and in Membrane Environments*

*DOI: http://dx.doi.org/10.5772/intechopen.91681*

*Transmission electron micrographs of glucagon (18 mg/ml) in 0.015 M acetic acid solution at pH 3.3. (a) Taken approximately 2 hrs after the dissolution of glucagon. The bar indicates 20 nm. (b) Taken 1 week after dissolution of glucagon. The bar indicates 50 nm. (c) Taken 6 months after dissolution of glucagon.*

*Electron microscope image of morphology of different types of glucagon fibrils. The scale bar is 50 nm. (a) Type A: The glucagon concentration, >5 mg/ml, 50 mM glycine, pH 2.5, low agitation. (b) Type Bunagitated: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, pH 2.5, low agitation. (c) Type Bagitated: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, vigorous agitation. (d) Type D: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, pH 2.5+, 150–250 mM Cl. (e) Type S: Glucagon concentration, <0.5 mg/ml,*

**Figure 2.**

**Figure 3.**

**125**

*0.01 N HCl, 1 mM SO4*

*<sup>2</sup> (ref. [30]).*

*The bar indicates 50 nm (ref. [36]).*

#### **Figure 1.**

*Time course behavior of glucagon aggregation in 0.01 M hydrochloric acid at 26°C and a glucagon concentration of 2.5 mg/ml. (a) Time course of reduced viscosity (msp/c); glucagon with no addition (*○*); glucagon seeded with preformed gel (*□*); glucagon solution containing 5% by volume dioxin (*●*). (b) Effect of glucagon concentration at 4 mg/ml (*□*); 2.5 mg/ml (*○*); and 1 mg/ml (*●*). (c) Effect of temperature on the aggregation rate of glucagon at a glucagon concentration of 2.5 mg/ml at 26°C (*○*); 30°C (*□*); and 35°C (*∇*) (ref. [4]).*

greater amounts of β-sheet [28]. Deconvolution and curve fitting of the amide I band showed that unaged glucagon contained 54% α-helix/random coil structure and 2% β-sheet structure, whereas aged glucagon comprised 22% α-helix/random coil structure and 49% β-sheet structure.

Detailed kinetic, spectroscopic, and morphological studies have revealed that glucagon can form several types of fibrils that differ at the level of molecular packing of the peptide [29–31, 43–46]. Each type forms through distinct

#### *Fibril Formation by Glucagon in Solution and in Membrane Environments DOI: http://dx.doi.org/10.5772/intechopen.91681*

nucleation-dependent aggregation pathways influenced by the solution conditions and can be self-propagated by seeding. Type A fibrils that form at high glucagon concentration (>5 mg/ml, pH 2.5) represent the least stable fibril type with a low melting midpoint (Tmapp < 32°C) and single protofilament fibrils observable by TEM (**Figure 3a**). Type B (Bunagitated and Bagitated) fibrils form under low glucagon concentration (<0.5 mg/ml, pH 2.5). Type Bunagitated fibrils grow by branching in the absence of agitation and appear as branched twisted fibrils by TEM (**Figure 3b**). Type Bagitated glucagon fibrils form when the solution is agitated, suggesting that agitation breaks the fibril creating more free fibril ends that align as parallel pairs (**Figure 3c**). Type D fibrils grow under low glucagon concentration (<0.5 mg/ml, pH 2.5) in the presence of 150–250 mM Cl and are twisted and tightly packed

#### **Figure 2.**

*Transmission electron micrographs of glucagon (18 mg/ml) in 0.015 M acetic acid solution at pH 3.3. (a) Taken approximately 2 hrs after the dissolution of glucagon. The bar indicates 20 nm. (b) Taken 1 week after dissolution of glucagon. The bar indicates 50 nm. (c) Taken 6 months after dissolution of glucagon. The bar indicates 50 nm (ref. [36]).*

#### **Figure 3.**

greater amounts of β-sheet [28]. Deconvolution and curve fitting of the amide I band showed that unaged glucagon contained 54% α-helix/random coil structure and 2% β-sheet structure, whereas aged glucagon comprised 22% α-helix/random

*Time course behavior of glucagon aggregation in 0.01 M hydrochloric acid at 26°C and a glucagon concentration of 2.5 mg/ml. (a) Time course of reduced viscosity (msp/c); glucagon with no addition (*○*); glucagon seeded with preformed gel (*□*); glucagon solution containing 5% by volume dioxin (*●*). (b) Effect of glucagon concentration at 4 mg/ml (*□*); 2.5 mg/ml (*○*); and 1 mg/ml (*●*). (c) Effect of temperature on the aggregation rate of glucagon at a glucagon concentration of 2.5 mg/ml at 26°C (*○*); 30°C (*□*); and 35°C (*∇*)*

Detailed kinetic, spectroscopic, and morphological studies have revealed that glucagon can form several types of fibrils that differ at the level of molecular packing of the peptide [29–31, 43–46]. Each type forms through distinct

coil structure and 49% β-sheet structure.

**Figure 1.**

*Molecular Pharmacology*

*(ref. [4]).*

**124**

*Electron microscope image of morphology of different types of glucagon fibrils. The scale bar is 50 nm. (a) Type A: The glucagon concentration, >5 mg/ml, 50 mM glycine, pH 2.5, low agitation. (b) Type Bunagitated: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, pH 2.5, low agitation. (c) Type Bagitated: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, vigorous agitation. (d) Type D: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, pH 2.5+, 150–250 mM Cl. (e) Type S: Glucagon concentration, <0.5 mg/ml, 0.01 N HCl, 1 mM SO4 <sup>2</sup> (ref. [30]).*

(**Figure 3d**). Type S fibrils grow under low glucagon concentration (<0.5 mg/ml, pH 2.5) in the presence of 1 mM Na2SO4 (7:1 ratio with glucagon) and appear as twisted mature fibrils by TEM (**Figure 3e**).

of glucagon in the fibril state exhibit signals at 167.2 and 21.0 ppm, respectively, and

the N-terminus of monomeric glucagon forms an α-helix structure, the center portion forms a random coil, and the C-terminus forms an α-helix structure, as shown in **Table 1** and **Figure 4** (right panels) in acetic acid solution. When the glucagon monomer aggregates to form fibrils, the N-terminal and C-terminal regions change from an α-helix to a β-sheet as seen with other amyloid-forming

The cytotoxicity of the glucagon fibril was assessed by exposing PC12 and NIH-3 T3 cells to 0.1–100 μM peptide aggregate for 72 hrs followed by cell viability determination under the WST-8 assay and released lactate dehydrogenase (LDH) [5]. A significant decrease in cell viability was observed in cultures exposed to 10–100 μM aged glucagon (P < 0.01) but not in cultures treated with 100 μM nonaged glucagon. It was determined whether the loss of cell viability was due to cell death by measuring the release of LDH. Treatment with 10 μM aged glucagon induced a significant increase in LDH release compared to control, whereas no significant increase in LDH release was observed in cultures treated with 100 μM nonaged glucagon or 1 μM or lower aged glucagon. Thus, glucagon fibrils were found to be highly toxic to PC12 cells, similar to the case of aged prion protein fragment (PrP)106–126 [50] and β-amyloid (Aβ)1–42 [51] (>10 mM). Aged salmon calcitonin also displayed significant cytotoxicity in PC12 cells, whereas nonaged

Next, signaling pathways for the cytotoxicity of peptide fibrils were investigated [5]. Caspase-3 activation is required for the early stages of apoptosis that include DNA fragmentation and morphological changes. To determine whether aged glucagon induces caspase-3 activation in PC12 cells, cells were exposed to 50 μM aged glucagon, and the caspase-3-like activities of the cell lysates were measured by cleavage of the fluorometric caspase-3 substitute Z-DEVD-rhodamine 110. The activity increased prior to the loss of membrane integrity, and 24 hrs after incubation, maximum caspase-3 activity was detected (160% of the control level). In contrast, no significant elevation of casepase-3 activity was observed in cells treated with 50 μM nonaged glucagon. These results indicate that the exposure of PC12 cells to peptide fibrils induces a rapid (within 24 hrs) and significant elevation

in casepase-3 activity prior to the loss of cell viability 72 hrs after exposure. In summary, the misfolding of the therapeutic peptide glucagon generates amyloidogenic fibrils, leading to cytotoxicity mediated by the activation of the

As shown in **Figure 5**, glucagon monomers (A) first aggregate to form weakly coupled oligomers (An) akin to the micelle state. Next, glucagon oligomers (An) form fibril intermediates (nuclei) (Bn) through a homogeneous nucleation process with a rate constant k1. Fibril intermediates (Bn) then react with monomer (A) to form elongated fibrils with a rate constant k2. This is called the inhomogeneous fibril elongation process. The B form plays a role in the catalysis of A to B, and therefore this is an autocatalytic reaction. Since the nucleation and elongation

**4. Kinetic analysis of the glucagon fibrillation process**

Conformationally dependent chemical shift values [47–49] clearly indicate that

indicate that the vicinities of Gly4 and Ala19 form β-sheet structures.

*Fibril Formation by Glucagon in Solution and in Membrane Environments*

peptides such as human calcitonin [14] in acetic acid solution.

salmon calcitonin did not induce significant cell death [5].

**3. Cytotoxicity of glucagon fibril**

*DOI: http://dx.doi.org/10.5772/intechopen.91681*

apoptotic enzyme caspase-3 in vitro.

**127**

Solid-state 13C NMR spectra were observed for 18 mg/ml [1-13C]Gly4 and [3-13C]Ala19-glucagon in 0.015 M acetic acid solution, pH 3.3 [36]. The 13C direct excitation with dipolar decoupling and magic angle spinning (DD-MAS) signal indicates monomeric glucagon, and the 13C cross polarization with magic angle spinning (CP-MAS) signal indicates fibril glucagon. The DD-MAS spectra (**Figure 4A** and **C**) of [1-13C]Gly4 and [3-13C]Ala19 exhibit signals at 171.7 and 16.4 ppm, respectively, consistent with the monomeric state and indicate that the region near the Gly4 and Ala19 residues forms α-helix structures, as shown by the conformationally dependent chemical shift values [47–49]. The experimentally determined chemical shift values and secondary structures are summarized in **Table 1**. The 13C CP-MAS spectra of [1-13C]Gly4 and [3-13C]Ala19 (**Figure 4B** and **D**)

#### **Figure 4.**

*13C DD-MAS and CP-MAS NMR spectra of [1-13C]Gly4 (A and B) and [3-13C]Ala19 (C and D) of [1-13C]Gly4 and [3-13C]Ala19-glucagon in acetic acid solution at pH 3.3. Schematic structures of glucagon in the monomeric and fibril states (right panels) (ref. [36]).*


*\* The structure around each amino acid residues was determined by comparing the experimentally obtained 13C chemical shift values (δiso) with typical 13C chemical shift values (δiso) of α-helix and β-sheet, reported as 171.6 and 168.5 for [1-13C]Gly and 14.9 and 19.9 for [3-13C]Ala, respectively [47–49].*

#### **Table 1.**

*Structural transitions during glucagon fibrillation in various conditions as determined by conformationdependent 13C chemical shifts (ppm)\* .*

#### *Fibril Formation by Glucagon in Solution and in Membrane Environments DOI: http://dx.doi.org/10.5772/intechopen.91681*

of glucagon in the fibril state exhibit signals at 167.2 and 21.0 ppm, respectively, and indicate that the vicinities of Gly4 and Ala19 form β-sheet structures.

Conformationally dependent chemical shift values [47–49] clearly indicate that the N-terminus of monomeric glucagon forms an α-helix structure, the center portion forms a random coil, and the C-terminus forms an α-helix structure, as shown in **Table 1** and **Figure 4** (right panels) in acetic acid solution. When the glucagon monomer aggregates to form fibrils, the N-terminal and C-terminal regions change from an α-helix to a β-sheet as seen with other amyloid-forming peptides such as human calcitonin [14] in acetic acid solution.

#### **3. Cytotoxicity of glucagon fibril**

(**Figure 3d**). Type S fibrils grow under low glucagon concentration (<0.5 mg/ml, pH 2.5) in the presence of 1 mM Na2SO4 (7:1 ratio with glucagon) and appear as

Solid-state 13C NMR spectra were observed for 18 mg/ml [1-13C]Gly4 and [3-13C]Ala19-glucagon in 0.015 M acetic acid solution, pH 3.3 [36]. The 13C direct excitation with dipolar decoupling and magic angle spinning (DD-MAS) signal indicates monomeric glucagon, and the 13C cross polarization with magic angle spinning (CP-MAS) signal indicates fibril glucagon. The DD-MAS spectra (**Figure 4A** and **C**) of [1-13C]Gly4 and [3-13C]Ala19 exhibit signals at 171.7 and 16.4 ppm, respectively, consistent with the monomeric state and indicate that the region near the Gly4 and Ala19 residues forms α-helix structures, as shown by the conformationally dependent chemical shift values [47–49]. The experimentally determined chemical shift values and secondary structures are summarized in **Table 1**. The 13C CP-MAS spectra of [1-13C]Gly4 and [3-13C]Ala19 (**Figure 4B** and **D**)

*13C DD-MAS and CP-MAS NMR spectra of [1-13C]Gly4 (A and B) and [3-13C]Ala19 (C and D) of [1-13C]Gly4 and [3-13C]Ala19-glucagon in acetic acid solution at pH 3.3. Schematic structures of glucagon in*

*The structure around each amino acid residues was determined by comparing the experimentally obtained 13C chemical shift values (δiso) with typical 13C chemical shift values (δiso) of α-helix and β-sheet, reported as 171.6 and*

*Structural transitions during glucagon fibrillation in various conditions as determined by conformation-*

**(structure)**

Monomer 171.7 (α-helix) 16.4 (α-helix) [36]

Fibril 169.2 (β-sheet) 21.0 (β-sheet) [36]

Fibril 171.5 (α-helix) 19.3 (β-sheet) [36]

Monomer 171.5 (α-helix) 15.9 (α-helix) [42]

Intermediate 168.9 (β-sheet) 20.5 (β-sheet) [42] Fibril 166.9 (β-sheet) 21.5 (β-sheet) [42]

Monomer 171.5 (α-helix) 16.4 (α-sheet) [36]

**[3-13C]Ala19 (structure)**

**Ref.**

**Fibril formation condition Fibril type [1-13C]Gly4**

*168.5 for [1-13C]Gly and 14.9 and 19.9 for [3-13C]Ala, respectively [47–49].*

*.*

twisted mature fibrils by TEM (**Figure 3e**).

*Molecular Pharmacology*

*the monomeric and fibril states (right panels) (ref. [36]).*

Acidic solution (0.015 M acetic acid

Acidic solution in the presence of bicelle

Glucagon inside lipid bilayer (DMPC) in

solution pH 3.3)

neutral solution

*\**

**Table 1.**

**126**

(DMPC+DHPC pH 3.3)

*dependent 13C chemical shifts (ppm)\**

**Figure 4.**

The cytotoxicity of the glucagon fibril was assessed by exposing PC12 and NIH-3 T3 cells to 0.1–100 μM peptide aggregate for 72 hrs followed by cell viability determination under the WST-8 assay and released lactate dehydrogenase (LDH) [5]. A significant decrease in cell viability was observed in cultures exposed to 10–100 μM aged glucagon (P < 0.01) but not in cultures treated with 100 μM nonaged glucagon. It was determined whether the loss of cell viability was due to cell death by measuring the release of LDH. Treatment with 10 μM aged glucagon induced a significant increase in LDH release compared to control, whereas no significant increase in LDH release was observed in cultures treated with 100 μM nonaged glucagon or 1 μM or lower aged glucagon. Thus, glucagon fibrils were found to be highly toxic to PC12 cells, similar to the case of aged prion protein fragment (PrP)106–126 [50] and β-amyloid (Aβ)1–42 [51] (>10 mM). Aged salmon calcitonin also displayed significant cytotoxicity in PC12 cells, whereas nonaged salmon calcitonin did not induce significant cell death [5].

Next, signaling pathways for the cytotoxicity of peptide fibrils were investigated [5]. Caspase-3 activation is required for the early stages of apoptosis that include DNA fragmentation and morphological changes. To determine whether aged glucagon induces caspase-3 activation in PC12 cells, cells were exposed to 50 μM aged glucagon, and the caspase-3-like activities of the cell lysates were measured by cleavage of the fluorometric caspase-3 substitute Z-DEVD-rhodamine 110. The activity increased prior to the loss of membrane integrity, and 24 hrs after incubation, maximum caspase-3 activity was detected (160% of the control level). In contrast, no significant elevation of casepase-3 activity was observed in cells treated with 50 μM nonaged glucagon. These results indicate that the exposure of PC12 cells to peptide fibrils induces a rapid (within 24 hrs) and significant elevation in casepase-3 activity prior to the loss of cell viability 72 hrs after exposure.

In summary, the misfolding of the therapeutic peptide glucagon generates amyloidogenic fibrils, leading to cytotoxicity mediated by the activation of the apoptotic enzyme caspase-3 in vitro.

#### **4. Kinetic analysis of the glucagon fibrillation process**

As shown in **Figure 5**, glucagon monomers (A) first aggregate to form weakly coupled oligomers (An) akin to the micelle state. Next, glucagon oligomers (An) form fibril intermediates (nuclei) (Bn) through a homogeneous nucleation process with a rate constant k1. Fibril intermediates (Bn) then react with monomer (A) to form elongated fibrils with a rate constant k2. This is called the inhomogeneous fibril elongation process. The B form plays a role in the catalysis of A to B, and therefore this is an autocatalytic reaction. Since the nucleation and elongation

where *f* is the fraction of B-glucagon fibrils in the system.

*Fibril Formation by Glucagon in Solution and in Membrane Environments*

glucagons. The relevant kinetic equation is given by

time by using an established protocol [52] (**Figure 6C**).

**Fibril formation condition k1 (s**�**<sup>1</sup>**

Eq. (5) can be integrated to give

*DOI: http://dx.doi.org/10.5772/intechopen.91681*

constants are given in **Table 2**.

*reaction mechanism under a variety of conditions.*

**solution**

**Table 2.**

**129**

Neutral solution (20% acetonitrile pH 7.5)

be given by

The second heterogeneous fibril elongation reaction can be given by k2

A þ Bn ! k2

where Bn and Bn+1 are elongated fibrils with n and n + 1 number of B-form

where a is the initial concentration of glucagon. The overall kinetic equation can

*<sup>f</sup>* <sup>¼</sup> *<sup>ρ</sup>*f g exp 1 ½ �� ð Þ <sup>þ</sup> *<sup>ρ</sup> kt* <sup>1</sup>

where *f* is the fraction of glucagon molecules in the fibril form at time *t* and ρ represents a dimensionless value describing the ratio of k1 (the rate constant for the first nucleation process) to k, namely, ρ = k1/k and k = ak2 (k2 is the rate constant for the second elongation process of the fibrils, and a is the initial peptide concentration) [14]. The best fits of Eq. (6) are shown in **Figure 6B** (solid lines), and the analyzed rate constants are summarized in **Table 2**. The k1 and k2 values were obtained experimentally from the intensity variation of the 13C CP-MAS NMR signals of [1-13C]Gly4 as shown in **Figure 6B** and **Table 2** for glucagon in acidic solution. In comparison, rate constants of fibril formation were obtained for glucagon in neutral solution by plotting the turbidity of the solution against the elapsed

The intensity of the 13C CP-MAS NMR signal of [1-13C]Gly4 was plotted against the elapsed time for glucagon in the presence of bicelles in acidic solution and for glucagon embedded inside lipid bilayers in neutral solution. The obtained rate

Acidic solution (0.015 M acetic acid pH 3.3) 2.6 � <sup>10</sup>�<sup>8</sup> 1.8 � <sup>10</sup>�<sup>2</sup> [36]

Acidic solution (pH 3.3) in the presence of bicelle (DMPC+DHPC) 2.3 � <sup>10</sup>�<sup>6</sup> 2.8 � <sup>10</sup>�<sup>3</sup> [36] Inside a lipid bilayer (DMPC) in neutral solution (pH 7.5) 2.8 � <sup>10</sup>�<sup>7</sup> 5.7 � <sup>10</sup>�<sup>4</sup> [42]

**5. Fibril formation by glucagon in the presence of bicelles in acidic**

*Rate constants for glucagon fibril nucleation (k1) and fibril elongation (k2) for a two-step autocatalytic*

In the presence of bicelles (DMPC-DHPC; 3:1), the N-terminus of glucagon forms an α-helix, the center portion forms a random coil, and the C-terminus forms

ð Þ¼ d*f =*d*t* ð Þ d*f =*d*t* <sup>1</sup> þ ð Þ d*f =*d*t* <sup>2</sup> ¼ k1ð Þþ 1 � *f* k2a 1ð Þ � *f* , (5)

Bnþ1, (3)

ð Þ d*f =*d*t* <sup>2</sup> ¼ k2a*f*ð Þ 1 � *f* , (4)

f g <sup>1</sup> <sup>þ</sup> *<sup>ρ</sup>* exp 1 ½ � ð Þ <sup>þ</sup> *<sup>ρ</sup> kt* (6)

**) k2 (s**�**<sup>1</sup> M**�**<sup>1</sup>**

1.2 � <sup>10</sup>�<sup>24</sup> 4.4 � 10 [42]

**) Ref.**

#### **Figure 5.**

*Schematic of the glucagon fibril formation process in acidic solution. Several monomers (A) aggregate to form weakly coupled micelles (An). Micelles (An) form a fibril nucleus (Bn) through a homogeneous nucleation process with a rate constant k1. Fibril nuclei react with monomers (A) to form elongated fibrils (Bn+1) with a rate constant k2. In this reaction, B acts as a catalyst to change the A form to the B form. Overall, this fibril formation reaction is a two-step autocatalytic reaction mechanism.*

#### **Figure 6.**

*(A) Time course of changes in the 13C CP-MAS and DD-MAS signals of [1-13C]Gly-glucagon during the fibril formation processes [36]. (B) Plot of normalized CP-MAS signal intensity against elapsed time for glucagon fibril formation in acidic solution at pH 3.3 [36]. (C) Plot of normalized turbidity intensities against elapsed time for glucagon fibril formation in neutral solution (20% acetonitrile solution, pH 7.5) [42].*

processes are rate-determining steps, fibril formation is a two-step autocatalytic reaction.

The rate constants of glucagon fibril formation were determined by observing the signal intensities of [1-13C]Gly4 in [1-13C]Gly4 and [3-13C]Ala19-glucagons by 13C CP-MAS NMR spectroscopy with time (**Figure 6A** and **B**). The signal intensities increased after a delay time. The increase in 13C CP-MAS signal intensities corresponds to the increase in fibril components, and thus we obtained the rate constants, k1 and k2, for the two-step autocatalytic reaction mechanism, in which k1 is the rate constant for the fibril nucleation process and k2 is the rate constant for the fibril elongation process [14].

The first reaction step is homogeneous nuclear formation given by

$$\mathbf{A}\_{\rm no} \xrightarrow{\mathbf{k}\_{\rm l}} \mathbf{B}\_{\rm no},\tag{1}$$

where Ano is the micelles formed by no number of A-form glucagon monomers and Bno is the fibrils formed by no number of B-form glucagon fibrils. The kinetic equation for Reaction (1) can be given by

$$(\mathbf{df}/\mathbf{dt})\_1 = \mathbf{k}\_1(\mathbf{1} - f),\tag{2}$$

*Fibril Formation by Glucagon in Solution and in Membrane Environments DOI: http://dx.doi.org/10.5772/intechopen.91681*

where *f* is the fraction of B-glucagon fibrils in the system. The second heterogeneous fibril elongation reaction can be given by k2

$$\mathbf{A} + \mathbf{B}\_{\mathrm{n}} \xrightarrow{\mathbf{k}\_{2}} \mathbf{B}\_{\mathrm{n+1}},\tag{3}$$

where Bn and Bn+1 are elongated fibrils with n and n + 1 number of B-form glucagons. The relevant kinetic equation is given by

$$(\mathbf{df}/\mathbf{dt})\_2 = \mathbf{k}\_2 \mathbf{a} f(\mathbf{1} - f),\tag{4}$$

where a is the initial concentration of glucagon. The overall kinetic equation can be given by

$$(\mathbf{df}/\mathbf{dt}) = (\mathbf{df}/\mathbf{dt})\_1 + (\mathbf{df}/\mathbf{dt})\_2 = \mathbf{k}\_1(\mathbf{1} - f) + \mathbf{k}\_2\mathbf{a}(\mathbf{1} - f),\tag{5}$$

Eq. (5) can be integrated to give

$$f = \frac{\rho \{ \exp \left[ (1 + \rho)kt \right] - 1 \}}{\{ 1 + \rho \exp \left[ (1 + \rho)kt \right] \}} \tag{6}$$

where *f* is the fraction of glucagon molecules in the fibril form at time *t* and ρ represents a dimensionless value describing the ratio of k1 (the rate constant for the first nucleation process) to k, namely, ρ = k1/k and k = ak2 (k2 is the rate constant for the second elongation process of the fibrils, and a is the initial peptide concentration) [14]. The best fits of Eq. (6) are shown in **Figure 6B** (solid lines), and the analyzed rate constants are summarized in **Table 2**. The k1 and k2 values were obtained experimentally from the intensity variation of the 13C CP-MAS NMR signals of [1-13C]Gly4 as shown in **Figure 6B** and **Table 2** for glucagon in acidic solution. In comparison, rate constants of fibril formation were obtained for glucagon in neutral solution by plotting the turbidity of the solution against the elapsed time by using an established protocol [52] (**Figure 6C**).

The intensity of the 13C CP-MAS NMR signal of [1-13C]Gly4 was plotted against the elapsed time for glucagon in the presence of bicelles in acidic solution and for glucagon embedded inside lipid bilayers in neutral solution. The obtained rate constants are given in **Table 2**.


#### **Table 2.**

processes are rate-determining steps, fibril formation is a two-step autocatalytic

*time for glucagon fibril formation in neutral solution (20% acetonitrile solution, pH 7.5) [42].*

*Schematic of the glucagon fibril formation process in acidic solution. Several monomers (A) aggregate to form weakly coupled micelles (An). Micelles (An) form a fibril nucleus (Bn) through a homogeneous nucleation process with a rate constant k1. Fibril nuclei react with monomers (A) to form elongated fibrils (Bn+1) with a rate constant k2. In this reaction, B acts as a catalyst to change the A form to the B form. Overall, this fibril*

*formation reaction is a two-step autocatalytic reaction mechanism.*

The first reaction step is homogeneous nuclear formation given by

Ano ! k1

where Ano is the micelles formed by no number of A-form glucagon monomers and Bno is the fibrils formed by no number of B-form glucagon fibrils. The kinetic

Bno, (1)

ð Þ d*f =*d*t* <sup>1</sup> ¼ k1ð Þ 1 � *f* , (2)

The rate constants of glucagon fibril formation were determined by observing the signal intensities of [1-13C]Gly4 in [1-13C]Gly4 and [3-13C]Ala19-glucagons by 13C CP-MAS NMR spectroscopy with time (**Figure 6A** and **B**). The signal intensities increased after a delay time. The increase in 13C CP-MAS signal intensities corresponds to the increase in fibril components, and thus we obtained the rate constants, k1 and k2, for the two-step autocatalytic reaction mechanism, in which k1 is the rate constant for the fibril nucleation process and k2 is the rate constant for the

*(A) Time course of changes in the 13C CP-MAS and DD-MAS signals of [1-13C]Gly-glucagon during the fibril formation processes [36]. (B) Plot of normalized CP-MAS signal intensity against elapsed time for glucagon fibril formation in acidic solution at pH 3.3 [36]. (C) Plot of normalized turbidity intensities against elapsed*

reaction.

**128**

**Figure 6.**

**Figure 5.**

*Molecular Pharmacology*

fibril elongation process [14].

equation for Reaction (1) can be given by

*Rate constants for glucagon fibril nucleation (k1) and fibril elongation (k2) for a two-step autocatalytic reaction mechanism under a variety of conditions.*

#### **5. Fibril formation by glucagon in the presence of bicelles in acidic solution**

In the presence of bicelles (DMPC-DHPC; 3:1), the N-terminus of glucagon forms an α-helix, the center portion forms a random coil, and the C-terminus forms an α-helix in the monomeric state (**Table 1**). In contrast to glucagon in acetic acid solution, the aggregation of monomers into the fibrils in the presence of bicelles results in the N-terminus maintaining an α-helix structure and the center portion remaining in a random coil structure, whereas the C-terminus changes from an αhelix to a β-sheet structure (**Figure 7** and **Table 1**). There is therefore significant difference in the structural transition between monomer and fibril in the presence and absence of bicelles, since the N-terminus maintains an α-helix structure in the process of fibril formation in the presence of bicelles. This result suggests that the N-terminal portion of a glucagon fibril significantly interacts with the lipid bilayer surface.

The fibril intermediates grow into longer fibrils on the surface of the lipid bilayer and protrude outside the lipid bilayers, as shown schematically in **Figure 7D** (right)

The k1 rate constant for nuclear formation in the presence of lipid bilayers is faster than in the absence of lipid bilayers (**Table 2**) because glucagon monomers associate with the surface of the lipid bilayer, migrate laterally on the surface of the lipid bilayer to form oligomeric intermediates, and then subsequently change to fibril intermediate through a homogeneous nucleation reaction. This two-

dimensional process may be faster than nuclear formation in the three-dimensional

The k2 rate constant for fibril elongation in the presence of lipid bilayers is slower than in the absence of lipid bilayers. As discussed previously, the N-terminal part of the glucagon molecule in a fibril in the presence of a lipid bilayer remains in an α-helix which may be stabilized when the helix interacts with the lipid bilayer. However, after the fibril grows and is released from the lipid bilayer, the N-terminal α-helix becomes more unstable than the N-terminal β-sheet formed in a fibril that protrudes from the lipid bilayer. This unstable fibril can grow to the outside of the lipid bilayer because it potentially acts as a template to form structures identical to the fibril nuclei formed on the surface of the lipid bilayer. The instability of the fibril state outside the lipid bilayer results in a decrease in the k2 value for fibril

These results show that glucagon molecules significantly interact with lipid bilayers during the fibril formation processes. To understand the fibril formation process under physiological condition, a variety of lipid bilayer systems including a

Fibril formation processes were investigated for glucagon embedded inside a lipid bilayer in neutral solutions (i.e., essentially physiological conditions) [42]. Glucagon-induced morphological changes of lipid bilayers and fibril formation process in a glucagon containing lipid bilayer are shown in **Figure 8**. The monomers may aggregate with each other to form oligomers (**Figure 8B**) likely driven by the amphipathic nature of the N-terminal and C-terminal α-helices. These oligomers then change into ellipsoidal fibril intermediates (nuclei) (**Figure 8C**) through a homogeneous reaction, as observed by TEM (**Figure 8C**, bottom). At this stage, the glucagon intermediates strongly interact with the lipid bilayer to form discoidal lipid bilayer particles. Subsequently, this ellipsoidal fibril intermediate (nucleus) interacts with monomeric glucagon inside the lipid bilayer to allow elongation of the fibril by changing from an α-helix to a β-sheet through a heterogeneous elongation process (**Figure 8D**). After a long time standing, fibril networks are formed, and lipid molecules are compartmentalized (**Figure 8E**), resulting in increased lipid molecule mobility and the induction of a gel-like state throughout the sample, now

The kinetic of fibrillation was thus analyzed using a two-step autocatalytic mechanism as summarized in **Table 2**. The k1 rate constant for nuclear formation in neutral solution is much slower than that in a DMPC bilayer in neutral conditions (**Table 2**). In a DMPC/glucagon bilayer, glucagon molecules are condensed inside the DMPC bilayer, and thus the nucleation rate of glucagon in a DMPC/glucagon bilayer is much faster than that in neutral solution. The k1 rate constant for nuclear formation in a lipid bilayer in a neutral solution is slower than in a lipid bilayer in an

**6. Fibrillation mechanism of glucagon inside the lipid bilayer**

and in the TEM picture (**Figure 7D**; right bottom).

*DOI: http://dx.doi.org/10.5772/intechopen.91681*

*Fibril Formation by Glucagon in Solution and in Membrane Environments*

elongation as compared to the absence of a lipid bilayer.

lipid bilayer in neutral solution were investigated.

**in the neutral condition**

in an amyloidogenic gel state [53, 54].

**131**

solution state.

The above findings provide insights into the mechanism of fibril formation in the presence and absence of lipid bilayers, as shown in **Figure 7**. In the absence of lipid bilayers, monomers may aggregate with each other to form oligomeric intermediates (similar to micelles) through a homogeneous reaction (**Figure 7B**; left), likely driven by the amphipathic natures of the N-terminal and C-terminal α-helices. These oligomeric intermediates then change into spherical fibril intermediates (**Figure 7C**; left) as observed by TEM (**Figure 2a** and **b**). Subsequently, these spherical fibril intermediates may form fibril nuclei and interact with monomeric glucagon to allow elongation of the fibril by changing from an α-helix to a β-sheet through a heterogeneous elongation process (**Figure 7D**; left).

In the presence of lipid bilayers, monomers form a structure similar to that in the absence of lipid bilayers. The monomers likely associate quickly with the lipid bilayer and subsequently associate with other monomers to form weakly coupled oligomeric intermediates (**Figure 7B**; right). These oligomeric intermediates may change their structure to form fibril intermediates on the surface of the lipid bilayer and are observed as ellipsoid-shaped fibril intermediates (**Figure 7C**; right and TEM picture) on the surface of the lipid bilayer. The elliptical shape is due to the Nterminal region retaining an α-helix structure even in the fibril intermediates.

#### **Figure 7.**

*Schematic diagram of the fibrillation processes of glucagon in acetic acid solution (left) and acidic solution in the presence of bicelles (center). (A) Monomeric form. (B) Weakly coupled oligomer. (C) Fibril intermediate. (D) Elongated fibril. TEM pictures of glucagon fibril intermediates with ellipsoidal shapes and of disk-type bicelles are seen in the top right photo. An elongated fibril is seen attached by its end to a bicelle in the bottom right photo (ref. [36]).*

*Fibril Formation by Glucagon in Solution and in Membrane Environments DOI: http://dx.doi.org/10.5772/intechopen.91681*

The fibril intermediates grow into longer fibrils on the surface of the lipid bilayer and protrude outside the lipid bilayers, as shown schematically in **Figure 7D** (right) and in the TEM picture (**Figure 7D**; right bottom).

The k1 rate constant for nuclear formation in the presence of lipid bilayers is faster than in the absence of lipid bilayers (**Table 2**) because glucagon monomers associate with the surface of the lipid bilayer, migrate laterally on the surface of the lipid bilayer to form oligomeric intermediates, and then subsequently change to fibril intermediate through a homogeneous nucleation reaction. This twodimensional process may be faster than nuclear formation in the three-dimensional solution state.

The k2 rate constant for fibril elongation in the presence of lipid bilayers is slower than in the absence of lipid bilayers. As discussed previously, the N-terminal part of the glucagon molecule in a fibril in the presence of a lipid bilayer remains in an α-helix which may be stabilized when the helix interacts with the lipid bilayer. However, after the fibril grows and is released from the lipid bilayer, the N-terminal α-helix becomes more unstable than the N-terminal β-sheet formed in a fibril that protrudes from the lipid bilayer. This unstable fibril can grow to the outside of the lipid bilayer because it potentially acts as a template to form structures identical to the fibril nuclei formed on the surface of the lipid bilayer. The instability of the fibril state outside the lipid bilayer results in a decrease in the k2 value for fibril elongation as compared to the absence of a lipid bilayer.

These results show that glucagon molecules significantly interact with lipid bilayers during the fibril formation processes. To understand the fibril formation process under physiological condition, a variety of lipid bilayer systems including a lipid bilayer in neutral solution were investigated.

#### **6. Fibrillation mechanism of glucagon inside the lipid bilayer in the neutral condition**

Fibril formation processes were investigated for glucagon embedded inside a lipid bilayer in neutral solutions (i.e., essentially physiological conditions) [42]. Glucagon-induced morphological changes of lipid bilayers and fibril formation process in a glucagon containing lipid bilayer are shown in **Figure 8**. The monomers may aggregate with each other to form oligomers (**Figure 8B**) likely driven by the amphipathic nature of the N-terminal and C-terminal α-helices. These oligomers then change into ellipsoidal fibril intermediates (nuclei) (**Figure 8C**) through a homogeneous reaction, as observed by TEM (**Figure 8C**, bottom). At this stage, the glucagon intermediates strongly interact with the lipid bilayer to form discoidal lipid bilayer particles. Subsequently, this ellipsoidal fibril intermediate (nucleus) interacts with monomeric glucagon inside the lipid bilayer to allow elongation of the fibril by changing from an α-helix to a β-sheet through a heterogeneous elongation process (**Figure 8D**). After a long time standing, fibril networks are formed, and lipid molecules are compartmentalized (**Figure 8E**), resulting in increased lipid molecule mobility and the induction of a gel-like state throughout the sample, now in an amyloidogenic gel state [53, 54].

The kinetic of fibrillation was thus analyzed using a two-step autocatalytic mechanism as summarized in **Table 2**. The k1 rate constant for nuclear formation in neutral solution is much slower than that in a DMPC bilayer in neutral conditions (**Table 2**). In a DMPC/glucagon bilayer, glucagon molecules are condensed inside the DMPC bilayer, and thus the nucleation rate of glucagon in a DMPC/glucagon bilayer is much faster than that in neutral solution. The k1 rate constant for nuclear formation in a lipid bilayer in a neutral solution is slower than in a lipid bilayer in an

an α-helix in the monomeric state (**Table 1**). In contrast to glucagon in acetic acid solution, the aggregation of monomers into the fibrils in the presence of bicelles results in the N-terminus maintaining an α-helix structure and the center portion remaining in a random coil structure, whereas the C-terminus changes from an αhelix to a β-sheet structure (**Figure 7** and **Table 1**). There is therefore significant difference in the structural transition between monomer and fibril in the presence and absence of bicelles, since the N-terminus maintains an α-helix structure in the process of fibril formation in the presence of bicelles. This result suggests that the N-terminal portion of a glucagon fibril significantly interacts with the lipid bilayer

The above findings provide insights into the mechanism of fibril formation in the presence and absence of lipid bilayers, as shown in **Figure 7**. In the absence of lipid bilayers, monomers may aggregate with each other to form oligomeric intermediates (similar to micelles) through a homogeneous reaction (**Figure 7B**; left), likely driven by the amphipathic natures of the N-terminal and C-terminal α-helices. These oligomeric intermediates then change into spherical fibril intermediates (**Figure 7C**; left) as observed by TEM (**Figure 2a** and **b**). Subsequently, these spherical fibril intermediates may form fibril nuclei and interact with monomeric glucagon to allow elongation of the fibril by changing from an α-helix to a β-sheet

In the presence of lipid bilayers, monomers form a structure similar to that in the absence of lipid bilayers. The monomers likely associate quickly with the lipid bilayer and subsequently associate with other monomers to form weakly coupled oligomeric intermediates (**Figure 7B**; right). These oligomeric intermediates may change their structure to form fibril intermediates on the surface of the lipid bilayer and are observed as ellipsoid-shaped fibril intermediates (**Figure 7C**; right and TEM picture) on the surface of the lipid bilayer. The elliptical shape is due to the Nterminal region retaining an α-helix structure even in the fibril intermediates.

*Schematic diagram of the fibrillation processes of glucagon in acetic acid solution (left) and acidic solution in the presence of bicelles (center). (A) Monomeric form. (B) Weakly coupled oligomer. (C) Fibril intermediate. (D) Elongated fibril. TEM pictures of glucagon fibril intermediates with ellipsoidal shapes and of disk-type bicelles are seen in the top right photo. An elongated fibril is seen attached by its end to a bicelle in the bottom*

through a heterogeneous elongation process (**Figure 7D**; left).

surface.

*Molecular Pharmacology*

**Figure 7.**

**130**

*right photo (ref. [36]).*

fibril intermediates that glow into elongated fibrils. Glucagon intermediates are formed on the surface of the lipid bilayer in the presence of bicelle. These fibrils are cytotoxic through their activation of apoptotic processes, similar to β-amyloid and salmon calcitonin. Kinetic analyses of glucagon fibril formation are performed using a two-step autocatalytic reaction mechanism comprising fibril nucleation and elongation processes. It is revealed that glucagon forms fibril intermediates and grows into elongated fibrils inside the lipid bilayer under neutral conditions. These properties of glucagon fibril formation indicate that the interaction of the glucagon fibril with lipid bilayers is strongly dependent on the process of fibril formation. A neutral system is thus considered to reflect the fibril formation process in biological cells and provides insight into the mechanism underlying cytotoxicity of glucagon

*Fibril Formation by Glucagon in Solution and in Membrane Environments*

*DOI: http://dx.doi.org/10.5772/intechopen.91681*

This work was supported by Grants-in-Aid for Scientific Research in an Innovative Area (JP16H00756 to AN) and by Grants-in-Aid for Scientific Research (C) (JP15K06963 to AN) from the Ministry of Culture, Sports, Science and Technology of Japan. The author wishes to thank Izuru Kawamura and Yoshiteru Makino for the discussion on this study and Izumi Yamane, Ayano Momose, Hideki Fujita, Eri Yoshimoto, Akie Kikuchi-Kinoshita, and Kazumi Haya for their experimental

Graduate School of Engineering, Yokohama National University, Yokohama, Japan

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

fibrils.

assistances.

**Acknowledgements**

**Conflict of interest**

**Author details**

Akira Naito

**133**

The authors declare no conflict of interest.

\*Address all correspondence to: naito@ynu.ac.jp

provided the original work is properly cited.

#### **Figure 8.**

*Schematic diagrams of the morphological states of glucagon-DMPC bilayer systems (DMPC/glucagon; 50/1) at temperatures above and below the phase transition temperature (Tc = 23°C for DMPC). (A) Glucagon-DMPC bilayer state 1 day after sample preparation. (B) Glucagon-DMPC bilayer state 2 days after sample preparation. (C) Glucagon-DMPC bilayer state 4 days after sample preparation with the corresponding TEM picture shown below. (D) Glucagon-DMPC bilayer state 7 days after sample preparation with the corresponding TEM picture shown below. (E) Glucagon-DMPC bilayer system 10 days after sample preparation. The corresponding TEM picture is shown below (ref. [42]).*

acidic solution. In an acidic solution, glucagon locates on the surface of lipid bilayer and then migrates laterally on the surface of the lipid bilayer to form oligomers and subsequently changes to fibril nuclei through homogeneous nucleation reaction. In contrast, glucagon is deeply embedded inside a lipid bilayer in neutral solution and thus migrates more slowly inside the lipid bilayer, as reflected in the lower k1 value.

The k2 rate constant for the elongation of glucagon fibrils in a DMPC/glucagon bilayer is much slower than that in neutral solution. Glucagon molecules in a DMPC/glucagon bilayer interact strongly with the DMPC bilayer. It therefore takes a long time to disrupt the interaction with the lipid bilayer and form interaction with glucagon fibrils. The k2 rate constant for fibril elongation in the presence of lipid bilayers under neutral condition is significantly slower than that in lipid bilayers under acidic conditions. Under neutral conditions, glucagon is embedded deep inside the lipid bilayer, and hence it takes longer to release monomeric glucagon from the lipid bilayer. Therefore, the k2 values for fibril elongation decrease as compared to the case in a lipid bilayer in acidic conditions.

#### **7. Conclusions**

It is demonstrated that glucagon forms fibrils in acidic solution and in the presence of lipid bilayer (bicelle) in acidic solution. Glucagon aggregates to form *Fibril Formation by Glucagon in Solution and in Membrane Environments DOI: http://dx.doi.org/10.5772/intechopen.91681*

fibril intermediates that glow into elongated fibrils. Glucagon intermediates are formed on the surface of the lipid bilayer in the presence of bicelle. These fibrils are cytotoxic through their activation of apoptotic processes, similar to β-amyloid and salmon calcitonin. Kinetic analyses of glucagon fibril formation are performed using a two-step autocatalytic reaction mechanism comprising fibril nucleation and elongation processes. It is revealed that glucagon forms fibril intermediates and grows into elongated fibrils inside the lipid bilayer under neutral conditions. These properties of glucagon fibril formation indicate that the interaction of the glucagon fibril with lipid bilayers is strongly dependent on the process of fibril formation. A neutral system is thus considered to reflect the fibril formation process in biological cells and provides insight into the mechanism underlying cytotoxicity of glucagon fibrils.

#### **Acknowledgements**

This work was supported by Grants-in-Aid for Scientific Research in an Innovative Area (JP16H00756 to AN) and by Grants-in-Aid for Scientific Research (C) (JP15K06963 to AN) from the Ministry of Culture, Sports, Science and Technology of Japan. The author wishes to thank Izuru Kawamura and Yoshiteru Makino for the discussion on this study and Izumi Yamane, Ayano Momose, Hideki Fujita, Eri Yoshimoto, Akie Kikuchi-Kinoshita, and Kazumi Haya for their experimental assistances.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

acidic solution. In an acidic solution, glucagon locates on the surface of lipid bilayer and then migrates laterally on the surface of the lipid bilayer to form oligomers and subsequently changes to fibril nuclei through homogeneous nucleation reaction. In contrast, glucagon is deeply embedded inside a lipid bilayer in neutral solution and thus migrates more slowly inside the lipid bilayer, as reflected in the lower k1 value. The k2 rate constant for the elongation of glucagon fibrils in a DMPC/glucagon

*Schematic diagrams of the morphological states of glucagon-DMPC bilayer systems (DMPC/glucagon; 50/1) at temperatures above and below the phase transition temperature (Tc = 23°C for DMPC). (A) Glucagon-DMPC bilayer state 1 day after sample preparation. (B) Glucagon-DMPC bilayer state 2 days after sample preparation. (C) Glucagon-DMPC bilayer state 4 days after sample preparation with the corresponding TEM*

*picture shown below. (D) Glucagon-DMPC bilayer state 7 days after sample preparation with the corresponding TEM picture shown below. (E) Glucagon-DMPC bilayer system 10 days after sample*

*preparation. The corresponding TEM picture is shown below (ref. [42]).*

bilayer is much slower than that in neutral solution. Glucagon molecules in a DMPC/glucagon bilayer interact strongly with the DMPC bilayer. It therefore takes a long time to disrupt the interaction with the lipid bilayer and form interaction with glucagon fibrils. The k2 rate constant for fibril elongation in the presence of lipid bilayers under neutral condition is significantly slower than that in lipid bilayers under acidic conditions. Under neutral conditions, glucagon is embedded deep inside the lipid bilayer, and hence it takes longer to release monomeric glucagon from the lipid bilayer. Therefore, the k2 values for fibril elongation decrease as

It is demonstrated that glucagon forms fibrils in acidic solution and in the presence of lipid bilayer (bicelle) in acidic solution. Glucagon aggregates to form

compared to the case in a lipid bilayer in acidic conditions.

**7. Conclusions**

**132**

**Figure 8.**

*Molecular Pharmacology*

Akira Naito Graduate School of Engineering, Yokohama National University, Yokohama, Japan

\*Address all correspondence to: naito@ynu.ac.jp

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Itoh Y, Endo H, et al. Role of aromatic residues in amyloid fibril formation of human calcitonin by solid-state 13C NMR and molecular dynamics simulation. Physical Chemistry Chemical Physics. 2013;**15**:8890-8901.

*DOI: http://dx.doi.org/10.5772/intechopen.91681*

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structure in Alzheimer's β-amyloid fibrils. Biochemistry. 2006;**45**:498-512.

[22] Xiao Y, Ma B, McElheny D,

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[26] Boesch C, Bundi A, Oppliger M,

resonance studies of the molecular conformation of monomeric glucagon in aqueous solution. European Journal of Biochemistry. 1978;**91**:209-214. DOI: 10.111/j.1432-1033.1978.tb2095.x

[27] Braun W, Winder G, Lee KH, Wüthrich K. Conformation of glucagon

in a lipid-water interphase by <sup>1</sup>

magnetic resonance. Journal of Molecular Biology. 1983;**169**:921-948. DOI: 10.1016/50022-2836(83)80143-0

[28] Onoue S, Iwasa S, Kojima T, Katoh F, Debari K, Koh K, et al.

H nuclear-magnetic-

H nuclear

10.1021/jacs.6b05129

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Wüthrich K. <sup>1</sup>

Parthasarathy S, Long F, Hoshi M, et al. Aβ(1-42) fibril structure illuminates self-recognition and replication of amyloid in Alzheimer's disease. Nature Structural & Molecular Biology. 2015; **22**:499-505. DOI: 10.1038/nsmb.2991

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Nakakoshi M, Sato M, et al. Characterization of the spherical intermediates and fibril formation of hCT in HEPES solution using solid-state 13C-NMR and transmission electron microscopy. Physical Chemistry

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10.1021/bi027378p

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[16] Itoh-Watanabe H, Kamihira-Ishijima M, Kawamura I, Kondoh M,

Chemical Physics. 2013;**15**:16956-16964.

[17] Kamgar-Parsi K, Hong L, Naito A, Brooks CL III, Ramamoorthy A. Growth-incompetent monomers of human calcitonin lead to a noncanonical direct relationship between peptide concentration and aggregation lag time. The Journal of Biological Chemistry. 2017;**292**:14963-14976. DOI: 10.1074/

[18] Gorman PM, Chakrabartty A. Alzheimer β-amyloid peptides: Structures of amyloid fibrils and alternate aggregation products. Peptide

Science. 2001;**60**:381-394. DOI: 10.1002/1097-0282(2001)60:5< 381::AID-BIP/0173>3.0.CO;2-U

[19] Tycko R. Insights into the amyloid folding problem from solid-state NMR. Biochemistry. 2003;**42**:3151-3159. DOI:

[20] Tycko R. Application of solid state NMR to the structural characterization of amyloid fibrils: Methods and results.

[21] Petkova AT, Yan W-M, Tycko R. Experimental constraints on quaternary

Progress in Nuclear Magnetic Resonance Spectroscopy. 2003;**42**: 53-68. DOI: 10.1016/S0079-6565(03)

[9] Cooper GJS, Willis AC, Clark A, Turner RC, Sim RB, Reid KBM. Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proceedings of the National Academy of Sciences of the United States of America. 1987;**84**: 8628-8632. DOI: 10.1073/pnas.95.23. 13363

[10] Vines G. Alzheimer's disease – From cause to cure? Trends in Biotechnology. 1993;**11**:49-55. DOI: 10.1016/0167-7799 (93)90122-p

[11] Scherzinger E, Lurz R, Turmaine M, Mangiarini L, Hollenbach B, Hasenbank R, et al. Huntingtonencoded polyglutamine expansions from amyloid-like protein aggregates in vitro and in vivo. Cell. 1997;**90**: 549-558. DOI: 10.1016/S0092-8674(00) 80514-0

[12] Sipe JD. Amyloidosis. Annual Review of Biochemistry. 1992;**61**: 947-975. DOI: 10.1146/annurev. bi.61.070192.004503

[13] Sipe JD, Cohen AS. History of the amyloid fibril. Journal of Structural Biology. 2000;**130**:88-98. DOI: 10.1004/ jsbi.2000.4221

[14] Kamihira M, Naito A, Tuzi S, Nosaka AY, Saitô H. Conformational transition and fibrillation mechanism of human calcitonin as studied by highresolution solid-state 13C NMR. Protein Science. 2000;**9**:867-877. DOI: 10.1110/ ps.9.5.867

[15] Itoh-Watanabe H, Kamihira-Ishijima M, Javkhlantugs N, Inoue R, *Fibril Formation by Glucagon in Solution and in Membrane Environments DOI: http://dx.doi.org/10.5772/intechopen.91681*

Itoh Y, Endo H, et al. Role of aromatic residues in amyloid fibril formation of human calcitonin by solid-state 13C NMR and molecular dynamics simulation. Physical Chemistry Chemical Physics. 2013;**15**:8890-8901. DOI: 10.1039/c3cp44544e

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Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques. Journal of Chromatography. A. 2006;**1109**:167-173. DOI: 10.1016/j. chroma.2005.11.130

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Structural transition of glucagon in the concentrated solution observed by electrophoretic and spectroscopic techniques. Journal of Chromatography. A. 2006;**1109**:167-173. DOI: 10.1016/j.

[35] Matsuzaki K. How do membranes initiate Alzheimer's disease? Formation of toxic amyloid fibrils by the amyloid β-protein on ganglioside clusters. Accounts of Chemical Research. 2014; **47**:2397-2404. DOI: 10.1021/ar50012721

[36] Yamane I, Momose A, Fujita H, Yoshimoto E, Kikuchi-Kinoshita A, Kawamura I, et al. Fibrillation

2019.01.008

(81)90165-6

mechanism of glucagon in the presence of phospholipid bilayers as revealed by 13C solid-state NMR spectroscopy. Chemistry and Physics of Lipids. 2019; **219**:36-44. DOI: 101016/j.chemphyslip.

[37] Epand RM, Jones AJS, Schreier S. Interaction of glucagon with dimyristoyl glycerophosphocholine. Biochimica et Biophysica Acta. 1977;**491**:296-304. DOI: 10.1016/0005-2795(77)90065-4

[38] Epand RM. Studies on the effect of the lipid phase transition on the

interaction of glucagon with dimyristoyl glycerophosphocholine. Biochimica et Biophysica Acta. 1978;**514**:185-197. DOI:

[39] Epand RM, Epand RF, Stewart TP, Hu SW. The condensing effect of glucagon on phospholipid bilayers. Biochimica et Biophysica Acta. 1981; **649**:608-615. DOI: 10.1016/0005-2736

[40] Naito A, Nagao T, Norisada K,

[41] Toraya S, Nagao T, Norisada K, Tuzi S, Saitô H, Izumi S. Morphological behavior of lipid bilayers induced by melittin near the phase transition temperature. Biophysical Journal. 2005;

Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state 31P and 13C NMR spectroscopy. Biophysical Journal. 2000;**78**:2405-2417. DOI: 10.1016/

Mizuno T, Tuzi S, Saitô H.

S0006-3495(00)76784-1

**89**:3214-3222. DOI: 10.1529/

biophysj.105.059311

10.1016/0005-2736(78)90290-0

[29] Pedersen JS, Dikov D, Flink JI, Hijuler HA, Christiansen G, Otzen DE.

The changing face of glucagon fibrillation: Structural polymorphism and conformational imprinting. Journal of Molecular Biology. 2006;**355**:501-523.

DOI: 10.1016/j.jmb.2006.09.100

4591-4601. DOI: 10.1111/j.1742-

4658.2010.07888.x

10.1038/srep09005

bbamem.2007.03.025

[33] Brender JR, Salamekh S,

DOI: 10.1021/ar200189b

10.1039/c3cs60431d

**136**

Ramamoorthy A. Membrane disruption and early events in the aggregation of the diabetes related peptide IAPP from a molecular perspective. Accounts of Chemical Research. 2012;**45**:454-462.

[34] Kotler SA, Walsh P, Brender JR, Ramamoorthy A. Differences between amyloid-β aggregation in solution and on the membrane: Insights into

elucidation of the mechanistic details of Alzheimer's disease. Chemical Society Reviews. 2014;**43**:6692-6700. DOI:

[30] Pedersen JS, Andersen CB, Otzen DE. Amyloid structure–one but not the same: The many levels of fibrillar polymorphism. FEBS Journal. 2010;**277**:

[31] Košmrlj A, Cordsen P, Kyrsting A, Otzen DE, Oddershede LB, Jensen MH. A monomer-trimer model supports intermittent glucagon fibril growth. Scientific Reports. 2015;**5**:9005. DOI:

[32] Naito A, Kawamura I. Solid-state NMR as a method to reveal structure and membrane-interactions of amyloidogenic protein and peptide. Biochimica et Biophysica Acta. 1768; **2007**:1900-1912. DOI: 10.1016/j.

chroma.2005.11.130

*Molecular Pharmacology*

[43] Andersen CB, Yagi H, Manno M, Martorana V, Ban T, Christiansen G, et al. Branching in amyloid fibril growth. Biophysical Journal. 2000;**96**:1529-1536. DOI: 10.1016/j.bpj.2008.11.024

[44] Andersen CB, Otzen D, Christiansen G, Rischel C. Glucagon amyloid-like fibril morphology is selected via morphology-dependent growth inhibition. Biochemistry. 2007; **46**:7314-7324. DOI: 10.1021.bi6025374

[45] Andersen CB, Hicks MR, Vetri V, Vandahl B, Rahbek-Nielsen H, Thogersen H, et al. Glucagon fibril polymorphism reflects differences in protofilament backbone structure. Journal of Molecular Biology. 2010;**397**: 932-946. DOI: 10.1016/j. jmb.2010.02.012

[46] Jong KLD, Incledon B, Yip CM, DeFelippis MR. Amyloid fibrils of glucagon characterized by highresolution atomic force microscopy. Biophysical Journal. 2006;**91**:1905-1914. DOI: 10.1529/biophysj.105.077438

[47] Saitô H. Conformation-dependent 13C chemical shifts: A new means of conformation characterization as obtained by high-resolution solid-state 13C NMR. Magnetic Resonance in Chemistry. 1986;**24**:835-852. DOI: 10.1002/mrc.1260241.002

[48] Saitô H, Ando I. High-resolution solid-state NMR studies of synthetic and biological macromolecules. Annual Reports on NMR Spectroscopy. 1989;**21**: 209-290. DOI: 10.1016/50066-4103(08) 60124-6

[49] Saitô H, Ando I, Ramamoorthy A. Chemical shift tensor – The heart of NMR: Insight into biological aspect proteins. Progress in Nuclear Magnetic Resonance Spectroscopy. 2010;**57**: 181-228. DOI: 10.1016/j.pnmrs.2010. 04.005

[50] Onoue S, Ohshima K, Endo K, Yajima T, Kashimoto K. PACAP protects neuronal PC12 cells from the cytotoxicity of human prion protein fragment 106-125. FEBS Letters. 2002; **522**:65-72. DOI: 10.1016/50014-5793 (02)02886-7

[51] Onoue S, Endo K, Ohshima K, Yajima T, Kashimoto K. The neuropeptide PACAP attenuates bamyloid (1-42)-induced toxicity in PC12 cells. Peptides. 2002;**23**:1471-1478. DOI: 10.1016/S0196-9781(02)00085-2

[52] Kamihira-Ishijima M, Nakazawa H, Kira A, Naito A, Nakayama T. Inhibitory mechanism of pancreatic amyloid fibril formation: Formation of the complex between tea catechins and the fragment of residue 22-27. Biochemistry. 2012;**51**:10167-10174. DOI: 10.101021/bi3012274

[53] Azakami H, Mukai A, Kato A. Role of amyloid cross β-structure in the formation of soluble aggregate and gel in heat-induced ovalbumin. Journal of Agricultural and Food Chemistry. 2005; **53**:1254-1257. DOI: 10.1021/jf049325f

[54] Corrigan AM, Donald AM. Particle tracking microrheology of gel-forming amyloid fibril networks. European Physical Journal E: Soft Matter and Biological Physics. 2009;**28**:457-462. DOI: 10.1140/epje/12008-10439-7

**139**

**Chapter 8**

Cancer

**Abstract**

approach to treat cancer.

nanomedicine

**1. Introduction**

Integrating Nanotherapeutic

Approaches for Management of

*Mohd Muazzam Khan, Md. Faheem Haider and Juber Akhtar*

Cancer is a leading cause of mortality worldwide, accounting for 8.8 million deaths in 2015. The landscape of cancer therapeutics is rapidly advancing with development of new and sophisticated approaches to diagnostic testing. Treatment plan for early diagnosed patients include radiation therapy, tumor ablation, surgery, immunotherapy and chemotherapy. However the treatment can only be initiated when the cancer has been diagnosed thoroughly. Theranostics is a term that combines diagnostics with therapeutics. It embraces multiple techniques to arrive at comprehensive diagnosis, molecular images and an individualized treatment regimen. Recently, there is an effort to tangle the emerging approach with nanotechnologies, in an attempt to develop theranostic nanoplatforms and methodologies. Theranostic approach to management of cancer offers numerous advantages. They are designed to monitor cancer treatment in real time. A wide variety of theranostic nanoplatforms that are based on diverse nanostructures like magnetic nanoparticles, carbon nanotubes, gold nanomaterials, polymeric nanoparticles and silica nanoparticles showed great potential as cancer theranostics. Nano therapeutic platforms have been successful in integrating image guidance with targeted

**Keywords:** nanomedicine, theranostics, targeted delivery, cancer, functionalized

Cancer has a major impact on society across the world. Estimated number of new cases of cancer, current cases of cancer, deaths, survival rate, mortality and in depth information, symptoms of cancer, its early detection, prevention and treatment all are provided by American Cancer Society. Nearly 13% of cancer diagnosed in 2017 was in the young at age of 20. The new review statistics shows 28 types of rare cancer which talks about mortality rate, survival, diagnosis and also provides an idea about symptoms and risk factors related to different types of cancer [1]. Therapeutic approaches such as development of nanoemulsions, liposomes, microspheres and nanoparticles have facilitated in fighting cancer. Among these,

Platforms to Image Guided

*Asad Ali, Zeeshan Ahmad, Usama Ahmad,* 

#### **Chapter 8**

## Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer

*Asad Ali, Zeeshan Ahmad, Usama Ahmad, Mohd Muazzam Khan, Md. Faheem Haider and Juber Akhtar*

#### **Abstract**

Cancer is a leading cause of mortality worldwide, accounting for 8.8 million deaths in 2015. The landscape of cancer therapeutics is rapidly advancing with development of new and sophisticated approaches to diagnostic testing. Treatment plan for early diagnosed patients include radiation therapy, tumor ablation, surgery, immunotherapy and chemotherapy. However the treatment can only be initiated when the cancer has been diagnosed thoroughly. Theranostics is a term that combines diagnostics with therapeutics. It embraces multiple techniques to arrive at comprehensive diagnosis, molecular images and an individualized treatment regimen. Recently, there is an effort to tangle the emerging approach with nanotechnologies, in an attempt to develop theranostic nanoplatforms and methodologies. Theranostic approach to management of cancer offers numerous advantages. They are designed to monitor cancer treatment in real time. A wide variety of theranostic nanoplatforms that are based on diverse nanostructures like magnetic nanoparticles, carbon nanotubes, gold nanomaterials, polymeric nanoparticles and silica nanoparticles showed great potential as cancer theranostics. Nano therapeutic platforms have been successful in integrating image guidance with targeted approach to treat cancer.

**Keywords:** nanomedicine, theranostics, targeted delivery, cancer, functionalized nanomedicine

#### **1. Introduction**

Cancer has a major impact on society across the world. Estimated number of new cases of cancer, current cases of cancer, deaths, survival rate, mortality and in depth information, symptoms of cancer, its early detection, prevention and treatment all are provided by American Cancer Society. Nearly 13% of cancer diagnosed in 2017 was in the young at age of 20. The new review statistics shows 28 types of rare cancer which talks about mortality rate, survival, diagnosis and also provides an idea about symptoms and risk factors related to different types of cancer [1].

Therapeutic approaches such as development of nanoemulsions, liposomes, microspheres and nanoparticles have facilitated in fighting cancer. Among these, the simplest platforms are the nanoemulsion having size range of 100 to 500 nm which are kinetically stabilized having high content of oil and low amount of surfactant [2]. Solubility of poorly soluble drugs [3, 4] and its bioavailability can be increased by converting the drug into forms like capsule and gels [5] or can be used in their original form. The method used for the fabrication of nanoemulsion are high energy methods (microfluidization and sonication) and low energy emulsification method [6–8].

Theranostics is a term originally coined to define an approach that combine's diagnostics with therapeutics [9]. It embraces multiple techniques to arrive at comprehensive diagnosis, molecular images and an individualized treatment regimen [10, 11]. Recently, there is an effort to tangle the emerging approach with nanotechnologies, in an attempt to develop theranostic nanoplatforms and methodologies [12]. Given that cancer is a highly heterogeneous and adaptable disease, diverse types of treatment options need to be chosen depending on patient's characteristics and disease progression.

#### **2. Theranostics**

Drugs or methods that are used for accompanying diagnosis and cure [13] are referred to as Theranostics. One of the achievements of nanotechnology is the fabrication of theranostic nanomedicine for the preparation of these types of drugs. The term defines "a nanotherapeutic system which can deliver targeted therapy and diagnose". This aspect provides help when fabricating nano based image contrasting agent and also in image guided therapeutics [14].

Rapid drug development, advanced disease management, reduced associated risk and cost are assumed to be the result of mutual techniques. Such type of investigation which involves quick diagnosis and treatment are very helpful in disease which are a major cause of morbidity and/or mortality and cancer is one of the disease and coincidently the initial research in theranostic is dedicated to oncology. Sound knowledge, core understanding of detection and therapy mechanism are required for the formation of theranostic agents. In order to fabricate theranostic agents one should have understanding of diagnostic strategies, molecular mechanism adverse effect and toxicity of material and techniques for nanoparticles preparation for therapy and diagnostic purpose.

Research in theranostics has rapidly improved in the past decade resulting into preparation of different contrast media and active ingredient with different method of preparation. Preparation of dual purpose nanoparticle system is the main aim of theranostics. Therefore it is important to put attention on all factors that influences the process, right from the preparation of nanoemulsion/nanoparticle till the removal of metabolites of the active molecules and other materials used. The factors can be the compatibility between chemicals, the condition in which the formulation is prepared, modification in formulations because of selected route of administration, the toxicity, metabolites of active ingredient, its biocompatibility and biodegradability and evaluation of pharmacodynamic and pharmacokinetic parameter and eventually the disadvantage and benefits of the process.

The basis of diagnosis in theranostics depends upon using different contrast agent during imaging. MRI is the most studied and used technique among all different imaging mechanism and a lot has been spent on research related to magnetic particles used as contrasting agent. Metals like gold, silver, iron oxide have been studied with the object of finding suitable particle for imaging with least toxicological effect. Diseased tissue and healthy tissue are differentiated in MRI by the use of these particles.

**141**

*Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer*

siRNA MRI plus RNA

**Drug used Applications Size Zeta** 

delivery

and PTT

Targeting, MRI and therapy

Drug carrier, X-ray/CT imaging, Photodynamic therapy

Diagnosis, DNA and drug delivery

and sensing

*Abbreviations: siRNA: short interfering ribonucleic acid, CNTs: carbon nanotubes, QDs: quantum dots, DOX: Doxorubicin, HPPH: 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-alpha, MTX: Methotrexate, PTX:* 

**potential**

— — [15]

30 nm −5 mV [19–21]

— — [26]

−3.54 mV [16–18]

−39 mV [22, 23]

— [24, 25]

45.97 nm and 6.3 nm

30 nm and 126 nm

20 nm and 120.6 nm **References**

As stated one of the brutal disease is cancer and hence theranostic research has put an eye on this area. Day by day the research is going on in positive direction and much useful research has already been carried out. In order to understand the concept of diagnosis of cancer and therapy related to it the use of nanoparticle agents is in progression [27, 28]. One such example of theranostic agent is manganese oxide nanoparticle carrying drugs and contrast agents [29] and silica nanoparticle with magnetic and fluorescent tags [30]. In the past few years, combination of metal nanoparticle or shells [31–33] with magnetic components has yielded different theranostic agents for biomedical applications which are widely used. Some

Theranostic nanosystems comprise of platforms/nanocarriers that are used as imaging as well as therapeutic agents via a single entity. Nanotheranostic devices can be made by many types of inorganic and organic nanomaterials. Preclinical implementations make use of nanotheranostic system because they might provide a way or method of understanding many crucial aspects of drug delivery and how

At present radiotherapy, surgery and chemotherapy are possible treatments for cancer patients. The purpose of the theranostic is to use these therapeutic strategies and reduce the risk associated with chemotherapy and radiotherapy and in addition to it avoid complications related to surgery and trauma. In addition, with the help

these systems can assist in knowing the power of personalized medicines.

examples of theranostic agents are given in **Table 1**.

**3. Nano theranostics**

**4. Therapeutic agents**

*DOI: http://dx.doi.org/10.5772/intechopen.94391*

Iron oxide siRNA, DOX,

Silica Pyropheophorbide

CNTs DNA plasmid, DOX,

Gold DOX Diagnosis tumor targeting

docetaxel

(HPPH), DOX

PTX

QDs DOX, MTX Imaging, therapy

*Paclitaxel, MRI: Magnetic resonance imaging, CT: Computed topography.*

*Different theranostic agents used for biomedical applications.*

**Contrast agent**

Manganese oxide

**Table 1.**

*Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.94391*


*Abbreviations: siRNA: short interfering ribonucleic acid, CNTs: carbon nanotubes, QDs: quantum dots, DOX: Doxorubicin, HPPH: 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-alpha, MTX: Methotrexate, PTX: Paclitaxel, MRI: Magnetic resonance imaging, CT: Computed topography.*

#### **Table 1.**

*Molecular Pharmacology*

cation method [6–8].

**2. Theranostics**

characteristics and disease progression.

agent and also in image guided therapeutics [14].

preparation for therapy and diagnostic purpose.

and eventually the disadvantage and benefits of the process.

the simplest platforms are the nanoemulsion having size range of 100 to 500 nm which are kinetically stabilized having high content of oil and low amount of surfactant [2]. Solubility of poorly soluble drugs [3, 4] and its bioavailability can be increased by converting the drug into forms like capsule and gels [5] or can be used in their original form. The method used for the fabrication of nanoemulsion are high energy methods (microfluidization and sonication) and low energy emulsifi-

Theranostics is a term originally coined to define an approach that combine's diagnostics with therapeutics [9]. It embraces multiple techniques to arrive at comprehensive diagnosis, molecular images and an individualized treatment regimen [10, 11]. Recently, there is an effort to tangle the emerging approach with nanotechnologies, in an attempt to develop theranostic nanoplatforms and methodologies [12]. Given that cancer is a highly heterogeneous and adaptable disease, diverse types of treatment options need to be chosen depending on patient's

Drugs or methods that are used for accompanying diagnosis and cure [13] are referred to as Theranostics. One of the achievements of nanotechnology is the fabrication of theranostic nanomedicine for the preparation of these types of drugs. The term defines "a nanotherapeutic system which can deliver targeted therapy and diagnose". This aspect provides help when fabricating nano based image contrasting

Rapid drug development, advanced disease management, reduced associated risk and cost are assumed to be the result of mutual techniques. Such type of investigation which involves quick diagnosis and treatment are very helpful in disease which are a major cause of morbidity and/or mortality and cancer is one of the disease and coincidently the initial research in theranostic is dedicated to oncology. Sound knowledge, core understanding of detection and therapy mechanism are required for the formation of theranostic agents. In order to fabricate theranostic agents one should have understanding of diagnostic strategies, molecular mechanism adverse effect and toxicity of material and techniques for nanoparticles

Research in theranostics has rapidly improved in the past decade resulting into preparation of different contrast media and active ingredient with different method of preparation. Preparation of dual purpose nanoparticle system is the main aim of theranostics. Therefore it is important to put attention on all factors that influences the process, right from the preparation of nanoemulsion/nanoparticle till the removal of metabolites of the active molecules and other materials used. The factors can be the compatibility between chemicals, the condition in which the formulation is prepared, modification in formulations because of selected route of administration, the toxicity, metabolites of active ingredient, its biocompatibility and biodegradability and evaluation of pharmacodynamic and pharmacokinetic parameter

The basis of diagnosis in theranostics depends upon using different contrast agent during imaging. MRI is the most studied and used technique among all different imaging mechanism and a lot has been spent on research related to magnetic particles used as contrasting agent. Metals like gold, silver, iron oxide have been studied with the object of finding suitable particle for imaging with least toxicological effect. Diseased tissue and healthy tissue are differentiated in

**140**

MRI by the use of these particles.

*Different theranostic agents used for biomedical applications.*

As stated one of the brutal disease is cancer and hence theranostic research has put an eye on this area. Day by day the research is going on in positive direction and much useful research has already been carried out. In order to understand the concept of diagnosis of cancer and therapy related to it the use of nanoparticle agents is in progression [27, 28]. One such example of theranostic agent is manganese oxide nanoparticle carrying drugs and contrast agents [29] and silica nanoparticle with magnetic and fluorescent tags [30]. In the past few years, combination of metal nanoparticle or shells [31–33] with magnetic components has yielded different theranostic agents for biomedical applications which are widely used. Some examples of theranostic agents are given in **Table 1**.

#### **3. Nano theranostics**

Theranostic nanosystems comprise of platforms/nanocarriers that are used as imaging as well as therapeutic agents via a single entity. Nanotheranostic devices can be made by many types of inorganic and organic nanomaterials. Preclinical implementations make use of nanotheranostic system because they might provide a way or method of understanding many crucial aspects of drug delivery and how these systems can assist in knowing the power of personalized medicines.

#### **4. Therapeutic agents**

At present radiotherapy, surgery and chemotherapy are possible treatments for cancer patients. The purpose of the theranostic is to use these therapeutic strategies and reduce the risk associated with chemotherapy and radiotherapy and in addition to it avoid complications related to surgery and trauma. In addition, with the help

of nanotechnology, theranostics may support the diversification of therapeutic approaches like PTT, PDT and immunotherapy. Here we report some of these therapeutic strategies often used in theranostics, like radiotherapy, chemotherapy, PDT, PTT and immunotherapy.

#### **5. Chemotherapy for management of cancer**

Anticancer drugs have proven beneficial in improving survival rate of cancer patients [34]. There are huge numbers of clinical anti-cancer drugs which are broadly applied to theranostics. On the basis of structure and resource of chemotherapy drugs, cancer therapeutic agents can be classified into six types: alkylating agent, antitumor antibiotic, phytogenic anticarcinogen, antimetabolites, hormone and miscellaneous anti-cancer drugs. Thermo DOX for liver cancer, Doxil for ovarian cancer therapy and Myocet for metastatic breast cancer are few cancer nanomedicines that have been approved by the FDA. Theranostic systems also make use of prodrugs like platinum (IV) prodrug to reduce the toxicity of drug and by increasing the active hits to the cells of tumors site. Due to their broad availability these prodrugs are very popular option. The UV light is transformed from NIR light by UCNP which activates the prodrugs to highly toxic platinum (II) complexes that enters the cell by endocytosis after grafted onto up converting nanoparticles (UCNP) [35]. In order to attain best therapeutic efficacy of drug delivery systems a high payload is essential. In theranostics, in order to maintain the original size and solubility in aqueous media a carrier with large pore volume and surface area are given preferences so that more therapeutic agent can be carried [36]. For example, Sorafenib with a loading ration of 28.2% can be loaded on porous silica nanoparticles and may release the therapeutic agent in sustained fashion [37]. GO, Ws2 and MoS2 are some of the popular 2-D nanomaterials that have a very high drug payload as they can bear chemotherapeutics on both sides of sheet. Some of the example of high drug payload include 118% for 7-ethyl-10-hydroxycamptothecin (SN38) and approx 239% for DOX were observed on MoS2 [38], DOX (approximately 400%) on GO [39] was also significant. Cancer cells show multidrug resistance (MDR) often when they are treated by single drug which can be overcome by employing efficient strategies of theranostics. By combining P-glycoprotein (P-gp) reversing agent with anticancer drug the hurdle of MDR can be resolved [40]. The function of P-gp reversing agent is to avoid the pumping of chemotherapeutic drugs out of cancer cells due to over expression of P-gp. One way to overcome MDR is by covering the positive change that is present on anticancer drugs. DOX alone cannot produce significant cancer effect but when it is adsorbed on the surface of polymeric nanoparticle more chance are there that cancer cell may readily uptake it and accumulate within cancer cell and produce more cytotoxicity to cancer cell. Nanocarriers loaded with combination of anticancer drugs provide synergistic effect thereby improving overall management of cancer [41, 42].

#### **6. Photothermal therapy for management of cancer**

Microwave, light irradiation or magnetic field can potentiate the effect of thermal therapy which in turn employs hyperthermia to kill cancer cells. Among all the above mentioned therapies photothermal therapy has the maximum capacity to destroy cancer cell while causing least damage to nearby healthy cells. Localized hyperthermia under light irradiation at tumor site is generated by using NIR absorbing agent in photothermal therapy [43]. In MR region an ideal PTT agent

**143**

upon them.

phototoxicity is caused by PTT.

*Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer*

should show strong absorbance and must exhibit less fluorescence quantum yield thereby promoting efficient conversion of absorbed light energy into heat via non-

Inorganic nanoparticles and organic dyes are extensively employed as PTT agents. Examples of organic dyes include Prussian blue, IR780, ICG, IR820, Cypate, IR825. These organic dyes have an added advantage of ease of loading on nanoplatforms and ideal NIR absorbance [44]. In order to improve the photostability and targeting ability of organic dyes they are being encapsulated into nanocarriers [45]. Carbocyanine dyes namely cypate and ICG were loaded into the polymeric micelle with loading rate of 50% and 20% for cypate and ICG respectively. Upon comparing loaded theranostic polymeric micelle with carbocyanine dye alone showed marked cellular uptake and longer retention time at the site of tumor. Remarkable PTT results were observed along with increased photothermal effect and photostability of organic dyes when nanomaterials like graphene derivatives absorbing strongly in the NIR regions were employed [46]. In NIR and PTT imaging tech-

Photothermal conversion efficiency will decrease in presence of high fluorescence quantum yield and fluorescence imaging is disturbed in case of low quantum yield hence there is not much surety in theranostic application of organic dyes. Apart from the organic dyes, a wide range of inorganic nanoparticles have been fabricated for theranostic applications. Inorganic nanoparticles exhibit strong photothermal conversion efficiency and NIR absorption for PTT. It encompasses customized gold nanostructure like nanoshell, nanocages, nanorods and nanotubes. On comparing the gold nanorods alone against gold nanorods coated with Pt nanodots, the latter showed significant better photothermal effect than the former [47]. And the better results were due to the presence of Pt shell in the endosomes which not only prevented the original sharp LSPR band of gold nanorods from shifting and dampening but also prevented the aggregation of gold nanorods. Carbon nanotubes [48], carbon dots [49], GO are some of the other nanomaterials that can used for PTT. GO for in-vivo PTT was used for the first time by Liu group. Further they reduced the GO to rGO which had 7 times more NIR absorption than GO hence increasing the PTT effect [50]. PTT for now might only be used for treating skin cancer and not for internal cancer because of limiting light penetration depth but its noteworthy therapeutics capacities with minimum possible side effect cannot be ignored. Further study is required to get deeper insight about how

Apart from PTT, hyperthermia induced magnetically is also one of the noninvasive procedures to treat cancer [51]. Dielectric constant and microwave frequency between malignant tissue and normal tissue in breast can be employed for the detection and treatment of breast cancer. Dielectric contrast is used for scattering of an illuminating microwave signal and the incident microwave pro-

Photosensitizers (PSs) used in PDT plays a vital role in the treatment of cancer and possess enormous potential. Cytotoxic reactive oxygen species or free radicals are generated when the molecular oxygen surrounding the diseased cell reacts with the absorbed light that is being transferred by PSs under laser irradiation which finally causes cell apoptosis and damage to cancerous cells [53]. No side effects are observed from photosensitizes and generate ROS only when laser light is irradiated

duces hyperthermia thereby treating malignant tissues [52].

**7. Photodynamic therapy for management of cancer**

*DOI: http://dx.doi.org/10.5772/intechopen.94391*

radiative transition rather than fluorescence emission.

niques both cypate and ICG can be used as theranostic agents.

#### *Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.94391*

should show strong absorbance and must exhibit less fluorescence quantum yield thereby promoting efficient conversion of absorbed light energy into heat via nonradiative transition rather than fluorescence emission.

Inorganic nanoparticles and organic dyes are extensively employed as PTT agents. Examples of organic dyes include Prussian blue, IR780, ICG, IR820, Cypate, IR825. These organic dyes have an added advantage of ease of loading on nanoplatforms and ideal NIR absorbance [44]. In order to improve the photostability and targeting ability of organic dyes they are being encapsulated into nanocarriers [45]. Carbocyanine dyes namely cypate and ICG were loaded into the polymeric micelle with loading rate of 50% and 20% for cypate and ICG respectively. Upon comparing loaded theranostic polymeric micelle with carbocyanine dye alone showed marked cellular uptake and longer retention time at the site of tumor. Remarkable PTT results were observed along with increased photothermal effect and photostability of organic dyes when nanomaterials like graphene derivatives absorbing strongly in the NIR regions were employed [46]. In NIR and PTT imaging techniques both cypate and ICG can be used as theranostic agents.

Photothermal conversion efficiency will decrease in presence of high fluorescence quantum yield and fluorescence imaging is disturbed in case of low quantum yield hence there is not much surety in theranostic application of organic dyes. Apart from the organic dyes, a wide range of inorganic nanoparticles have been fabricated for theranostic applications. Inorganic nanoparticles exhibit strong photothermal conversion efficiency and NIR absorption for PTT. It encompasses customized gold nanostructure like nanoshell, nanocages, nanorods and nanotubes. On comparing the gold nanorods alone against gold nanorods coated with Pt nanodots, the latter showed significant better photothermal effect than the former [47]. And the better results were due to the presence of Pt shell in the endosomes which not only prevented the original sharp LSPR band of gold nanorods from shifting and dampening but also prevented the aggregation of gold nanorods. Carbon nanotubes [48], carbon dots [49], GO are some of the other nanomaterials that can used for PTT. GO for in-vivo PTT was used for the first time by Liu group. Further they reduced the GO to rGO which had 7 times more NIR absorption than GO hence increasing the PTT effect [50]. PTT for now might only be used for treating skin cancer and not for internal cancer because of limiting light penetration depth but its noteworthy therapeutics capacities with minimum possible side effect cannot be ignored. Further study is required to get deeper insight about how phototoxicity is caused by PTT.

Apart from PTT, hyperthermia induced magnetically is also one of the noninvasive procedures to treat cancer [51]. Dielectric constant and microwave frequency between malignant tissue and normal tissue in breast can be employed for the detection and treatment of breast cancer. Dielectric contrast is used for scattering of an illuminating microwave signal and the incident microwave produces hyperthermia thereby treating malignant tissues [52].

#### **7. Photodynamic therapy for management of cancer**

Photosensitizers (PSs) used in PDT plays a vital role in the treatment of cancer and possess enormous potential. Cytotoxic reactive oxygen species or free radicals are generated when the molecular oxygen surrounding the diseased cell reacts with the absorbed light that is being transferred by PSs under laser irradiation which finally causes cell apoptosis and damage to cancerous cells [53]. No side effects are observed from photosensitizes and generate ROS only when laser light is irradiated upon them.

*Molecular Pharmacology*

PDT, PTT and immunotherapy.

**5. Chemotherapy for management of cancer**

effect thereby improving overall management of cancer [41, 42].

Microwave, light irradiation or magnetic field can potentiate the effect of thermal therapy which in turn employs hyperthermia to kill cancer cells. Among all the above mentioned therapies photothermal therapy has the maximum capacity to destroy cancer cell while causing least damage to nearby healthy cells. Localized hyperthermia under light irradiation at tumor site is generated by using NIR absorbing agent in photothermal therapy [43]. In MR region an ideal PTT agent

**6. Photothermal therapy for management of cancer**

of nanotechnology, theranostics may support the diversification of therapeutic approaches like PTT, PDT and immunotherapy. Here we report some of these therapeutic strategies often used in theranostics, like radiotherapy, chemotherapy,

Anticancer drugs have proven beneficial in improving survival rate of cancer patients [34]. There are huge numbers of clinical anti-cancer drugs which are broadly applied to theranostics. On the basis of structure and resource of chemotherapy drugs, cancer therapeutic agents can be classified into six types: alkylating agent, antitumor antibiotic, phytogenic anticarcinogen, antimetabolites, hormone and miscellaneous anti-cancer drugs. Thermo DOX for liver cancer, Doxil for ovarian cancer therapy and Myocet for metastatic breast cancer are few cancer nanomedicines that have been approved by the FDA. Theranostic systems also make use of prodrugs like platinum (IV) prodrug to reduce the toxicity of drug and by increasing the active hits to the cells of tumors site. Due to their broad availability these prodrugs are very popular option. The UV light is transformed from NIR light by UCNP which activates the prodrugs to highly toxic platinum (II) complexes that enters the cell by endocytosis after grafted onto up converting nanoparticles (UCNP) [35]. In order to attain best therapeutic efficacy of drug delivery systems a high payload is essential. In theranostics, in order to maintain the original size and solubility in aqueous media a carrier with large pore volume and surface area are given preferences so that more therapeutic agent can be carried [36]. For example, Sorafenib with a loading ration of 28.2% can be loaded on porous silica nanoparticles and may release the therapeutic agent in sustained fashion [37]. GO, Ws2 and MoS2 are some of the popular 2-D nanomaterials that have a very high drug payload as they can bear chemotherapeutics on both sides of sheet. Some of the example of high drug payload include 118% for 7-ethyl-10-hydroxycamptothecin (SN38) and approx 239% for DOX were observed on MoS2 [38], DOX (approximately 400%) on GO [39] was also significant. Cancer cells show multidrug resistance (MDR) often when they are treated by single drug which can be overcome by employing efficient strategies of theranostics. By combining P-glycoprotein (P-gp) reversing agent with anticancer drug the hurdle of MDR can be resolved [40]. The function of P-gp reversing agent is to avoid the pumping of chemotherapeutic drugs out of cancer cells due to over expression of P-gp. One way to overcome MDR is by covering the positive change that is present on anticancer drugs. DOX alone cannot produce significant cancer effect but when it is adsorbed on the surface of polymeric nanoparticle more chance are there that cancer cell may readily uptake it and accumulate within cancer cell and produce more cytotoxicity to cancer cell. Nanocarriers loaded with combination of anticancer drugs provide synergistic

**142**

PDT requires low light density to cause damage to cancer cell unlike the PTT which requires high density laser light to generate hyperthermia that can cause damage to cancer cells [54]. PDT encompasses noteworthy advantages like very less invasiveness, on repeating the therapy is show no cumulative toxicity, very less damage to immune and hemopoietic system thereby improving the overall health and contributing to quality life for the patient. An ideal PS must have following properties like triplet state formation of high quantum yield and a good amount of triplet lifetime so that interaction with ground state oxygen is possible thereby generating sufficient ROS. However many PSs does not have good tumor selectivity, good amount of photosensitivity and absorption maxima above 700 nm [55]. A distinctive NIR absorption at 700 nm was observed by the help of extra axial mob linkers in monosubstituted phthalocyanine [56] that produced 20 nm red shift of characteristic Q band. PEG functionalized iron oxide nanocluster surface when loaded with Ce6 the absorption peak of chlorine e6 (Ce6) showed red shift from 650 nm to 704 nm [57]. The energy transferred from UCNPs to PSs are able to excite the combination of PSs and UCNP's, therefore inhibiting the growth of tumor by generation of cytotoxic singlet oxygen [58].

#### **8. Radiation therapy for management of cancer**

Radiation therapy has become an integral part to treat many sarcomas. The mechanism of action of radiation therapy is that the radiation damages strings of DNA in the nucleus of cells which stops the cell multiplication. Apart from aforementioned functions of radiation therapy, it also produces reactive oxygen therapy (ROS) which indirectly damages the tumor cell and also damages the DNA of mitochondria and other organelle of cell. In case of surgical resection, the survival could be prolonged by employing radiation therapy. However due to frequent and repeated high dose of X-ray irradiation that causes systemic side effects and resistance to radiation had been noticed in cancerous cells.

Metal nanoparticles in strong association with strong capacities of photoelectric absorbance are used as radiation dose enhancing agents. For example research shows that radio sensitization is being mediated by Gold NP due to greater energy deposition and absorption in surrounding tissue from photoelectrons. Radiotherapy with prolonged circulation time in blood has been demonstrated by Auger electrons and characteristic X-rays [59] and polyethylene glycosylation modified gold nanoparticle (N GNPs). Radiotherapy can relieve the symptoms and prolong the lives of terminal cancer patients. However radiotherapy is not an easy task and may cause loss of organ functions also as it may also induce many complications. Moreover, it cannot completely remove cancer cells. In coming future we may see highly accurate and precise exposure of tumor site to high radiation by the application of radiation wave knife for much better clinical results.

#### **9. Immunotherapy for management of cancer**

After radiotherapy, surgery or chemotherapy it has been observed that a small number of cancer cells may still remain alive and in addition to it the overall treatment quality is also decreased due to drug resistance. Immunotherapy has great potential to treat cancer as it acts on the immune system rather than on the tumor itself. Immunotherapy is considered as a unique and promising strategy for cancer therapy [60] and the main advantages include its specific promotion on immune cells only aiming on target cells or target tissues. So far, the related investigations

**145**

*Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer*

have been gradually transformed from laboratory research to clinical practice. For clinical treatment the use of immunotherapeutic drugs such as immune checkpoint inhibitors and T cells have been approved by FDA and have great potential for cancer treatment. Improved immunotherapeutic nanomaterials loaded with antigens, immune adjuvants and nucleic acids have been demonstrated to be helpful. The nanoplatforms may affect and alter immune cell actions and response nonspecifically. They may easily damage the cancer cells and achieve tumor targeting with pathogens factors. For e.g. repetitive and homogenous antigens conjugated with gold nanoparticles are able to trigger immune response in an in-vitro setting even without the use of adjuvant. Recently a combination of IR phthalocyanine dye IR700 with monoclonal antibodies had been fabricated and this novel technique is known as Photoimmunotherapy [61]. Least side effect and significantly fast cell necrosis rate is observed when antibodies bind to target tumor cells during the PIT and is activated by NIR light irradiation. Hence for monitoring and treating cancer

in a highly selective manner PIT is a good theranostic approach.

Conventional cancer therapies often do not succeed to eradicate tumor completely. In order to recover anticancer efficacy, the arrangement of two or more therapeutic modalities such as chemo photodynamic, photothermal photodynamic, chemo photo thermal synergistic formulations have been explored. Thermomotherapeutic characteristics in association with theranostic methods result in development of anticancer drug that possess synergistic therapeutic effect [62]. Chemotherapy could be improved by the use of photothermal effect which aids the intracellular translocation of anti-cancer drugs [63]. Risk of overtreatment could be minimized along with the reduction in dose of therapeutic agent with less laser exposure time. All these can be attained by combination of PDT/PTT. Synergistic effect of PTT/PDT have been seen when GO was loaded with methylene blue [64]. In this system, lesser dose of nano GO was applied, as compared to the particular PTT treatment of nano GO. In addition, the PTT and PDT combinational treatment could be spoil both superficial and deep regions of the tumor, and thus overcome the drawbacks of single treatments [65]. To further progress cancer therapy efficacy, numerous types of theranostic platforms were developed to combine chemotherapy, PTT and PDT simultaneously [66]. Treating cancer with combinational therapy has become an essential trend in cancer therapeutics. Compared to single modality therapy, the combined therapy can reduce the dosage of the drugs and thus decrease the side effects in treatment. More prominently, the combined therapy has the potential to decrease multidrug resistance of tumor cells, thus improving the therapeutic efficacy. The combined therapy may bring a novel

**10. Multimodal therapy for management of cancer**

opportunity to the next invention of cancer treatment [67].

**11. Imaging-guided therapy for management of cancer**

The theranostic nanoparticles have an ever increasing consideration for image guided therapy in current years because these nanoparticles can follow the pharmacokinetic process, guide the treatment and monitor therapeutic process and outcome. They could be employed to imagine and quantify the performance of drug delivery systems for numerous special purposes such as biodistribution and pharmacokinetics of nanocarriers, metabolic response and drug release process of the nanocarriers. Koukourakis group and Harrington group engaged Technetium

*DOI: http://dx.doi.org/10.5772/intechopen.94391*

*Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.94391*

have been gradually transformed from laboratory research to clinical practice. For clinical treatment the use of immunotherapeutic drugs such as immune checkpoint inhibitors and T cells have been approved by FDA and have great potential for cancer treatment. Improved immunotherapeutic nanomaterials loaded with antigens, immune adjuvants and nucleic acids have been demonstrated to be helpful. The nanoplatforms may affect and alter immune cell actions and response nonspecifically. They may easily damage the cancer cells and achieve tumor targeting with pathogens factors. For e.g. repetitive and homogenous antigens conjugated with gold nanoparticles are able to trigger immune response in an in-vitro setting even without the use of adjuvant. Recently a combination of IR phthalocyanine dye IR700 with monoclonal antibodies had been fabricated and this novel technique is known as Photoimmunotherapy [61]. Least side effect and significantly fast cell necrosis rate is observed when antibodies bind to target tumor cells during the PIT and is activated by NIR light irradiation. Hence for monitoring and treating cancer in a highly selective manner PIT is a good theranostic approach.

#### **10. Multimodal therapy for management of cancer**

Conventional cancer therapies often do not succeed to eradicate tumor completely. In order to recover anticancer efficacy, the arrangement of two or more therapeutic modalities such as chemo photodynamic, photothermal photodynamic, chemo photo thermal synergistic formulations have been explored. Thermomotherapeutic characteristics in association with theranostic methods result in development of anticancer drug that possess synergistic therapeutic effect [62]. Chemotherapy could be improved by the use of photothermal effect which aids the intracellular translocation of anti-cancer drugs [63]. Risk of overtreatment could be minimized along with the reduction in dose of therapeutic agent with less laser exposure time. All these can be attained by combination of PDT/PTT. Synergistic effect of PTT/PDT have been seen when GO was loaded with methylene blue [64]. In this system, lesser dose of nano GO was applied, as compared to the particular PTT treatment of nano GO. In addition, the PTT and PDT combinational treatment could be spoil both superficial and deep regions of the tumor, and thus overcome the drawbacks of single treatments [65]. To further progress cancer therapy efficacy, numerous types of theranostic platforms were developed to combine chemotherapy, PTT and PDT simultaneously [66]. Treating cancer with combinational therapy has become an essential trend in cancer therapeutics. Compared to single modality therapy, the combined therapy can reduce the dosage of the drugs and thus decrease the side effects in treatment. More prominently, the combined therapy has the potential to decrease multidrug resistance of tumor cells, thus improving the therapeutic efficacy. The combined therapy may bring a novel opportunity to the next invention of cancer treatment [67].

#### **11. Imaging-guided therapy for management of cancer**

The theranostic nanoparticles have an ever increasing consideration for image guided therapy in current years because these nanoparticles can follow the pharmacokinetic process, guide the treatment and monitor therapeutic process and outcome. They could be employed to imagine and quantify the performance of drug delivery systems for numerous special purposes such as biodistribution and pharmacokinetics of nanocarriers, metabolic response and drug release process of the nanocarriers. Koukourakis group and Harrington group engaged Technetium

*Molecular Pharmacology*

generation of cytotoxic singlet oxygen [58].

**8. Radiation therapy for management of cancer**

tance to radiation had been noticed in cancerous cells.

tion of radiation wave knife for much better clinical results.

**9. Immunotherapy for management of cancer**

PDT requires low light density to cause damage to cancer cell unlike the PTT which requires high density laser light to generate hyperthermia that can cause damage to cancer cells [54]. PDT encompasses noteworthy advantages like very less invasiveness, on repeating the therapy is show no cumulative toxicity, very less damage to immune and hemopoietic system thereby improving the overall health and contributing to quality life for the patient. An ideal PS must have following properties like triplet state formation of high quantum yield and a good amount of triplet lifetime so that interaction with ground state oxygen is possible thereby generating sufficient ROS. However many PSs does not have good tumor selectivity, good amount of photosensitivity and absorption maxima above 700 nm [55]. A distinctive NIR absorption at 700 nm was observed by the help of extra axial mob linkers in monosubstituted phthalocyanine [56] that produced 20 nm red shift of characteristic Q band. PEG functionalized iron oxide nanocluster surface when loaded with Ce6 the absorption peak of chlorine e6 (Ce6) showed red shift from 650 nm to 704 nm [57]. The energy transferred from UCNPs to PSs are able to excite the combination of PSs and UCNP's, therefore inhibiting the growth of tumor by

Radiation therapy has become an integral part to treat many sarcomas. The mechanism of action of radiation therapy is that the radiation damages strings of DNA in the nucleus of cells which stops the cell multiplication. Apart from aforementioned functions of radiation therapy, it also produces reactive oxygen therapy (ROS) which indirectly damages the tumor cell and also damages the DNA of mitochondria and other organelle of cell. In case of surgical resection, the survival could be prolonged by employing radiation therapy. However due to frequent and repeated high dose of X-ray irradiation that causes systemic side effects and resis-

Metal nanoparticles in strong association with strong capacities of photoelectric

After radiotherapy, surgery or chemotherapy it has been observed that a small number of cancer cells may still remain alive and in addition to it the overall treatment quality is also decreased due to drug resistance. Immunotherapy has great potential to treat cancer as it acts on the immune system rather than on the tumor itself. Immunotherapy is considered as a unique and promising strategy for cancer therapy [60] and the main advantages include its specific promotion on immune cells only aiming on target cells or target tissues. So far, the related investigations

absorbance are used as radiation dose enhancing agents. For example research shows that radio sensitization is being mediated by Gold NP due to greater energy deposition and absorption in surrounding tissue from photoelectrons. Radiotherapy with prolonged circulation time in blood has been demonstrated by Auger electrons and characteristic X-rays [59] and polyethylene glycosylation modified gold nanoparticle (N GNPs). Radiotherapy can relieve the symptoms and prolong the lives of terminal cancer patients. However radiotherapy is not an easy task and may cause loss of organ functions also as it may also induce many complications. Moreover, it cannot completely remove cancer cells. In coming future we may see highly accurate and precise exposure of tumor site to high radiation by the applica-

**144**


#### **Table 2.**

*Theranostic technologies for cancer treatment [68].*

and Indium labeled PEGylated liposomes respectively to monitor drug targeting to the sarcomas and breast cancer sites [69]. In the clinical practice, surgical resection is a regular and inevitable procedure for cancer therapy. Theranostics gives a possibility in intraoperative imaging to guide the operation process. During the surgery,

**147**

*Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer*

physicians could congregate the diagnostic information for precise imaging as well as visualized therapy. In theranostic platform, DOTA-Gd act as a MRI contrast agent for preoperative finding and surgical planning; the Raman molecules visualized the excellent margin of tumor, allowing precise resection for the duration of operation process. The multimodal NP could recognize tumor edge for precise resection of tumor. This approach could be planned for simple intraoperative navigation and real-time imaging [70]. Theranostic technologies commonly utilized for cancer

Theranostic approach to management of cancer offers numerous advantages. They are designed to monitor cancer treatment in real time. A wide variety of theranostic nanoplatforms that are based on diverse nanostructures like magnetic nanoparticles, carbon nanotubes, gold nanomaterials, polymeric nanoparticles, or silica nanoparticles showed great potential as cancer theranostics. Nano therapeutic platforms have been successful in integrating image guidance with targeted

Authors declare no conflict of interest related to this manuscript.

*DOI: http://dx.doi.org/10.5772/intechopen.94391*

treatment are given in **Table 2**.

**12. Conclusion**

approach to treat cancer.

**Conflict of interest**

**Abbreviations**

PTT Photothermal therapy PDT Photodynamic therapy

CT Computed topography UNCPs Up converting nanoparticles

GO Grapheme oxide MDR Multidrug resistance MoS2 Molybdenum disulfide ICG Indocyanine green WS2 Tungsten disulfide Ce6 Chlorine e6

MRI Magnetic resonance imaging PET Positron emission tomography

DOX Doxorubicin NIR Near infrared *Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.94391*

physicians could congregate the diagnostic information for precise imaging as well as visualized therapy. In theranostic platform, DOTA-Gd act as a MRI contrast agent for preoperative finding and surgical planning; the Raman molecules visualized the excellent margin of tumor, allowing precise resection for the duration of operation process. The multimodal NP could recognize tumor edge for precise resection of tumor. This approach could be planned for simple intraoperative navigation and real-time imaging [70]. Theranostic technologies commonly utilized for cancer treatment are given in **Table 2**.

#### **12. Conclusion**

*Molecular Pharmacology*

Dicyanomethylene-4H-

Cy7, 111In Cyclophosphamide,

pyran

UCNP

(β-NaYF4:Yb3+,Er3+)

**Imaging method**

Optical Imaging

MR imaging

CT imaging

PET Imaging

US imaging

**146**

**Table 2.**

*Theranostic technologies for cancer treatment [68].*

and Indium labeled PEGylated liposomes respectively to monitor drug targeting to the sarcomas and breast cancer sites [69]. In the clinical practice, surgical resection is a regular and inevitable procedure for cancer therapy. Theranostics gives a possibility in intraoperative imaging to guide the operation process. During the surgery,

**Imaging agent Therapeutic agent Function**

etoposide

Cy5.5 Paclitaxel Real time tracking of NP

FITC-coumarin pair Doxorubicin Drug release monitoring

Cy5.5-BHQ pair Doxorubicin Real time imaging of apoptosis Ce6 Ce6 Real time tracking of NP

Ce6-BHQ pair Ce6 Drug release monitoring &

UCNP (NaYF4:Er) TPGSd Dual imaging (optical, CT) &

UCNP (NaYF4:Yb/Er) Ce6, doxorubicin Imaging of particle location &

Gd Doxorubicin Real time monitoring of drug

SPION SPION Detection & hyperthermia

SPION/FITC siRNA MR imaging & gene therapy

GNR GNR Dual imaging (X-ray/CT) &

64Cu Doxorubicin Quantitative biodistribution

64Cu siRNA Quantitative determination of

Perfluoropentane Docetaxel Triggered drug release &

CaCO3 Doxorubicin Tumor imaging & triggered

Perfluorooctyl bromide Camptothecin Chemotherapy & ablation

Perfluorohexane CPT11m Tumor imaging &

GNP Doxorubicin CT imaging of cancer &

SPION Doxorubicin Tumor detection &

location

location & PDT

PDT

Cisplatin prodrug Imaging of NP location

Real time imaging of apoptosis

reducing multidrug resistance

chemotherapy/PDT

treatment of tumor

chemotherapy

chemotherapy

siRNA NPs

chemotherapy

drug release

chemotherapy/ablation

therapy

Therapy

PTT/radio sensitization

analysis & Chemotherapy

biodistribution & efficacy of

delivery

Camptothecin Drug release monitoring

Theranostic approach to management of cancer offers numerous advantages. They are designed to monitor cancer treatment in real time. A wide variety of theranostic nanoplatforms that are based on diverse nanostructures like magnetic nanoparticles, carbon nanotubes, gold nanomaterials, polymeric nanoparticles, or silica nanoparticles showed great potential as cancer theranostics. Nano therapeutic platforms have been successful in integrating image guidance with targeted approach to treat cancer.

#### **Conflict of interest**

Authors declare no conflict of interest related to this manuscript.

#### **Abbreviations**


*Molecular Pharmacology*

#### **Author details**

Asad Ali1 , Zeeshan Ahmad<sup>2</sup> , Usama Ahmad1 \*, Mohd Muazzam Khan1 , Md. Faheem Haider1 and Juber Akhtar1

1 Faculty of Pharmacy Integral University, Lucknow, India

2 Makams Industries Private Limited, Bhiwadi, Alwar, Rajasthan, India

\*Address all correspondence to: usamaahmad.10@outlook.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**149**

*Integrating Nanotherapeutic Platforms to Image Guided Approaches for Management of Cancer*

[10] Lukianova-Hleb EY, Oginsky AO, Samaniego AP,

Theranostics. 2011;**1**:3

Shenefelt DL, Wagner DS, Hafner JH, et al. Tunableplasmonicnanoprobes for theranostics of prostate cancer.

[11] Zhu L, Xie J, Swierczewska M, Zhang F, Quan Q, Ma Y, et al. Real-time video imaging of protease expression in

[12] Fang C, Zhang M. Nanoparticlebased theragnostics: Integrating diagnostic and therapeutic potentials

[13] Ahmed N, Fessi H, Elaissari A. Theranostic applications of

Today. 2012;**17**(17-18):928-934

[14] Sumer, B. and Gao, J., 2008. Theranosticnanomedicine for Cancer.

manganese oxide nanoparticles for cancer targeted siRNA delivery and magnetic resonance imaging. Biomaterials. 2011;**32**(1):176-184

[16] Huang P, Bao L, Zhang C, Lin J, Luo T, Yang D, et al. Folic acidconjugated silica-modified gold nanorods for X-ray/CT imagingguided dual-mode radiation and photo-thermal therapy. Biomaterials.

[17] Chen W, Bardhan R, Bartels M, Perez-Torres C, Pautler RG, Halas NJ, et al. A molecularly targeted theranostic probe for ovarian cancer. Molecular Cancer Therapeutics. 2010;**9**(4):1028-1038

[18] Prabaharan M, Grailer JJ, Pilla S, Steeber DA, Gong S. Gold nanoparticles

2011;**32**(36):9796-9809

[15] Bae KH, Lee K, Kim C,

nanoparticles in cancer. Drug Discovery

Park TG. Surface functionalized hollow

vivo. Theranostics. 2011;**1**:18

in nanomedicine. Journal of Controlled Release: Official Journal of the Controlled Release Society.

2010;**146**(1):2

*DOI: http://dx.doi.org/10.5772/intechopen.94391*

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[3] Ahmad, U., Akhtar, J., Singh, S.P., Badruddeen, Ahmad, F.J., Siddiqui, S. and Wahajuddin, 2017. Silymarin nanoemulsion against human

hepatocellular carcinoma: development and optimization. Artificial cells, nanomedicine, and biotechnology,

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Md. Faheem Haider1

, Zeeshan Ahmad<sup>2</sup>

provided the original work is properly cited.

, Usama Ahmad1

2 Makams Industries Private Limited, Bhiwadi, Alwar, Rajasthan, India

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Juber Akhtar1

1 Faculty of Pharmacy Integral University, Lucknow, India

\*Address all correspondence to: usamaahmad.10@outlook.com

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### *Edited by Angel Catala and Usama Ahmad*

This book concentrates on recent developments related to the application of original structural biology, biochemistry, biophysics, physiology, genetics, and molecular biology as well as basic pharmacological problems that offer mechanistic insights that are generally significant for the field of pharmacology. Written by experts, chapters cover such topics as drug transport mechanisms and drug–receptor complexes. This volume offers up-to-date, expert reviews of the fast-moving field of molecular pharmacology.

Published in London, UK © 2021 IntechOpen © setixela / iStock

Molecular Pharmacology

Molecular Pharmacology

*Edited by Angel Catala and Usama Ahmad*