Synthesis and Investigation of Colon Specific Polymeric Prodrug of Budesonide with Cyclodextrin

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P

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I

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PAST EDITORS

• Dr. Nagavi B.G.

Mysore

1997 - 2006

• Dr. Rao M.N.A.

Manipal

1995-1996

• Dr. Gundu Rao P.

Manipal

1985-1995

• Dr. Kasture A.V.

Nagpur

1981 - 1984

• Dr. Saoji A.N.

Nagpur

1980 - 1980

• Dr. Lakhotiya C. L.

Nagpur

1979 - 1980

• Dr. Chopde C.T.

Nagpur

1978 - 1978

• Dr. Gundu Rao P.

Manipal

1975 - 1978

• Dr. Mithal B. M.

Pilani

1967 – 1974

EDITOR–IN–CHIEF

Dr. Sanjay Pai P.N.

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EDITORIAL OFFICE

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• Pharmacognosy - Dr. Ganapaty S., Dr. Salma Khanam,

Dr. Swati S.Patil

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Dr. Shobha Rani R.H.

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Vol. 43(3), Jul-Sep, 2009

CONTENTS

Review Articles

• Obesity: Current Therapy and Emerging Targets

Kashaw Sushil Kumar, Jatav Varsha, Gupta Neeraj, Gupta Vivek, Jain Rupsee, Rajak Harish and Mishra

Pradeep.........................................................................................................................................................219-231

• USFDA Generic Approval Process

Nitin Saigal, Sanjula Baboota, Alka Ahuja and Javed Ali………………………………………………...232-240

• Stimuli Sensitive Hydrogels: A Review

S.S. Bushetti, Vinod Singh, S.Appala Raju, Atharjaved,Veermaram…………………………………………..241-250

• Transdermal Drug Delivery: Evolving Technologies and Expanding Opportunities

Geeta Aggarwal, Ashish Garg, Sanju Dhawan……………………………………………………………………251-259

Research Articles

• Microwave Assisted Fast Extraction of Mucilages and Pectins

B Geetha, K P Shivalinge Gowda, G T Kulkarni and Shrishailappa Badami………………………………...260-265

• Synthesis of Some 2-(Substituted)-5-[(N-Benzotriazolomethyl)-1, 3, 4-Thiadiazolyl]-4-Thiazolidinones for their

Anti-Fungal Activity

K.P.Namdeo, V.K.Singh and S.K.Prajapati……………………………………………………………………….266-271

• Effect of Milling on Correlation between Interquartile Coefficient of Skewness and Coefficient of Kurtosis in

Pharmaceutical Powders – II

Dabas Sunny, Shakya Pragati, Sharma Vijay and Pathak Kamla…………………………………...…………272-279

• Evaluation of Antidiabetic Activity of Caesalpinia bonduc (L.) Roxb Leaves (Caesalpianceae) in Alloxan

Induced Diabetic Mice

P.B.Aswar, S.S. Khadabadi, U.A. Deokate, S.P. Jain, K.R.Biyani, R.D. Jawarkar……………………….…280-283

• Rationalization of Physico Chemical Characters of 2-Phenyl-3-hydroxy-4(1H)-quinolinone-7-carboxylic acid

Analogs as Topoisomerase Inhibitors: A QSAR Approach.

P.Valentina, K.Ilango and Maida Engels………………………………………………………………………….284-289

• Formulation and Evaluation of Bilayer Tablets of Propanolol Hydrochloride

Aisha Khanum, Vinay Pandit and Shyamala Bhaskaran……………………………………………………..….290-294

• Synthesis and Investigation of Colon Specific Polymeric Prodrug of Budesonide with Cyclodextrin

Prabhakara Prabhu, D.Satyanarayana, Harish Nairy M, Mohd.Gulzar Ahmed, R.Narayanacharyulu, E.V.S.

Subrahmanyam……………………………………………………………………………………………………..…295-299

• Development and Evaluation of Ziprasidone Hydrochloride Fast Disintegrating/ Dissolving Tablets using

Complexation Techniques

Deshmukh Sambhaji S., Potnis Vaishali V., Mahaparale Paresh R. Kasture Pramod V. and Gharge Vikram

S………………………………………………………………………………………………………………………….300-307

• Synthesis and Antimicrobial Evaluation of Some New Pyrimidine Derivatives

Shashikanth R. Pattan, A.B. Khade, M.A. Khade, J.S. Pattan, S.G. Jadhav, N.S.Dighe, S.A. Nirmal, S.V.

Hiremath, A.N. Merekar……………………………………………………………………………………………..308-312



INVITED EDITORIAL

Wearing Eye Protection Goggles Should be Made Mandatory in all

College Laboratories

Dr. M.N.A.Rao

Divis Laboratories limited, Hyderabad

Professor K.Barry Sharpless won the Nobel Prize in Chemistry in 2001 for his work on chiral catalysts. Dr.

Sharpless, like all good chemists, always wore safety glasses while working in the laboratory. One day, shortly after

joining MIT as an assistant professor, he removed his safety glasses and was going home. While walking to the

door, he saw a graduate student sealing an NMR tube. Dr. Sharpless went to his student’s bench and picked up the

tube from the liquid nitrogen bath. The tube immediately frosted. He wiped it to see the content better and held it

near his face. Suddenly he saw the solvent level in the tube falling, and the tube exploded. Glass fragments shredded

his cornea, penetrated the iris, and caused partial collapse of one eye. Other injuries were superficial facial cuts.

After two weeks in hospital, he was discharged having lost the vision in one eye. If he had lost both eyes, the world

would have missed out on his scientific contributions.

This shows how important it is to wear eye protection goggles at all times while working in the lab, especially the

chemistry lab. Today, safety goggles must be compulsorily worn in all laboratories of schools and universities

abroad. This is also a compulsory safety practice in many Indian universities and central laboratories and in almost

all Indian industries. Abroad, it is compulsory even for school children to wear them while working in a laboratory.

Unfortunately, it is not practiced in any of the pharmacy institutions in the country. This is, in my opinion, a major

safety lapse. A student of pharmacy spends a significant amount of time in the laboratories. Teachers and Principals

must insist on students wearing safety goggles through out their stay in the laboratory. Teachers must also practice

this safety habit. The national and state level regulatory authorities should take the lead in inculcating this practice

among students and teachers. It is high time we start this practice to protect our students.

Like the white apron, safety goggles must be part of the laboratory attire in all pharmacy institutions. Approved

safety goggles MUST BE WORN AT ALL TIMES.

Dr. M.N.A. Rao is the former Editor, Indian Journal of Pharmaceutical Education and is currently holding the

position of General Manager, R&D, Divis Laboratories, Hyderabad

Indian J.Pharm. Educ. Res. 43(3), Jul-Sept, 2009

219

Obesity: Current Therapy and Emerging Targets Kashaw Sushil Kumar*, Jatav Varsha, Gupta Neeraj, Gupta Vivek, Jain Rupsee, Rajak

Harish and Mishra Pradeep

*Pharmaceutical Chemistry Division, Department of Pharmaceutical Sciences, Dr. H.S.Gour University, Sagar,

(M.P.), 470003; email - [email protected]

Abstract

The increasing prevalence of obesity worldwide has prompted the World Health organization (WHO) to classify

it as a global epidemic. A round the globe, more than a half a billion people are overweight, and the chronic

disease of obesity represents a major threat to health care system in developed and developing countries.

Energy homeostasis is accomplished through a highly integrated and reductant neurohumoral system.

Adrenergic and serotonergic agents enjoyed before is now disfavored due to abuse and lack of exact receptor

subtype profile respectively. β3-adrenergic receptor agonists acting, as thermogenic agents are new approach

and its value will become apparent once data are available from relevant clinical evaluation. Some drugs from

this class are under clinical trials. Transgenic technology has provided new opportunities to modify the

complex body weight regulation system and to assess the relative importance of the individual components.

Certain peptides have been used successfully as antiobesity agents. They reduce gastrointestinal absorption and

affect feeding behavior. Since obesity results from genetic predisposition, combined with the proactive

environment situation, we discuss new potential targets for generation of drugs that may help people in gaining

control over appetite as well as increase total energy expenditure and fat oxidation.

Keywords: Obesity, BMI, Themogenic agents, GIT peptides

INTRODUCTION

In the present era, it has been proved beyond doubt that obesity is one of the main threats to mankind, as it is the cause of many ailments. It results from increased energy in take and decreased energy expenditure. Only in United States 30,000 deaths are attributed annually to this cause. It is a chronic condition characterized by an over abundance of adipose tissue and can be measured in a variety of different ways. Increase in cell size, cell number or both can lead to increase in an adipose tissue mass. Increased size alone result in hypertrophic obesity, which is relatively mild. Increase in fat cell number, however, cause hyperplastic obesity characteristic of more severe condition. While the volume of lipid an individual adipose cell can accumulate is finite, the capacity of adipose tissue to expand is virtually limitlesss The most common way of measurement of obesity is the Body Mass Index (BMI) defined as the ratio of body weight (kg) and height (m).

It is difficult to set a definitive BMI level threshold to define obesity, particularly for women as excess weight in them is often found in the pelvis and not in the abdomen. Nevertheless, obesity is defined by the World Health Organization as a BMI of 30.0 kg/m and above and a BMI of 25.0 -29.9 kg/m classified as overweight or preobese. Workers have estimated the direct cost of obesity at $70 billion in 1995 whereas a recent study mandated by the American Obesity Association estimated the total cost of obesity to be $238 billion in 1999. Pharmaceutical companies now realize that there is an enormous economics opportunity if safe and efficacious drugs can be developed. This situation is analogous to what happened in the recent past with the development of drugs for mental health, which now benefit so many people. PATHOPHYSIOLOGY OF OBESITY Many factors, which may lead to development of obesity are genetic diseases, tumors and endocrine or hypothalamic disorders. In addition genetic factors also contributies. Each of the 24 human chromosomes except the Y chromosome contains obesity-related

Indian Journal of Pharmaceutical Education & Research Received on 3/5/2007 ; Modified on 14/8/2007 Accepted on 12/5/2009 © APTI All rights reserved

APTI ijper


220

gene. These include agonistic related neuropeptide Y (NPY) and its receptors (Y5 and Y6), proopiomelanocortin (POMC), uncouple proteins-2 (UCP-2), leptin and its receptors and peroxisomes

proliferator activated receptors (PPARγ). The regulation of adiposity by hypothalamus, plays a critical role in appetite and metabolism (Fig.1). Also abnormalities in the secretion and synthesis of the hormones secreted from peripheral tissues such as the gastrointestinal tract, pancreas and adipose tissue which partly control the hypothalamus reflexes have been responsible for obesity in many animal models. The control of feeding involves the central nervous system (CNS), adrenals, gastrointestinal tract and adipose tissues. Clinically relevant obesity is responsible for various adverse effects on health, being associated with an increase in morbidity and mortality form non-insulin dependent diabetes mellitus (NIDDM), hypertension, hypercholesterolemia, sleep apnea and other medical conditions . A large body of epidemiological data correlates increased body weight with risk such as high blood pressure, coronary heart disease, diabetes, altered steroid metabolism, gallstones, and certain forms of cancer. In one study, involving a cohort of >11500 female nurses over a period of 14-16 years, increased risks of cardiovascular disease and cancer were observed with increasing BMI. The study showed that, there was 100% greater risk of death from all causes for a BMI of 29.0 kg/m and above relative to risk of death for slender women (BMI of <19.0 kg/m). Insulin resistance due to obesity believed to play a role in other pathological states such as dyslipidemias, atherosclerosis, hypertension and other cardio-vascular disease. The incidence of obesity in Western society as well as in developing nation due to sedentary life-style now represents a major threat to human health. All the above complications due to obesity is also rising in India. Thus obesity is a disease and there should be an effort to counteract this disease. Research work is on to take care of this disease. Since in obesity drug therapy is likely to occur over a prolonged period of time, it is important that the therapeutic index of new agents be as high as possible. The role of current agents in therapeutic treatment

of obesity

Since obesity is the storage of excess energy, to reduce body fat there must be a period of negative energy balance. This can be achieved by reducing food intake or by increasing energy consumption (Fig 2). The search for antiobesity drugs has been divided into three basic approaches:

• Satiety drugs, which reduce the appetite, also called anorectic agents.

• Thermogenic drugs, which increases the amount of fat that is metabolised in the body by burning off excess calories.

• Drugs which can reduce the amount of fat absorbed in the gut.

SATIETY AGENTS

Adrenergic Agents: These agents act by releasing catecholamines from presynaptic nerve terminals and stimulating adrenergic receptors in the medial paraventricular nucleus in the hypothalamus (Table 1)1. Early anorectics such as amphetamines described in 1938, its isomer d-amphetamines, methamphetamine, phenametrazine and benzphetamines are no longer recommended for use because of their addiction potential and adverse cardiovascular properties including tachycardia and hypertension2. Serotonergic Agents: Serotonergic antiobesity drugs increases satiation, the process which brings a period of eating to an end, and satiety, the absence of hunger after eating14. Anorectics of this class can promote the release of serotonin in the CNS, prevent its reuptake, or act as serotonin receptor agonists (Table 1) 3.

Sibutramine is a β-phenethylamine that is a dual serotonin and norepinephrine reuptake inhibitor. Sibutramine reduces food consumption by both the inhibition of monoamine reuptake and enhanced thermogenesis. In human clinical trials, Sibutramine produced dose-dependent weight loss. It has minimal direct affinity for serotonergic, adrenergic, dopaminergic, muscarinic, glutamate, and benzodiazepine receptors (Fig. 2). Orlistat is a natural product from Streptomyces

toxytricini and is a potent inhibitor of pancreatic, gastric, and carboxylester lipases and phospholipase A2, which are required for the hydrolysis of dietary fat in the GI tract into fatty acids and monoacyl glycerol. Orlistat reacts with Ser-152 of pancreatic lipase, to form esters, which hydrolyze very slowly (Fig.3).


221

THERMOGENIC AGENTS

ββββ3-Adrenergic Receptor Agonists: The β3-adrenergic

receptors (β3-ARs) are found primarily on the cell surfaces of both white and brown adipocytes and mediates a variety of metabolic functions, including lipolysis and thermogenesis4,5. Recent studies suggest that thermogenesis in brown adipose tissue has an important role in the regulation of energy balance. Thermogenesis is effected by noradrenaline released from sympathetic nerve endings. The noradrenaline

stimulates β3-ARs causing increase in intracellular cyclic adenosine monophosphate stimulation of lipolysis. The released fatty acids, serves to regulate and activate the mitochondrial uncoupling protein (UCP-1), which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure6. UCP-1 is an integral component of the mitochondrial inner membrane of brown adipocytes and is a central to brown adipose tissue function (Fig.4)7.The large

number of β3-ARs agonists have been prepared and evaluated and these fall under following chemical classes8. 1. Arylethanolamines : Beecham company products

BRL-26830A (1) and BRL-35135 (2) were the first β3-ARs thermogenic agents disclosed and are potent stimulators of metabolic rate in rodents9. Despite high (80-90%) sequence homology, between rodent and

human receptors, β3-ARs agonists possess widely different functional effects at each receptor. After evaluation of a series of related structures, ZD2079 (3) was chosen for human clinical trials, but have only weak agonist activity in a human lipolysis assay in adipocytes. Compound (4) increased metabolic rate and reduced body weight in clinical trials, it displayed a poor pharmacokinetic profile which necessitated the use of high dose10. Compounds containing cyclic ureidobenzensulfonamide moiety (5) [ EC50 = 14 nM;

binds > 850 fold to β3 than β1 & β2 receptors], imidazolidinone benzene sulfonamide (6) [EC50 = 5.7

nM; binds > 420 and 120 fold to β3 than β1 & β2 receptors, respectively], imidazolone benzenesulfonamide (7) [ EC50 = 14nM ; binds > 97-

10, 000 fold to β3 than to β1 and β2 receptors] are

potent and selective β3-ARs agonists but show poor

bioavailibility in dogs. Recently oxazole

benzenesulfonamide derivatives with potent β3-ARs

agonist and excellent selectivity against other β-receptors have been developed showing good bioavailability in dogs. 2. Aryloxypropanolamines : As a class aryloxypropanolamines are partial agonists or

antagonists at β2- and β3- ARs, which contributes to the selectivity of the in vivo biological effect11. Compounds

(8) and (9) are potent β3-ARs agonists chosen from a large number of related derivatives12. Aryloxypropanolamine with sulfonamide containing side chains has been prepared (10) showed a 6.3 nm

EC50 for the stimulation of human β3–Ars13.

Compound (11) is among the potent β3-ARs agonists reported, with a 0.43 nm EC50

12. The propionic acid

BMS-187257 (12) was optimal (β3 EC50 = 630 nm, intrinsic activity 0.72) though only2 to3 fold selective

over β1 and β2 14.

DRUGS REDUCING THE FAT ABSORPTION

FROM GASTROINTESTINAL TRACT: A large number of peptides are thought to affect feeding behavior when administered centrally or peripherally. Some recent endogenous peptides that effect feeding are listed in Table No.3 . Neuropeptide Y : In 1930s Neuropeptide Y (NPY), a 36-amino acid c-amidated peptide which is a member of the pancreatic polypeptide family, found in the central and peripheral nervous systems was shown to be a an important component in appetite and food intake15. NPY cause sustain increase in food intake, as well as reducing BAT activity and increasing insulin secretion, all of which lead to obesity. Y1, Y2, Y3, Y4 are receptor subtypes having high affinity to NPY16. Recently, a collaboration between an American Biotechnology Company, Synaptic pharmaceuticals, and Ciba-Geigy pharmaceuticals has led the team to clone, characterise and patent the novel NPY feeding receptor which has been called the Y5 type. Restricting the availability of food increases the activity of NPY is probably mediated through a Y5 receptor; however, the Y1 receptors may mediate the effect of exogenous NPY on intake of a highly palatable diet. The first potent nonpeptide structure (13) was designed using the c-


222

Table 1: List of sympathomimetic amines that have been marketed as anorectic agents

Table 2: Newer antiobesity drugs

Drug Mechanism Structure

Sibutramine

monoamine reuptake inhibitor:serotonin, norepinephrine receptor affinity

Orlistat gastric lipase inhibitor

System Mechanism Drug Structure

Adrenergic agents Stimulates norepinephrine release

Amphetamine

α1 agonist Phenylpropanolamine

Stimulates norepinephrine release

Phentermine

Stimulates norepinephrine release

Diethylpropion

Blocks norepinephrine reuptake

Mazindol

Serotonergic agents Stimulates 5-HT release

Fenfluramine

Stimulates 5-HT release

Dexfenfluramine

NH 2

Me

NHEt

F C 3

Me

NH 2

Me

OH

NH 2

Me Me

NHEt

F C 3

Me

NEt 2

Me

O

Cl

HO

N

N

Cl

N

Me

Me Me Me

Me H

HN

O O

O

nC6H

nC6H

O

O

Me


223

Table 3: Some recent endogenous agents that effects feeding

Increase Feeding Decrease feeding Galanin Amylin ; cholecystokinin ; Cytokines Gamma-amino butyric acid Corticotropin releasing hormone Growth hormone releasing factor Dopamine ; Enterostatin ; Glucagon Neuropeptide Y Glucagon like peptide -1 Norepinephrine Insulin ; Leptin ; Neurotensin

Opiates : Dynorphin, β-endrophin Serotonin

Fig. 1: Control of feeding in humans illustrating the redundant and interconnected nature of central and

peripheral mechanisms

Fig. 2: The role of leptin and neuropeptide Y (NPY) in the signalling pathway are shown, as are antiobesity targets

at various stages of the cycle (in circle)

Hypothalamus Neuropeptide Y Orexins Pituitary

α-MSH

CNS

Periphery Adrenals Cortisol

Adipose Tissue Leptin PPARγ β3-adrenergic receptors UCP-1,2

GI Tract CCK Bombesin GLP-1 Enterostatin


224

Ser-152 of Ser-152 and orlistat acylderivative;

pancreatic Lipase very slowly hydrolyzed.

Fig. 3.

Fig. 4: When activated by cold, uncoupling proteins (UCPs) let hydrogen ion pass through the inner mitochondrial

membrane, thereby abolishing the hydrogen ion gradient needed to drive ATP Synthesis. Nucleotide

binding prevents the UCP from doing that

(1) (2) (3)

(4)

OH

N

O

OO

nC6H

13

O

OH O

nC6H

13

O

N

H

Me

NClOH H

C OMe

O

Me

NClOH H

O C

O

OMeO

NOH H COOH

H

O

O

COONa

COONa

NOH

Cl

MeO

ON RN

N

N

OH H

NS

O

H


225

N N

N

N NS O

O

O

OH

H

H

(5)

N N

N

N

OH H

NS

O

H

O

O

(CH2)5 CH3

(6) (7)

O

HS N

OH

N

CH3

(CH2)3COOH

O

OH

H

10 R = H

11 R = NHC(O)NH-n-C6H13

O C

O

OH

NR

(12)

N

N

M eO C H2O

Ph

C2H

5

N H

CN

O H

NH2

N

N H O

CO

P hP h

H

(13) 14 (14)

O N

O

O H

O H H

R

O

N H ( C H2) O M e

8 R = O H

9 R =


226

N

N

N

N

O

O

Ci

H

(15)

Gly TrpMet

OHOSO

3

-Met-Asp-(N-Met)-Phe-NH2

(22)

Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH

2

(19)

Nle

OHOSO3

Gly Trp Nle-(N-Me)Asp Phe NH2

(23) (24)

NN

N

NO

OPh

H

N

MeO O

i-Pr

N

N

N N HPh

O

OH

Ph

Ph(i-Pr)N

O

O

N

N

NH

NH

OMe

MeO(CH2)2O

(18)

(20)

NH

N

NH

H

O

MeO

OMeH

NHN

NO

O

N

O

CH2

H

(17)

(16)

((21)

NH

NH

CH3

CONH

O

Asp (N-Me)Phe NH2Tryboc


227

terminal region of NPY. Cyclohexyl compound (14) have 39 nm IC50 at the Y1 receptor. Benzimidazole (15) has a 1.7 nm IC50 at the Y1 receptor. Quinazoline (16) had low nM Y5 receptor affinity. Aminopyrazole (17) has 8.3 nM IC50 at the Y5 receptor. The amide derivative (18) is the most potent NPY5 receptor antagonist described in the literature. Glucagon-Like Peptide-1 Agonist: Glucagon like peptide-1 (7-36) amide (GLP-1) is a 29 amino acid peptide prepared by differential posttranslational processing of the preglucagon gene product found in the hypothalamus of the preglucagon gene product found in the hypothalamus and produces reductions in feeding in fasting animals17. GLP-1 is important in glucose homeostasis after the ingestion of meals comprised of carbohydrates via modulation of gastric emptying and alteration of the secretion of insulin and glucagons from the pancreas. Receptors for GLPI are concentrated in the paraventricular nucleus of the hypothalamus, the amygdala, and regions of the brain stem. GLP-1 may act as an anorectic agent and reduce food intake when injected intracarebroventricularly in rats. Pretreatment with GLP-1 antagonist, exendin (9-36) amide, blocks this effect18. Galanin: Human galanin is a 30 amino acid peptide found in the brain19. When galanin is injected into the hypothalamus of satiated rats, it stimulates food intake, with a preference for the intake of dietary fact. Galanin also reduces energy expenditure and sympathetic activation of BAT. However, chronic administration of galanin does not appear to induce the obese state in rodents. Antagonists such as M35 and M40 are very potent and can block galanin induced and natural food consumption in rats. Enterostatin: Enterostatin is the active pentapeptide derived from procolipase, a 101 amino acid peptide that is synthesized in the pancreas and stomach. It

selectively inhibits fat intake during both normal feeding and in experimental paradigm that involved dietary choice. By either peripheral or central administration, it selectively reduces the intake of food20. Small molecule enterostatin agonists may find promise in the treatment for obesity. Bombesin: Bombesin is a tetradecapeptide originally isolated from the skin of the European frog Bembina bombina, and it is found throughout the mammalian CNS and GI tract. Administration of bombesin, either centrally or peripherally, inhibits food intake in animal models. Bombesin and bombesin like peptides bind to specific GPRCs and it was recently reported that mice lacking the bombesin-3 receptor developed a mild obesity, associated with hypertension and impairment of glucose metabolism. Thus, this receptor subtype is likely to be required for proper regulation of energy homeostasis and adiposity. Bombesin agonists acting at this site may be useful new therapeutics, perhaps for increasing metabolic rate in those individuals with a genetic propensity for a lower basal rate of energy expenditure21. Cholecystokinin-A Agonists: Cholecystokinin (CCK) the 33 amino acid peptide found in the CNS and GI Tract reduces meal size by the inhibition of gastric emptying and by activation of the vagal afferents which exert control over the hypothalamus mediated through CCK-A and CCK-B receptor subtypes. While a variety of biologically active forms of CCK have been identified, the truncated C-terminal octapeptide CCK-8 (19) retains full biological activity. CCK-B receptors are located primarily in the CNS whereas CCK is involved in anxiety and pain. CCK-A receptors are found mainly in the periphery and also in the brain. CCK aids the digestion of nutrients by inducing gallbladder contraction, pancreatic secretion, delayed gastric emptying, and satiety. The release of CCK-8


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from the small intestine following nutrient ingestion triggers these effects at CKK-A receptors on vagal afferents which relay the satiety signal to the brain. A 71378 (20) was potent and selective CCK-A agonist. The tetrapeptide A71623 (21) is a potent and selective agonist at the CCK-A receptor22 Compound ARL 15849 (22) is 6600 fold selective for CCK-A versus CCK-B with improved stability and longer duration23 1,5-Benzodiazepine was first nonpeptide CCK-agonist reported (23). Replacing C-3 phenylamide of (23) with an indazolylmethyl group lead to GW5823 (24). The compound is the first CCK-A agonist to demonstrate oral activity in a rat-feeding model. CCK-Inactivating Peptidase Inhibitors: A CCK-inactivating serine peptidase has recently been identified. It is a membrane- bound isoform of tripeptidyl peptidase II. Whose function was not previously known. The peptidase was found in neurons that responded to CCK as well as in nonneuronal cells. It inactivates CCK-8 in two steps that corresponded to CCK-8 → CCK-5 (gly Trp- Met- Asp-Phe-Nh2) → (Gly-Trp-Met). Both fragments are biologically inactive. Inhibitors of the peptidase have been designed in a rational manner resulting in the discovery of butabindide. Butabindide (25) inhibits the CCK-inactivating peptidase competitively and selectively without activity in CCK receptor binding assays. When 25 was administered intravenously to starved mice, a significant satiating effect was observed that was selectively blocked by devazepide, a CCK–A receptor antagonist. Butabindide was also shown to significantly reduce food intake in rats24. Amylin: Amylin, a 37 amino acid peptide, is a hormone which is released by the β- cells in the pancreas along with insulin. It is similar in structure to calcitonin and the calcitonin gene related peptides, and these peptides have similar effects on carbohydrate metabolism. Amylin inhibits food intake in a variety of rodent-feeding models, which provides support for the role of amylin as a peripherally acting satiety factor. Leptin: The product of the Lep gene, leptin, discovered in 1994, is a 167-amino acid protein expressed in white adipose tissue, in gastric epithelium and placenta. When leptin was administered to obese mice, which are leptin-deficient, a sharp decrease in weight occurred via reduced food intake, increased oxygen consumption, and body temperature elevation.

No weight loss was observed when leptin was administered to diabetic mice (Leptin receptor-deficient). Obesity in humans is generally associated with high leptin levels, which suggests that human obesity correspond more to the diabetic mouse leptin-resistant state then to the obese mouse leptin-deficient state25 . One notable difference between obese and lean individuals is that the majority of leptin in plasma is bound to plasma proteins in lean subjects and is free in obese subjects. Insulin , steroid hormones and noradrenaline are important regulators of leptin production and secretion. Circulating leptin signals the CNS first to rapidly increase sympathetic outflow and then to inhibit food intake. The sympathetic nervous system increase lipolysis, thermogenesis and energy expenditure while it suppresses pancreatic insulin secretion and leptin expression in white adipose tissue

through the β3- adrenergic receptors . Mice in which the leptin gene was disrupted, and mice and rats with genetic disturbances of leptin receptor signaling show hyperphagia, hyperinsulinemia and decreased sympathetic outflow, leading to obesity early in life. Recently, another anorectic peptide affecting the leptin system has been described. The hypothalemic peptide cocaine- and amphetamine- regulated transcript (CART) has been shown to be a satiety factor that is closely associated with the actions of leptin and NPY . FUTURE DRUG TARGETS

MCH-1 antagonist: Melanin-concentrating hormone (MCH) is a cyclic, 19-amino-acid neuropeptide produced in the lateral hypothalamus. MCH is implicated in a number of physiological processes including feeding behavior, energy balance, reproductive function, memory, sleep- wake regulation and emotional states. Since the discoveries in the mid-1990s that MCH is an appetite stimulant in rodents and mice that lacking MCH become lean, together with the findings that MCH receptor 1 (MCHIR) is responsible for these functions, most research has been focused on the roles of the MCH system in feeding and energy homeostasis. Some pharmaceutical companies have reported nonpeptidic MCHIR antagonists with diverse structural features in the treatment of obesity. Substituted phenyl biaryl urea derivatives were synthesized and evaluated as MCH-R1 antagonists for the treatment of obesity26.


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MC-4 Receptor agonists: Studies using nonselective agonists and antagonists of melanocortin-3 receptor (MC3R) and MC4R point to the importance of the CNS melanocortin system in the control of food intake. α-Melanocyte stimulating hormone (MSH), synthesized within ARCPOMC neurons, is an agonist of MC3R and MC4R that, after exogenous central administration, robustly decreases food intake in rats. MT II is a synthetic analog of α-MSH that also has agonistic properties at both MC3R and MC4R and decreases food intake and body weight of mice and rats. Results support the hypothesis that the brain MC4R is intimately involved in the control of food intake and body weight and provide evidence that selective activation of MC4R causes anorexia that is not secondary to aversive effects27. Growth hormone fragments: A small synthetic peptide sequence of human growth hormone (hGH), hGH (177-191) or AOD-9401, has lipolytic and antilipogenic activity similar to that of the intact hormone when C57BL/6J (ob/ob) mice were orally treated with either saline (n=8) or AOD-9401 (n=10) for 30 days, from day 16 onward, body weight gain in AOD-9401-treated animals was significantly lower than that of saline treated controls. Food consumption did not differ between the two groups. Analyses of adipose tissue exvivo revealed that AOD-9401 significantly reduced lipogenic activity and increased lipolytic activity in this tissue. Increased catabolism was also reflected in an acute increase in energy expenditure and glucose and fat oxidation in ob/ob mice treated with AOD-9401. In addition, AOD-9401 increased in vitro lipolytic activity and decreased lipogenic activity in isolated adipose tissue from obese rodents and humans. Together these findings indicate that oral administration of AOD-9401 alter lipid metabolism in adipose tissue resulting in a reduction of weight gain in obese animals. The maked lipolytic and antilipogenic action of AOD-9401 in human adipose tissues suggest that this small synthetic hGH peptide has potential in the treatment of human obesity. PTP-1B inhibitors: Coordinated tyrosine phosphorylation is essential for signaling pathways regulated by insulin and leptin. Obesity and type-2 diabetes are characterized by resistance to hormones insulin and leptin, possibly due to attenuated or diminished signalling from the receptors.

Pharmacological agents capable of inhibiting the negative regulators of the signalling pathways are expected to potentiate the action of insulin and leptin and therefore be beneficial for the treatment of obesity and type-2 diabetes. A large body of data from cellular, biochemical, mouse and human genetic and chemical inhibitor studies have identified protein tyrosine phosphatase 1B (PTP1B) as a major negative regulator of both insulin and leptin signalling. In addition, evidence suggests that insulin and leptin action can be enhanced by the inhibitors of PTP1B28 Consequently PTP1B has emerged as an attractive novel target for the treatment of obesity and Type-2 diabetes. DGAT inhibitors: Because the ability to make triglyceride is essential for the accumulation of adipose tissue, inhibition of triglyceride synthesis may ameliorate obesity and its related medical consequences. Acyl Co A: diacylglycerol acyltransferase (DGAT1) is a key enzyme in the mammalian triglyceride synthesis pathway. Mice lacking DGAT1 are resistant to obesity and have increased sensitivity to insulin and leptin. DGAT-1 deficient mice are also resistant to diet induced hepatic steatosis. The effect of DGAT1 deficiency on energy and glucose metabolism result in part from the altered secretion of adipocyte- derived factors. Although complete DGAT1 deficiency causes alopecia and impairs development of mammary gland, these abnormalities are not observed in mice with partial DGAT1 dificiency29. These findings suggest that pharmacological inhibition of DGAT1 may be a feasible therapeutic strategy for human obesity and type-2 diabetes. Thyroid hormone β3 receptor agonists: Thyroid hormones (TH) are potent modulators of adaptive thermogenesis and can potentially contribute to development of obesity . The decrease of T3 in association with reduction of calorie intake is centrally regulated via decreases in leptin and melanocortin concentrations and peripherally via a decrease in deiodinase activity, all aimed at protein and energy sparing. The use of TH in the treatment of obesity is hardly justified except in cases of elevated thyrotropin (TSH) with low/normal T3 and T4 and/or a low T3 or a high TSH/T3 ratio. TH treatment with small doses of T3

can also be exceptionally applied in obese patients resistant to dietary therapy who are taking β-adrenergic


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blockers or with obesity developed after cessation of cigarette smoking and with hyperlipidemia and a concomitant high thyrotropin/T3 ratio. The increased β-3 receptor expression in BAT (Brown Adipose Tissue) is reversed by administration of T3 within 24h in analogy to the situation in thyrotoxicosis where β-3 adrenoceptor virtually disappear as a part of a compensatory mechanism preventing overstimulation of thermogenesis. In addition to the interaction taking place in the periphery, interactions at the central level may also contribute to thermoregulation. Hypothyroidism is associated with increased sympathetic activity as reflected by increased plasma nor epinephrine concentration and urinary excretion, the opposite being observed in hyperthyroidism. In the last case TH, by increasing obligatory thermogenesis reduces the need for facultative thermogenesis30. Conclusion: Agents which reduce body weight have been actively sought for many decades. Because of the multitude and complexity of disturbances in energy intake, expenditure and partitioning that are associated with obesity, it has been difficult to determine which abnormalities are causative versus less important phenomenon that are consequences of the altered neuroendocrine and metabolic milieu. Due to risk of abuse and chemical dependence, the amphetamine type mechanism (adrenergic agents) is now disfavoured. Activation of serotonergic system or inhibiting serotonergic reuptake has proved successful. The major

problem with β3-adrenergic receptor agonists is

its cross reactivity at β1− and β2− ARs and its value will become apparent once clinical evaluations are available. Transgenic approach has provided new paths to modify the complex body weight regulating system and to assess the relative importance of the individual components. CCK agonist, bombesin and NPY antagonist appears to act at more fundamental level and might be useful as therapeutics. Increasing help of molecular biology and advanced gene targeting strategies will obviously led to a better understanding of the role gene plays in body weight regulation. Regardless of whether the impact of obesity is judged by the personal dissatisfaction of obese individuals and their families with their weight and shape, or by the cost to public healthcare system, the problem needs to be taken seriously.

Acknowledgement: Authors wish to thank the Director, CDRI, Lucknow for providing library facilities. References : 1. Triscari J, Tilley J, Hogan S. The pharmacological

treatment of obesity. Annu. Rep. Med. Chem. 1988; 23: 191-200.

2. Silverston T. Appetite Suppressants: a review.

Drugs. 1992; 43, 820-836. 3. Grignaschi G, Samanin R. Role of 5-HT receptors

in the effect of d-fenfluramine on feeding patterns in the rat. Eur. J. Pharmacol. 1992; 212: 287-289.

4. Berkowitz DE, Nordone NA, Smiley RM, Price DT, Kreutter DK, Fremeou RT, Schwinn DA. Distribution of β3 adrenoceptor m-RNA in human tissue. Eur. J. Pharmacol. 1995; 289: 223-228.

5. Emorine LJ, Marullo S, Sutren MMB, Patey G, Tate K, Klutchko CD, Strosberg AD. Molecular characterization of the human β3 adrenoceptor . Science. 245: 1118-1121.

6. Gura T. Uncoupling proteins provide a clue to obesity causes. Science. 1998; 280: 1369-1370.

7. Strosberg AD. Structure and function of β3 adrenergic receptor. Annu. Rev. Pharmacol.Toxicol. 1997; 37: 421-450.

8. Dow RL. β3- Adrenergic Agonist: Potential therapeutics for obesity. Exp. Opin. Invest. Drugs. 1997; 6: 1811-1825.

9. Arch JRS, Ainsworth AT, Cowthorne MA, Piercy V, Sennitt MV, Thody VE, Wilson C, Wilson S. Atypical β adrenoceptor on brown adipocyte as target for anti-obesity drugs. Nature. 1984; 309: 163-165.

10. Umekawa T, Yoshida T, Sakane N, Kondo M. A highly specific β3 adrenoceptor agonist, on lypolysis of human and rat adipocytes. Horm-Metab. Res. 1996; 28: 394-396.

11. Leo A, Mueller M. Phenoxypropanolamines. Eur. Pat. Appl. EP14023, 1985; Chem. Abstr. 1986; 104: 68565.

12. Howe R, Rao BS, Holloway BR, Stribling D. Selective β3- Adrenergic Agonists of brown adipose tissue and thermogenesis. J. Med. Chem. 1992; 35: 1751-1759.

13. Weber AE, Mathvink RJ, Perkins L, Hutchins JE, Candelore MR, Tota L, Strader CD, Wyvratt MJ, Fisher MH. Potent, selective benzenesulphonamide


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agonists of the human β3 adrenergic receptor. Bioorg. Med. Chem. Lett. 1999; 9: 749-754.

14. Fisher LG, Sher PM, Skwish S, Michel IM, Seiler SM, Dickinson KEJ, A potent, selective and novel heterocyclic β3 adrenergic receptor. Bioorg. Med. Chem. Lett. 1996; 6: 2253-2258.

15. Cheryl P Kordik, Allen B Reitz, Pharmacological treatment of obesity: Therapeutic strategies. J. Med. Chem. 1999; 42: 193-194.

16. Doods HN, Wieland HA, Engel HA, Engel W. The first selective neuropeptide y1

receptor antagonist: A review of its pharmacological properties. Regul. Pept., 1996; 65: 71-77.

17. Alessio DA, Thirlby R, Laschansky EC, Zebroski H, Ensinck JW. Response of GLP-1 to nutrients in humans. Digestion. 1993; 54: 377-379.

18. Goke R, Lorsen PJ, Mikkelsen SP, Skeikh SP. Distribution of GLP-1 binding sites in the rat brain: Evidence that exendin is a ligand of brain GLP-1 sites. Eur. J. Neurosci. 1995; 2294-2300.

19. Kask K, Berthold M, Bartfai T. Galanin receptors: Involvement in feeding, pain, depression and Alzheimer’s disease. Life Sci. 1997; 60: 1523-1533.

20. Erlanson-Albertsson C. Enterostatin- A peptide regulating fat intake. Obes. Res. 1997; 5: 360-372.

21. Ohlki-Hamazaki H, Watase K, Yamamoto K, Ogura H, Yamano M, Yamada K, Maeno H, Imaki J, Kikuyama S, Wada E, Wade K. Mice lacking bombesin receptor subtype-3, develop metabolic defects and obesity. Nature. 1997; 390: 165-169.

22. Lin CW, Holladay MW, Witte DG, Miller TR, Wolfram CAW, Bianchi BR, Bennett MJ, Nadzan AM. CCK agonist with high potency and selectivity for CCK-A receptors, Am. J. Physiol. 1990; 258: G648-G651.

23. Simmons RD, Kaiser FC, Pierson ME, Rosemond JR. A selective CCK-A agonist with anorectic

activity in the rat and dog. Pharmacol. Biochem. Behav. 1998; 59: 439-444.

24. Rose L, Vargas F, Facchinetti P, Bourgeat P, Bambat RB, Bishop PB, Chan SMT, Moore ANJ, Ganellin CP, Schwartz JC. Characterization and inhibition of a cholecystokinin- inactivating serine peptidase. Nature. 1996; 380: 403-409

25. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM, Weight reducing effects of the plasma protein encoded by the obese gene. Science. 1995; 269: 543-546

26. Timothy J Kowalski, Mark D Mc Briar. Therapeutic potential of melanin- concentrating hormone-1 receptor antagonists for the treatment of obesity. Expert Opin. Investig. Drugs. 2004 ;13:1113-1122.

27. Stephen C Benoit, Michael W Schwartz et. al. A novel selective melanocortin-4 receptor agonist reduces food intake in rats and mice without producing aversive consequences. The J. of Neuroscience. 2000; 20(9): 3442-3448

28. Inada S, Ikeda Y, Suehiro T, Takata H, Osaki F,

Arii K, Kumon Y, Hashimoto K, Glucose enhances protein tyrosine phosphatase 1B gene transcription in hepatocytes. Molecular

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29. Ji H, Friedman M I, Reduced capacity for fatty acid oxidation in rats with inherited susceptibility to diet-induced obesity. Metabolism. 2007; 56:1124-1130.

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USFDA Generic Approval Process Nitin Saigal, Sanjula Baboota, Alka Ahuja and Javed Ali*

Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar,

New Delhi – 110 062, India.

*Author for correspondence

[email protected], [email protected]

ABSTRACT

The USFDA generic approval process is the most fundamental aspect of the pharmaceutical industry which

should be proverbial to every scientist stepping up in the generic or the innovator drug industry. The world

pharmaceutical industry orbits around the provisions made in the Hatch Waxman Act by the US congress in

1984 which gave birth to the generic drug industry. The act being passed with a vision of maintaining a balance

between the brand name and the generic drug industry and above both the consumers, still allows some

ambiguities and loopholes in the form of some anticompetitive practices which requires resolution. The purpose

of this manuscript is to bring into the light of the pharmaceutical scientists and academicians with the

provisions of this act, the deficiencies involved with case studies and the overall criteria and process of the

FDA for approving generic drug products.

KEYWORDS: Hatch Waxman Act, NDA, ANDA, Generic, Innovator, Brand name manufacturer, Generic

Manufacturer, Lawsuit, Infringement, ANDA Para certifications, f2 value, Bioequivalence

INTRODUCTION

The generic drug is a safe, effective and economical substitute of a brand name drug product. The act which surrounds the generic drug approval process of the USFDA is the “Hatch Waxman Act of 1984” which we also identify by the “Drug price control and Patent Term Restoration Act of 1984” which led to a plethora of generic drugs entering into the market. Early before the passage of the act the pharmaceutical market was ruled by the brand name drug manufacturers which we also affectionately call as the Innovators. The generic drug manufacturers seldom used to compete with them and invest their funds for the already existing brands in the market. There was a lack of competition and the innovator enjoyed his elite monopoly period within which no other brand of the drug can enter into the market. This exclusivity period also allowed the innovator to quote highest prices of the drugs. To stop these practices and to make a user friendly drug market, the US congress passed the Hatch Waxman Act in 1984 which was regarded as one of the most successful

segments of the US legislation at that time. The act was passed taking into consideration the interests of both the innovator and the brand name drug manufacturer. However the provisions of the act are maneuvered by some crafty brand name manufacturers and also associated it with some anticompetitive practices which are discussed later in the manuscript. Overview of the world pharmaceutical industry:

Global pharma market is worth $643 billion (2007) with a 7% annual growth rate with a projected size of $812 billion in 2010, the key contributors being the United States, Japan, Germany, France and UK. If we see the contribution of the generic market, we see an even better growth of 11% with an expected growth of 13-15% by 2010 and the projected size of the generic industry is $93 billion by 2010 from a $55 billion in 2006 with a 14% cumulative annual growth rate. In addition, the patent expired molecules valued at $102 billion in 2006 are expected to grow to $160 billion in 2010 and the products loosing patent protection in 2006 valued in excess of $18 billion. Emerging markets, including China, India, Brazil and Turkey, showed


APTI ijper


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growth over 10 percent in 2007, largely due to their growing economies and broader access to medications. Growth in China will be 15 - 16 percent and the market size will reach $15 - 16 billion in 2010. Generally, locally manufactured generics dominate these markets1. The data in Table 1 as per the year 2007 shows that the generic industry is growing at a rate faster than the overall pharmaceutical industry, the majority of reasons being economic changes, changing consumer demands for low prices high quality drugs, changing societal values, significant health-care demands, changing composition of workforces and the major contribution being the increase in competition amongst the generic and the brand name industry which has come due to the change in the USFDA approval process from a slow time consuming to a comparatively faster process. The driving force of the generic industry being the price control and quality management leading to easy acceptance by the consumer, market diversification, regulatory involvement and the provisions of the act to challenge the validity of the patents issued to the innovator drug companies. Indian relevance to the global generics industry: As per the Indian perspective, the knowledge of the generic approval process is mandatory for the scientists stepping up in the Indian pharmaceutical industry. Majority of companies in India namely Ranbaxy, Dr Reddys, Cipla, Wockhardt, Sun Pharmaceuticals, Glenmark, Lupin, Piramal, Torrent and Matrix laboratories etc. are working on generics and most of them run their ground operations in the US and other countries. Indian companies are committed to provide quality generics at affordable prices to the patients worldwide with a view to help bring down the healthcare costs. Whilst Indian companies continue to enhance the momentum of the generics business in over 125 markets, these companies are also accelerating drug discovery program through collaborations and alliances. India is a potential place for top quality generics due to which we have company like Ranbaxy placed amongst the top 10 global generics players with US being the company’s largest market. India remains an important market for the majority of Indian companies. The indigenous industry supplies around 70% of the country’s pharmaceuticals. The proportion of revenue derived from India depends largely on the strategy of

the individual company and its penetration into the generic markets overseas. BIRTH OF GENERICS: THE HATCH WAXMAN

ACT OF 1984

The act which gave rise to the generic drug industry was passed as amendments of the Federal Food, Drug, and Cosmetic Act (FFD & C). The main objectives of the act were to: 1. Offset the drug approval delay: Early before the

passage of this act, the innovators used to encounter a delay in approval of their New Drug Applications (NDA) for brand name product which eventually affected their brand entry in the market and financial losses.

2. Bring healthy competition and to avoid price

rise: Early before its passage there was no scope of the generics to enter the market until the patent term of the innovator has expired as no provisions of challenging the validity of the innovator patent was there. Also there were not many benefits expected by the generic in investing capital to re-launch another version of already existing brand of the drug after its patent expiry. This product monopoly kept competition away and the drug prices high as per the innovators will.

3. Make life easier for the generic: Generic manufacturers used to face a lot of difficulties in doing all the pre-clinical and clinical studies as done by the brand name manufacturers to get their generic approved.

The act was passed keeping one eye on the interests of both the brand name and the generic drug manufacturers providing almost equal benefits to both. The benefits received by the brand name manufacturers are: 1. Regulatory delays in brand approval were

deciphered. 2. The market exclusivity and the ‘effective patent

life’ (the time left when the drug comes into the market after drug development and FDA approval) was extended from 8.5 years (1980) to 13 years (late 1980s) and now it’s nearly 16-17 years.

The benefits received by the generic manufacturers are: 1. The generic could challenge the validity of the

patent issued to the innovator, 2. The act provided with the stipulation to file an

Abbreviated New Drug Application (ANDA) and


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to leave out the expensive pre-clinical and clinical trials as done by the innovator and provided with the provision of relying on the safety and efficacy data of the innovator drug and to perform a much cheaper substitutable study called ‘bioequivalence’ to establish equivalence with the innovator.

3. As per the act, once approved the generic gets an exclusivity period of six months to rule the market along with the innovator during which no other generic can be given approval.

The FDA publishes the information on every approved drug product in the “Approved Drug Products with Therapeutic Equivalence Evaluations” which we also know by the name of “Orange Book”. It lists the NDA’s and ANDA’s along with the expiry dates of patents and generic exclusivities. It is a ready reference of brand name drug products for the generic companies who usually use this information to identify the reference for developing their generic versions. The Orange book also contains certain therapeutic equivalence codes. An A- prefixing product is considered to be substitutable and B- prefixing is a safe and effective product for use but is regarded in-equivalent and non-substitutable with the brand name drug product2. ANDA CERTIFICATIONS

While filing an ANDA, the generic manufacturer is required to file one of the following four possible certifications on the subject of the reference brand name patent listed in the Orange Book. 1. Para I certification: There is no patent for the

drug listed in the orange book 2. Para II certification: Patent is listed but has

expired 3. Para III certification: Patent is listed, is valid but

the generic wants approval to market the drug once the pertinent patent expires

4. Para IV certification: The generic manufacturer either challenges the validity of the brand name listed patent asserting it to be invalid or fake, or it affirms not to cross the fringe of the brand name patent claims. Para IV certification is the most critical of all and gives rise to almost all the anti-competitive practices associated with the Hatch Waxman amendments of the FFD and C act.

USFDA APPROVAL REQUIREMENTS: BRAND

NAME (NDA) VS. GENERIC (ANDA)

Many of the points for the requirement for generic and brand name drug approval are the same (Table 2). The generic must have the same active ingredient, same route of administration, same dosage form, with same strength and condition of use. Chemistry (components and composition), Manufacturing, and Controls (specifications and tests, packaging and stability) are rigorously reviewed by both the new drug reviewers and the generic drug reviewers. Labeling is also reviewed and has to be identical in most aspects (generic may delete portions of labeling protected by patent or exclusivity, bottle size need not be the same). The same FDA field inspectors inspect the manufacturing facilities for generics and for the innovator products. These facilities must be up to date with respect to good manufacturing practices or GMPs and must have documentation that they are able to manufacture the products for which they have applications. The differences in the applications, as previously stated, are the clinical studies and animal studies required for the new drug application and the bioavailability studies that have to be conducted to define the drug’s interactions and adverse events. A surrogate for these studies is the bioequivalence study that has to be submitted for the ANDA. Showing in the bioequivalence study that the active ingredient is absorbed at the same rate and extent as the reference product allows the generic to rely on the findings of safety and efficacy of that product2. USFDA APPROVAL PROCESS (PARA IV

CERTIFICATION)

A generic manufacturer must warn the brand name manufacturer of his course of action of filing a Para IV certification against the innovator along with legal grounds explaining that the existing innovator patent (s) is/are invalid or will not be violated by the generic. This gives an immediate right to the innovator to file a patent infringement lawsuit against the generic manufacturer even if the innovator becomes aware of the authenticity of the generic claim through the legal grounds proposed by the generic manufacturer. The innovator can file this lawsuit within 45 days of the ANDA filing notice from the generic. If it does, this sets in a process which detains the FDA from giving approval to the generic for a period of 30 months, which means the innovator can


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enjoy the monopoly status for 2.5 years regardless of the virtues of the suit. Generic manufacturers should make Para IV certification only if they are sure of their genuineness or the innovator’s counterfeit, else will have to pay the associated damages to the patent holder for the litigation. FDA is free to grant approval after 30 months which depends upon the result of the pending litigation3. An important point to be noted is that USFDA only grants approval, the litigation cases are dealt within the courts and finally if everything goes right in favor of the generic, patent is granted by the USPTO. Generic is free to enter the market if the approval and patent is granted but may also opt to withhold his product from the market if the litigation is still pending. If the generic enters the market and then loses the court case, it will have to pay huge damages levied by the innovator for his market losses. The apprehensions and associated foreboding forces the generic to wait for the proceedings to be over, which adds on the time for which the innovator rules the market. The six month generic exclusivity:

The Hatch Waxman Act offers with an incentive to the ‘first approved generic’ after filing a Para IV certification. This incentive gives the first generic an exclusivity period of six months to market, earn profits and to recover the cost of litigation (if any). During this period the brand name is the only competition the generic has to face and the FDA cannot approve any other generic until this generic exclusivity period perishes. The 180 day cycle begins on the day the generic comes into the market for the first time or the day of courts decision in favor of the generic. It is entirely up to the generic when to start this clock; he may enter the market before or after the litigation is over. If the generic manufacturer does not market his drug because of the pending court decision, it may lead to protracted and indefinite delays in the beginning of the generic exclusivity period which directly will benefit the innovator. So the risk is more or less associated with generic manufacturer opening one of the discrepancies in the Hatch Waxman Act LOOPHOLES IN HATCH WAXMAN ACT AND

CASE STUDIES

The Hatch Waxman act was introduced to benefit the consumers with a vision of providing equally safe and effective medicines as is the brand name at a cheaper

cost. However some devious brand name manufacturers have contrived the provisions to take advantages of the pitfalls leaving the generic manufacturers and the consumers sniveling. The major loopholes3 in the act which have been explored by the brand name manufacturers to gather full advantage are: 1. Warehousing patents and frivolous lawsuits:

The Buspar case (BMS vs Mylan Pharmaceuticals relates to both the Para III and Para IV certifications. Bristol Mayer Squibb (BMS) Co had patent number 4182763 with a primary claim of buspirone as an anxiolytic agent. This patent expired on July 21, 2000, and Mylan Pharmaceuticals filed a Para III certification and obtained a tentative approval to market the generic version of the drug not before July 22, 2000. However, BMS obtained another patent 365 just 12 hours before the expiry of its first (763) patent claiming the active metabolite of buspirone as responsible for the anxiolytic effect. The patent was for the combination of the chemical compound in the drug with stomach acid which was produced naturally. The patent was listed in the Orange Book and as per the laws was informed to Mylan that the ANDA was incomplete and needed justification that its generic version of buspirone would not infringe upon the new 365 patent. Mylan in anticipation filed a Para IV certification under the Hatch Waxman Act which led to the patent infringement lawsuit against Mylan immediately halting the final market approval of the generic and the associated stays and delays. FDA could not approve the marketing of the generic that Mylan had already loaded on trucks for shipment4, 5, 6. It is also referred to as Evergreening or

Warehousing of patents. An innovator may patent multiple attributes of a product (may be color, manufacturing process or the chemicals produced when the drug is ingested in the body) and keeps on adding patents in the Orange Book, essentially forcing the generic to hose between waiting for the patents to expire or file a Para IV certification which brings along the risks of litigation and associated costs and delays. The provision of filing a patent infringement lawsuit gives the brand name manufacturer at least an additional two and a half


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years of product monopoly has resulted in a wave of such lawsuits. 2. Maneuvering the six month Generic

Exclusivity: The practices commonly referred to as ‘Sweetheart deals’ allow the first generic manufacturer and the innovator to enter into an agreement and to have a control when to introduce the generic. This would not trigger the 180 days generic exclusivity period offered to the first generic and the FDA is bound not to approve another generic until the 180 day generic exclusivity period perishes. This will suspend the competition for an indefinite period of time. This extended brand name monopoly allows the brand name and the generic manufacturer to share the financial awards keeping other generic competitors in an indeterminate state. Cardizem CD (diltiazem hydrochloride) is a prescription drug manufactured by Hoechst-Marion Roussel, Inc. (“HMRI”) that is used to treat angina and hypertension. The Cardizem litigation arises primarily out of an interim “settlement” of patent infringement litigation brought by HMRI against Andrx Pharmaceuticals, Inc., the first ANDA filer for Cardizem CD. In September 1997, the FDA tentatively approved Andrx’s generic Cardizem product. Thus, Andrx would have been expected to be able to enter the market upon the expiration of the 30-month stay in July 1998. However, in settling the patent infringement litigation, HMRI and Andrx allegedly agreed that, beginning upon final FDA approval of Andrx’s generic product, HMRI would pay Andrx $10 million per quarter not to enter the market with its generic product until the conclusion of the patent litigation7. Hytrin (terazosin hydrochloride) is a drug manufactured by Abbott Laboratories (“Abbott”) for the treatment of high blood pressure and enlarged prostate. The Hytrin litigation involves agreements between Abbott and Geneva Pharmaceuticals, Inc. (“Geneva”) and between Abbott and Zenith Goldline Pharmaceuticals, Inc. (“Zenith”) to settle patent infringement litigation relating to Hytrin. The agreement between Abbott and Geneva was an “interim”settlement agreement that did not finally resolve the patent issues. Geneva allegedly agreed to accept $4.5 million per

month from Abbott to refrain from marketing any generic Hytrin product (including Geneva’s approved capsule, which was not at issue in the infringement lawsuit) until another drug maker sold a generic version of Hytrin in the United States or Geneva received a final, unappealable judgment that its proposed generic tablet did not infringe Abbott’s patents. As part of this agreement, Geneva and Abbott allegedly agreed to continue the patent infringement litigation on Geneva’s generic tablet8. Cipro (Ciprofloxacin hydrochloride) between Bayer Corporation (“Bayer”), Barr Laboratories (“Barr”), the Rugby Group (“Rugby”), and Hoechst-Marion Roussel, Inc. (“HMRI”) to settle patent litigation related to Bayer’s patent covering ciprofloxacin hydrochloride (the “‘444 patent”)9. Monodox (doxycycline monohydrate) is an antibiotic manufactured by Watson Pharmaceuticals, Inc. (“Watson”). This litigation arises out of an agreement between Watson and Halsey Drug Co. (“Halsey”), in which Watson acquired Halsey’s ANDA for generic Monodox10. Procardia XL (nifedipine) is a prescription drug manufactured and sold by Pfizer, Inc. (“Pfizer”) to treat angina and hypertension. The antitrust claims in this litigation involve an alleged agreement between Pfizer and Mylan Pharmaceuticals, Inc. (“Mylan”) to settle patent infringement litigation in 200011. Nolvadex (tamoxifen citrate) is a drug sold by AstraZeneca that is used to treat breast cancer. The tamoxifen litigation involves an agreement between Imperial Chemical Industries, PLC (“Imperial”) (an affiliate of AstraZeneca) and Barr Laboratories (“Barr”) to settle patent infringement litigation relating to Imperial’s patent for tamoxifen (the “‘516 patent”)12.

FDA GENERIC APPROVAL CRITERIA:

ESTABLISHING IN VITRO EQUIVALENCE AND

BIOEQUIVALENCE WITH THE REFERENCE

LISTED PRODUCT (RLP)

The generic aspirant must establish equivalence with the RLP or the reference formulation developed by the innovator. FDA has established two basic criteria for the generic to be equivalent with the innovator.


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1. In vitro equivalence criteria:

Comparison of the dissolution profiles of generic and brand name drugs by pharmaceutical researchers is done by Similarity Factor or f2 value estimations. After introduction of this factor by Moore and Flanner, it has been adopted by the Centre of Drug Evaluation and Research (USFDA) and by the Human Medicines Evaluation Unit of the European Agency for the Evaluation of Medicinal Products (EMEA) as a criterion for the assessment of the similarity between two dissolution profiles. It is defined by the USFDA as the logarithmic reciprocal square root transformation of one plus the mean squared (the average sum of squares) differences of drug percent dissolved between the test and reference products.

( ) ( )

∗

−+∗=

−

=

∑ 10011log5025.0

1

2n

t

tt TRn

f

where n is the number of dissolution time point s, and Rt and Tt are the reference and test dissolution values (mean of at least 12 dosage units) at time t. In dissolution profile comparisons, especially to assure similarity in product performance, regulatory interest is in knowing how similar the two curves are, and to have a measure which is more sensitive to large differences at any particular time point. For this reason, the f2 comparison has been the focus in Agency guidances. When the two profiles are identical, f2=100. An average difference of 10% at all measured time points results in a f2 value of 50. FDA has set a public standard of f2 value between 50-100 to indicate similarity between two dissolution profiles. For a dissolution profile comparison at least 12 units should be used for each profile determination. Mean dissolution values can be used to estimate the similarity factor, f2. To use mean data, the % coefficient of variation at the earlier point should not be more than 20% and at other time points should not be more than 10%. The dissolution measurements of the two products (test and reference, pre- and post- change, two strengths) should be made under the same test conditions. The dissolution time points for both the profiles should be the same, e.g., for immediate release products 15, 30, 45 and 60 min, for extended release products 1, 2, 3, 5 and 8 h. Since f2 values are sensitive to the number of dissolution time

points, only one measurement should be considered after 85% dissolution of the product. For products which are rapidly dissolving, i.e., more than 85% in 15 min or less, a profile comparison is not necessary. A f2 value of 50 or greater (50-100) ensures sameness or equivalence of the two curves and, thus, the performance of the two products13, 14. An f2 value less than 50 does not necessarily mean lack of similarity. One may use a different method for establishing linearity such as statistical analysis may be provided, e.g., 90% confidence intervals. 2. In vivo bioequivalence

The generic manufacturer has to conduct a bioequivalence study in place of the pre-clinical and clinical studies (performed by the innovator) to establish equality with the innovator. The purpose of bioequivalence is to have a generic drug product which is substitutable for a reference or brand name drug product ‘without any additional prescriber intervention’. This means the patient who is receiving and is very well controlled on the brand name drug walks into his pharmacy and the pharmacist could substitute an A-prefixed FDA approved generic for his next course of therapy and there could effectively be no observable difference in the therapeutic outcome for that patient. There should not be any change in the efficacy and no additional incidents of side effects. For bioequivalence studies, the dosage form is given to the patient to swallow and the drug which was in the solid form goes into the solution form in the GI tract. This step of the formulation falling apart in the GIT and the drug going into the solution state is the most critical and rate limiting step as far as the formulations’ performance is concerned as this is the only step in which the formulators have any control, rest of the steps such as disposition of the drug are more or less dependent on the patient’s physiology. The drug after going into the solution state goes through the biotransformation phase in the liver and is finally available for absorption in the blood which then carries the drug to the site of activity to produce a therapeutic effect or some other pharmacological effects in the form of adverse effects. The drug’s appearance in the blood is the most favorable and sensitive point at which we can measure the drug concentration because of the reasons of blood being a carrier, an intermediate between the drug being absorbed and the eventual


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therapeutic effect and the simplicity of sampling and monitoring in comparison to the other fluids in the body. Other less favorable choices of measuring drug concentrations may be the in vivo pharmacologic affects which may or may not be studied in a controlled environment. The usual study designs for conducting bioequivalence studies are single dose; two-way crossover fasted or fed state studies. This means all the test subjects receive both the generic and the brand name product. This is done in a cross-over manner and products are given once to each subject on different occasions and the pharmacokinetic data are compared between the two treatments between each subject. This study gives us an idea of the inter-subject variability. However another design known as the replicate design gives an idea of the variability within-subject. Replicate design gives the same product to each subject more than once. The overall number of subjects is reduced but the number of times each subject has to come increases. The two most common parameters which are studied and compared are the Cmax and the AUC which are related to the rate and extent of drug absorption in the blood respectively. These parameters are accessed using the 90% confidence intervals that must fit between 80-125%. The difference seems to be as much as 46% but since the CI is involved the data never gets even close to these bounds and the mean always lies in the center of these bounds. The bounds seem to be unbalanced when we see the 80-125% bioequivalence limits. Does it mean that the FDA allows the 90% CI of the test vs. sample to be 25% above and on the other hand doesn’t allow the 90% CI to be less than 20% below the reference? This actually is not the case as is explained by the Office of Generic Drugs (OGD). FDA based on a lot of clinical experience and inputs have come up with the significant difference for statistics to be 20% with an alpha level of 0.05 significant level. Now if we express this mathematically, if the test statistically is 20% less than the reference, it means the T/R would be 80/100, which is 80%. In other case if the reference is 20% less than the test, it becomes 100/80 which gives a value of 125%. This means that the test can’t be 20% less than the reference and the reference can’t be 20% less than the test2.

Biowaivers: FDA allows the generic manufacturers to not have to study every single strength of a product and requires some in vitro tests for the same. The example being the testing on the low strength version of an already bioequivalence established product. The lower strengths of the test and reference product are proportional to their matching higher strengths and FDA only requires some in vitro testing to assure that it is true. The FDA grants waivers of such bioequivalence studies15, 16. Other types of products which may be granted biowaivers are the ones in which the bioequivalence seems to be self-evident, parenteral solutions, inhalational anesthetics, topical solution and oral solutions which do not require a transition from solid state to liquid state. CONCLUSION

US congress passed the Hatch Waxman Act in the year 1984 for the benefits of the consumers in an attempt to stop the dominance and monopoly of the innovator and to encourage the use of cheaper and equally effective drugs as the innovator. This has promoted the generic drugs and encouraged the generic manufacturers to bring cheaper versions of the brand name drugs without compromising the quality of the product. But still there are some ambiguities which need to be dealt with as the generic manufacturers still find many obstacles to market their products due to the anticompetitive practices associated with the act as discussed earlier. The brand name manufacturer being the pioneer of the product has got every minute information about the product which allows him to prepare himself in advance with the ways to tackle the generic assertions. Moreover the associated anti-competitive practices have proven to be extremely profitable to brand name drug manufacturers and in some cases to the generics as well. Both the generic and the brand name industries have equal rights to earn profits but in this process in some way or the other the consumers are being reprimanded and are not receiving what they are worthy of due to the drug prices being kept artificially high. There have been ways to counter the industry anti-competitive practices such as the Federal Trade Commission forcing the industry to play fair by punishing manufacturers indulging in anti-competitive practices, The Greater access to Affordable Pharmaceuticals act and The Consumers Access to


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Prescription Drug Improvement Act are some of the legislations passed to deal with the same. Above all there is a need to educate the consumers and physicians in US about the realities of the generic drugs to prompt the use of generic drugs and to make an endeavor to

keep the rising drug prices in control. As per the Indian drug industry the know-how of the principles of generic approval is necessary as the country has a major contribution to the circulation of top quality generics to the world especially in the US market.

Table 1: Comparison the global pharmaceutical industry with the global generic industry (2007).

Parameters Global pharmaceutical industry Generic share

Size $643 bn $55 bn Growth rate 7% 11% Key players US ($245 bn)

Japan ($60 bn) Germany ($27 bn) France ($24 bn) UK ($15 bn)

US ($28 bn) Germany ($6 bn) UK ($3.4 bn) France ($2 bn)

Expected growth 5-7% 13-15% Projected size (by 2010) $812 bn at 6% growth rate 93 bn at 14% growth rate

Table 2: NDA vs ANDA review process. (Source: Centre for drug evaluation and research, USFDA).

Brand Name drug (NDA) requirements Generic drug (ANDA) requirements

Chemistry Chemistry Manufacturing Manufacturing Controls Controls Labeling Labeling Testing Testing Animal Studies

Bioequivalence study Clinical Studies Bioavailability Studies

REFERENCES

1. Rajbhanshi N. “Overview of the World Pharmaceutical Industry”. Presented at the IPA conference “Workshop on GMP and Regulatory Affairs”, Indian Pharmaceutical Association Delhi Branch, 28 October 2007, India Habitat Centre, New Delhi.

2. Buehler GJ, Conner D. The FDA Process of Approving Generic Drugs, Office of Generic Drugs [online]. [cited 2008 August 18] Available from: URL: www.fda.gov/CDER/meeting/CTP2005/Generic.ppt.

3. Mossinghoff GJ. Overview of the Hatch Waxman Act and its impact on the drug development process. Food and Drug Law Journal Vol. 54. [online]. 1999 [cited 2008 August 24]. Available

from: URL: www.fdli.org/pubs/Journal%20Online/54_2/art2.pdf.

4. Meade T. Case Analysis-In Re Buspirone Patent and Antitrust Litigation. The Richmond Journal of Law and Technology Vol IX, Issue 1 [online]. Fall 2002 [cited 2008 August 26]. Available from: URL: law.richmond.edu/jolt/v9i1/article1.pdf.

5. Himes JL, Zain S. Anti-competitive Innovation: Is There a Role for Antitrust In Evaluating Product Line Extensions? American Conference Institute, Pharmaceutical Antitrust, New York [online] May 15-16, 2007 [cited 2008 August 20] Available from: URL: www.oag.state.ny.us/bureaus/antitrust/pdfs/anti_competitive_innovation_ProductLine_Extension_by_himes.pdf.


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6. Flor VS. The Approaching Storm. Intellectual

Asset Management [online]. October/November 2006 [cited 2008 July 24]. Available from: URL: www.skgf.com/media/news/news.286.

7. Consent Agreement Resolves Complaint Against Pharmaceutical Companies Hoechst Marion Roussel, Inc. and Andrx Corp. Commission Alleged that Companies' Agreement Likely to Stifle Competition in U.S. Drug Market. Federal Trade Commission. Protecting America’s Consumers [online] 2004 [cited 2008 July 25]. Available from: URL: http://www.ftc.gov/opa/2001/04/hoechst.shtm.

8. Terazosin Hydrochloride Antitrust Litigation. Intellectual Property Update [online]. January 2004 [cited 2008 July 20]. Available from: URL: www.townsend.com/files/AntitrustIPU.pdf.

9. In re Ciprofloxacin Hydrochloride Antitrust Litigation [online]. 2008 [cited 2008 July 15]. Available from: URL: www.ftc.gov/os/2008/01/ciprobrief.pdf.

10. Gordon GG. Recent Pharmaceutical Industry Antitrust Cases Raising Intellectual Property antitrust Issues Antitrust Enforcement: Cutting Edge Lessons from the Pharmaceutical Industry American Bar Association, Section of Antitrust Law, Spring Meeting [online]. 2003 [cited 2008 July 21] Available from: URL: www.abanet.org/antitrust/committees/intell_property/recentpharmcases.doc.

11. Pfizer Inc vs Shalala Donna E. [online] [cited 2008 July 28] Available from: URL: http://caselaw.lp.findlaw.com/scripts/getcase.pl?navby=search&case=/data2/circs/DC/985151a.html.

12. In re: Tamoxifen Citrate Antitrust Litigation, Another "Reverse Payments" Case, Appealed to Supreme Court [online]. December 14, 2006 [cited 2008 July 22]. Available from: URL: http://www.orangebookblog.com/2006/12/in_re_tamoxifen.html.

13. Gohel MC, Panchal MK. Refinement of Lower Acceptance Value of the Similarity Factor f2 in Comparison of Dissolution Profiles. Dissolution Technologies [online]. 2002 February [cited 2008 April 12]. Available from: URL:

www.dissolutiontech.com/DTresour/0202art/DTFeb2002_art3.pdf.

14. Guidance for Industry: Dissolution Testing of Immediate Release Solid Dosage Forms. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) [online]. August 1997 [cited 2008 July 15]. Available from: URL: www.fda.gov/cder/Guidance/1713bp1.pdf.

15. Fassen F, Vromans H. Biowaivers for Oral Immediate Release Products. Implications of Linear Pharmacokinetics. Clin Pharmacokinet 2004; 43 (15): 1117-1126.

16. Guidance for Industry: Waiver of In Vivo Bioavailability and Bioequivalence studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) [online] August 1997 [cited 2008 June 12]. Available from: URL: www.cop.ufl.edu/safezone/pat/pha5128/resources/waiver.pdf.


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Stimuli Sensitive Hydrogels: A Review S.S. Bushetti*a, Vinod Singhb, S.Appala Rajuc

, Atharjaved d,Veermarame

Luqman College of Pharmacy, Gulbarga, Karnataka (India)a,d

SBS PG Institute of Biomedical Sciences & Research, Dehradun.(India) b,e

HKE’s College of Pharmacy, Gulbarga, Karnataka( India)c

For correspondence: [email protected]

ABSTRACT:

Hydrogels are hydrophilic, three-dimensional networks, which are able to imbibe large amounts of water or

biological fluids, and resembles to a biological tissue and undergoes a phase change transition after receiving

a specific stimulus. They are insoluble due to the presence of chemical (tie-points, junctions) and/or physical

crosslinks such as entanglements and crystallites. These are used as Water gel explosives ,as sustained-release

delivery system and provides absorption, desloughing and debriding capacities of necrotics and fibrotic tissue.

Hydrogels that are responsive to specific molecules, such as glucose or antigens can be used as biosensors as

well as in DDS.These materials can be synthesized to respond to a number of physiological stimuli present in

the body, such as pH, ionic strength and temperature. These can be classified as neutral or ionic, on the basis of

nature of the side groups. The availability of large molecular weight protein- and peptide-based drugs due to

the recent advances in the field of molecular biology has given us new ways to treat a number of diseases.

Hydrogels are present in disposable diapers where they "capture" urine. Hydrogels are having wide

applications in the field of pharmaceutical and medicinal field such as transdermal, ocular, subcutaneous,

rectal, gastrointestinal tract and oral tract.

Keywords: Stimuli sensitive hydrogels, pH-sensitive hydrogels; Temperature-sensitive hydrogels; Applications

in drug delivery; Polymer network structure

INTRODUCTION

In this changing scientific and educational world, pharmaceutical industry serves us in developing major new solutions of significant medical problems. These solutions/treatments may be in different forms such as Hydrogels which can be used as contact lenses, membranes for biosensors, linings for artificial hearts, materials for artificial skin, and drug delivery devices1,2,3,4 .Hydrogel is a network of polymer chains that are water-insoluble, sometimes found as a colliadal gel in which water is the dispersion medium. Hydrogels are superabsorbent (they can contain over 99% water) natural or synthetic polymers. Hydrogels possess also a degree of flexibility very similar to natural tissue, due to their significant water content. Uses for hydrogel are

• environmentally sensitive hydrogels. These hydrogels have the ability to sense changes of pH, temperature, or the concentration of

metabolite and release their load as result of such a change.

• Water gel explosives

• as sustained-release delivery system

• provide absorption,desloughing and debriding capacities of necrotics and fibrotic tissue.

• hydrogels that are responsive to specific molecules, such as glucose or antigens can be used as biosensors as well as in DDS.

• In disposable diapers where they "capture" urine, or in sanitary towels

• contact lenses (silicone hydrogels, polyacrylamides

• Medical electrodes using hydrogels composed of cross linked polymers (polyethylene oxide,polyAMPS and polyvinylpyrrolidone)

• currently used as scaffolds in tissue engineering. In case of scaffolds, hydrogels may contain human cells in order to repair tissue.


APTI ijper


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Hydrogels are three-dimensional, hydrophilic, polymeric networks capable of imbibing large amounts of water or biological fluids 5,6. Hydrogels are composed of homopolymers or copolymers, and are insoluble due to the presence of chemical crosslinks i.e tie-points and junctions, or physical crosslinks e.g entanglements or crystallites7,8,9,10.Hydrogels exhibit a thermodynamic compatibility with water which allows them to swell in aqueous media.5,6,11,12,13 Hydrogels resemble natural living tissue because of their high water contents and soft consistency which is similar to natural tissue. Several stimuli which causes the triggering effects in hydrogels include physical stimuli, such as light 14, magnetic field 15, electric current 16 and ultrasound 17, which can be applied to the systems externally, and chemical stimuli, like ionic species 18, certain chemical substances and biological compounds 19. In some cases, their effective applications can be found in engineering, rather than pharmaceutical fields. Recent developments on hydrogels addresses the use of water-swollen, crosslinked biomedical materials as carriers for the development of novel pharmaceutical formulations and for the delivery of drugs, peptides and proteins, as targeting agents for site specific delivery, or as components for the preparation of protein or enzyme conjugates.Network structure and the thermodynamic nature of hydrogels play a key role in their diffusional behavior, molecular mesh size changes (especially in environmentally responsive hydrogels), and the associated molecular stability of the incorporated bioactive agents. 1. Classification of hydrogels Hydrogels can be classified as neutral or ionic, based on the nature of the side groups. According to their mechanical and structural characteristics, they can be classified as affine or phantom networks. Classification of hydrogels is based on the nature of the side groups; they can be either neutral or ionic. The chemical nature and number of these pendent groups can be precisely controlled by the choice of the chemical entities used in the polymer synthesis. Additionally, they can be homopolymer or copolymer networks, based on the method of preparation. Further , Classified is based on the physical structure of the networks as amorphous, semicrystalline, hydrogen-bonded structures, supermolecular20 structures and hydrocolloidal

aggregates. (5,6,7,8,9) Hydrogels show a swelling behavior dependent on the external environment and termed as physiologically- responsive hydrogels 21, where polymer complexes can be broken or the network can be swollen as a result of the changing external environment. These systems tend to show drastic changes in their swelling ratio as a result. Some of the factors affecting the swelling of physiologically-responsive hydrogels include pH, ionic strength, temperature and electromagnetic radiation. 21

2. Monomers

Monomers most often used for the synthesis of mucoadhesive polymers include acrylic and methacrylic acid (MAA). The idea of adhesion promoters diffusing across the polymer/mucin interface has also been introduced 22. Chains of polymerized ethylene glycol, either freely loaded in the carrier or grafted to the polymer surface, have been utilized as adhesion promoters22. The `stealth' properties of poly(ethylene glycol), known also as PEG, have also been used to reduce the uptake of particulate carriers by the reticuloendothelial system23. PEG has also been shown to both lengthen the biological half-life and reduce the immunogenicity of high molecular weight. The literature on the synthesis of new polymer materials has exploded since the first biomedical application of poly (hydroxyethyl methacrylate), known also as PHEMA, by Wichterle and Lim24 in 1961. Novel materials based on polymers such as PHEMA, poly(N-isopropylacrylamide) (PNIPAAm), and poly(vinyl alcohol) (PVA), are synthesized primarily by new and innovative preparation techniques, a number of new monomers have been prepared for production of polymers with desired properties as well. Hydrogels are also used as carriers that can interact with the mucosa lining in the gastrointestinal (GI) tract, colon, vagina, nose and other parts of the body due to their ability to prolong their residence time at the delivery location25. The interaction between such carriers and the glycoproteins in the mucosa is thought to occur primarily via hydrogen bonding. 3. Network structure

Parameters used to characterize the network structure of hydrogels are the polymer volume fraction in the swollen state (y 2,s), molecular weight of the polymer chain between two neighboring crosslinking points (Mc), and the corresponding mesh size (j ). The


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polymer volume fraction in the swollen state is a measure of the amount of fluid imbibed and retained by the hydrogel. The molecular weight between two consecutive crosslinks, which can be either of a chemical or physical nature, is a measure of the degree of crosslinking of the polymer. It is important to note that due to the random nature of the polymerization process itself, only average values of Mc can be calculated. The correlation distance between two adjacent crosslinks, j , provides a measure of the space available between the macromolecular chains available for the drug diffusion; again, it can be reported only as an average value. Two methods that are prominent among the growing number of techniques utilized to elucidate the structure of hydrogels due to their frequent use, are the equilibrium swelling theory and the rubber elasticity theory. Hydrogels resemble natural rubbers in their remarkable property to elastically respond to applied stresses. A hydrogel subjected to a relatively small deformation, less than 20%, will fully recover to its original dimension in a rapid fashion. Elastic behavior of hydrogels can be used to elucidate their structure by utilizing the rubber elasticity theory originally developed by Treloar 26 and Flory 27,28 for vulcanized rubbers and modified to polymers by Flory 13. In order to perform an analysis of the structure of hydrogels using the rubber elasticity theory, experiments need to be performed using a tensile testing system. The rubber elasticity theory has not only been used to analyze chemically, but physically, crosslinked hydrogels 29,30,31 as well as hydrogels exhibiting temporary crosslinks due to hydrogen bonding. Mechanism of drug release of many drugs from hydrogels is diffusion that occurs through the space or ‘pore’ available between macromolecular chains. Depending upon the size of these pores, hydrogels can be conveniently classified as macro-porous, micro-porous, non-porous. Parameter used in describing the size of the pores is the correlation length, j , which is defined as the linear distance between two adjacent crosslinks. 4. Properties of hydrogels

4.1. Factors affecting swelling of hydrogels

Highly crosslinked hydrogels have a tighter structure, and will swell less compared to the same hydrogels with lower crosslinking ratios. Crosslinking ratio is defined as the ratio of moles of crosslinking agent to the

moles of polymer repeating units. The higher the crosslinking ratio, the more crosslinking agent is incorporated in the hydrogel structure. Crosslinking hinders the mobility of the polymer chain, hence lowering the swelling ratio. Chemical structure of the polymer also affects the swelling ratio of the hydrogels. Hydrogels containing hydrophilic groups swell to a higher degree compared to those containing hydrophobic groups. Hydrophobic groups collapse in the presence of water, thus minimizing their exposure to the water molecule. As a result, the hydrogels will swell much less compared to hydrogels containing hydrophilic groups. Swelling of temperature-sensitive, pH sensitive and Ion-activated hydrogels can be affected by changes in the temperature of the swelling media, pH and Ionic strength. 4.2. Dynamics of swelling

Swelling kinetics of hydrogels is classified as diffusion-controlled (Fickian) and relaxation-controlled (non-Fickian) swelling. When water diffusion into the hydrogel occurs much faster than the relaxation of the polymer chains, the swelling kinetics is diffusion-controlled.Dynamics of swelling is explained by Peppas and Colombo 32. 4.3. Mechanical properties

Hydrogels designed to protect a sensitive therapeutic agent, such as protein, must maintain its integrity to be able to protect the protein until it is released out of the system. Changing the degree of crosslinking can be utilized to achieve the desired mechanical property of the hydrogel and thus by Increasing the degree of crosslinking of the system a stronger gel will be obtained. However, a higher degree of crosslinking creates a more brittle structure. Thus, Optimum degree of crosslinking is maintained to achieve a relatively strong and elastic hydrogel. Copolymerization is utilized to achieve the desired mechanical properties of hydrogels. Incorporation of a co-monomer contributes to increase in H-bonding that leads to increase in the strength of the hydrogel. 4.4. Cytotoxicity and in-vivo toxicity

Cytotoxicity tests are used to evaluate the toxicity of hydrogels. Extract dilution, direct contact and agar diffusion are common assay method used to evaluate toxicity of hydrogels. Most of the problems with toxicity associated with hydrogel carriers are the unreacted monomers, oligomers and initiators that leach


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out during application. The relationship between chemical structures and the cytotoxicity of acrylate and methacrylate monomers has been studied extensively33. Several measures have been taken to solve this problem, including modifying the kinetics of polymerization in order to achieve a higher conversion, and extensive washing of the resulting hydrogel. The formation of hydrogels without any initiators has been studied to eliminate the problem of the residual initiator. Commonly used technique is gamma irradiation 35,36,37. Hydrogels of PVA have been also made without the presence of initiators by using thermal cycle to induce crystallization 34. The crystals formed act as physical crosslinks and crystals are able to absorb the load applied to the hydrogels. 5. Stimuli-sensitive swelling-controlled release

systems

5.1. pH-sensitive hydrogels

Hydrogels exhibiting pH-dependent swelling behavior can be swollen from ionic networks. The ionic networks contain either acidic or basic pendant groups38,39. Ionic hydrogels are swollen polymer networks containing pendent groups, such as carboxylic or sulfonic acid, which show sudden or gradual changes in their dynamic and equilibrium swelling behavior as a result of changing the external pH. In pH sensitive hydrogels, ionization occurs when the pH of the environment is above the pKa of the ionizable group38,39. As the degree of ionization increases, the number of fixed charges increases, resulting in increased electrostatic repulsions between the chains. This causes an increased hydrophilicity of the network, and greater swelling ratios.In case of cationic materials containing pendent groups such as amines38,39,40,41,42. These groups ionize in media which are at a pH below the pKb of the ionizable species. Thus, in a low pH environment, ionization increases, causing increased electrostatic repulsions. The hydrogel becomes increasingly hydrophilic and will swell to an increased level. The swelling of polyelectrolyte gels is significantly affected by the ionic strength of the swelling agent38,41. As the ionic strength of the swelling agent increases, the concentration of ions within the gel must increase in order to satisfy the Donnan equilibrium. The swelling force is reduced due to increased gelcounter ion

interaction and a decrease in the osmotic swelling forces. Katchalsky38 suggested that the collapse and expansion of PMAA) gels occurred reversibly by simply adjusting the pH of the fluid. Ohmine and Tanaka43 observed the sudden collapse of ionic networks in response to sudden changes in the ionic strength of the swelling medium. Khare and Peppas44 observed pH- and ionic strength-dependent swelling kinetics in these gels and examined the swelling kinetics of poly(MAA) or poly(acrylic acid) with poly(hydroxyl ethyl methacrylate). Hydrogels composed of lightly crosslinked N-isopropylacrylamide (NIPAAm) and MAA were synthesized and characterized for their sensitivity to external conditions and their ability to control the release of two antithrombotic agents, heparin and streptokinase. PNIPAAm is noted for its sharp change in swelling behavior across the lower critical solubility temperatures of the polymer, while PMAA shows pH-sensitive swelling due to ionization of the pendant carboxylic groups in the polymer. Hydrogel copolymers of NIPAAm and MAA with appropriate compositions were designed to sense small changes in blood stream pH and temperature to deliver antithrombotic agents, such as streptokinase or heparin, to the site of a blood clot45. 5.2. Temperature-sensitive hydrogels

Temperature sensitive hydrogels are having ability to swell or deswell as a result of changing the temperature of the surrounding fluid and these thermosensitive systems are classified as positive or negative temperature-sensitive systems. A positive temperature- sensitive hydrogel has an upper critical solution temperature (UCST). Such hydrogels contract upon cooling below the UCST. Negative temperature-sensitive hydrogels have a lower critical solution temperature (LCST). These hydrogels contract upon heating above the LCST. Some work with temperature-sensitive hydrogels was done by the group of Tanaka46. PNIPAAm is the best example of a negative temperature-sensitive hydrogel. Hitotsu et al.47. worked with crosslinked PNIPAAm and determined that the LCST of the PNIPAAm gels was 34.38C. They found that the LCST could be increased by mixing small amounts of ionic copolymers in the gels. Hoffman48 proposed the application of PNIPAAm and its copolymers for temperature-modulated drug release by


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bulk squeezing and surface regulation. In the bulk squeezing system, the drug that is distributed evenly inside the matrix is squeezed out of the system due to the deswelling of the hydrogel as a result of increasing the temperature of the environment above the volume phase transition temperature. In the surface regulation system, the swelling ratio of the skin layer is increased as the temperature of the system is lowered below the volume phase transition temperature, and hence, the drug molecules will be able to diffuse through the skin layer. Chen and Hoffman49 prepared P(NIPAAm-g-AA) gels which exhibited temperature- and pH-sensitive behavior. These gels were able to respond rapidly to both temperature and pH changes. Jeong et al.50 synthesized a new biodegradable triblock copolymer of poly(ethylene glycol-b-(dl-lactic acid-coglycolic acid)-b-ethylene glycol) (PEG±PLGA±PEG). This copolymer shows sol-to-gel or gel-to-sol transitions as temperature increases monotonically. This thermoreversible copolymer is very important for drug delivery applications because it can be injected as a free-flowing solution (sol) at room temperature. Upon injection into the body, the copolymer becomes a gel at body temperature. Kim and associates51 developed a Hydrogel of PNIPAAm and PAA which was able to effectively release the protein drug, calcitonin, in response to temperature and pH changes. Various applications of these hydrogels are now a days important in pharmaceutical and medical fields such as on±off drug release regulations, biosensors and intelligent cell culture dishes52. 5.3. Other stimuli-sensitive hydrogels

Lavon and Kost 53 suggested mass transport enhancement in non-erodible polymeric controlled release systems by ultrasound for a better understanding of the ultrasound-enhancing drug release phenomenon. They explained that the enhancing effect of ultrasound on drug release from the systems is due to the contribution of a convective term, generated by cavitation, without any destructive effect of the morphology of the polymers. Hsu and Block 54 explained electrokinetic phenomena in three types of anionic gels (agarose, agarose ±carbomer 934P, and agarose±xanthan gum) under an applied electric current for electrically-modulated drug delivery and further suggested that electrical current strength and gellant content could influence both syneresis and drug

migration. Miyazaki et al. 55,56 developed a novel reversibly antigen-responsive hydrogel. This hydrogel was an AAm-based semi-interpenetrating polymer network (IPN), consisting of an antibody-grafted linear PAAm and the crosslinked PAAm grafted with the corresponding antigen. In the absence of a free antigen, the hydrogel can shrink due to the intra-chain antigen±antibody binding in the polymer network, while it swells in the presence of the free antigen because of dissociation of the intra-chain binding by exchange of the grafted antigen for free antigen. This swelling/shrinking process was shown to be reversible. Due to this property, the hydrogel membrane allowed the antigen-responsive change of hemoglobin permeation through the membrane in response to stepwise changes in the antigen concentration.. Antibiotics covalently attached to the PVA-based hydrogel via the thrombinsensitive peptide linkers can be released from the Hydrogel only in the presence of infection, because of thrombin-sensitive cleavage of the peptide linkers. This hydrogel is used for a wound dressing with microbial infection-responsive controlled release of antibiotics. 6. Applications of hydrogels in drug delivery. 6.1. Peroral drug delivery

In peroral administration, hydrogels can deliver drugs to four major specific sites; mouth, stomach, small intestine and colon. By controlling their swelling properties or bioadhesive characteristics in the presence of a biological fluid, hydrogels can be a useful device for releasing drugs in a controlled manner at these desired sites. Hydrogels also adhere to certain specific regions in the oral pathway, leading to a locally increased drug concentration, and enhancing the drug absorption at the release site. 6.2 Drug delivery in the oral cavity

Periodontal disease, stomatitis, fungal and viral infections, and oral cavity cancers can be treated by the oral delivery of hydrogels. Long-term adhesion of the drug containing hydrogel against copious salivary flow, which bathes the oral cavity mucosa, is required to achieve this local drug delivery. Nagai et al. 57 developed a bioadhesive tablet, composed of a double layer, with a bioadhesive layer made of hydroxypropyl cellulose and poly(acrylic acid) and a lactose non-adhesive backing layer. It is a local delivery system of triamcinolone acetonide for the treatment of aphthous


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ulcers. Petelin et al. 58 investigated three different hydrogel-based ointments as vehicles for liposome delivery into the oral cavity tissues by electron paramagnetic resonance (EPR). Vehicles used were Orabasew (a sodium carboxymethylcellulose, pectin and gelatin combination in a polyethylene ±paraffin base), Carbopol 934Pw and neutralized poly(MAA-co-methyl methacrylate (MMA)). Liposome containing mucoadhesive ointments were prepared by simply mixing multilamellar liposomes with each ointment prediluted with phosphate-buffered saline of pH 7.4 in the volume ratio of 1:4. Kitano et al. 59 proposed a hydrogel ointment containing absorption enhancers for the buccal delivery of 17 b-estradiol (E2) to treat osteoporosis. It is well known that the oral administration of E2 results in very low availability due to its high first-pass effect. Ethanol solution containing E2, and glyceryl monolaurate as an absorption enhancer, and an aqueous solution of a commercial carboxyvinyl polymer (Hiviswako 103) and triethanolamine were mixed together to produce the Hydrogel ointment. In-vivo studies using hamsters demonstrated that the buccal administration of E2 with this formulation allowed the maintenance of the E2 plasma level at over 300 ng/ml per cm3 for 7 h, while no primary morphological change of buccal membrane was observed 7 h after application. 6.3. Drug delivery in the GI tract

Patel and Amiji 60 proposed stomach-specific antibiotic drug delivery systems for the treatment of Helicobacter pylori infection in peptic ulcer disease. For localized antibiotic delivery in the acidic environment of the stomach, they developed cationic hydrogels with pH-sensitive swelling and drug release properties. The hydrogels were composed of freeze-dried chitosan±poly (ethylene oxide) (PEO) IPN. pH-dependent swelling properties and the release of two common antibiotics, amoxicillin and metronidazole, entrapped in the chitosan±PEO semi-IPN were evaluated in enzyme-free simulated gastric fluid (SGF; pH 1.2) and simulated intestinal fluid (SIF; pH 7.2). The swelling ratio of the hydrogels after 1 h in SGF was found to be 16.1, while that in SIF was only 8.60. Akiyama et al. 61 developed hydrogels with protease inhibitory activities using Carbopolw (C934P), a poly(acrylic acid) product, which shows an inhibitory effect on the hydrolytic activity of trypsin, and its

neutralized freeze-dried modification (FNaC934P). They demonstrated that two-phase formulations, consisting of the rapid gel-forming FNaC934P and the efficient enzyme-inhibiting, but more slowly swelling, C934P, had the most profound effect on trypsin activity inhibition. Lowman et al. 62 developed an Oral insulin delivery using pH-responsive hydrogels. The hydrogels used to protect the insulin in the harsh, acidic environment of the stomach before releasing the drug in the small intestine were crosslinked copolymers of PMAA with graft chains of polyethylene glycol (P(MAAg-EG)). The insulin-containing P(MAA-g-EG) microparticles demonstrated strong dose-dependent hypoglycemic effects in in-vivo oral administration studies using both healthy and diabetic rats. The blood glucose levels in these animals were decreased significantly for at least 8 hrs due to the absorption of insulin in the GI tract. 6.4. Rectal delivery

Miyazaki et al. 63 suggested the potential application of xyloglucan gels with a thermal gelling property as vehicles for rectal drug delivery. Xyloglucan sol±gel transition temperature is around 22±278C, and thus, it gels at body temperature; on the other hand, it can be easily administered since it behaves as a liquid at room temperature. Rectal administration of xyloglucan hydrogels containing indomethacin using rabbits showed a well controlled drug plasma concentration-time profile without reduced bioavailability, when compared to commercial indomethacin suppositories. Watanabe et al. 64 reported reduced irritation by rectal hydrogels prepared with water-soluble dietary fibers, xanthan gum and locust bean gum. 6.5 Ocular delivery

Suspensions and ointments, can be retained in the eye, although these give patients an unpleasant feeling such as blurring, grittyness because of the characteristics of solids and semi-solids. In ocular drug delivery, many physiological constraints prevent a successful drug delivery to the eye due to its protective mechanisms, such as effective tear drainage, blinking and low permeability of the cornea. Thus, conventional eye drops containing a drug solution tend to be eliminated rapidly from the eye, and the drugs administered exhibit limited absorption, leading to poor ophthalmic bioavailability. The short-term retention results in a


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frequent dosing regimen to achieve the therapeutic efficacy for a sufficiently long duration. Due to their elastic properties, hydrogels represent an ocular drainage-resistant device. In addition, they may offer better feeling, with less of a gritty sensation to patients.In-situ-forming hydrogels are attractive as an ocular drug delivery system because of their facility in dosing as a liquid, and their long-term retention property as a gel after dosing. Rheological evaluation of Gelritew, deacetylated gellan gum which gels upon instillation in the eye due to the presence of cations, was studied by Carlfors et al. 65 Their study indicated that a high rate of the sol/gel transition of Gelritew in-situ gels results in long precorneal contact times. Chetoni et al.66 reported silicone rubber/Hydrogel composite ophthalmic inserts. Poly(acrylic acid) or poly (MAA) IPN was grafted on the surface of the inserts to achieve a mucoadhesive property. The ocular retention of IPN-grafted inserts was significantly higher with respect to ungrafted ones. An in-vivo study using rabbits showed a prolonged release of oxytetracycline from the inserts for several days.Cohen et al.67 developed an in-situ-gelling system of alginate with high guluronic acid contents for the ophthalmic delivery of pilocarpine. This system significantly extended the duration of the pressure-reducing effect of pilocarpine to 10 h, compared to 3 h when pilocarpine nitrate was dosed as a solution. 6.6 Transdermal delivery

Transdermal delivery is useful for delivering drug for a long duration at a constant rate, and drug delivery can be easily stopped by simply removing the devices, and that drugs can bypass hepatic first-pass metabolism. Hydrogels provide a better feeling for the skin in comparison to conventional ointments and patches because of their high water content. Carbopol 934w-based formulation containing phosphatidylcholine liposomes (liposome-gel) was developed by Kim et al.68. In their study, the skin absorption behavior of hydrocortisone-containing liposome gel was assessed. Gayet and Fortier 69 developed hydrogels obtained from the copolymerization of bovine serum albumin (BSA) and PEG. Due to their high water content over 96%, allowing the release of hydrophilic and hydrophobic drugs, their use as controlled release devices in the field of wound dressing was proposed as the potential application of the BSA±PEG hydrogels.

Sun et al.70 devised composite membranes comprising of crosslinked PHEMA with a nonwoven polyester support. Depending on the preparation conditions, the composite membranes can be tailored to give a permeation flux ranging from 4 to 68 mg/cm2 per h for nitroglycerin. Sodium nonivamide acetate 71, and luteinizing hormone releasing hormone 72 were studied for the improved transdermal penetration. 6.7. Subcutaneous delivery

A bioerodible hydrogel based on a semi-IPN structure composed of a poly (1-caprolactone) and PEG macromer terminated with acrylate groups was devised by Cho et al.73. Long-term constant release over 45 days of clonazepam entrapped in the semi-IPN was achieved in vivo. Zhao and Harris 74 developed two types of PEG hydrogels i.e First is prepared by a polycondensation reaction between difunctional PEG acids and branched PEG polyols. Upon hydrolysis of the resulting ester linkages, these gels degrade into only PEG and PEG derivatives. Second is PEG-based hydrogels having functional groups in which protein drugs can be covalently attached to the gel network via ester linkage.Thus, the release of the protein drugs immobilized would be controlled by the hydrolysis of the ester linkage between the gel and the protein, followed by the diffusion of the protein out of the gel, and by the degradation of the gel. Biodegradable crosslinked dextran hydrogels containing PEG (PEG-Dex) was reported by Moriyama and Yui 75 and Insulin release from these hydrogels was regulated by the surface degradation of PEG-Dex microdomain-structured. CONCLUSION:

Hydrogels can be seen as optimized tools for facing various medicinal and pharmaceutical problems by producing sustained and prolonged effects with diminished side effects. Various hydrogels prepared are able to produce the desired and required sustained effects. In case of ophthalmic drug delivery, elastic property of hydrogels provides a reduction in lacrimal drainage. Transdermal preparations used to produce a constant penetration of a flux into skin for a longer period of time. The super adsorbent property of hydrogels helps in absorbing urine fro the diapers of the childrens.


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Transdermal Drug Delivery: Evolving Technologies and Expanding

Opportunities Geeta Aggarwal*, Ashish Garg$, Sanju Dhawan#

Dept. of Pharmaceutics, Rayat Institute of Pharmacy, Railmajra, Distt. Nawanshahr, Punjab, India 144533 $Eli Lilly and Company Pvt Ltd, Gurgaon, Haryana, India 122001

#University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India 160014

Email: [email protected]

ABSTRACT

Until very recently, the only drugs that could permeate transdermally were those possessing a very narrow and

specific combination of physicochemical properties. However, rapid advances in bioengineering have led to the

emergence of various new "active" enhancement technologies designed to transiently circumvent the barrier

function of the stratum corneum. These novel systems, using iontophoresis, sonophoresis, electroporation,

microneedle arrays, RF (radiofrequency) micro channels, pressure waves, or needleless injections will greatly

expand the range of drugs that can be delivered transdermally. Crucially, the delivery of macromolecules will

become possible and the transdermal flux of other molecules could be enhanced by several orders of

magnitude. This article sequentially reviews the basis of each of the new enhancement techniques and discusses

how these emerging technologies will influence transdermal delivery in the coming years.

Key Words: Transdermal drug delivery, iontophoresis, sonophoresis, electroporation, microneedle technology,

radiofrequency microchannels, pressure waves, needleless injections

INTRODUCTION

The past twenty five years have seen an explosion in the creation and discovery of new medicinal agents. Related innovations in drug delivery systems have not only enabled the successful implementation of many of these novel pharmaceuticals, but have also permitted the development of new medical treatments with existing drugs. These innovations offer substantial clinical advantages, including reduced dosing frequency and improved patient compliance, minimized fluctuation of the concentrations and maintenance of blood levels within a desired range, localized drug delivery and potential for reduced adverse effects. The creation of transdermal delivery systems has been one of the most important of these innovations, offering a number of advantages over the oral route1. Patients often forget to take their medicine and even the most faithfully compliant get tired of swallowing pills. So transdermal delivery offers convenience, especially notable in patches that require only once weekly application. Such a simple dosing regimen can aid in

patient adherence to drug therapy. It can be used as an alternative route of administration to accommodate patients that cannot tolerate oral dosage forms. Drugs that cause gastrointestinal upset can be good candidates for transdermal delivery because this method avoids direct effects on stomach and intestine. Drugs that are degraded by enzymes and acids in gastrointestinal system may also be good targets. First pass metabolism, an additional limitation to oral drug delivery can be avoided with transdermal administration. Another advantage, steady permeation of the drug across the skin allows for more consistent serum drug levels, often a goal of therapy. The lack of peaks in plasma concentration can reduce the risk of side effects. Thus drugs that require relatively consistent plasma levels are very good candidates for transdermal drug delivery2-4. The active ingredients currently available on the market in the form of transdermal devices are fentanyl for moderate or severe pain, nitroglycerine for angina pectoris, nicotine for smoking cessation, testosterone for hypogonadism in males, clonidine for hypertension,


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lidocaine as anaesthetic, scopolamine for motion sickness, estrogen/progesterone for hormone replacement therapy, norelgestromin/estradiol for birth control and estradiol for postmenstrual syndrome5. In addition drugs like rivastigmine for Alzheimer’s and Parkinson dementia, rotigotine for Parkinsonism, fentanyl iontophoretic for patient controlled pain management, fentanyl generic as analgesic, methylphenidate for attention deficit hyperactive disorder and selegiline for depression are recently approved as transdermal drug delivery systems6. US FDA approved the first transdermal patch in 1981. This patch delivered scopolamine, a drug which suppresses nausea and vomiting in motion sickness3. Over the last two decades, more than 35 transdermal products have been approved generating sales of $ 3.2 billion in 2002, which is predicted to rise to $ 4.5 billion in 2008. This rapid increase in market value has led to transdermal drug delivery becoming one of the fastest growing sectors within pharmaceutical industry7. The main limitation of transdermal drug delivery is the lipoidal barrier of stratum corneum. For a drug to be delivered passively via the skin it needs to have adequate lipophilicity and also a molecular weight <500Da. These requirements have limited the number of commercially available products based on transdermal delivery. However, recent advances and emerging technologies can be used to deliver drugs transdermally so that a wide range of drugs can be used as transdermal drug delivery systems8-9. Currently, market growth is fuelled by the launch of new products within existing applications such as hormone replacement and pain management therapies. The introduction of Androgel, the first transdermal testosterone gel, has resulted in unprecedented growth of this segment in a short span of time, clearly demonstrating the available market potential10-12. This review gives in brief, the technologies and the emerging technologies in transdermal delivery of drugs. PERMEATION ENHANCERS

Substances that reduce the skin’s ability to perform its barrier function are collectively known as penetration enhancers. These substances make the skin more permeable and allow drug molecules to cross the skin at faster rate13-14. Over the last 15 years, a tremendous amount of work has been directed towards the search for specific chemicals, combination of chemicals,

which can act as penetration enhancers. Some of the works have been enlisted in Table 1. However, the amount of drug that can be delivered using these methods is still limited since the barrier properties of skin are not fundamentally changed15. ETHOSOMES

Newer advancements in the patch technology have lead to the development of ethosomal patch, which consists of drug in ethosomes. These drug carrying vesicular systems are then embedded in polymer matrix. Transdermal delivery of testosterone from an ethosomal patch was found to be more effective both in the in

vitro and in vivo systems than the commercially available patches. Ethosomal systems are made up of soya phosphatidylcholine, ethanol and water. They may form multilamellar vesicles and have a high entrapment capacity for molecules of various lipophilicities16. The elastic vesicles and transferosomes have also been used as drug carriers for a range of small molecules, peptides, proteins and vaccines17. IONTOPHORESIS

To expand the number of compounds that can be delivered via the skin, researchers are developing novel transdermal technologies, including iontophoresis which uses an electric current to cause charged particles to move18. A pair of adjacent electrodes placed on the skin set up an electrical potential between the skin and the capillaries below. At the positive electrode, positively charged drug molecules are driven away from the skin’s surface toward the capillaries. Conversely, negatively charged drug molecules would be forced through the skin at the negative electrode. Transdermal iontophoresis has been extensively applied to delivery of anti-inflammatory agents and other compounds for local effects in the context of physical therapy. Iontophoresis has the potential to expand the range of compounds available for transdermal delivery to include proteins and peptides and enhance skin transport. Because the current can be literally switched on and off and modified, iontophoretic delivery enables rapid onset and offset. Hence drug delivery is highly controllable and programmable19-21. Various companies like Vyteris, based in Fair Lawn, New Jersey, ALZA, General Medical Company of Los Angeles, California and Birch Point Medical in Oakdale, Minnesota are developing iontophoretic delivery systems. Clinical trial results, presented by


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Vyteris, at the recent “Drug Delivery – Latest Technology & Strategic Partnerships” conference in Munich, Germany, have shown successful delivery of peptides up to a molecular weight of 3,500 Daltonsi. ALZA has now developed an E-TRANS formulation of fentanyl. In clinical trials, E-TRANS fentanyl administered for 20 minutes every hour for 24 hours resulted in mean serum levels greater than those achieved from intravenous fentanyl. According to ALZA, more than 90% of patients who used E-TRANS indicated they would like to use the E-TRANS fentanyl system for subsequent surgery, given the convenience, ease-of-use and the ability to achieve pain relief within a few minutes of pressing the button22. With further advances in formulation science, electrode design and battery technology, iontophoresis may also become a viable alternative for the delivery of other drugs that are used over a long term23. ELECTROPORATION

Another transdermal technology being developed is electroporation which involves the creation of aqueous pores in lipid bilayers by the application of a short (microseconds to milliseconds) electric pulse24-25. Various parameters governing the performance of electroporative delivery to the skin are voltage, pulse length, number of pulses and electrode area26. Anionic phospholipids, but not cationic or neutral phospholipids, have been found to enhance the transdermal transport of molecules by electroporation27. Ichor Medical Systems is dedicated to the clinical application and commercialization of electroporation mediated DNA drug delivery28. Skin electroporation continues to be an active area of research. Larger macromolecules have also been delivered including heparin, insulin and vaccines29-31. RF MICROCHANNELS

Radio frequency skin ablation is an established medical technique that has been commonly used for surgical procedures. This technique is modified into RF microchannel technology to create passage through the skin that allows a novel and unique approach for transdermal drug delivery. RF microchannels are a universal solution that can expand the range of molecules that can be delivered transdermally32-33. The Israeli company, TransPharma Medical, is using alternating current at radio frequencies (RF) to create aquatic throughways, about 100 micrometers wide,

across the stratum corneum. TransPharma’s system consists of a reusable, hand-held electronic unit and a disposable microelectrode array consisting of hundreds of closely spaced electrodes. The array is snapped on to the handset and pressed lightly against treatment site. This action activates the handset to apply alternating RF current to the electrodes for several milliseconds, causing microchannels to begin forming. The number of active electrodes determines the number of pores and thus, amongst other factors, the rate at which drug will cross the skin. When the channels are formed, the current stops automatically, and the user is alerted by audible and visual signals. A closed loop electronic feedback system controls the duration of current delivery by monitoring the current being applied, detecting when the microchannels have reached the desired depth. Importantly, the channels only reach as far as the epidermis, where there are no nerves or blood vessels, and the RF is too high to stimulate muscles or nerves, so there is no significant pain and trauma from the procedure. Following microchannel formation, the electrode array is lifted away from the skin and discarded. The patch containing the drug can then be applied. The microchannels will remain for up to 24 h, allowing for prolonged duration of drug delivery if desired34. ULTRASOUND/ SONOPHORESIS: PAINLESS

DRUG DELIVERY

Still another transdermal technology under development is low frequency sonophoresis, which enhances the transport of permeants, such as drugs through cell membranes as a result of ultrasonic energy35. Ultrasonic sound waves cause acoustic cavitation, the resultant effects of which microscopically disrupt the lipid bilayers of the stratum corneum and thereby influencing the influx of permeants. Thus sonophoresis is able to increase the penetration of various low molecular weight drugs as well as high molecular weight proteins36. Dermisonics is an intellectual property company and advanced technology incubator has patented U-Strip™ system which employs proprietary microelectronics and ultrasonic technologies with a drug-carrying patch to enable the painless delivery of drugs through the skin's natural pores and hair follicles. The U-Strip™ Insulin Patch alone could improve the lives of insulin-dependent diabetics, reaching 55 million diabetics, or


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nearly 30% of the total 185 million diabetic population worldwide, who endure painful needle injections to survive this disease37. PRESSURE WAVES

Pressure waves, which are generated by intense laser radiation, can also permeabilize the stratum corneum as well as the cell membrane. These pressure waves are compression waves and thus exclude biological effects induced by cavitations. Their amplitude is in the hundreds of atmospheres (bar) while the duration is in the range of nanoseconds to a few microseconds. The pressure waves interact with cells and tissue in ways that are probably different from those of ultrasound. Furthermore, the interactions of the pressure waves with tissue are specific and depend on their characteristics, such as peak pressure, rise time and duration. A single pressure wave is sufficient to permeabilize the stratum corneum and allow the transport of macromolecules into the epidermis and dermis. In addition, drugs delivered into the epidermis can enter the vasculature and produce a systemic effect. For example, insulin delivered by pressure waves resulted in reducing the blood glucose level over many hours. The application of pressure waves does not cause any pain or discomfort and the barrier function of the stratum corneum always recovers38. MICRONEEDLES

Recently, the use of micron-scale needles in increasing skin permeability has been proposed and shown to dramatically increase transdermal delivery, especially for macromolecules9, 39. Although microneedles were first proposed in 1970s, the technology needed to make microneedles did not become widely available until 1990s. Using the tools of the microelectronics industry, microneedles have been fabricated with a range of sizes, shapes and materials, which create micrometer scale holes in the outer skin layer, thereby allowing passage of large molecules and other compounds that ordinarily could not traverse the skin. Most drug delivery studies have emphasized solid microneedles, which are painless because they are too small to touch the nerves located deeper in the skin. To address practical applications of microneedles, the ratio of microneedle fracture force to skin insertion force (i.e. margin of safety) should be optimal for needles with small tip radius and large wall thickness40. Some scientists recently used this technique to administer

insulin to diabetic rats. Over a 4 h time period, blood glucose levels steadily dropped by as much as 80 percent41. Transdermal delivery using microneedles is also developed by ALZA Corporation and 3M (St. Paul, MN). ALZA’s Macroflux technology uses a thin titanium screen that consists of 200 µm projections that create pathways to deliver the drug through the stratum corneum. Similar to the Macroflux transdermal technology is 3M’s Microstructured Transdermal System (MTS) that also incorporates micro needle technology for targeted delivery of drug molecules across the skin membrane42. The drug molecule can be coated onto the micro projections to achieve a bolus delivery or using a reservoir to ensure continuous delivery of the drug over a specified time9. Because people would require minimum training to apply microneedles, these devices may prove useful for immunization programs in developing countries or for mass vaccination or antidote administration in bioterrorism incidents. NEEDLELESS INJECTIONS

This method of administering drugs circumvents issues of safety, fear and pain associated with the use of hypodermic needles. Transdermal delivery is achieved by firing the liquid or solid particles at supersonic speeds through the outer layers of the skin using a suitable energy source. The mechanism involves forcing compressed gas through the nozzle, with the resultant drug particles entrained within the jet flow reportedly traveling at sufficient velocity for skin penetration43-44. The powerjet injector has been reported to successfully deliver testosterone, lidocaine hydrochloride and insulin45. Some of the drugs used by the recent transdermal technologies are enlisted in Table 2. CONCLUSION

The recent transdermal technologies challenge the paradigm that there are only few drug candidates for transdermal drug delivery. With the active transdermal technologies, molecular size, melting point, solubility, dose size is not a limiting factor. The opportunities for transdermal drug delivery are expanding through the application of new formulation technologies and active delivery systems. Now a much wider set of compounds, including macromolecules, can be delivered transdermally at therapeutic levels46. However, for


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Table No. 1 - Examples of permeation enhancers

Category Subcategory Examples Drugs used References

Solvents Short chain alcohols

Ethanol Anemonia 47

Long chain alcohol

(i) Octanol (ii) Methanol (iii) C6 - C12 alcohols

Nicotinamide Testosterone, diclofenac Benzoyl peroxide, Retinoic acid

48

Polyalcohols Propylene glycols Ethinylestradiol 49

Alkylmethyl Sulphoxide

Dimethysulphoxide (DMSO)

Salbutamol, Steroids, Salicylate, Antiviral, Antimycotic, Methadone

50

Pyrrolidones 2-pyrrolidone and N-methyl-2-pyrrolidone

Zidovudine, Steroids, Caffeine, β-estradiol, NSAIDs

51-52

Azone Laurocapram (1-dodecylazacloheptane-2-one)

Fluoxetine 53

Surfactants Sodium lauryl sulphate, Benzalkonium chloride, Span 20

Diclophenac, Lorazepam 54-55

Essential oils

Menthol d-limonene Caraway oil, Lemon oil Linoleic acid

Quercetin Nicorandil Piroxicam

56 57-58 59

Miscellane-ous Urea

Indomethacin 60

Table No. 2 - List of drugs used by active transdermal drug delivery technologies

Drug Delivery methods Drugs used References

Iontophoresis

Haloperidol Verapamil Salicylic acid Insulin Chlostridium botulinum toxin type A Lidocaine

61 62 63 64 65

66-67 Electroporation

Heparin Lidocaine Insulin Vaccines DNA Oligonucleotides Vitamin C

29 68 69 31 30 70 71

RF Microchannels Proteins 32


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Ultrasound

Insulin Heparin Mannitol

72 73 74

Pressure waves Insulin 38 Microneedles

Insulin Calcein

75 76

Needle less injections Insulin 45

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Disclaimer : The views expressed are those of authors

only and Eli lilly and Company India Pvt. Ltd. does

not subscribe to any statements/views of the authors.


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Microwave Assisted Fast Extraction of Mucilages and Pectins B Geethaa, K P Shivalinge Gowdab, G T Kulkarnia and Shrishailappa Badami*a

aJ S S College of Pharmacy, Rocklands, Ootacamund 643 001, Tamilnadu, bJ S S College of Pharmacy, Mysore – 571 008, Karnataka

*For correspondence : [email protected]

ABSTRACT

A microwave assisted extraction technique was developed to optimize the extraction of mucilages and pectins

from several commonly used plant sources. Microwave extraction at 180 W intensity and 40 m heating period

produced higher yields of mucilages when compared to 1 h conventional heating in all the seven plant sources

used. The yields of drumstick, passion fruit mucilages obtained by microwave method were found to be 211.34

and 173.46% more than that obtained by conventional method, respectively. Similarly, 350 W intensity and 20

m heating period gave higher yields of pectins when compared to 1 h conventional heating in all the seven plant

sources tested. The percentage increase in the yields in case of guava and butter fruit by microwave method

were found to be 191.11 and 121.26, respectively. The products obtained by both the methods were of similar

nature both physically and chemically. The developed microwave procedure can be used successfully in

commercial and routine laboratory isolation of mucilages and pectins.

Key Words: Microwave, Mucilage, Pectin, Fenugreek, Drum Stick, Asparagus, Mustard, Guava, Orange-peel,

Mango

INTRODUCTION

In the past few years, microwave heating has been found to be a convenient source of energy not only in kitchen, but also in chemical laboratories1. Many of the basic principles of green chemistry are best suited for microwave processes2. It is one of the simple, fast, clean, eco-friendly and efficient method. It is economic in saving energy, fuel and electricity. A very short response time and better yields of the products are the main advantages of microwave heating. Commercial microwave ovens are used as a source of energy in chemical laboratories for efficient heating of water, moisture analysis, wet ashing of biological and geological materials, waste material management, sterilization of pharmaceutical preparations, inactivation of enzymes in food products, etc. In the past few years, a large number of reports have appeared on the use of this technique in acceleration of organic reaction for rapid and green synthetic procedures1. Extraction is one of the most crucial point in the analytical chain in the effort of achieving a complete recovery of target compounds. Recently, microwave

energy is being used for extraction of phytoconstituents from plants3,4. Microwave extraction follows the same principle as maceration or percolation, but the speed of breaking up of plant cells and plant tissues is much higher. This reduced processing time is an economic advantage and also there is less risk of decomposition or disintegration and oxidation of the valuable plant constituents. Microwave assisted extraction methods require shorter time, less solvents, higher extraction rate and better products with lower costs4. The microwave consists of a number of radiation chambers in order to manipulate the required energy. The energy requirement can be controlled much better than with conventional thermal energy. A great deal of work is required to standardize experimental procedures using this technique. Today, the whole world is turning towards finding suitable alternatives for the synthetic compounds used in pharmaceutical industries from natural sources2. The synthetic polymers used as excipients suffer from many disadvantages such as high cost, toxicity, non-biodegradability and environmental pollution caused during their synthesis5. Natural polymers like mucilages Indian Journal of Pharmaceutical Education & Research

Received on 12/7/2008 ; Modified on 11/2/2009 Accepted on 13/3/2009 © APTI All rights reserved

APTI ijper


261

and pectins are easy to isolate, purify and are non-toxic and biocompatible. They are biodegradable and will not cause environmental pollution5. They are found as common ingredients in cosmetic, food and non-food industries. They are used as binding, thickening, emulsifying, suspending and stabilizing agents in pharmaceutical industries and used as matrices for sustained release of drugs, because of their higher water swellability, non-toxicity, low cost and free availability5. Conventionally, mucilages and pectins are isolated by heating for 1 h, in aqueous solvents6,7. In order to reduce the duration involved in their extraction and study the yield, mucilages and pectins from several commonly available plant products were isolated in the present study using microwave method. MATERIALS AND METHODS

Collection and Authentication:

Fenugreek (Trigonella foenum-graecum Linn, Leguminosae, seeds), Drum stick (Moringa oleifera Lam, Moringaceae, fleshy mass with pods), Wild asparagus (Asparagus racemosus Willd, Liliaceae, tuberous roots), Passion fruit (Passiflora edulis, Sims, Passifloraceae, pericarp), Indian mustard (Brassica

juncea Czern. and Coss., Brassicaceae, seeds), Black gram (Vigna mungo Linn. Hepper, Fabaceae, seeds) and Holy basil (Ocimum sanctum Linn, Lamiaceae, seeds) were used for the isolation of mucilages. Orange (Citrus reticulata, Blanco, Rutaceae, dried peels), Common guava (Psidium guajava, Linn, Myrtaceae, pericarp), Mango (Mangifera indica, Linn, Anacardiaceae, pericarp), Butter fruit (Persea

Americana, Mill, Lauraceae, pericarp), Passion fruit, Tree tomato (Cyphomandara betacea, Linn, Solanaceae, pericarp) and Tight skinned orange (Citrus

sinensis, Linn. Osbeck, Rutaceae, fresh peels) were used for the isolation of pectins. These materials were collected from in and around Ootacamund and authenticated by Medicinal Plants Survey and Collection Unit, Ootacamund. Isolation of Mucilage by Conventional Procedure:

Fenugreek seeds (10 g) were powdered for 5 m in a mechanical blender and soaked in distilled water (300 ml) for 24 h in a RB flask. It was boiled for 1 h under reflux with occasional stirring and kept aside for 2 h for the release of mucilage into water. The material was filtered through a muslin bag and hot distilled water (50 ml) was added through the sides of the marc and

squeezed well in order to remove the mucilage completely. Equal volume of ethanol was added to the filtrate to precipitate the mucilage and kept inside a refrigerator for one day for effective settling. It was filtered and dried completely in an incubator at 37º C, powdered and weighed. It was subjected to chemical tests to confirm its identity6,7. Similarly, mucilages were isolated from all the other plant sources and chemical tests were carried out. Isolation of Mucilage by Microwave Procedure:

Fenugreek seeds (10 g) were powdered in a mechanical blender for 5 m and soaked in distilled water (300 ml) for 24 h in a 1000 ml beaker. It was kept in a microwave oven (LG Health Wave System, MG-605AP, 900 W, 230 V, 50 Hz) along with a glass tube inside to prevent bumping. It was subjected to microwave irradiation at 540 W intensity for 7 m. The beaker was removed from the oven and kept aside for 2 h for the release of mucilage into water. It was processed in a similar way as explained in the conventional procedure, weighed and chemical tests were carried out. The experiment was repeated several times using various intensities and different durations as shown in Table 1. In each case, the yield was calculated. Fenugreek seeds gave highest yield at 180 W intensity and 40 m heating period. Hence, using these parameters, mucilages were isolated from all the other plant sources and chemical tests were performed. Isolation of Pectin by Conventional Procedure:

Dried orange peel (25 g) was cut into smaller pieces and soaked in distilled water (200 ml) for 2 h. It was adjusted to pH 4.5 with tartaric acid solution (10%) and boiled under reflux for 1 h with occasional stirring. It was filtered while hot and the filtrate cooled and poured into a beaker containing acetone (600 ml) to precipitate out the pectin. It was filtered using a nylon sieve and washed with acetone to make the pectin free from acidic ions6. Pectin thus obtained was completely dried in an incubator at 37º C, powdered and weighed. It was subjected to chemical tests to confirm its identity8. Isolation of Pectin by Microwave Procedure:

Dried orange peel (25 g) was cut into small pieces and soaked in distilled water (200 ml) for 2 h in a 1000 ml beaker. Its pH was adjusted to 4.5 with tartaric acid and kept in a microwave oven along with a glass tube inside to avoid bumping. It was subjected to microwave


262

irradiation at 160 W for 10 m. It was processed in a similar way as explained in the conventional procedure, weighed and chemical tests were performed. The experiment was repeated several times using various intensities and different durations as shown in Table 2. In each case, the yield was calculated. Orange peel gave highest yield of pectin at 350 W intensity when heated for a duration of 20 m. Hence, using these parameters, pectins were isolated from all the other plant sources and chemical tests were performed. RESULTS AND DISCUSSION

Using fenugreek seeds and dried orange peel, respectively for the isolation of mucilage and pectin, microwave standardization was carried out at several intensities and durations (Tables 1 and 3). Using 180 W intensity and 40 m heating duration, 30% increase in the yield of mucilage was obtained under microwave irradiation when compared to the conventional heating method. Similarly, for the isolation of orange peel pectin, use of 350 W intensity and 20 m heating period produced 52% increase by microwave method.

All the fourteen plants, when subjected to microwave irradiation using the above parameters produced high yields of mucilages and pectins. The yields of drumstick and passion fruit were found to be 211.34 and 173.46% more by microwave method, respectively (Table 3). The yields of Asparagus, Indian mustard, holy basil and black gram mucilages obtained by microwave method were found to be 25-85% more than the conventional yields. Similarly, the yields of guava and butter fruit pectins obtained by microwave method were found to be 191.11 and 121.26% more than the corresponding conventional yields, respectively (Table 4). The other four plant sources also produced 30-70% increase in yields by microwave method. All the mucilages and pectins isolated by both the methods were found to be identical in nature and gave positive results for all the chemical tests performed. Thus, all the selected plant sources produced high yields of mucilages and pectins in shorter duration by the developed microwave method when compared to conventional heating.

Table 1 – Standardization of microwave method for the isolation of mucilage from Fenugreek seeds

S. No.

Conventional Method Microwave Method

Duration Yield (g) Intensity (W) Duration (m,

sec) Yield (g)

Percent increase in

yielda

1 1 h 2.642 540 7, 0 2.464 --

2 -- -- 540 10, 0 Product charred

--


--


--

5 -- -- 360 10, 0 1.512 -- 6 -- -- 360 20, 0 2.644 -- 7 -- -- 180 30 2.867 8.5

8 -- -- 180 40 3.424 29.6 a In comparison to conventional method


263

Table 2 – Comparison of duration of isolation and yields of mucilages isolated by conventional and microwave

methods

S. No.

Plant source Yield by conventional methoda,b Yield by microwave methoda,c

Percent increase in

yieldd

Weight (g) Percent Weight (g) Percent

1 Fenugreek 2.642 ± 0.069 26.42 3.424 ± 0.029 34.24 29.59 2 Drum stick 0.247 ± 0.022 2.47 0.769 ± 0.014 7.69 211.34 3 Asparagus 0.111 ± 0.013 1.11 0.205 ± 0.016 2.05 84.68 4 Passion fruit 0.569 ± 0.036 5.69 1.556 ± 0.059 15.56 173.46

5 Indian mustard

0.936 ± 0.038 9.36 1.610 ± 0.047 16.10 72.00

6 Black gram 3.152 ± 0.086 31.52 3.946 ± 0.056 39.46 25.19 7 Holy Basil 2.310 ± 0.096 23.10 3.146 ± 0.074 31.46 36.19

aAverage of three determinations ± SD ; b Reaction time: 1 h ; cIntensity: 180 W, reaction time: 40 m d In comparison to conventional method

Table 3 – Standardization of microwave method for the isolation of pectin from dried orange peel

S. No.

Conventional Method Microwave Method

Duration Yield (g) Intensity (W) Duration (m,

sec) Yield (g)

Percent increase in

yielda

1 1 h 0.432 160 10 0.234 -- 2 -- -- 350 10 0.368 -- 3 -- -- 500 10 0.372 -- 4 -- -- 350 15 0.484 12.03 5 -- -- 350 20 0.658 52.31 6 -- -- 500 15 0.386 -- 7 -- -- 650 5 Charred

product --

a In comparison to conventional method

Table 4 – Comparison of duration of isolation and yields of pectins isolated by conventional and microwave

methods

Sl. No.

Plant source Yield by conventional methoda,b Yield by microwave methoda,c

Percent increase in

yieldd

Weight (g) Percent Weight (g) Percent

1 Dried orange peel

0.432 ± 0.018 1.728 0.658 ± 0.036 2.632 52.31

2 Mango 0.579 ± 0.021 2.316 0.749 ± 0.035 2.996 29.36 3 Guava 0.349 ± 0.026 1.396 1.016 ± 0.082 4.064 191.11 4 Passion fruit 0.734 ± 0.051 2.936 1.246 ± 0.076 4.984 69.75


264

5 Tree tomato 0.962 ± 0.072 3.848 1.608 ± 0.066 6.432 67.15 6 Butter fruit 0.268 ± 0.018 1.072 0.593 ± 0.026 2.372 121.26

7 Tight skinned orange

0.486 ± 0.024 1.944 0.764 ± 0.032 3.056 57.20

aAverage of three determinations ± SD ; b Reaction time: 1 h ; cIntensity: 350 W, reaction time: 20 m ; d In

comparison to conventional method In recent years, modern techniques are effectively being used for the extraction and isolation of phytoconstituents. Ways to minimize the consumption of energy and developing efficient isolation and purification processes is of utmost global importance today. Plant mucilages and pectins are found as common ingredients in cosmetic, pharmaceutical, food and non-food industries due to their low cost compared to the synthetic polymers9. In view of the rising costs and fluctuations in availability of the synthetic polymers, scientists are engaged in finding suitable alternatives to these10. Such an effort would be welcomed both locally and internationally. In India, only few units are manufacturing mucilages and pectins on commercial scale. USA, Switzerland and other European countries are producing these in large scale. Most of the Indian needs are mainly met by import. The conventional isolation of mucilages and pectins from plants consumes more time and gives low yields. Hence, the cost of production of these is increasing. In the present study, mucilages and pectins were isolated in high yields and in lesser durations in all the fourteen plant sources by the developed microwave method when compared to the conventional heating. Same quantities of raw materials were used for both the methods. The yields of drumstick and passion fruit mucilages by microwave method were found to be double than the conventional heating. The developed microwave methods save 20 and 40 m heating duration respectively, for the isolation of mucilages and pectins. The speed of breaking up of plant cells and plant tissues is much higher under microwave conditions4. The present study also proves the same. Efforts have not been made to utilize the vast agricultural resources for the isolation of mucilages and pectins in India. Among the seven plant sources used for the isolation of mucilages, fenugreek, black gram, and holy basil were found to be very good sources due to their high contents of mucilage, which was in the range of 32-40%. Passion fruit and Indian mustard were

also found to be ideal sources due to the presence of around 15% of mucilage in them. Similarly, all the seven plant sources used for the isolation of pectin were found to be good sources of pectin, as their pectin content was found to be almost identical and in the range of 2.4-6.4%. Usually only a few of these are used for the commercial isolation of mucilages and only the citrus fruits are preferred for the commercial isolation of pectins. Hence, the present work identifies new sources of raw materials for the commercial isolation of mucilages and pectins. CONCLUSION

In the present study, a microwave assisted method for rapid extraction of mucilages and pectins from different sources has been optimized. The developed method improved the yield of mucilages as well as pectins from the selected sources. In conclusion, the developed microwave procedure can be used successfully in commercial and routine laboratory isolation of mucilages and pectins. ACKNOWLEDGEMENT

The authors thank Jagadguru Sri Shivaratri Deshikendra Mahaswamiji of Suttur Mutt for providing the facilities. One of the authors (GB) thank AICTE, New Delhi for the award of QIP fellowship. REFERENCES

1. Sharma SV, Ramasarma GVS, Suresh B. MORE chemistry: an eco-friendly technology. Indian J Pharm Sci 2002; 64: 337-344.

2. Anastas PT, Warner JC. Green Chemistry: Theory and Practice. New York: Oxford Press; 1998.

3. Mattina NJI, Berges WAI, Denson CL. Microwave assisted extraction of taxanes from Taxus biomass. J Agric Food Chem 1997; 45: 4691-4696.

4. Fulzale DP, Satdive RK. Comparison of technique for the extraction of the anticancer drug camptothecin from Nothapodytes foetida. J Chromatogr-A 2005; 1063: 9-13.

5. Kulkarni GT, Gowthamarajan K, Satish Kumar MN, Suresh B. Gums and mucilages: therapeutic


265

and pharmaceutical applications. Nat Prod Rad 2002; 1(3): 10-17.

6. Kokate CK. Practical Pharmacognosy. New Delhi: Vallabh Prakashan; 1999.

7. Kulkarni GT, Gowthamarajan K, Brahmajirao G, Suresh B. Evaluation of binding properties of selected natural mucilages. J Sci Ind Res (India) 2002; 61: 529-532.

8. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. Pune: Nirali Prakashan; 2002.

9. Franz G. Polysaccharides in pharmacy: current applications and future concepts. Planta Med 1989; 55: 493-497.

10. Verma PRP, Razdan B. Studies on Leucaena

leucocephala seed gum: evaluation of emulsifying properties. J Sci Ind Res (India) 2003; 62: 198-206.


266

Synthesis of Some 2-(Substituted)-5-[(N-Benzotriazolomethyl)-1, 3,

4-Thiadiazolyl]-4-Thiazolidinones for their Anti-Fungal Activity K.P.Namdeo1*, V.K.Singh2 and S.K.Prajapati2

SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur (CG) 1

Institute Of Pharmacy, Bundelkhand University, Jhansi (UP) 2

Address for correspondence: [email protected]

ABSTRACT

Benztriazole, Thiadiazole and Thiazolidinone showed diversified pharmacological activities. In view of

potential biological activities of Benztriazole, Thiadiazole and Thiazolidinone, it was considered worthwhile

to synthesize some 2-(substituted) - 5- [(N-benzotriazolomethyl)-1, 3, 4-thiadiazolyl] - 4 - thiazolidinone and

evaluate for their possible antifungal activity.

Key Words: Benztriazole, Thiadiazole, Thiazolidinone, Antifungal

INTRODUCTION

1,2,3-Benztriazoles were reported to have potential fungicidal1 and antiparasitic2 activity. Similarly 1,3,4-Thiadiazole derivatives were also reported to possess fungicidal, herbicidal, bactericidal3, pesticidal, insecticidal, antihistaminic and antiamoebic4, CNS stimulant, anticholinergic, hypoglycemic5, cadiotonic6, antihypertensive7, anticonvulsant, hypnotic8, antianthelmintic, analgesic, anti-inflammatory and antioxidant activity 9. 4-Thiazolidinone nucleus has also occupied a unique place in the field of medicinal chemistry due to wide range of biological activities like antischistomal, antibacterial, anticancer, potassium channel inhibitor, cardiovascular10, anticonvulsant, sciatic nerve blocking, local anesthetic, sedative, cholerectic, antitubercular11, antiviral, antitumour, CNS depressant12, hypoglycemic13, antiallergic14, antihypertensive, antianthelmintic, anti-inflammatory, antiparkinsonian15, analgesic, hypnotic16, antithyroidal, fungicidal17, cysticidal, antileukemic18 and antioxidant activity19. In view of potential biological activities of Benztriazole, Thiadiazole, 4-Thaizolidinone and some similar activities of Thiadiazole and 4-Thiazolidinone, they were fused together and synthesized (X1-X6 ) to probe how far these combinations could enhance the fungicidal action, as out lined in the Scheme-1.

MATERIAL AND METHOD

All the Chemicals were procured from Qualigen Mumbai and CDH New Delhi. They were of an analytical grade. Melting Points were determined in open glass capillaries using Tempo melting point apparatus and were uncorrected. 2-(substituted)-5-[(N-Benzotriazolomethyl)-1,3,4-Thiadiazolyl]-4-Thiazolidinones were prepared as per the method described in the literature20,21,22 and the synthetic procedure involved the six steps as stated below. STEP I- The Synthesis of Benztriazole (i)

In a mixture of 11.5 mL glacial acetic acid and 30 ml water, 0.1M o-phenylenediamine was dissolved and then added a solution of 0.1M NaNO2 in 15ml of water, stirred continuously for 15 minutes. The temperature was maintained at 12 oC, chilled in ice bath and product (i) was collected by filtration. The yield obtained was 65% and M.P. was 99 oC. STEP II- The synthesis of N-Benztriazolacetate (ii) A mixture of product (i) (0.1M), ethyl acetate (0.1M) and 0.3g of K2CO3 in 60 ml of acetone was stirred for 10 hrs. The solvent was removed under reduced pressure. A solid mass was produced and then needle shaped brown crystals were obtained after recrystallization from the mixture of chloroform and ether (8:2%V/V).The yield obtained was 60% and M.P. was 40 oC. STEP III - The synthesis of N-Benztriazol acetyl thio

semicarbazide (iii)


APTI ijper


267

The crystals obtained from step II (0.08M) and thiosemicarbazide (0.08M) were taken in 50 mL of ethanol, stirred for 6 hrs and then refluxed for 3 hrs. The yellow colored compound was obtained after recrystallization from the mixture of chloroform and hexane (9:1V/V). The yield was 57% and MP 102-103oC. TEP IV- The synthesis of 2-amino 5-(N-

Benztriazolomethyl)-1,3,4-Thiadiazole (iv)

Compound iii (0.08M) was added in Conc.H2SO4 and kept over night at room temperature, then neutralized with ammonia and extracted with ether. The ether was distilled off and the product recrystallised from methanol, the yield was 52% & M.P.121 oC. STEP V- Synthesis of 2-Benzylidenylamino-5-(N-

Benztriazolomethyl)-1, 3, 4-Thiadiazole (v)

(0.2M) compound iv, (0.2M) carbonyl compound (R1R2 C=O) and (2 ml) glacial acetic acid were refluxed in 50 mL methanol for 8 hrs. Solvent was distilled off and product recrystallised from mixture of benzene and chloroform (1:6v/v). M.P. was 129 oC and yield was 50%. STEP VI- The synthesis of 2-Phenyl 5-(N-

Benztriazolomethyl)-1, 3, 4-thiadiazolyl]-4-

thiazolidinone (vi) The compound (v) (0.1M) and mercapto acetic acid (10 mm) with a pinch of anhydrous ZnCl2 were added in 30 mL of tetrahydrofuran (THF) and refluxed for 12 hrs on water bath. The product was separated and recrystallised from ethanol. The M.P. was 138 oC and yield was 58%. The purity of the synthesized compounds were established by TLC using 2% silica gel G, n-butanol: glacial acetic acid: water (4:1:5) as eluents. Iodine chamber and UV light used for detecting the compounds. MP of the compounds X1 to X6 were 138oC, 130oC, 163oC, 156 oC, 122 oC & 126 oC respectively. Structures of the compounds were established on the basis of C, H and N analysis reports, IR and NMR spectra. DETERMINATION OF ANTI-FUNGAL

ACTIVITY

All the synthesized compounds were screened for antifungal activity on agar plates using Saboraud’s medium by Cup-plate technique23, 24. The fungal

cultures were C. albicans, A.niger, A. flavus, Alternaria

alternata. The standard drug used for the present study was Streptomycin. The concentration of the synthesized compounds and standard drugs were taken as 50 µg/ml .All the compounds were dissolved in DMF. In order to account for the effect due to DMF, a blank was also performed. RESULT AND DISCUSSION

A perusal of the table–2 and figure–1 shows that compared to the standard drug, all the synthesized compounds were effective against all the four strains but compound X4 was least effective and compound X2 was most effective. The compound X3 was most effective against C. albicans and A. flavus. In case of C.

albicans compound X1 has shown activity similar to Standard and Compound X5 was less effective. In A.

niger compound X2 was most effective and has shown more activity than the Standard. Compound X6 was less effective against A. niger. In A. flavus compound X3 was most effective and has shown more activity than the Standard. In A. alternata compound X2 was most effective comparable to standard and Compound X4 was less effective. CONCLUSION

These compounds were synthesized with the objective of developing better antifungal molecules with maximum percentage of yield and optimal antifungal activity. It was observed that halogen substituted aromatic compounds were more active than unsubstituted aromatic compounds and aromatic compounds were more active than alkyl substituted compounds12. All the synthesized compounds were effective against all the four strains; this activity might be due to the presence of three nuclei; Benztriazole, Thiadiazole and 4-Thaizolidinone in one structure. Compound X2 was most effective; the activity could be due to the presence of phenyl and para-bromophenyl ring. Compound X3 was less effective than compound X2; it might be due to presence of only para-chlorophenyl ring. Compounds X4 and X5 exhibited less antifungal activity; the activity could be due to the absence of aromatic nucleus or presence of alkyl groups. Further investigations with appropriate structural modification of title compound may result in therapeutically useful products.


268

NH2

NH2

HNO2

N

N

N

N

NN

N

NN

CH2

N

NN

CH2CH2COONHNHCSNH2

NN

SCH2

NN

S

HSCH2COOH,

H2SO4O=C

R1R2

O=C S

CNR2

NH2NH2CSNH2

C2H5OH

R1

NN

S NH2

N=CR1

R2

N

NN

CH2COOC2H5

NH

NN ClCH2COOC2H5

O-Phenylenediamine (i)

K2CO3, CH3COCH3

THF and Anh.ZnCl2

(vi)(v)

(iii)

(ii)

(iv)

Where, R1 R2

X1 - H -C6H5

X2 - -C6H5 -4-BrC6H4 X3 - H -4Cl-C6H5

X4 - CH3 -C6H5 X5 - CH3 -C2H5

X6 - C6H5 -C6H5

Scheme-I

CH3COOH

Table-1 Spectral data of 2-(Substituted)-5-[(N-Benzotriazolomethyl)-1,3,4-Thiadiazolyl]-4-Thiazolidinone

IR Spectra

Type Vibration Frequency in cm-1

Thiazolidinone ring C=O Str 1739

C -S Str 752

C -N Str 1333

C=N Str 1670

Thiadiazole ring N -N Str 1626.7

C=N Str 1670 C -S Str 641 Benzotriazole Ring C -N Str 1271.39 C=C Str 1536.15 Ar-H Str 3044.19 N =N Str 1382


269

PMR Spectrum

Signal position Relative No of protons Inference 7.68-7.36 9H Ar-H 4.65 2H -CH2 of Thiazolidinone 8.00 1H -CH of Thiazolidinone 3.97-3.85 2H -S-CH2 –C Thiazolidinone

Table-2 Antifungal activity of synthesized compounds

Compound Code Zone of Inhibition (mm)

C.albicans A.niger A.flavus Alternaria

alternata

X1 12 09 08 08 X2 07 15 10 11 X3 10 07 15 08 X4 08 08 08 07 X5 06 08 10 10 X6 09 06 10 10 Standard 12 10 12 12

Figure: 1 Antifungal activity of synthesized compounds

ACKNOWLEDMENT

Authors are very thankful to the Head, SAIF, Punjab University, Chandigarh for carrying out IR & NMR Spectroscopy and Head, Department of Biotechnology, Janta college, Chandarpur, Maharastra for providing Microbial Strains. Dr. Raghuveer Irchhaiya, Institute of Pharmacy, Bundelkhand University, Jhansi (UP) is also

acknowledged for providing necessary facilities to carry out this work. REFERENCES

1. Rai A.K., Mishra A.R., Abushma and Tripathi, D.S., Synthesis of Triaryl-s-Triazolo-[3,4-b]-1,3,4-Thiazolo[3,2-b]-s-Triazine-5-thione as Possible Fungicides, Indian J. Heterocyclic Chem., 2006; 16: 121-124.


270

2. Jaiswal K.P. and Patel H.D., Synthesis and Characterization of Arylidene Acetohydrazido Benztriazole Derivatives and study of their antibacterial and antifungal activities, Oriental J. Chemistry, 2004; 20(1): 169.

3. Tripathi, D.S., Mishra A.R., Mishra R.M., Singh D. and Dwivedi A.K., Synthesis and Fungitoxicity of Some New Thiadiazolo-s-Triazine Derivatives, Indian J. Heterocyclic Chem., 2006; 16: 117-120.

4. Sinha R., Garg S.P. and Sah P., Synthesis and Biological activities of some 5-(2-arylamino -1,3,4-Thiadiazolo)-Thiazo substituted sulphonamides and related compounds, Oriental J. Chemistry, 2004; 20(1): 131.

5. Jagadhani S.G., Kale S.B., Dalvi N.R., More M.S. and Karale B.K., Synthesis of 4- ( (Benzo (d) Isoxazol -3yl ) Methyl ) N-Phenyl-1,3,4-Thiadiazol- 2- amines and 5- (Benzo (d) Isoxazol -3yl ) Methyl) 4-Phenyl - 4H- 1,2,4-Triazole-3-Thiols, Indian J. Heterocyclic Chem., 2006; 15: 335.

6. Alex M. and Gallagher R. J., inventors; American Cyanamid Co, Assignee, 1H-1,2,4 - thiadiazolo (3,4-b) quinazolin -5 - one-2, 2 - dioxides and a method for increasing the cardiac output of a mammal with them, United States Patent, 4904667, 1972, Nov 7.

7. Krishna B.G., Venkateshan A., Srinivas B., Rao J.V. and Sarangapani M., Synthesis of 5-(Substituted Benzoxazol-5-yl)–2-Methyl thio/Benzylthio/arylaminocarbonylmethylthio-1,3,4-Thiadiazoles as Anti-inflammatory agents, Indian J. Heterocyclic Chem., 2005; 15:110.

8. Siddiqui N., Ali S., Khan S.A., Drabu S., Rana A. and Alam M., Synthesis of 3 - Arylamino-4-aryl-5-(N-arylthiocarbonylimino)- 4,5 - Dihydro- 1,2,4-Thiadiazoles as Anticonvulsant agents, Indian J. Heterocyclic Chem., 2004; 14: 159.

9. Chhajed M.R., Khedekar P.B. and Mudhey A.S., Synthesis and free-radical activity of Some 1,3,4-Thiadiazoles derivatives, Indian J. Heterocyclic Chem., 2007; 16: 259.

10. Dobariya A.V., Patel J.R., and Parekh H.H., Thiazolidinones Bearing Chloroquinoline nucleus as potential Antimicrobial Agents, Indian J. Heterocyclic Chem., 2001, 11:115.

11. Solankee A., Kapadia K.,Thakor I., Patel J. and Lad S., Synthesis and Biological assay of some New 2,3-Diaryl -5methyl-4Thiazolidinones, Oriental J. Chemistry, 2004; 20(1): 127.

12. Mishra P., Namdeo K.P., Jain S.K. and Jain S., Synthesis and Antimicrobial Activity of 4-Thiazolidinones, Asian J. Chem., 1999; 11(1): 55.

13. Gaikwad N.J. and Gautam P., Mannich reaction products of 5 – Benzylidene - 2-Thioxo-4-Thiazolidinone as hypoglycemic agents, Indian Journal of Heterocyclic Chemistry, 2002,12: 181-182.

14. Kaneria D.J., Datta N.J., Khunt R.C. and Parekh H.H., Synthesis and Pharmacological Activity of some new 4-Thiazolidinone derivatives, Indian J. Heterocyclic Chem., 2003; 12: 277.

15. RamaRao N, Ramu B. and Gopinath C., Synthesis and Characterization of some biological Thiazolidinones, Asian J. Chem., 2004; 16(1): 531.

16. Nair D.S. and Shah A.C., Synthesis of 5-Thiazolidinone derivatives of (R) & (S) -2 -aminobutanols, Indian Journal of Heterocyclic Chemistry, 2007; 16: 231.

17. Daulatabad C.D. and Bhat G.G., Synthesis and Antimicrobial studies of 4-Thiazolidinone derivatives from 2-Bromohexanoic acid (part-II), Indian J. Heterocyclic Chem., 1999; 9: 157.

18. Giri S and Khan MH, Synthesis and Antifungal Activity of some 3-[5’-aryl-3’- Mercapto-1’,2’,4’-Triazol-4’-yl]- 4-Thiazolidinones, Asian J. Chem., 1992; 4(4): 812.

19. Panetta J.A., Shadle J.K., Philips M.L., Benslay D.N. and Ho P.P.K., 4-Thiazolidinones as potent antioxidants, as Anti-inflammatory agent, Annals of the New York Academy of Sciences, 1993; 696: 415-416.

20. Eurniss B.S, Hannaford A.J., Smith P.W.G. and Tatchell A.R., Vogel’s Textbook of Practical Organic Chemistry, 5th edition, Addison Wesley Longman limited, Edinburgh Gate, Harlow, England, 1989; 1163.

21. Ulmann’s Encyclopedia of Industrial Chemistry, A2, 1985, 41.

22. Soloman G. and Fryhle G., Organic Chemistry, 7Th

edition, John Willey and Sons Inc, Delhi, 2000; 958.


271

23. Pharmacopoeia of India, Vol.2, 3rd Edition, Ministry of Health and family welfare, government of India, Delhi, 1985; 85.

24. Pelczar M.J., Chan E.C.S. and Krieg N.R., Microbiology, 5th edition, Tata Mc.Graw Hill

publishing Company Limited, New Delhi,1993; 536.


272

Effect of Milling on Correlation between Interquartile

Coefficient of Skewness and Coefficient of Kurtosis in

Pharmaceutical Powders – II Dabas Sunny, Shakya Pragati, Sharma Vijay and Pathak Kamla*

Department of Pharmaceutics, Rajiv Academy for Pharmacy,

N.H. # 2, Chattikara, Mathura 281001.

*Author for Correspondence: [email protected]

ABSTRACT

In the present study, pharmaceutical powders categorized as inorganic and organic powders were subjected to

size classification by sieving, quantized by coefficient of skewness (IQCS) and coefficient of kurtosis (β2)

determination and the correlation between the two statistical parameters was assessed. Additionally, effect of

milling on the IQCS and β2 was studied as variable. Frequency distribution curves exhibited asymmetrical

positively skewed distribution for both categories of powders and the asymmetric particle size distribution was

confirmed by the IQCS values greater than zero and β2 that was either less than or greater than 3. A large

negative correlation between IQCS and β2 for inorganic powders and a fairly large positive correlation was

observed for organic powders. Upon milling, with variable sized balls the asymmetry increased and that was

ascertained by the correlation between the two statistical parameters, which in general improved with finer

milling.

Keywords: Powders, Sieving, Interquartile Coefficient of skewness, Coefficient of kurtosis, Coefficient of

correlation, Effect of milling

INTRODUCTION

Today, the pharmaceutical industry pays much attention to the role and variability of the physical properties of particulate material, not only active ingredients, but also pharmaceutical powders. Pharmaceutical powders that are usually poly disperse with variable particle size and shapes thus it is necessary to know not only the size of particles, but also how many particles of the same size exist in the sample. In the rapidly growing field of pharmaceutical powders and solid dosage form, particle size and shape are known to affect the product performances including processing characteristics, content uniformity, solubility, stability, bioavailability and consequently therapeutic efficiency1. Many techniques for the measurement of particle size are available, but their choice and application may prove difficult. Most often a powder is composed of three

types of particles, elementary particles, aggregates and agglomerates and offers a variety of particle sizes, and also particle shapes2. For better handling of manufacturing process it is important to determine the particle size distribution of pharmaceutical powders3. Quantization of the particle size distribution can be done by defining various related statistical parameters, namely coefficient of kurtosis, Yule coefficient of Skewness and intra quartile coefficient of Skewness (IQCS). Skewness and kurtosis are used to investigate particle size distributions. The degree of symmetry of particle size distribution may be defined by coefficient of kurtosis β2, that can be given by the equation β 2 = µ4/ µ2

2, where µ2 is the second moment about mean and µ4 is

the fourth moment about the mean and moments can be used to describe the properties of a distribution. For a given frequency function kurtosis is a measure of how sharply the function peaks around its mode. Coefficient of kurtosis is a measure of kurtosis.


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Kurtosis is a measure of whether the data are peaked or flat relative to a normal distribution. That is, data sets with high kurtosis tend to have a distinct peak near the mean, decline rather rapidly, and have heavy tails. Data sets with low kurtosis tend to have a flat top near the mean rather than a sharp peak. For univariate data Y1, Y2, ..., YN, the formula for kurtosis is:

Where is the mean, is the standard deviation, and N is the number of data points. The kurtosis for a standard normal distribution is three. For this reason, some sources use the following definition of kurtosis:

This definition is used so that the standard normal distribution has a kurtosis of zero. In addition, with the second definition positive kurtosis indicates a "peaked" distribution and negative kurtosis indicates a "flat" distribution. Skewness is a measure of symmetry, or more precisely, the lack of symmetry. A distribution, or data set, is symmetric if it looks the same to the left and right of the center point. For univariate data Y1, Y2, ..., YN, the formula for skewness is:

Where is the mean, is the standard deviation, and N is the number of data points. The skewness for a normal distribution is zero, and any symmetric data should have skewness near zero. Negative values for the skewness indicate data that are skewed left and positive values for the skewness indicate data that are skewed right. By skewed left, we mean that the left tail is long relative to the right tail. Similarly, skewed right means that the right tail is long, relative to the left tail. Some measurements have a lower bound and are skewed right. Numerous reports on the use of these parameters are cited in literature 4,5,6 for characterization of the pharmaceutical powders and their impact on processing variables has been interpreted. Skewness and kurtosis of force-time profile obtained from compression of lactose, glucose and mannitol granules are reported as

significant measures of departure from the normal frequency7. Based on the relation between the skewness and kurtosis coefficient an approach was developed to get the distribution pattern of aerosol8 .The influence of particle size distribution on the flow properties of limestone powders has been characterized by the average particle size, the variation coefficient and the skewness9. A quantitative method has been introduced to distinguish between a gamma distribution and an exponential (e.g. Marshall-Palmer) distribution10

. Skewness has been used to investigate the particle size, shape, particle surface roughness and water loss on drying of lactose monohydrate carrier particle on the aerodynamic properties of dry powder inhalation11. For assessment of the impact of long term orally administered probiotic strain Enterococcus faecium M-74(EF M-74) enriched with selenium on lipid profile in humans, coefficient of kurtosis and skewness were used to test data distribution12 and for testing the homogeneity of the granules in tablets13. The skewness coefficient of particle size distribution and tapped density of the granulated mixture were chosen as the model input variables in optimization of high shear wet granulation wetting process using fuzzy logic modeling14. Polycarpou and Tayebi modeled the effect of skewness and kurtosis on the static friction coefficient of rough surfaces of the powders15. It was predicted that surfaces with high kurtosis and positive skewness exhibit lower static friction coefficient compared to the Gaussian case. More importantly, it was predicted that, for high kurtosis values, the static friction coefficient decreases with decreasing external force rather than increasing as seen with increasing skewness. The analyses of micronised and controlled atomization and sonocrystallization of paracetamol and beclomethasone dipropionate to produce particles with lower skewness (i.e. a more symmetrical distribution) and a higher kurtosis (i.e. a less dispersed distribution) led to improved utility of pharmaceutical compositions16. Kurtosis has also been used as the criterion for lot of quality pharmaceutical unit doses based on the percentage of defectives has been generally used in the evaluation of tests determining content uniformity17. The degree of skewness of the particle population may also be defined by IQCS (interquartile coefficient of skewness). IQCS can be determined by the following


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equation, IQCS = (c-a)-(a-b)/ (c-a) + (a -b) where, a is the median diameter, b and c are the lower and upper quartile points. The value of IQCS lies between -1 to +1 and for a symmetrical particle size distribution IQCS is zero18. The literature reports the use of IQCS for assessment of a spectrum for pharmaceutical uses that are cited in our previous report19. In the earlier report by the authors, microscopy was used for quantitative evaluation of particle size distribution studies of pharmaceutical powders categorized as fillers, natural polymers, and synthetic

polymers by determining IQCS and β2. Correlation was attempted between the two statistical parameters for

both categories. Linear regression of IQCS and β2

revealed a negative correlation for all categories of excipients and maximum linearity was observed for natural polymers. In the present study, pharmaceutical powders categorized as inorganic and organic powders were subjected to size classification by sieving, quantized by

IQCS and β2 determination and the correlation between the two statistical parameters was assessed.

Additionally, effect of milling on the IQCS and β2 was studied as variable. MATERIALS AND METHODS

The following powders were used as supplied – ascorbic acid, poly vinyl alcohol form Qualikem fine chemicals, New Delhi,, sodium nitrate form CDH, New Delhi, citric acid, sodium dichromate and potassium chloride were procured from Qualigens fine chemical, Mumbai. Particle size analysis

Particle size distribution study was carried out by the method of sieving using a nest of BSS sieves arranged in order of (# 10, 16, 22, 44, 60, 85, 100, 120, 200 and receiver) on a mechanical shaker (Jindal Scientific Industries, Ambala, India), for 20 min. The weight of powder retained on each sieve was used for calculation of various micromeritic parameters. Representation of Particle Size Distribution

The data obtained from all the experimentations were plotted as frequency distribution curve (percentage frequency versus mean particle diameter) and cumulative frequency distribution curve (cumulative percentage frequency versus mean particle diameter).

Calculation of IQCS and ββββ2

IQCS was calculated by using the equation, IQCS = (c-a) – (a-b) / (c-a) + (a-b), where a is the median diameter and b and c are the lower and upper quartile points in a

cumulative frequency distribution curve and β2 can be

calculated by using the equation, β2 = µ4 / (µ2)2, where

µ2 is the second moment about mean and µ4 is the fourth moment about mean16. Effect of milling

10g potassium chloride was placed in a ball mill, capacity 1 kg (HICON, New Delhi) and was subjected to milling by varying both the ball size i.e. larger (diameter 1.1cm ± 0.03), medium (diameter 0.8cm ± 0.04) and smaller (diameter 0.5cm ± 0.03), and the time of milling as 5, 10, and 20 min respectively. At the end of milling the powder was collected and subjected to sieve analysis as described in the previous section. RESULTS AND DISCUSSION

Sieving, as a method for size classification is widely used for characterizing the range of grain sizes present in the powder. In this technique a quantity of powder is separated into fractions on a surface containing holes of uniform size. The two problems associated with sieve characterization are difficulty of determining the point at which fractionation process is complete and coping with the variation in sieve apertures present in new and worn sieves. Uses of electrochemical sieves, normalization of apertures etc. are few approaches that can be used to alleviate the effects of variation in aperture sieve sizes20, 21. Statistical characterization of the particle size distribution data is one important tool that can account for the variation in aperture sizes. Thus in the present report, six pharmaceutical powder samples categorized as inorganic powders (sodium dichromate, potassium chloride and sodium nitrate) and organic powders (poly vinyl alcohol, ascorbic acid and citric acid) were subjected to sieving particle size analysis to get the particle size distribution data. For symmetrical normal size distribution the peak frequency value, known as mode separates the frequency distribution curve into two identical halves. But in case of asymmetrical particle size distribution, frequency distribution curve is skewed, either positively or negatively depending on the proportion of particles present in the powder. If larger proportion of fine particles is present positively skewed frequency


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distribution curve is obtained. The significance of plotting cumulative percent frequency distribution curve is that it is possible to compare two or more particle population. Frequency distribution curves exhibited asymmetrical distribution of the particle population that was positively skewed for both categories of powders. A bell shaped frequency distribution curve is obtained22 when the particles are normally distributed, the frequency distribution curve for ascorbic acid was closer to the normal frequency distribution curve. Amongst the organic powders citric acid and sodium dichromate from the inorganic category displayed bimodal distribution (Fig 1, 2) that is a reflection of the unit operation used in the manufacture. Bimodal frequency distribution curves18 are observed for the powders that are subjected to milling due to which some particles get fractured and some remain unbroken, that results in the formation of two modes in frequency distribution curve. Figures 3 and 4 are the percentage cumulative oversize frequency distribution curves of organic and inorganic powders respectively. Though the curves were not sigmoidal in shape as expected for a normal distribution, these were spread beyond the upper and lower quartiles permitting calculation of IQCS. Quantitative statistical analysis of the data was done to

get the values of IQCS and β2 and results obtained are summarized in table 1. All the powders confirmed asymmetric particle size distribution as their IQCS values were observed to be either greater than zero (sodium nitrate) or less than zero for sodium dichromate, potassium chloride, ascorbic acid, poly vinyl alcohol and citric acid. IQCS can have values from -1- 0- +1 and 0 value indicates normal distribution23. It is the numerical value that signifies the extent of deviation from the normal distribution whereas negative and positive signs are associated with flatness or peakedness of the distribution respectively. Thus sodium nitrate with positive value of 0.250 showed maximum peakedness in the frequency distribution curves.

The coefficient of kurtosis (β2) was less than 3 for potassium chloride suggesting platykurtic distribution,

while β2 values greater than 3 for sodium dichromate, sodium nitrate, polyvinyl alcohol, ascorbic acid and citric acid favored leptokurtic distribution but none

displayed mesokurtic distribution. The curve of normal frequency distribution is described as mesokurtic when

β2 equals to zero. If the curve of a frequency distribution is more peaked than normal curve, it is

called leptokurtic frequency distribution curve (β2 > 3) and if it is more flat topped than the normal curve, it is

called platykurtic frequency distribution curve (β2 < 3)24. The IQCS and β2 values obtained for powder samples were subjected to linear regression and correlation coefficient equal to -0.6451 and 0.8897 for the inorganic and organic powders respectively were deduced. A large negative correlation between IQCS

and β2 for inorganic powders and a fairly large positive correlation was observed for organic powders. Correlation, (often measured as a correlation coefficient), indicates the strength and direction of a linear relationship between two random variables. In general statistical usage, correlation or co-relation refers to the departure of two variables from independence. It is important to remember that "large" and "small" (Table 2) correlation should not be taken as synonyms for "good" and "bad" in terms of determining that a correlation is of a certain size. For example, a correlation of 1.0 or −1.0 indicates that the two variables analyzed are equivalent modulo scaling 25. The effect of milling was experimented on potassium chloride (inorganic) and citric acid (organic) both of

which showed least values of IQCS and β2 (least asymmetrical distribution) in their respective categories. The results obtained are summarized in tables 3 and 4. Both the micromeritic statistical parameters were significantly modified on milling when compared to the unmilled powders. For potassium chloride powder (crystal lattice is a face-centered cubic structure) IQCS changed from initial negative (-0.238) to positive values with increase in milling time irrespective of the ball diameter(s) and maximum values for IQCS were observed when milling was done with balls of largest diameter followed by medium sized balls and small sized balls consecutively. In fact larger balls crush the feed by breaking large particles without interference by the fines and the smaller ones grind small particles to form the fine product 26 that may be attributed to the increase in small


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Table 1: Comparative Statistical Parameters of Different Powder Samples based on particle size distribution data

obtained on sieve analysis.

Sample Powder

Category

Calculated Value

Coefficient of

Correlation

(r)

Geometric

mean diameter

(d’g)

Geometric

standard

deviation

(σ’g)

IQCS ββββ2

Sodium dichromate Potassium chloride Sodium nitrate

Inorganic powders

300 280 250

1.304 1.333 1.190

-0.385 -0.238 0.250

10.04 2.81 3.18

- 0.6451

Ascorbic acid Citric acid Poly vinyl alcohol

Organic powders

260 270 330

1.238 1.285 1.375

-0.500 -0.316 -0.166

8.30 3.36

22.51

0.8897

Table 2: Guideline for interpretation of correlation coefficient

Correlation Negative Positive

Small -0.3 to -0.1 0.1 to 0.3 Medium -0.5 to -0.3 0.3 to 0.5

Large -1.0 to -0.5 0.5 to 1.0

Table 3: Effect of milling on IQCS and β2 using variable sized balls for varying time on potassium chloride

powder.

S. No. Time

interval

(min)

Larger Balls Medium Balls Smaller Balls

IQCS β2 IQCS β2 IQCS β2

1 0 -0.238 2.81 -0.238 2.81 -0.238 2.81 2 5 0.548 4.43 0.090 15.05 0.090 15.36 3 10 0.466 1.25 0.040 8.00 0.076 15.38 4 20 0.517 0.71 0.179 9.69 0.109 10.55

Coefficient of correlation (r) 0.1865 0.7692 0.9217

Type of correlation Small positive correlation

Large positive correlation Large positive correlation

Table 4: Effect of milling on IQCS and β2 using variable sized balls for varying time on citric acid powder.

S. No. Time

interval

(min)

Larger Balls Medium Balls Smaller Balls

IQCS β2 IQCS β2 IQCS β2

1 0 -0.316 3.36 -0.316 3.36 -0.316 3.36 2 5 -0.217 5.21 -0.120 4.82 -0.166 9.11 3 10 -0.217 6.23 -0.043 6.12 0.043 9.16. 4 20 -0.460 9.45 -0.043 7.14 0.200 8.65

Coefficient of correlation (r) -0.6502 0.9234 0.7088

Type of correlation Medium negative correlation

Large positive correlation Large positive correlation


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Fig 1. Comparative frequency distribution curves of

citric acid, ascorbic acid and poly vinyl alcohol

powders

Fig 2. Comparative frequency distribution curves of

sodium dichromate, potassium chloride and sodium

nitrate powders.

Fig 3. Comparative percentage cumulative frequency

oversize distribution curves of poly vinyl alcohol,

ascorbic acid and citric acid powders.

Fig 4. Comparative percentage cumulative frequency

oversize distribution curves of sodium dichromate,

potassium chloride and sodium nitrate.

increasing ball diameter but the effect of milling sized particle population on milling. Larger the size of balls more is size reduction. With increase in the time length for milling the values

of IQCS did not change significantly. β2 increased with

time and the increase in β2 was not very consistent

except in case of large balls were a decrease in β2 was recorded with increased milling time. However the correlation between these two statistical parameters improved consistently with increasing ball diameters. For citric acid powder (orthorhombic crystal structure) IQCS changed from initial higher negative (-0.238) to lower negative values with increase in milling time till 10 min irrespective of the ball diameter(s) suggesting a

tendency to approach normal distribution especially on milling with small sized balls for 10 minutes. A maximum value for IQCS was observed when milling was done with balls of largest diameter followed by medium sized balls and small sized balls, a pattern similar to the one recorded for potassium chloride.

β2 increased with decreasing ball diameters, in contrast to that observed for potassium chloride powder,

however the effect of milling time on β2 was variable.

For larger sized balls an increase in β2 was recorded with increase in milling time suggesting an increase in peakedness for particle size distribution in contrast to that observed with potassium chloride powder where an increase in flatness of the frequency distribution curve


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with milling was observed. For milling operation on potassium chloride, on applying two way ANOVA calculated at 5% α level (95% confidence level), the F(cal)2,6 = 0.97 was less than F(tab)2,6 = 5.14 23, suggesting insignificant difference between IQCS values obtained using different sized balls at all time intervals, also the F(cal)3,6 = 2.36 was less than F(tab)3,6 = 4.76, indicating insignificant difference between IQCS values obtained on increasing the time of milling. The F(cal)2,6 of 7.39 was greater than F(tab)2,6 of 5.14,indicating significant difference between values of β2 calculated using different sized balls and insignificant difference between values of β2 on increasing the time of milling was interpreted by the F(cal)3,6 = 3.36 that was less than F(tab)3,6 = 4.76. For citric acid powder, the IQCS values did not change significantly on varying either the size of ball or on increasing the time of milling [ F(cal)2,6 = 2.72 is less than F(tab)2,6 = 5.14, for different sized balls; F(cal)3,6 = 1.41 is less than F(tab)2,6 = 4.76, on increasing the time of milling irrespective of the ball diameters].These results are in consistence with those obtained for potassium chloride powder where the IQCS value did not change significantly but the same was not observed for coefficient of kurtosis,β2. Insignificant difference between the values was

recorded on milling with different sized balls as the F(cal)2,6 = 3.82was less than F(tab)2,6 = 5.14, whereas an increase in milling time documented a highly significant difference between values of β2 (F(cal)3,6 = 10.38 is much greater than F(tab)2,6 = 4.76)

The IQCS and β2 values obtained on milling were subjected to linear regression and the type of correlation is shown in tables 3 and 4. Invariably the correlation improved as the ball size decreased that was distinctly visible in case of potassium chloride powder while in citric acid powder a deviation was seen with the least sized balls. This may be attributed to asymmetric bimodal distribution of citric acid powder with heavy tail of fines that got size reduced by the smaller balls thus approaching normal distribution. CONCLUSION

The IQCS and β2 values of the inorganic and organic pharmaceutical powders demonstrated skewness with large negative and positive correlation respectively.

Upon milling the asymmetry increased and that was ascertained by the correlation between the two statistical parameters, which in general improved with finer milling. The results presented are those obtained by machine sieving based on simple vibratory action performed by stacking sieves in ascending order of aperture and placing the powder at the top sieve. Studies on sieving with Rotap sifter, that combines a gyratory and jolting movement for size classification are in progress. REFERENCES

1. Martin A. Physical Pharmacy. 4th ed. New Delhi: B. I. Waverly (P) Ltd; 1999.

2. Andres C, Bracconi P, Reginault P, Blouquin P, Rochat MH, Pourcelot Y. Assessing the particle size of a broadly dispersed powder by complementary techniques. Int. J. Pharm. 1998; 167: 129-138.

3. Andres C, Bracconi P, Reginault P, Blouquin P, Rochat MH, Pourcelot Y. Particle-size distribution of a powder: comparison of three analytical techniques. Int. J. Pharm. 1996; 144; 141-146.

4. Sharma V, Philip AK, Pathak K. Modified Polysaccharides as Fast Disintegrating Excipient for Orodispersible Tablets of Roxithromycin. AAPS Pharm. Sci.Tech. 2008; 9(1): 87-94. doi: 10.1208/s12249-007-9026-4.

5. Delphine M, Jacques L, Esteban C, Severian D. Physicochemical and structural characterization of a polyionic matrix of interest in biotechnology, in the pharmaceutical and biomedical fields. Carbohydrate Polymers, 2004; 55: 437-453.

6. Chonara YE, Pillay V, Car Micheal T, Danckwerts MP. Studies on a novel doughnut shaped mini tablet for intra occular drug delivery. AAPS Pharm. Sci. Tech. 2007; 8(4): Article 118.doi:10.208/pt0804118.

7. Juppo AM, Kervinen L, Yliruusi J. Skewness and kurtosis of force time profile obtained from compression of lactose, glucose and mannitol granules Eur. J. Pharm. Biopharm. 1995; 41: 374-81.

8. Liu Y, Liu F. On the description of aerosol particle size distribution. Atmospheric Res., 1994; 31(1-3): 187-198. doi:10.1016/0169-8095(94)90043-4

9. Kurz H P, Munz G. The influence of particle size distribution on the flow properties of limestone


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powder. Powder Tech. 1975; 11(1): 37-40. doi:10.1016/0032-5910(75)80020-9

10. Yangang L. Skewness and Kurtosis of measured raindrop size distribution. Atmos. Environ. Part A. General Topics, 1992; 26(15): 2713-2716. doi:10.1016/0960-1686(92)90005-6

11. Podczeck F. The relationship between physical properties of lactose monohydrate and the aerodynamic behavior of adhered drug particles. Int. J. Pharm. 1998; 160: 119-130.

12. Hlivak P, Odraska J, Ferencik M, Ebringer L, Jahnova E, Mikes Z. One-year application of probiotic strain Enterococcus faecium M-74 decrease serum cholesterol levels. Bratisl Lek Listy. 2005; 106(2): 67-72.

13. Thiel WJ, Nguyen LT. Fludized bed film coating of an ordered powder mixture to produce micro escapsulated ordered units. J. Pharm. Pharmacol. 1984; 36,145-152.

14. Belohlav Z, Brenkova L, Kalcikova J, Hanika J, Durdil P, Tomasek V, Palatova M. Optimization of the high-shear wet granulation wetting process using Fuzzy Logic Modeling. Pharm. Dev. Tech. 2007; 12(4): 345-352. doi: 10.1080/10837450701369428

15. Polycarpou A A, Tayebi N. Modeling the effect of skewness and kurtosis on the static friction coefficient of rough surfaces. Tribology Int. 2004; 37: 491-505.doi: 10.1016/j.triboint.2003.11.010

16. Cardinal LB. Technological Innovation in the Pharmaceutical Industry: The Use of Organizational Control in Managing Research and Development. Organization Science 2001; 12: 19-36

17. Bruce F. Comparison of criteria for content uniformity J. Pharm. Sci.1973; 63: 183-199.

18. Aulton ME. Pharmaceutics: The Science of Dosage form Design, 2nd ed. New York: Churchill Livingstone; 2002.

19. Chamoli K, Jain D, Pathak K. Correlation between interquartile coefficient of skewness and coefficient of kurtosis in pharmaceutical powders, Ind. J. Pharm Ed. Res. Vol. 42, 4, October – December, 2008 .(In Press)

20. Kaye BH. Characterization of powders and aerosols. 1st ed. Weinheim, New York: Wiley- VCH; 1999.

21. Allen T. Particle Size Analysis. 4th ed. London: Chapman and Hall; 1992.

22. Agarwal AK, Vasishtha AR. Advanced Mathematics for Pharmacists, 1st ed. Meerut: Krishna Prakashan Media (P) Ltd; 2004; pp109-119.

23. Bolton S. Pharmaceutical: Practical and Clinical Application, 3rd ed. New York: Marcel Dekker; 1997.

24. Filliven JJ. NIST/SEMATECH e-Handbook of statistical methods. 2003 [cited 2006 July 18]. Available from: URL: http:// www.itl.nist.gov/ div 898/handbook / eda/ section 3 /eda 3661.htm

25. Cohen J. Statistical power analysis for the behavioral sciences, 2nd ed, 1988.

26. Carter SJ. Cooper and Gunn’s Tutorial Pharmacy. 6th ed. Belfast: The Universities Press. 1972. 188-189.


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Evaluation of Antidiabetic Activity of Caesalpinia bonduc (L.) Roxb

Leaves (Caesalpianceae) in Alloxan Induced Diabetic Mice P.B.Aswar*1, S.S. Khadabadi1, U.A. Deokate1, S.P. Jain2, K.R.Biyani2, R.D. Jawarkar3.

1 Government College of Pharmacy, Shivaji nagar, Amravati-444603. 2Anuradha College of Pharmacy, Chikali, Dist-Buldhana.

3 I.B.S.S.B.S. College of Pharmacy, Malkapur, Dist- Buldhana-443101.

* Present address for correspondance :[email protected].

Abstract

Caesalpinia bonduc (L.) Roxb leaves extract (ethanolic and aqueous 500mg/kg body wt.) were evaluated for its

effect on blood sugar level in normal and alloxan induced wistar albino mice at various time points comparing

it with standard drug metformin (75 mg/kg). The studies indicate that the crude ethanolic extract exhibited

statistically significant hypoglycemic (P < 0.01) and anti-hyperglycemic (P < 0.001) activities in normal and

alloxan-induced diabetic albino mice respectively. The study reports for the first time the hypoglycemic activity

of leaves extract of Caesalpinia bonduc (L.) Roxb. in mice.

Keywords: Caesalpinia bonduc, alloxan, wistar albino, metformin.

INTRODUCTION:

Diabetes mellitus is a medical disorder characterized by varying or persistent hyperglycemia (high blood sugar levels) resulting from the defective secretion or action of the hormone insulin. Although diabetes has been recognized since antiquity, and treatments were known since the middle ages, the elucidation of the pathogenesis of diabetes occurred mainly in the 20th century. Though different types of oral hypoglycemic agents available along with insulin for treatment of diabetes mellitus,there is agrowing interest in herbal remides,due to the side effects associated with these therapeutic agents.Because of perceived effectiveness,minimal side effects in clinical experience and relatively low cost,herbal drugs are widely prescribed even when their biologically active compunds are unknown1. Plant Caesalpinia bonduc (L.) Roxb was found throughout the hotter parts of India, particularly along the seacoast up to an altitude of 830m., common in West Bengal and South India and cultivated as a hedge plant2. From extensive literature survey it was found that Caesalpinia boduc (L.) Roxb leaves shows contractile smooth muscle activity in pregnant rats3 and seed extract showed antidiabetic activity4, antitumor

activity5, antioxidant activity5 and antibacterial activity6 but no evidence was found on leaves about antidiabetic activity. Phytochemical screening of Caesalpinia boduc (L.) Roxb leaves showed presence of carbohydrates, glycoside, proteins, saponins, flavonoids and tannins7. As flavonoids, sterols/triterpenoids, alkaloids and phenolics are known to be bioactive antidiabetic principles8.Flavonoids are known to regenerate the damaged beta cells in the alloxan diabetic rats9.Phenolics are found to be effective antihyperglycemic agents10. The present work was undertaken to study the effect of leaves extract of Caesalpinia bonduc (L.) Roxb plant on blood glucose following administration to normal and hyperglycemic mice. MATERIALS AND METHODS: The leaves of Caesalpinia bonduc (L.) Roxb. were collected from Village Kathora, Dist-Amravati (Maharashtra) and authenticated by Dr. Mrs. P.Y. Bhogaonkar, Head of Botany Department, Government Vidarbha Institute of Science and Humanities, Amravati (Maharashtra).A voucher specimen was deposited in the herbarium of the department of Pharmacognosy and Phytochemistry, Government College Of Pharmacy, Amravati . Preparation of Extract

The dried powdered leaves were extracted with


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petroleum ether (40-60 0 C) to remove lipids and waxy materials and then successively extracted with ethanol 50% and water in soxhlet extractor11. The extract was concentrated under vaccum to get residue. The residue was dried in vaccum dessicator. Both aqueous and ethanolic extract were selected for hypoglycemic screening. Animals Used:

Wistar Albino mice of both sexes, weighing between 20-30 gms, were used. They were housed in polypropylene cages and were fed on standard laboratory diet and water ad libitum12. Animals described as fasted were deprived of food for 16 hr, but had free access to water. All the drugs (standard and test) as well as vehicle were administered per-orally, using infant feeding tube. The project was undertaken after the prior approval from the Animals Ethics Committee. Acute and short-term Toxicity Study:

The ethanolic and aqueous extracts were tested for its acute and short- term toxicity in mice. To determine acute toxicity of different doses of the drug (1.0, 3.0 and 5.0 g/kg) were administrated to different groups of mice (2 mice were used for each group; control mice received 0.5% CMC). Mortality and general behavior of the animals were observed periodically for 48 h. The animals were observed continuously for the initial 4 h and intermittently for the next 6 h and then again at 24 h and 48 h following drug administration. The parameters observed were grooming, hyperactivity, sedation, loss of righting reflex, respiratory rate and convulsion13.

To study short- term toxicity, 3 groups of mice used. 3 male mice (20-25 g, body weight) were used in each group. Group I was kept as control and group II and III received 500 mg/kg ethanolic and aqueous extracts respectively in 0.5% CMC. The drug was administrated daily for 7 days (p.o.). Control group received 0.5% CMC in an identical manner. The behavior of the animals was observed daily for 1h in the forenoon (10 to 11 A.M.) for 7 days. Acute Toxicity Study and Determination of Test

Dose:

During preliminary toxicity study, no adverse effect or mortality was observed in albino mice with oral administration of ethanolic and aqueous. extracts up to a high dose of 5 gm/kg body weight observed for 24 hr.

Hence a high dose of 500 mg/kg body weight was selected as a test dose. Standard Drug and basis of selection of its Dose:

Metformin tablet (500 mg / tab.) “Glycomet” manufactured by USV Limited was used as a standard drug. The dose was selected on the basis of adult human effective dose. (75 mg/kg body weight)14. Determination of Blood Glucose Level:

For blood glucose determination, blood was obtained by snipping tail with the help of sharp razor. Blood glucose level was monitored by using Hypo guard Advance Blood Glucose Meter, (Nicholas Piramal Ltd.India) Each time the tail of the mice was sterilized with spirit. ANTIDIABETIC SCRENNING:

Induction of Experimental Diabetes:

Mice were fasted for 18 hrs and experimental diabetes was then induced by administration of three doses of alloxan monohydrate (150 mg/kg) each i.p. at intervals of 48 hrs. Seven days after the last administration, the animals were fasted for 18 hrs and blood glucose levels were determined. Animals with fasting blood glucose levels ranging from 200-300 mg/dl (mild diabetic mice) were considered as diabetic and used for the study15.

Determination of efficacy of ethanolic 50% and

aqueous extract in alloxan induced diabetic mice:

The diabetic mice were divided into four groups of six each. Group one received vehicle only (0.5 % CMC) and served as control group. Group two received standard drug Metformin (75 mg/kg) and served as standard group. Remaining two groups received ethanolic and aqueous extracts at a dose of 500 mg/kg body weight. Study for the acute hypoglycemic activity involved determination of blood glucose levels at 0, 1, 2, 3, 5 and 24 hrs after administration of single dose. Effect of various extracts of Caesalpinia bonduc (L) Roxb. leaves on blood glucose level in alloxan induced diabetic wistar mice were given in table no.1. Hypoglycemic study in normal fasted mice:

Animals were fasted overnight and were divided in to four groups of five each as follows Group1- Received 3% CMC suspension as control group Group2- Received metformin (75mg/ kg) body weight as Std. group Group3- Received aqueous extract (500mg/ kg) body weight


282

Group4- Received ethanolic 50% extract (500mg/ kg) body weight Blood Samples were collected after 120 min of drug administration16. Results are elaborated in table no. 2 . RESULTS:

Preliminary phytochemical screening reveals presence of carbohydrates, glycoside, proteins, saponins, flavonoids and tannins. During preliminary toxicity study, no adverse effect or mortality was observed in albino mice with oral administration of ethanolic 50% and aqueous extracts up to a high dose of 5 gm/kg body weight observed for 24 hr. Hence a high dose of 500 mg/kg body weight was selected as a test dose. Single administration (single dose) of ethanolic 50% extract (500 mg/kg) of leaves of Caesalpinia bonduc in diabetic wistar albino mice, showed significant reduction on fall in blood glucose level (FBGL) after 3 and 5hr interval. Maximum reduction in FBGL (39.09 %, 41.35 %) was seen after 3 and 5 hr of administration of dose with a significance level of p< 0.01. Metformin (75 mg/kg) showed maximum reduction (38.37 %, 43.91 %) after 3 and 5 hr with a significance level of p< 0.01.

Acute study in normal fasting mice ethanolic 50% extract (500mg/kg body weight) showed 42.85% reduction in blood glucose level and aqueous extract (500mg/kg body weight) showed 30.95% reduction in blood glucose level while metformin treatment resulted in 40.78% reduction in blood glucose level at 2 hr interval.

DISCUSSION :

The results obtained in the present study clearly indicate that the ethanolic extract (500mg/kg body weight) of leaves of C. bonduc has significant hypoglycemic activity in both the normal and alloxan induced diabetic mice. Flavonoids, sterols/triterpenoids, alkaloids and phenolics are known to be bioactive antidiabetic principles .The antidiabetic effect of ethanolic extract of C. bonduc leaves may be due to the presence of more than one antihyperglycemic principles mentioned above. Further studies will be focused on determination of the mechanism(s) of action and isolation of bioactive principles responsible for hypoglycemic activity.

Table 1: Effect of ethanolic and aqueous extracts of Caesalpinia bonduc (L) Roxb. leaves on blood glucose level

in alloxan induced diabetic Wistar albino mice.

Groups 0 hr BGL (mg/dL)

1 hr BGL (mg/dL)

2 hr BGL

(mg/dL)

3hr BGL (mg/dL)

5 hr BGL (mg/dL)

24 hr BGL

(mg/dL)

Control 225.67±19.70 227.33±16.80 220.33±13.20 224.5±11.90 213±14.70 233.5±23.40

Standard 271.33±22.50 240±19.05 192.8±19.70** 167±17.32* 152±21.03* 274±26.70 Ethanol Extract

266.16±21.82 244.16±21.82 201.83±21.82** 162.83±21.24* 156±25.32* 265.33±22.74

Aqueous Extract

271.16±29.13 229.16±19.90 182.1667±17.68 177.1667±3.20 174.1667±5.65 272.6667±10.46

Values are mean±SD, n=5 in each group, *p<0.01 when compared with vehicle treated group (Dunnett’s test) BGL-Blood Glucose Level.

Table 2: Effect of ethanolic and aqueous extracts of Caesalpinia bonduc (L) Roxb. leaves on blood glucose level

in normal fastened Wistar albino mice.

Groups 0 hour 2 hours Control 107±11.640 98.4±11.104

Standard 102±7.949 64±9.471 * Ethanol extract 89.6±11.88697 51.2±8.927486 * Aqueous extract 98.2±7.190271 67.8±7.085196 Values are mean±SD, n=5 in each group, *p<0.01 when compaired with vehicle treated group (Dunnett’s test)


283

ACKNOWLEDGEMENT:

Authors are thankful to the Principal, Anuradha College of Pharmacy, Chikali, Dist-Buldana (Maharashtra) for allowing us to conduct animal studies. REFERENCES:

1) Valiathan, M.S. Healing plants. Current Science 1998. 75 (11), 1122–1126.

2) Kirtikar K.R., Basu B.D. Indian Medicinal Plants. Vol.-II. Dehradun. International Book distributors, 1996; 839-852.

3) J.Y.Datte,A.Traore,A.M.Offoumou,A.ziegler “Effects of leaf extract of Caesalpinia bonducella

(Caesalpiniaceae) on the contractile activity of uterine smooth muscle of pregnant rats” J.Pharmacol.Vol.60,Issue2,March 1998,pp.149-155.

4) D.M. Kannur, V.I. Hukkeri, K.S. Akki. Antidiabetic activity of Caesalpinia bonducella

seed extracts in rats. Fitoterapia 77 (2006) 546–549 5) Gupta M, Mazumder UK, Kumar RS, Sivakumar

T, Vamsi ML. “Antitumor activity and antioxidant status of Caesalpinia bonducella against Ehrlich ascites carcinoma in Swiss albino mice.” J Pharmacol Sci. 2004 Feb; 94(2):177-84.

6) Saeed MA, Sabir AW “Antibacterial activity of Caesalpinia bonducella seeds.” Fitoterapia. 2001 Nov; 2(7):807-9.

7) Canonica.L, Jommi.G, Manitto.P, Pelizzoni.F, 1963.Bitter principles of Caesalpinia bonducella. Tetrahedron Letts.29, 2079-2086.

8) Oliver-Bever, B., 1986. Medicinal Plants in Tropical West Africa. Cambridge University Press, London, 245–267.

9) Chakravarthy, B.K., Gupta, S., Gambir, S.S., Gode, K.D., 1980. Pancreatic beta cell regeneration. A

novel antidiabetic mechanism of Pterocarpus

marsupium Roxb. Indian Journal of Pharmacology 12, 123–127.

10) Manickam, M., Ramanathan, M., Farboodinay Jahromi, M.A., Chansouria, J.P.N., Ray, A.B., 1997. Antihyperglycemic activity of phenolics from Pterocarpus marsupium. Journal of Natural Products 60, 609–610

11) Harbone J.B. “A Guide to Modern Techniques in Plant Analysis,” First Edition, 1973, 52- 116-182

12) S. Kumar, D. Kumar, R.R. Deshmukh, P.D. Lokhande, S.N. More, V.D. Rangari. Antidiabetic potential of Phyllanthus reticulatus in alloxan-induced diabetic mice. Fitoterapia 79 (2008) 21–23

13) L.S.R. Arambewela, L.D.A.M. Arawwawala, W.D. Ratnasooriya. An antidiabetic activity of aqueous and ethanolic extracts of Piper betle leaves in rats. Journal of Ethnopharmacology 102 (2005) 239–245

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arvensis in alloxan diabetic rat . Indian Drugs 41(11) November 2004.

15) S.Badole, N.Patel, S.Bodhankar, B.Jain, S.Bhardwaj .Antihyperglycemic activity of aqueous extract of leaves of Cocculus hirsutus (L.) Diels in alloxan – induced diabetic mice. Indian J. Pharmacol February2006, Vol38, Issue-1, 49-53.

16) B. Kameswara Rao, P. Renuka Sudarshan, M.D. Rajasekhar, N. Nagaraju, Ch. Appa Rao. Antidiabetic activity of Terminalia pallida fruit in alloxan induced diabetic rats. Journal of Ethnopharmacology 85 (2003) 169–172


284

Rationalization of Physico Chemical Characters of 2-Phenyl-3-

hydroxy-4(1H)-quinolinone-7-carboxylic acid Analogs as

Topoisomerase Inhibitors: A QSAR Approach. P.Valentina*, K.Ilango and Maida Engels

* Department of Pharmaceutical Chemistry, S.R.M.College of Pharmacy,

S.R.M University, Kattankulathur – 603203, Tamil Nadu, India.

E-mail: [email protected]

Abstract

A 3D QSAR model has been developed for a series of 19 compounds of 2-Phenyl-3-hydroxy-4(1H)-quinolinone-

7-carboxylic acid and their phenacyl esters as cytotoxic agent. Various physico chemical parameters and

cytotoxic activities against five cancer cell lines were used as independent variables and dependent variables

respectively. The best models were validated by leave-one-out procedure. Further, bootstrapping method was

adopted to assess the robustness of the model. It is revealed that thermodynamic parameters were found to have

overall significant correlation ship with cytotoxic activity and these studies provide insight to design new

molecules.

Key words - QSAR, Quinolinone, Topoisomerase, Cytotoxic.

INTRODUCTION

A large number of 4-hydroxy quinolinone derivatives are known for their inhibition of eukaryotic DNA Topoisomerase II, DNA gyrase and Topoisomerase IV enzymes that participate in cell division1-2. The mammalian enzyme, topoisomerase II, has the ability to change DNA topology by breaking the double helix of DNA and allowing a chain transition3-4 by forming a covalent linkage between the enzyme and the DNA. Many studies revealed that quinolone derivatives either stabilize the enzyme–DNA complex and block the rejoining of DNA or prevent the enzyme binding with DNA by interacting with the topoisomerase enzyme or DNA. This mechanism was the basis for the development of new antitumor agents, which are able to block the topoisomerase enzyme I and II.A large number of drugs were developed following this strategy. The currently available drugs like Camphothecin, Doxorubicin, Etoposide, Ellipticine and Amascarine 5 are Topoisomerase I and II inhibitors. Many antibacterial quinolinones were assessed regarding their ability to induce mammalian Topoisomerase II mediate DNA cleavage invitro and

were found to form a promising new class of anti tumor agents 6-10.Different types of quinolinones have been reported to possess the activity as inhibitor of Topoisomerase II, but none of the derivatives from this group has reached clinical trials. The purpose of this study is to gain insight into structural features related to the anticancer activity of the compound from the quinolinone series by applying the QSAR methodology and also to suggest a new substituent to enhance the anti cancer activity. The QSAR study is a useful tool for a retinal search of bioactive compounds. QSAR study describes a definite role in quantitative term of structural feature in molecule with a definite contribution to the activity of a particular physiochemical property of the structural feature. Thus QSAR provides deeper insight into the mechanism of drug receptor interactions. We therefore report here a QSAR study on 2-phenyl-3-hydroxy-4(1H)-quinolinone-7-carboxylic acid derivatives for

NH

O

OH

ArHOOCNH

O

OH

ArC

OCH 2

COAr

Fig. I Parent structure of 2-Phenyl -3-hydroxy -4- quinolinone -1-carboxylic acids and their phenyl acyl ester derivatives.

1-9 10 - 9

O


APTI ijper


285

their invitro cytotoxic activity against five cancer cell lines to explore their physiochemical properties required for inhibition and thus, may be helpful in designing new molecules. EXPERIMENTAL

A set of 19 compounds of 2-Phenyl-3-hydroxy-4(1H)-quinolinone-7-carboxylic acid and their phenacyl ester has been selected from the reported work of Soural. et al11.The biological activity data IC50 was converted to negative logarithmic dose in moles(pIC50) for QSAR analysis summarized in Table-1. Molecular modeling studies were performed using C.S.Chem Office12 version 8.0 (Cambridge Soft). All the molecules were constructed using Chem Draw Ultra version 8.0 and it was saved as template structure. For every compound, the template structure was suitably changed considering its structural feature, copied on 3D model and finally the model was cleaned up and subjected to energy minimization using Molecular Mechanics (MM2). The minimization was executed until the Root Mean Square (RMS) gradient value reaches a value smaller than 0.1

Kcal / Mol. A°. Minimized molecules were subjected to reoptimization via Austin model-1 method until RMS gradient attains a value smaller than 0.0001 Kcal / Mol.

A° using MOPAC. The geometry optimization of the lowest energy structure was carried out using Eigenvector Following (EF) routine. The descriptor values for all the molecules were calculated using “compute properties” module of program and tabulated in Table -2. The data was transferred to the statistical program in order to establish a correlation between physico chemical parameters as independent variable and pIC50 as dependent variable employing multiple regression analysis method. The regression analysis was carried out using a computer programme VALSTAT 13. All the possible combination of parameters was considered for the QSAR study. The best model was selected on the basis of various statistical parameters given in Table-3. The robustness and applicability of QSAR equation was further performed using the leave one out cross validation method, bootstrapping squared correlation coefficient (r2 bs), randomized biological data test (chance) and outliers (Z-score value). RESULTS AND DISCUSSION

Among the several models, one of the best model was

chosen from each cell line for further analysis based on statistical parameters which are summarized in Table-4 and further validated using “leave one out”(Loo) cross validation processes and the predicted values are shown in Table-5. All the final equations shows significance (F) more than 95% level. All the models have high statistical parameters like squared correlation coefficient (r2>0.8), cross-validation coefficient (Q2>0.3), standard deviation (S<0.3), Fischer values >99.9% and all the selected models explain more than 80% variance in the biological activity. The bootstrapping values (r2bs) for all the models showed that they were quiet robust. A close look at these equations suggests that thermodynamic parameters, boiling point (BP) and Van der Waal’s Energy (VWD) predominantly govern cytotoxic activity. The positive contribution of boiling point explains the potency of quinolinone derivatives, which have ability to interact with receptor by different attractive forces like hydrogen bonding and Van der Waal’s force. Few studies revealed that Topoisomerase inhibitors bind to DNA by hydrogen bond to form a stable DNA-drug-enzyme-complex. The VWD is a thermodynamic parameters defined as the sum of pair wise Van der Waal’s interaction energy terms for atoms separated by 3 chemical bonds, related to the structure of the molecule it self. The positive correlation of VWD and BP indicates that substitution of polarizable group in the quinolinone moiety or in the side chain may give potent compounds, which facilitate the stable drug-enzyme-DNA complex formation. So substitution of electron withdrawing groups and more polarizable atoms that have a dipole moment or induce a dipole moment may enhance the cytotoxic activity. The negative contribution of steric parameters Connoly Solvent Excluded Volume (CSEV) and Connoly Molecular surface Area (CMA) suggests that the bulky substituents are unfavorable for the cytotoxic activity and the bulkiness should be decreased. For all the above five models, the thermodynamic parameter BP and VWD are positively contributed to the biological activity. So it can be concluded that the group with strong thermodynamic influence in the substitution in quinolinone ring or in the side chain may significantly increase the cytotoxic activity. These structural features may be helpful in the development of more potent cytotoxic agent.


286

Table 1. Cytotoxic activity of 3-Hydroxy-4-oxo-2-phenyl-1,4-dihydroquinoline-7-carboxylic acid derivatives on

human malignant cell lines of different tissue origin and drug resistance profile

Compounds A 549 CEM CEM-DNR

bulk K 562 K 562-tax

Ar IC50

(µm) pIC50

IC50

(µM) pIC50

IC50

(µM) pIC50

IC50

(µM) pIC50

IC50

(µM) pIC50

Ph 236.2 -2.37 223.4 -2.35 171.2 -2.23 198.9 -2.30 196.3 -2.29

2-NO2 Ph 226.1 -2.35 100.8 -2.00 153.5 -2.19 130.0 -2.11 183.0 -2.26

2-I-Ph 191.8 -2.28 124.4 -2.09 134.1 -2.13 129.0 -2.11 174.9 -2.24

2-Cl-Ph 201.9 -2.31 156.1 -2.19 149.7 -2.18 161.1 -2.21 167.5 -2.22

3-Cl-Ph 239.1 -2.38 224.9 -2.35 163.4 -2.21 190.0 -2.28 197.5 -2.30

2- Br-Ph 192.4 -2.28 117.0 -2.07 145.5 -2.16 146.3 -2.17 160.6 -2.21

4-CH3-Ph 190.7 -2.28 195.2 -2.29 158.6 -2.00 172.0 -2.24 172.0 -2.24

3,5-Dichloro-4-NH2-Ph

9.8 -0.99 12.3 -2.17 12.0 -1.08 11.0 -1.04 11.7 -1.07

3-NO2-Ph 250.0 -2.40 227.2 -2.36 166.1 -2.22 200.1 -2.30 197.1 -2.29

Ph 250.0 -2.40 235.6 -2.37 167.2 -2.22 208.8 -2.32 192.9 -2.29

2-NO2 Ph 120.2 -2.08 147.0 -2.17 128.0 -2.11 132.4 -2.12 141.8 -2.15

2-I-Ph 46.6 -1.67 25.6 -1.41 105.2 -2.02 28.9 -1.46 40.3 -1.61

3-Cl-Ph 11.3 -1.05 5.5 -0.74 12.0 -1.08 5.00 -0.70 8.5 -0.93

2-Br-Ph 4.5 -0.65 8.0 -0.90 11.5 -1.06 5.6 -0.75 10.7 -1.03

2-Br-Ph 10.4 -1.02 4.9 -0.69 12.3 -1.09 5.2 -0.71 8.5 -0.93

4-CH3-Ph 2.8 -0.45 3.8 -0.58 4.9 -0.69 2.9 -0.46 2.8 -0.45

3,5-Dichloro- NH2 - Ph

0.7 0.17 0.8 -0.12 3.6 -0.56 1.1 -0.04 1.2 -0.08

3 NO2-Ph 162.5 -2.21 102.0 -2.01 189.9 -2.28 26.1 -1.42 43.2 -1.64

4-NO2-Ph 151.7 -2.18 182.6 -2.26 174.6 -2.24 48.6 -1.69 96.5 -1.98

Table 2. Descriptors used in present QSAR study

Descriptors Type Descriptors (units)

BP Thermodynamic Boiling point (Kelvin)

CP Thermodynamic Critical pressure (Kelvin)

CT Thermodynamic Critical temperature (bar)

TORERG Thermodynamic Torsion energy (kcal/mol)

LogP Thermodynamic Logarithmic partition coefficient

MP Thermodynamic Melting point (Kelvin)


287

Descriptors Type Descriptors (units)

MR Thermodynamic Molar refractivity (cm3/mol)

VWD Thermodynamic Van der Waals force (kcal/mol)

STERG Thermodynamic Stretch energy (kcal/mol)

STBERB Thermodynamic Strech bend energy (kcal/mol)

OVAL Steric Ovality (unit less)

CAA Steric Connolly accessible surface area (Å)

CMA Steric Connolly molecular surface area (Å)

CSEV Steric Connolly solvent-excluded volume (Å)

EM Steric Exact mass (g/mol)

MW Steric Molecular weight

B Electronic Bending energy (kcal/mol)

DIPOLE M Electronic Dipole moment (Debye)

TOT Electronic Total energy (ev)

Table 3.QSAR equation against various cell line.

Cell Line Equation

A 549 BA= [-3.56 (±0.74)]+VWD” [0.16(±0.06)] + BP [0.0019 (±0.0017)]- CSEV [0.04 (± 0.06)] CEM BA= [-3.62(±0.80)] +VWD” [0.11(±0.06)] +BP [0.003(±0.001)]- CSEV [0.03(±0.065)] CEM-DMR BA= [0.96 (±2.06)] + VWD” [0.004 (±0.01)] +BP [0.003 (±0.001)] – CMA [4.20453(±2.17049 K 562 BA= [-3.816(±0.49)] + VWD” [0.10(±0.043)]+ BP [0.003 (±0.001)] – CSEV [0.01 (±0.04)] K 562-tax BA= [-3.67(±0.77)]+ VWD” [0.12 (±0.06)] + BP [0.002 (±0.007)]- CSEV [0.032 (± 0.06)]

Table 4. Summary of Multiple Linear Regression (MLR) analysis with validation using various parameters

Equation n r2 Q2 Std F r2 bs PRESS SDEP Variance Chance

1 19 0.89 0.35 0.23 26.6 0.99 0.56 0.47 0.05 <0.001 2. 19 0.88 0.42 0.25 19.8 0.87 0.51 0.43 0.06 <0.001 3. 19 0.85 0.59 0.20 18.4 0.81 0.33 0.28 0.04 <0.001 4a. 18 0.95 0.67 0.15 56.0 0.90 0.38 0.31 0.02 <0.001 5. 19 0.88 0.44 0.24 23.6 0.89 0.51 0.43 0.05 <0.001

Q2 denotes cross – validated correlation coefficient. PRESS and SDEP denote predicted residual sum of squares and

standard deviation error of predictions respectively. Standard error of estimate (s), variance ratio (F) at specified

degree of freedom (df) and bootstrap (r2bs). a) Compound 8 is outlier

Table 5. Calculated and predicted pIC50 values of QSAR model exhibiting 2-4-(1H)-quinolinone-7-carboxylic

acids

A-549 CEM CEM-DNR

bulk K562 K 562-tax

Cal. Pred. Cal. Pred. Cal. Pred. Cal. Pred. Cal. Pred.


288

A-549 CEM CEM-DNR

bulk K562 K 562-tax

Cal. Pred. Cal. Pred. Cal. Pred. Cal. Pred. Cal. Pred.

-2.33 -2.41 -2.42 -2.43 -2.34 -2.37 -2.39 -2.50 -2.39 -2.41

-2.40 -2.33 -2.27 -2.30 -2.32 -2.37 -2.27 -2.29 -2.27 -2.29 -2.22 -2.18 -2.00 -1.93 -1.94 -1.92 -1.98 -1.89 -1.98 -1.89

-2.25 -2.21 -2.25 -2.29 -2.22 -2.23 -2.27 -2.31 -2.27 -2.31

-2.34 -2.33 -2.30 -2.29 -2.23 -2.38 -2.28 -2.28 -2.27 -2.27 -2.16 -2.15 -2.10 -2.09 -2.09 -2.07 -2.07 -2.06 -2.07 -2.06 -2.41 -2.44 -2.39 -2.41 -1.60 -1.29 -2.37 -2.40 -2.37 -2.40 -1.60 -1.7 -2.28 -1.77 -1.36 -1.55 - - -1.60 -1.71 -2.35 -2.35 -2.28 -2.27 -2.32 -2.35 -2.28 -2.28 -2.28 -2.28 -2.36 -2.35 -1.81 -2.27 -2.32 -2.35 -2.28 -2.28 -2.28 -2.28 -1.83 -1.79 -1.46 -1.76 -1.96 -1.93 -1.80 -1.75 -1.80 -1.75 -1.59 -1.55 -0.83 -1.48 -1.82 -1.65 -1.46 -1.45 -1.46 -1.47 -1.20 -1.61 -0.93 -1.07 -1.15 -1.30 -0.83 -1.20 -0.83 -1.20 -0.45 0.81 -2.42 -1.82 -1.22 -1.29 -0.78 -1.64 -0.78 -1.64

-1.40 -1.25 -0.98 -1.11 -1.25 -1.43 -1.05 -1.10 -1.15 -1.09 -1.83 -1.76 -1.59 -1.70 -1.49 -1.87 -1.45 -1.70 -1.37 -1.70 -0.62 -0.58 -0.63 -0.71 -1.18 -1.40 -0.72 -0.59 -0.48 -0.59 -1.87 -1.72 -1.27 -1.53 -1.57 -1.68 -1.47 -1.55 -1.67 -1.55

-1.72 -1.58 -1.64 -1.45 -1.70 -1.87 -1.35 -1.46 -1.26 -1.46

ACKNOWLEDGEMENTS

Authors are gratefully acknowledging the Principal for providing necessary facilities to carryout this work and Mr.A.K.Gupta for his relentless co-operation in this study. REFERENCES

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290

Formulation and Evaluation of Bilayer Tablets of Propanolol

Hydrochloride Aisha Khanum*, Vinay Pandit and Shyamala Bhaskaran

Al-Ameen college of Pharmacy, Lalbagh Main gate, Bangalore-560027

Author for Correspondence : [email protected]

ABSTRACT

Propranolol hydrochloride bi-layer tablets were formulated consisting of two layers such as fast releasing layer

and sustaining layer. Fast releasing layer was prepared using super-disintegrant such as sodium starch

glycolate and sustaining release layer was prepared using hydrogels like Hydroxy propyl methyl cellulose

(HPMCK4M) and Sodium Carboxy methyl cellulose (high viscosity grade) alone and in combination with an

aim to attain zero order release after the initial burst of release. Selected formulations were subjected to In-

vitro drug release in simulated gastric fluid and simulated intestinal fluid which revealed that dissolution

kinetics followed a first order release for formulations containing HPMC or NaCMC alone where as

formulations containing combination of polymers followed a zero order kinetics. Analysis of variance showed a

significant difference (p<0.05) in drug release among the formulations. Therefore hydrogels in appropriate

proportions are suitable for formulating controlled release tablets to have a zero order release for water-

soluble drugs like propranolol hydrochloride.

KEY WORDS: Propranolol hydrochloride, Hydrogels, Zero order release and Bilayer tablets.

INTRODUCTION

Multilayered tablet concept has long been utilized to develop sustained release formulations. Such a tablet has a fast releasing layer and may contain bi or triple layers, to sustain the drug release4. The pharmacokinetic advantage relies on the criterion that, drug release from the fast releasing layer leads to a sudden rise in blood concentration. However the blood level is maintained at steady state, as the drug is released from sustaining layer. Propranolol HCl, a nonselective beta-adrenergic blocker used widely in the treatment of hypertension, angina pectoris, pheochromocytoma and cardiac arrhythmias1. It is almost absorbed from gastrointestinal tract but subjected to hepatic tissue binding and first pass metabolism. Conventional form available is administered orally, 40 mg daily and is started with half to 1 tablet b.i.d and increase gradually as required up to a maximum of four tablets2. But due to raising problems with the conventional tablet resulting in fluctuations of drug plasma levels and also the drug has

short biological half-life (3-4 h)3 and first pass metabolism favors for sustained release dosage form. In recent years slow and sustained release formulation of Propranolol HCI has become available with claims that these formulations maintain beta adrenoreceptor blockade throughout a 24 h period and enable the drug to be given once daily. In the present study, Propanolol HCI bi-Layer tablets were formulated consisting of two layers such as fast releasing layer and sustaining layer. Fast releasing component was prepared using super-disintegrant such as sodium starch glycolate and sustaining release layer was prepared using hydrogels like Hydroxy propyl methyl cellulose (HPMCK4M) and sodium carboxy methyl cellulose (high viscosity grade) alone and in combination with an aim to attain zero order release after the initial burst release. MATERIALS AND METHODS

Propranolol hydrochloride was obtained as gift sample from Natco Pharma Ltd, Hyderabad. HPMCK4M, Sodium CMC and sodium starch glycolate was obtained as gift sample from Dr Reddy's laboratories, Indian Journal of Pharmaceutical Education & Research

Received on 31/1/2008 ; Modified on 12/8/2008 Accepted on 10//1/2009 © APTI All rights reserved

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Hyderabad. Lactose, magnesium stearate and talc were procured from Nice Chemicals, Nagpur. Other materials and solvents used were of analytical grade. Preparation of Bilayer Tablet

The bi-layer tablets were prepared by direct compression method. The drug and other excipients used were passed through sieve No. 85 before their use in the formulation. The sustaining layer was formulated with various amounts of hydro gels as given in Table 1. The dose in the formulation for fast releasing was 25 mg. The maintenance dose or sustaining dose of Propranolol hydrochloride was calculated as per reported method5, 6, 7. Formulation of Fast Release Layer

The fast release layer was formulated by uniformly mixing propranolol HCI (25mg) with sodium starch glycolate (15 mg) using talc and magnesium stearate as lubricant and glidant respectively. Formulation of Sustained Release Layer

The sustaining layer was formulated using hydrogels by mixing 55 mg of propranolol hydrochloride uniformly with HPMC and Sodium CMC, as per the formula given in Table 2. Talc (3 mg) and magnesium stearate (2 mg) were added respectively as lubricants. Compression of Bilayer Tablets

The quantity of powders for sustained release layer was compressed lightly using a single punch-tableting machine (Cadmach® Machinery Co.Pvt.Ltd., Mumbai) equipped with 6.5 µm circular, flat and plain punches. Over this compressed layer, required quantity of the fast release layer was placed and compressed to obtain hardness in the range of 6-7 kg/cm2 to form a bilayered tablet. Evaluation of Bilayer Tablets

The physical tests for the bilayer tablets of all the formulations were performed and average values were calculated. Weight variation was determined by weighing 20 tablets individually, the average weight was calculated and the percent variation of each tablet from the average weight of tablet was calculated. Hardness and friability of the tablet formulations were evaluated using Monsanto® hardness tester and Roche friabilator respectively. Drug Content Uniformity

Ten tablets were weighed individually, then placed in a mortar and powdered with a pestle. An amount equivalent to 50-mg drug was extracted with 50 ml of

methanol and sonicated for 15 minutes. Finally the volume was made up to 100 ml with methanol. The mixture was then filtered and 1 ml of the filtrate was suitably diluted and and then the drug content was measured at 290 nm6 using Elico Ind. Ltd. U.V. Visible double beam spectrophotometer. Mass Degree of Swelling

Mass degree of swelling of hydrogels was determined by equilibrating each tablet formulation with 100 ml of water for 5 h. The tablets were removed, blotted using tissue paper and weighed. The mass degree of swelling (Q) was calculated using the formula7 Q=Mass of swollen gel/Mass of dry polymer In vitro Release Study

Release of Propranolol HCl was determined using, USP (XXI) six stage dissolution rate test apparatus I (Thermolab®) at 100 rpm. The dissolution rate was studied using 900 ml of simulated gastric fluid for first 2 h followed by simulated intestinal fluid for the remaining hours. The temperature was maintained at 37± 0.2° C. Samples of 5 ml each were withdrawn at different time intervals i.e. 5,10,15,20,25,30,60,120 upto 720 min. Each sample withdrawn was replaced with an equal amount of fresh dissolution medium. Samples were suitably diluted and analyzed for Propranolol HCI content at 290 nm. The rate and mechanism of release of Propranolol HCI from the prepared bilayer tablets were analyzed by fitting the dissolution data into, zero order equation Q = Qo-Kot, where Q is the amount of drug released at time t and Ko is the release rate constant. First order equation, Ln Q = Ln Qo- K1t, where KI is the release rate constant and Higuchi's equation, Q = K2 t, where Q is the amount of drug released at time t and K2 is the diffusion rate constant. The dissolution data was further analyzed to define the mechanism of release by applying the dissolution data following the empirical equation, Mt/Ma = Knf

where Mt/Ma is the fraction of drug released at time t. K is a constant and n characterizes the mechanism of drug release from the formulations during dissolution process. FT-IR Study

Infrared spectrum was taken in the Perkin- Elmer FT - IR (spectrum RX) by scanning the optimized fonnu1ations in potassium bromide discs. The sample


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of pure drug, physical mixtures (1: 1) of both drug and polymers were scanned individually. Statistical Analysis

Release rate constant i.e. Ko (release rate constant of cumu1ative % drug release versus time plot) for bi-Iayer tablets of different fonnu1ations were calcu1ated. The Ko of different fonnu1ations were subjected to Analysis of Variance (ANOVA) test. The results were subjected to statistical validity using Microsoft Excel

software. RESULTS AND DISCUSSION

The prepared bilayer tablets were evaluated for various physical properties. All the batches were produced under similar conditions to avoid processing variables. These were evaluated for various physical parameters such as weight variation, thickness, hardness, friability and drug content. Hardness of tablets ranged from 6.0-6.8 kg/cm2, thickness of tablets ranged from 2.8-3.0 µm. The percentage friability of all the fomu1ations was between 0.26-0.64 %. The values of hardness test and percent friability indicates good handling property

of prepared bi-layered tablets. The drug content uniformity in the bi-layered tablets was within the range from 92.5 to 107.5%. The swelling degree was maximum for Fl i.e. 7.83, this is due to high swelling capacity of polymer indicating that the release is maximum for this fonnu1ation. In other fonnu1ations, from F2 to F6 the swelling degree was ranging from 4.95 to 6.75, F7 had a swelling degree of only 5.32, which is a combination of HPMC and NaCMC.8 Gel viscosity at the periphery increases owing to the high degree of cross-linking between HPMC, which is non-ionic, and NaCMC, which is ionic in nature. The release of Propranolol HCI from the prepared formulations was analyzed by plotting the cumulative percent drug released vs time as shown in fig 1. Simple visual observation of the plot shows an initial burst effect. From all formulations, over 30 % of the Propranolol HCI was released within the first hour of dissolution study.

Table 1: Formulation of sustained release bilayer tablets

Formulations PropranololHCI: HPMC:NaCMC

Ingredients (mg/tablet)

Propranolol HCI HPMC K4M

NaCMC Micro

crystalline cellulose

Fl 1: 0.5 55 27.5 - 66.5 F2 1:1 55 55 - 39 F3 1: 1.5 55 82.5 - 11.5 F4 1: 0.5 55 - 27.5 66.5 F5 1: 1 55 - 55 39 F6 1: 1.5 55 - 82.5 11.5 F7 1:0.5:0.5 55 27.5 27.5 39

Table 2: Evaluation parameters of propranolol HCL bilayer tablets

Formulations Drug Content (mg / tablet)

(± S.D)n

Swelling Degree (Q) (± S.D)n

Fl 78.21 ± 1.79 7.36 ±0.84 F2 79.25 ±0.7 55.93 ±0.37 F3 77.59 ± 1.2 45.02 ±0.28 F4 78.63 ±0.58 6.07 ±0.69 F5 79.68±2.83 5.48 ±0.77 F6 78.36 ±1.89 4.82 ±0.48 F7 79.58 ±1.25 5.26 ±0.56 S.D Standard Deviation n = 6


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Table 3: Kinetics of drug release from Propranolol HCL bilayer tablets

Formulations Drug release kinetics, Correlation coefficients 'r' Release

exponentional Zero Order First Order Higuchi Equation Fl 0.929 0.990 0.999 0.521 F2 0.949 0.992 0.997 0.565 F3 0.971 0.996 0.997 0.603 F4 0.917 0.998 0.998 0.553 F5 0.920 0.994 0.991 0.662 F6 0.966 0.998 0.998 0.683 F7 0.998 0.923 0.997 0.771

Figure 1: Dissolution profile of Propanolol HCL Bilayer Tablets

This initial high amount of Propranolol HCI release can be attributed to release of drug from the immediate release layer of the formulations. The initial release of Propranolol HCI with HPMC K4M 1:0.5 (40 %) and sodium CMC (high viscosity grade) 1:0.5 (38 %) was very high compared to other formulations. This high percent release can be attributed to release of the drug from fast release layer and also release of drug from the surface of sustaining layer of the tablet. The release rate was found to be decreasing as the concentration of polymer increased. This is due to the reason that the swelling degree is less because of higher concentration of polymer. In F7 cumulative percent drug release was about 96 % in 12 h. Drug release from F7 followed a zero order release whereas other formulations (F1-F6) followed a first order release as shown in Table -3. During the release of the drug from

the hydrogel-based tablets, two processes go on simultaneously. The first one being advancement of swelling front on glassy polymer and second is the attrition of the rubbery state polymer i.e. gel at the periphery which is devoid of drug. When the rates of these two processes are equal, the diffusion path length for the drug remains constant and zero order release is obtained. All the formulations exhibit best correlation by the Higuchi9 equation proving that the release is by diffusion mechanism as shown in table 3. Koresmeyer and Peppas10 equation revealed that all formulations have 'n' value from 0.521 to 0.771 indicating that they follow Non-fickian diffusion. FT -IR study indicated that there was no interaction between the drug and polymers used in the study. Analysis of variance (ANOVA) using Ko values for each formulation showed a highly significant difference among


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formulations (drug to polymer ratios) at P < 0.05 levels. Hence, Bilayer tablets showed initial burst effect to provide loading dose of the drug followed by extended zero order release (F7) and first order release (Fl - F6) for 12 h from the sustaining layer. Therefore, hydro gels in appropriate proportions are suitable to achieve a zero order release for water-soluble drugs like propranolol HCl. CONCLUSION

Thus it has been concluded that in a bilayer tablets it is possible to have a fast releasing layer and a sustaining layer so as to zero order release of water soluble drugs like propanolol can be achieved. REFERENCES

1. Martindale, The Extra Pharmacopoeia, 31st ed., The Pharmaceutical Press, London 1996, pp. 936–937.

2. Serlin MJ, Orme ML, Maciver M, Green GJ, Sibeon RG, Beckenridge AM. Pharmacodynamics and pharmacokinetics of conventional and long acting propranolol in patients with moderate hypertension, J. Clin. Pharmcol. 15 (1983) 519–526.

3. B. Taylan, Y. Capan, O. Guven, S. Kes and A. A. Hincal, Design and evaluation of sustained release and buccal adhesive propranolol hydrochloride tablets, J. Control. Rel. 38 (1996) 11–20; DOI:10.1016/0168-3654(95)00094-1.

4. Abraham MA, Shirwaikar A. Formulation of multilayered sustained release tablets using

insoluble matrix system, Indian J. Pharm. Sci. 59 (1997) 312–315.

5. Vercammen JP, Dauwe D, Brioen P. Possibility of use of Eudragit RS as a sustained release matrix agent for the incorporation of water soluble active compounds at high percentages, STP Pharma. Sci. 7 (1997) 491–497.

6. Rawlins EA, Bentley's Text Book of Pharmaceutics. 8th Edn. Bailliere Tindall ELBS; London 1996. pp. 663 -666.

7. Thummel KE, Shen DD, Isoherranen N, Smith HE. Design and Optimization Regimens; Pharmacokinetic Data, of dosage in Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 11th ed. (Eds. L. L. Bruton, J. S. Laro and K. L. Parker), McGraw-Hill Medical Publishing Division, London 2006, pp. 1863.

8. Jayaswa SB, Gode KD, Khanna SK. Sustained release tablet formulation of propranolol hydrochloride with Eudragit, Aust. J. Pharm. Sci. 9 (1980) 22–26.

9. Makhija SN, Vavia PR. Once daily sustained release tablets of venlafaxine, a novel antidepressant, Eur. J. Pharm. Biopharm. 54 (2002) 9–15; DOI: 10.1016/S0939-6411 (02) 00049-8.

10. Peppas NA, Analysis of Fickian and non Fickian drug release from polymers, Pharm. Acta Helv. 60 (1985) 110–111.


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Synthesis and Investigation of Colon Specific Polymeric Prodrug of

Budesonide with Cyclodextrin Prabhakara Prabhu*, D.Satyanarayana, Harish Nairy M, Mohd.Gulzar Ahmed,

R.Narayanacharyulu, E.V.S. Subrahmanyam

NGSM Institute of Pharmaceutical Sciences, Paneer, Deralakatte, Mangalore- 574 160

*Corresponding Author: prabhuprashu @ rediffmail.com.

ABSTRACT

Corticosteroid drug budesonide conjugated with β-cyclodextrin through a spacer molecule. The coupling

produced budesonide-cyclodextrin conjugate (polymeric prodrug) in which the drug is selectively introduced at

one of the secondary hydroxyl group of cyclodextrins through an ester linkage. The aqueous solubility of the

conjugate (>2 %w/v) was much higher than that of budesonide and no appreciable release of drug was

observed in the stomach and small intestinal homogenates of rats. On the other hand fast disappearance of

conjugate and appearance of the drug was observed in the cecal and colon contents of rats. The present

budesonide - cyclodextrin conjugate may be of the value as an orally administered colon specific polymeric

prodrug and hence the direct effect of the drug in localized area is of much useful in the treatment of colon

related diseases like ulcerative colitis and inflammatory bowel disease.

KEY WORDS: Budesonide, β-cyclodextrin, colon specific, polymer-prodrug.

INTRODUCTION:

Recently, numbers of cyclodextrin (CyD) derivatives have been prepared for many purposes, such as the construction of enzyme models, chiral separators, or recognizing agents. 1-3 Among these purposes, application of Drug-CyD conjugates to drug targeting is important. For example, CyD derivatives bearing small saccharide, such as glucose, galactose, mannose, fructose, etc., work as carriers for transporting active drugs to sugar receptors such as lectins on cell surfaces. 4-7 Cyclodextrins are cyclic oligo saccharides consisted of 6-8 glucose units through α-1, 4-glycosidic bonds and have been utilized for improvement of certain properties of drugs, such as solubility, stability, bioavailability, etc., by formation of inclusion complexes. CyDs are barely known to be hydrolyzed and only slightly absorbed in passage through the stomach and small intestine. Most bacteroids isolated from the human colon are capable of degrading CyDs, as evidenced by their ability to grow on CyDs using them as sole carbon source and by the stimulation of cyclodextranase activity by exposure to CyDs. This

biodegradable property makes CyDs useful as a colon-targeting carrier; thus, CyD prodrugs may serve as a source of site specific delivery of drugs to the colon.8 Chronic colitis, eg., ulcerative colitis and Chron’s disease, is currently treated with glucocorticoids and other anti-inflammatory agents. Administration of these agents by the oral route and intravenous route produces systemic side effects, including adreno suppression, immuno suppression, cushinoid symptoms and bone resorption. In theory, selective delivery of drug to colon could lower the required dose and hence reduce the side effects. Budesonide is an anti-inflammatory synthetic potent corticosteroid. Once absorbed, distribution of budesonide is extensive and protein binding is roughly 90%. Budesonide undergoes approximately 85% first pass metabolism. Plasma half life is approximately 2 hr and when absorbed systemically, it shows severe adverse effects. To overcome these drawbacks, the present study was undertaken to investigate the colon specific polymeric prodrug of budesonide. In the present study attempt has been made to to conjugate budesonide through a spacer molecule to hydroxyl group of β- CyD. Furthermore, enzymatic degradation of the conjugate was investigated to gain insight into the hydrolysis mechanism in colonic contents.


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MATERIALS

Budesonide was gift sample from Asrta-Zeneca Ltd, Bangalore. β- CyD was procured from HiMEDIA Mumbai, Carbonyldiimidazole and Silica gel 60 F254 were obtained from Merck, Germany. The other chemicals used in the study were of LR grade. METHODS

Preparation of CyD ester conjugate (BDS).

This synthesis method involved two steps. In the first step budesonide hemiester was synthesized as this drug lacks carboxyl group and in the subsequent step this hemiester was conjugated to hydroxyl group of cyclodextrin. The detailed procedure is as follows. Budesonide, succinic anhydride and 4-dimethylaminopyridine (DMAP) 2 m mol each were dissolved in 50 ml anhydrous acetone. After reacting for 30 min at 25º C the acetone was removed by vacuum evaporation in a rotating flask. Thus obtained product (budesonide hemisuccinate) was recrystallized from ethanol/water (30:70). Further, this hemisuccinate was conjugated to β cyclodextrin as follows. 1 m mol of hemiester was dissolved in 15 ml of anhydrous Dimethyl sulfoxide. To this 1 m.mol carbonyldiimidazole (CDI) was added and reacted for 30 min at room temperature. β cyclodextrin ( 1 m mol solution in 2 ml DMSO) and 1 m mol of triethylamine (TEA) were added, and reacted at room temperature for 24 hr. The β cyclodextrin conjugation with budesonide (BDS) was precipitated by addition of 25 ml of acetone. The conjugation reaction was monitored by thin layer chromatography using silica gel 60 F254 as stationary phase and the mobile phase was ethyl acetate/water/2-propanol in the ratio of 7:5:7v/v (indicator: p-anisaldehyde for cyclodextrin and UV at 254 nm for the steroid and conjugate). This reaction was carried out in presence of nitrogen atmosphere.9 Experimental:

Measurement of solubility: The screw capped vials containing the conjugate 250 mg in 10 ml of water were shaken at 25 ºC for 5 min. It was centrifuged at 2000 rpm for 5 min and the supernatant was analyzed by UV –Visible double beam spectrophotometer for the conjugate at 270 nm.8 Estimation of drug content of the conjugation:

Though, there is no official method to estimate the drug content of cyclodextrin prodrugs, in the present study

the hydrolysis was brought about in presence of rat cecal matter, as the chemical hydrolysis path way may not result in complete hydrolysis of conjugation. For the purpose of the complete hydrolysis of the prodrug, 5 ml solution of 1.0 X 10-3 M was prepared in isotonic phosphate buffer of pH 7.4 and incubated with the rat cecal matter preparation for 48 hr at 37ºC. The rat cecal matter preparation was made by mixing the contents of rat cecal matter (2%w/v) with chilled pH 7.4 isotonic phosphate buffer and then filtered through gauge to remove the visible particles. After centrifugation at 2000 rpm for 5 min the supernatant was analyzed for the free drug content at 244.3 nm after suitable dilution. In vitro hydrolysis study and determination of drug

content: In vitro hydrolysis study of compound BDS was studied in simulated gastric fluid of pH 4.4 acetate buffer and simulated intestinal fluid of pH 7.4 phosphate buffer. Initial concentration was 5 ml of 1.0 X 10-3 M conjugate solution was taken. The temperature was maintained at 25ºC and aliquots were removed at different time intervals for 24 hr and determined the free drug content (budesonide) by UV-Visible double beam spectrophotometer at 244.3 nm. Similar study was conducted simultaneously in presence of rat fecal matter (~0.5 g). The results are reported in Figure -1. Hydrolysis in rat gastro intestinal tract contents or

homogenates: Male Wistar rats, 150-200 g, were fed a standard diet. The rats were killed by decapitation. Stomach, small intestine, cecum and colon were removed and the contents were diluted by 20 % w/v with the following chilled isotonic buffers: Stomach (pH 4.4 acetate buffer), small intestine (pH 7.4 phosphate buffer), and cecum and colon (pH 6.8 phosphate buffer). The dispersions of the contents were filtered through gauge to remove the large visible particles. The conjugate solution (5 ml of 1 mg/ml strength in the corresponding isotonic buffers) was added to the filtrates (5 ml) in air tight containers. The pH of the contents were adjusted to 4.4 (stomach), 7.4 (small intestine) and 6.8 (cecum and colon) by the addition of 0.1 N NaOH and nitrogen gas was introduced into the containers and incubated at 37 ºC. At appropriate time intervals samples were withdrawn and equal amount of corresponding buffers were replaced. The withdrawn samples were diluted


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suitably and centrifuged. Supernatant liquid was subjected to spectral analysis for determining the release of free budesonide from conjugation by UV- Visible spectrophotometer at wavelength of 244.3nm10. RESULTS AND DISCUSSION

Preparation of CyD-budesonide was achieved as shown in Scheme of synthesis.

Initially budesonide hydroxyl group was esterified to prepare hemiester by using succinic anhydride, as the drug lacks carboxyl group to prepare ester with cyclodextrin. Then the carboxyl group of hemiester of budesonide was activated by reacting with carbonyldiimidazole at room temperature and then conjugated with cyclodextrin in presence of TEA as catalyst. Finally acetone was added to precipitate the conjugation. IR spectral values indicated that the peak at 1730 cm-1, presence of ester group (C=O) and broad peak at 3220 cm-1 indicated the presence –OH group in the molecule as cyclodextrins contains many -OH groups. Proton NMR spectra: δ 7.32 – 7.35 (m, 1 H, BDS 1–H ), δ 6.15- 6.18 (m, 1 H, BDS 2-H), 5.92 ( s, 1 H, BDS 4-H), δ 5.57 - 5.63 (m, 15 H, CyD 2,3 – OH), δ 4.82 – 4.89 (d, 7 H, CyD 1-H), δ 5.13- 5.14 (dd, 2 H, BDS 21-H), 4.26 (s, 1 H, BDS 11-H), δ 4.37 – 4.52 (m, 8 H, CyD 6-OH), δ 3.69 – 3.80 (m, overlaps with HOD CyD 2-, 3-, 4-, 5-, 6- H,), δ 3.37 – 3.42 (m, Cyd 2,4 –H), δ 2.56- 2.58 (m, overlaps with DMSO-d6 succinyl, BDS 6a-, 6b-, 16 b-H), δ 1.79 (s, 3 H, BDS 19-CH3), δ 1.37-1.44 (m, 3 H, BDS 6a, 12 b, 15 b-H), δ 0.93 (s, 3 H, BDS 18-CH3). Mass spectral data: Molecular ion peak at 1647 (M+) and M+1 peak at 1648. Peak corresponding to 1135 is that of cyclodextrin and at 430 belonging to Budesonide. The presence other peaks in the mass spectra at 973, 811, 649, 487, 325, 163 indicated the fragmentation pattern of cyclodextrin for each molecule of glucose. Thin layer chromatography: Stationary phase;- Silica gel 60

F254 , Mobile phase; Ethyl acetate : water : 2-propanol (7:5:7 v/v). Spot identification method: p-anisaldehyde for cyclodextrin and as well as by UV method at λmax254 nm for steroid and conjugate. Rf value: 0.73. Melting range: By open capillary in liquid paraffin bath method (Thiel’s apparatus) 210-213 º C. UV Scan: λmax at 270 nm in water. Solubility:

The conjugation BDS was found to be soluble in water though the drug is insoluble in water. It was observed that the property of conjugate was found to be different from budesonide as the drug is insoluble in water. It is of interest to note that the conjugate was soluble (2% w/v, 20 mg/ml) in water. This result was in sharp contrast to the decreased solubility of β- CyD derivatives conjugated at the primary hydroxyl positions. For example, the aqueous solubility of biphenylylacetic acid and n-butyric acid conjugated at the primary hydroxyl position of β- CyD was approximately 1/10 that of the parent compound. The decreased solubility has been attributed to strong intermolecular association between the drug moieties and the neighboring β- CyD cavity, forming a stable columnar packing structure in the solid state.9 Hence, It was found that the conjugation at secondary hydroxyl group resulted more soluble prodrug than the drug budesonide. Further, from the drug content estimation it was found that in ~ 8 mg of conjugate (5 ml of 1.0 X 10-3 M conjugate), ~1.5 mg drug was present. Hence, in every 100 mg of conjugate approximately ~ 20 mg of drug was found. In vitro hydrolysis study From the in vitro hydrolysis study it was observed that the conjugate BDS did not release the free drug budesonide significantly both in the simulated gastric and intestinal fluid. However, in presence of rat fecal matter in phosphate buffer it was observed that significant amount of drug release (~50%, Figure 1). This is attributed to the presence of micro-organisms in the fecal matter which in turn releases the enzymes glycosidases, causes the fermentation of cyclodextrins. Hence the drug release was observed. Hydrolysis in rat gastro intestinal tract contents or

homogenates

The amount of free budesonide released from the conjugates over a period of time in presence of stomach, small intestine and colon & cecum contents


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Table 1. Hydrolysis kinetics in rat gastro intestinal tract contents or homogenates

Time

in minutes

Percentage hydrolysis of conjugate in intestinal segment

Stomach Small intestine Cecum & Colon

30 1.3 2.2 5.7 60 6.5 7.5 12.5 90 10.5 12.5 26.2 120 11.8 14.8 35.5 150 9.2 22.2 43.3 180 8.9 23.0 57.6 210 8.9 26.0 65.2 720 8.8 28.8 89.3 1440 8.5 31.8 91.8

Fig 1. In vitro hydrolysis of conjugation BDS.

were given in Table-1. It can be observed that the drug budesonide in the contents of stomach and small intestine released marginally around 12% and 32 % respectively, which could be accounted for spontaneous hydrolysis in aqueous solution in the stomach and presence of smaller number of organisms in the small intestine. On the other hand, in the cecal and colonic contents the conjugates released the drug significantly (approximately 90 %) within 12 hr. The results indicate that the budesonide activation took place site-specifically in the rat cecal and colonic contents. This activation of drug from the conjugates may be

attributed to the presence of glycosidase enzymes released from the bacteria present in the cecum and colonic area of rat. CONCLUSION:

Colonic specific drug delivery can be achieved with carriers by making prodrugs that survive passage through stomach and small intestine, but active moiety is released by enzymes specifically produced in colon. A well known demonstration of this concept is the delivery of 5-aminosalicylic acid by the use of azo linked prodrugs. Further, Friend et al demonstrated the colon targeting of steroids by the use of glycoside


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prodrugs.11 The present study indicated that the ester type prodrug of budesonide with β- CyD released preferentially in cecal and colonic contents of rats after fermentation of CyD to smaller saccharides, suggesting that β- CyD can serve as a new class of site-specific drug carrier. Thus, the β- CyD conjugation approach can avoid the systemic side effects of the drug and may provide a versatile means for construction of colon specific delivery systems for drugs. ACKNOWLEDGEMENT:

The authors are grateful to Nitte Education Trust, Mangalore, Indian Institute of Sciences, Bangalore and Central Drug Research Institute, Lucknow, for providing the necessary facilities to carry out this study. REFERENCES

1. Chen Z, Bradshaw J.S, Shen Y, Habata Y, Lee ML, Novel method to prepare dihydroxy permethylated β-cyclodextrin isomeric intermediates for bifunctional enzyme mimics, J.Org.Chem. 1997, 67, 8529-8534.

2. VanEtten R.L, Sebastian J.F, Clowes G.A, Bender M.L, Acceleration of phenyl ester cleavage by cycloamyloses. A model for enzymatic specificity, J.Am.Chem .Soc. 1967, 89, 3242-3253.

3. Cramer F, Meckensen G, Cyclodextrin imidazol-Verbidengen, Chem.Ber. 1970, 103, 2138-2147.

4. Matsuda K, Inazu T, Haneda K, Mizuno M, The chemo-enzymatic synthesis and evaluation of oligosaccharide branched cyclodextrins, Bioorg.Med.Chem.Lett. 1997, 7, 2353-2356.

5. 5.Kassab R, Felix C, Parrot-Lopez H, Bonaly R, Synthesis of cyclodextrin derivatives carrying biorecognizable saccharide antennae, Tetrahedron.Lett. 1997, 38, 7555-7558.

6. Fruike T, Aiba S, Synthesis of novel cyclodextrin derivatives having oligosaccharide cluster, Chem.Lett. 1999, 69-70.

7. Garcia-Lopez J.J, Harnandaez-Mateo F, Isac-Garcia J, Kim J.M, Synthesis of perglycosylated β-cyclodextrins having enhanced lectin binding affinity, J.Org.Chem. 1999, 64, 522-531.

8. Hirayama F, Ogata T, Yano H, Arima H, Udo K, Takano M, Uekama K, Release characteristics of a short chain fatty acid, n-butyric acid, from its β-cyclodextrin ester conjugate in rat biological media, J.Pharm.Sci. 2000, 89, 1486-1495.

9. Uekama K, Yano H, Hirayama F, Arima H.

Preparation of Prednisolone- appended α-,-β- and -

γ- cyclodextrins: Substitution at secondary hydroxyl groups and In vitro hydrolysis behaviour. J.Pharm.Sci. 2004; 90: 493–503.

10. McLeod A.D., Friend D.R., Tozer T.N. Synthesis and chemical stability of glucocorticoid-dextran esters: potential prodrugs for colon specific delivery. Int.J. Pharm. 1993, 92, 105-114.

11. Friend D.R, Chang G.W, Drug glycosides: Potential prodrugs for colon-specific drug delivery, J.Med.Chem 1984; 40: 515–57.


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Development and Evaluation of Ziprasidone Hydrochloride Fast

Disintegrating/ Dissolving Tablets using Complexation Techniques Deshmukh Sambhaji S., Potnis Vaishali V. *, Mahaparale Paresh R.1 Kasture Pramod V.1

and Gharge Vikram S.2

Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune- 411018, Maharashtra, India. 1Dr. D. Y. Patil College of Pharmacy, Pimpri, Pune- 411018, Maharashtra, India.

2 Emcure Pharmaceuticals Ltd., Bhosari, Pune- 411026, INDIA

*For Correspondence : [email protected]

ABSTRACT

Ziprasidone Hydrochloride, a Class II drug is an atypical antipsychotic agent, which is poorly water soluble

with only 60% bioavailability. In present study attempt has been made to prepare Ziprasidone Hydrochloride

fast disintegrating/ dissolving tablets. For enhancing solubility of drug, inclusion complexes of drug were

prepared using βCD and HPβCD. These complexes were characterized by solubility study, differential

scanning calorimetry and X- ray diffractometry. To aid in faster disintegration of tablets, superdisintegrants in

different proportions were used and their effect on disintegration was studied. The inclusion complexes with

HPβCD prepared by microwave method exhibited highest enhancement in solubility (0.024g/100ml) and also

showed fastest dissolution profile (100% drug release in 5 min.). So this complex was worked further for

formulation of Ziprasidone Hydrochloride fast disintegrating/ dissolving tablets. Among the tablet formulations

prepared using different superdisintegrants, those with 5 % w/w Polyplasdone showed the lowest disintegration

time (21 sec.) and fastest dissolution profile.

KEY WORDS

Ziprasidone Hydrochloride, Solubility enhancement, Inclusion Complex, Fast dissolving tablets,

Superdisintegrants.

INTRODUCTION

The major problem faced by many patients with conventional tablet dosage form is difficulty in swallowing. This problem is more apparent when drinking water is not easily available to the patient taking medicine. Hence patients may not comply with prescription, which results in high incidence of ineffective therapy1. The fast dissolving drug delivery system is rapidly gaining acceptance as an important novel drug delivery system. This delivery system offers better patient compliance than conventional tablet dosage form2. Bioavailability of drug from this delivery system is significantly greater than from conventional tablet3. Ziprasidone Hydrochloride is a newer atypical antipsychotic drug used in treatment of schizophrenia,

which is an ongoing mental disease4. Frequent relapses occur in about 55 % of schizophrenic patients5, mostly because of medication noncompliance as it depends upon route of administration and type of dosage form used. Thus there is an obvious need for development of fast disintegrating /dissolving tablets to overcome patient noncompliance. Ziprasidone Hydrochloride is practically insoluble in water (BCS Class II)6. Like other drug candidates in this class, this drug also has solubility-limited bioavailability (60%). Hence prior to its formulation as mouth disintegrating tablets it was decided to improve its solubility by complexation with cyclodextrins7. EXPERIMENTAL Materials

Ziprasidone Hydrochloride (Sun Pharmaceuticals Ltd, Baroda), β-Cyclodextrin and HP-β-Cyclodextrin


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(Signet Chemical Corporation, Mumbai), Polyplasdone XL 10, Ac-Di-Sol (Signet Chemical Corporation, Mumbai) and Primojel (Liva Pharma, Sinnar) were used. All other chemicals and reagents used were of analytical grade. Methods

a) Phase solubility analysis for Ziprasidone

Hydrochloride8

Phase solubility studies were performed to determine stoichiometric proportions of Ziprasidone

Hydrochloride with βCD and HPβCD. Also this data was used to determine the stability constant of complexes. For this, the stock solutions of 0.01 M of βCD and HPβCD were prepared separately using distilled water. These stock solutions were appropriately diluted with distilled water to give molar solutions in the range of 0.002 to 0.01 for both βCD and HPβCD. Five ml of each molar solution was filled in screw-capped vials and the excess quantity of drug was added to each vial separately. The vials were kept for shaking at ambient temperature for 48 hrs using a lab shaker (Remi). The supernatant solutions were collected carefully and filtered using Whatman filter paper (No.41). The absorbances of resultant solutions were recorded at 317nm. b) Preparation of inclusion complexes by microwave

irradiation method9

The required molar (1:1) quantities of drug and cyclodextrins were weighed accurately and transferred to round bottom flask. Minimum amount of solvent mixture (Methanol: Water=1:1 v/v) was added to this. The mixture was reacted for two minutes at 600C in the microwave oven. After reaction was complete, adequate amount of solvent mixture was added to remove the residual free drug and βCD or HPβCD. Precipitate so obtained was separated using Whatman filter paper (No.41). It was dried in vacuum oven at 400C for 48 hrs. A faint pink colored powder was obtained which was then stored in airtight containers till further use. c) Characterization of Ziprasidone Hydrochloride

inclusion complexes

Inclusion complexes of Ziprasidone Hydrochloride were characterized by following analytical techniques. i) Estimation of drug content

The quantities of inclusion complex equivalent to 10mg of Ziprasidone Hydrochloride were dissolved in Water/Methanol mixture (1:1). Appropriate dilutions

were made and drug content of each complex was calculated. ii) Saturation solubility studies

Solubility study was performed according to method reported by Higuchi and Connors.10 Excess quantities of inclusion complexes were added to 25 ml distilled water taken in stoppered conical flasks and mixtures were shaken for 24 hrs in rotary flask shaker. After sufficient shaking to achieve equilibrium, 2 ml aliquots were withdrawn at 1 hr intervals and filtered through Whatman filter paper no. 41. The filtrate so obtained was analysed spectrophotometrically at 317 nm. Shaking was continued until three consecutive readings were same. iii) IR spectral analysis11

Infra red spectra of drug and its inclusion complexes were recorded by KBr method using Fourier Transform Infrared Spectrophotometer (FTIR-8400s). A base line correction was made using dried potassium bromide and then spectra of dried mixtures of drug and inclusion complexes with potassium bromide were recorded. iv) X – ray diffraction (XRD) study11

The X ray diffraction pattern of the selected inclusion complexes was compared with that of the pure Ziprasidone Hydrochloride. This was done by measuring 2ø in the range of 4 to 500 with reproducibility of ±0.0010 on a diffractometer (Philips). The XRD patterns were recorded automatically using rate meter with time constant of 2 × 102 pulse/second and with the scanning speed of 20 (2ø)/min. v) Differential scanning calorimetric (DSC)

analysis11

This study was performed using DSC model (Perkin Elmer). For this study, the samples were placed in a platinum crucible and the thermograms were recorded at a heating rate of 100C/min in the range of 200C to 3100C. vi) Dissolution studies of Ziprasidone Hydrochloride

and its inclusion complexes

The quantity of inclusion complex equivalent to 20 mg of Ziprasidone Hydrochloride was placed in dissolution medium and apparatus was run maintaining following test conditions. [Dissolution medium- 900 ml of phosphate buffer pH 7.4 containing 1% w/v sodium lauryl sulphate, Speed of paddle- 75 rpm., Temperature of dissolution medium- 370C ± 0.50C, Apparatus type - USP XXII (paddle)]. Aliquots of 10 ml were withdrawn


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at time intervals of 5, 10, 15, 20, 25, 30, 40, 50 and 60 min. The volume of dissolution medium was adjusted to 900 ml by replacing each 10 ml aliquot withdrawn with 10 ml of fresh dissolution medium. The concentrations of drug in samples were determined by measuring absorbances at 317 nm. Cumulative percent drug released was determined at each time point. Pure drug was used as control. d) Development of rapidly disintegrating tablets by

direct compression method12

Fast disintegrating tablets were prepared using HPβCD inclusion complexes of Ziprasidone hydrochloride. The formula included variable amounts of superdisintegrants and other excipients. The resulting powder blends were evaluated for flow parameters. The powder blends equivalent to the 20 mg of drug were directly compressed into tablets using 8.45 mm flat faced round punches. The superdisintegrants used were Polyplasdone XL 10, Ac Di Sol and Primojel in concentration range of 3 %w/w, 4 %w/w and 5 %w/w individually. Table I gives compositions of these tablet formulations. M10 was a control formulation containing no superdisintegrant. Final weight of each tablet was kept constant (200mg) by varying the weight of microcrystalline cellulose i.e. Avicel (PH 102). Tablets were evaluated for various parameters.

Evaluation of powder blends

The powder blends were evaluated for the flow parameters such as angle of repose, tapped density, bulk density and compressibility index. Evaluation of tablet characteristics

The tablets were evaluated for weight variation, uniformity of drug content, hardness, friability, in vitro disintegration time, disintegration time in the oral cavity and in vitro dissolution behaviour (Table II). Dissolution study

The tablets from the formulation (M1-M10) were subjected to dissolution test using USP Dissolution Test Apparatus Type II maintaining following parameters [Dissolution medium- 900 ml of phosphate buffer pH 7.4 containing 1% w/v sodium lauryl sulphate, Apparatus type - USP XXII (paddle), Speed 75 rpm, Temperature of dissolution medium- 370C ± 0.50C.] All the dissolution tests were carried out in triplicate. RESULTS AND DISCUSSION

a) Phase solubility analysis of Ziprasidone

Hydrochloride

Phase solubility study was done to determine the stoichiometric proportion of Ziprasidone Hydrochloride with complexing agent βCD and HPβCD. The phase solubility analysis indicated formation of a 1:1 molar

inclusion complex of drug with βCD and HPβCD. Apparent stability constant (KC) was found to be 674 K-

1 and 1218 K-1 for βCD and HPβCD complexes respectively. b) Preparation of inclusion complexes of

Ziprasidone Hydrochloride

Inclusion complexes of Ziprasidone Hydrochloride with βCD and HPβCD prepared by microwave irradiation method found to be slightly pink, free flowing powders. c) Characterization of Ziprasidone Hydrochloride

inclusion complexes

Inclusion complexes of Ziprasidone Hydrochloride with βCD and HPβCD showed consistency in drug content (almost 100 %). Cyclodextrins and their derivatives are proved to be powerful solubilizers for many poorly water-soluble drugs.8 In present work also significant enhancement in the solubility of drug was observed for inclusion complexes with cyclodextrins. The saturation solubility of inclusion complexes of Ziprasidone Hydrochloride with βCD and HPβCD were found to be

0.020 ± 0.91 g/100ml and 0.024 ± 0.80 g/100ml respectively which is far superior to solubility of pure

drug 0.0024 ± 0.23 g/100ml. i) IR spectral analysis IR spectra of inclusion complexes of Ziprasidone Hydrochloride with βCD and HPβCD are given in Fig.1.Analysis of IR spectra of inclusion complexes revealed the disappearance of characteristic peaks of aromatic C-H stretching and N-H stretching at 3250 cm-

1 and 3400 cm-1 respectively. While the peak at 1750 cm-1 (carbonyl stretching of lactum ring) remained intact, it therefore, suggests that vibrating and bending movements of guest molecule i.e. Ziprasidone hydrochloride were restricted due to formation of inclusion complexes. It may be the aromatic ring portion of Ziprasidone Hydrochloride which has been included into the cavity of cyclodextrin. ii) X – ray diffraction study of inclusion complexes

of Ziprasidone Hydrochloride

Ziprasidone Hydrochloride, HPβCD and inclusion complexes of drug with HPβCD prepared by microwave method were subjected to XRD analysis


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Table I: Composition of various formulations of Ziprasidone Hydrochloride fast dissolving tablets

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10

Complex* (mg) 80 80 80 80 80 80 80 80 80 80

Avicel PH102 (mg)

103 101 99 103 101 99 103 101 99 109

Polyplasdone 3% 4% 5% - - - - - - - Ac-Di-Sol - - - 3% 4% 5% Primojel - - - - - - 3% 4% 5% - Mannitol 4% 4% 4% 4% 4% 4% 4% 4% 4% 4%

Na. Saccharin 1% 1% 1% 1% 1% 1% 1% 1% 1% 1% Magnesium

Stearate 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5%

* Ziprasidone Hydrochloride-hydroxypropyl betacyclodextrin complexes prepared by microwave method.

Table II: Data for evaluation of tablet properties of Ziprasidone Hydrochloride fast

disintegrating/dissolving tablets

Formulation Content Uniformity (%) Weight Variation

(mg)

Disintegration Time (In Vitro)

(sec.)

Disintegration Time (In Vivo)

(sec.)

M1 99.33 ± 0.241 198.43 ± 1.99 24.67 ± 1.53 30.67±0.58 M2 96.96 ± 0.199 197.86 ± 2.70 18.67 ± 1.15 27.33±1.15 M3 97.49 ± 0.158 199.00 ± 2.07 15.33 ± 2.08 21.67±2.52 M4 95.90 ± 0.137 196.29 ± 3.53 26.33 ± 1.53 32.67±2.52 M5 97.22 ± 0.045 197.29± 1.91 21.67 ± 1.15 29.00±1.73 M6 97.49 ± 0.209 197.00 ± 4.11 17.00 ± 2.00 25.67±2.08 M7 98.28 ± 0.209 198.43 ± 2.61 29.67 ± 2.08 38.67±0.58 M8 98.01 ± 0.120 197.71 ± 2.81 25.00 ± 1.00 31.67±0.58 M9 98.28 ± 0.079 198.43 ± 2.19 27.00 ± 1.00 32.33±0.58 M10 98.01 ± 0.164 198.57 ± 3.81 50.67 ± 1.53 59.33±3.06

Fig. 1. IR Spectra of inclusion complexes of

Ziprasidone Hydrochloride with ββββCD and HPββββCD

prepared by microwave irradiation method Drug- Ziprasidone Hydrochloride, ββββ CD- Beta cyclodextrin, HPββββ CD- Hydroxypropyl Beta

cyclodextrin, Drug ββββ CD- Ziprasidone Hydrochloride Beta cyclodextrin inclusion complex

Drug HPββββ CD- Ziprasidone Hydrochloride Hydroxypropyl Beta cyclodextrin inclusion

complex

Fig. 2. XRD pattern for Ziprasidone Hydrochloride

(pure), HPββββCD (pure) and their inclusion complex

prepared by microwave irradiation method


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Fig. 3. DSC pattern for Ziprasidone Hydrochloride

(pure), HPββββCD (pure) and their inclusion complexes

prepared by microwave method

Fig. 4. Dissolution profiles of Ziprasidone

Hydrochloride (pure), HPββββCD (pure) and their

inclusion complexes prepared by microwave method


Hydrochloride fast dissolving tablets containing

Polyplasdone XL 10 as superdisintegrant


Hydrochloride fast dissolving tablets containing Ac-

Di-Sol as superdisintegrant


Hydrochloride fast dissolving tablets containing

Primojel as superdisintegrant

Fig. 8. Effect of type of superdisintegrant on

dissolution profiles of fast dissolving tablets at 3%

concentration


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Fig. 9. Effect of type of superdisintegrant on dissolution

profiles of fast dissolving tablets at 4% concentration

Fig. 10. Effect of type of superdisintegrant on

dissolution profiles of fast dissolving tablets at 5%

concentration

(Fig 2). From the figure, it was evident that pure drug existed as microcrystalline particles as many broad peaks of very low intensity were observed. However no sharp peaks were detected. The X-ray diffraction pattern for inclusion complexes was characterized by complete absence of any diffraction peak for the drug, suggesting probable transformation of microcrystalline form into an amorphous state.11

iii) Differential scanning calorimetric analysis

Ziprasidone Hydrochloride, HPβCD and inclusion complexes of drug with HPβCD prepared by microwave method were subjected to DSC analysis. The thermograms are given in Fig. 3. The thermogram of pure drug showed broad endothermal peak ranging from 400C to 1300C due to loss of water of hydration. It confirms monohydrate salt form of drug. It also showed small endothermal peak at 3000C corresponding to its melting point. The DSC thermogram of HPβCD was characterized by an endothermic peak at about 2800C. DSC thermogram of inclusion complex of drug with

HPβCD showed sharp endothermal peak at around 2600C corresponding to melting of HPβCD. Also broad endotherm of drug ranging from 500C to 1400C was observed, it might be due to loss of water molecule from drug. The formation of inclusion complex is suggested by disappearance of endothermal peak at melting point of drug. iv) Dissolution studies of Ziprasidone Hydrochloride

and its complexes

The inclusion complexes of Ziprasidone Hydrochloride with both cyclodextrins produced pronounced enhancement in its dissolution (Fig. 4). HPβCD (100 %

drug release within 5 min.) was found to be more effective than βCD (100 % drug release within 25 min.) in enhancing the dissolution rate of drug as compared to pure drug alone (only 55 % drug release within 60 min.) d) Development of rapidly disintegrating tablet

formulation by direct compression method

The present work was undertaken to formulate and evaluate fast disintegrating Ziprasidone Hydrochloride tablets by direct compression method. Superdisintegrants at different concentration levels (3 % w/w, 4 % w/w, 5 % w/w) were included to assist faster disintegration. The absorption of water is an important step for subsequent disintegration process of tablets. Bi et.al.12 have reported that when higher concentrations of superdisintegrant were added to tablet formulations, they absorbed considerable amount of water and resulted in increase in viscosity of fluid within the tablet mass. This delayed further water penetration into the tablets. Therefore, it was decided to use superdisintegrant concentrations only up to 5% w/w. Bi et.al.13 also reported that tablets containing excessive large amounts of water-soluble fillers such as Tablettose exhibited slower disintegration of tablets. The probable reason cited was that after dissolution of Tablettose, the channels in Ac-Di-Sol fiber structure widened considerably allowing it’s swelling which then offered less help in rapid disintegration of tablets. Hence it was decided to use water insoluble filler like microcrystalline cellulose (Avicel PH 102) to overcome this problem. Mannitol was used for its sweet taste and


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negative heat of solution, which leaves a pleasant cooling sensation in mouth. Evaluation of powder blends

Bulk densities of powder blends were found between 0.33-0.53 g/ml. The angle of repose values varied from 190 to 260. Carr’s Index values varied from 11 % to 19 %. From these values it was evident that all these blends had excellent flow properties.

e) Evaluation of tablets

All batches of prepared tablets were evaluated for the different parameters. Weight variation for prepared tablets was found within specifications of Indian Pharmacopoeia. Average weight for tablet was in the range of 196 mg –199 mg. Hardness values for tablets of all formulations were in the range of 4-4.5 Kg/cm2. Thickness and diameter values for of all tablets were in the range of 3.95-4.01 mm and 8.43-8.46 mm respectively. Friability of all the formulations was in the range of 0.20-0.45 %. Drug contents for all the formulations were found in the range of 95.90-99.35 % (Table II). i) Evaluation of effect of concentration of

superdisintegrant on disintegration time of tablets

All tablet formulations indicated disintegration time in between 15.33 to 29.67 sec (Table II). The control formulation (M10) showed disintegration time of 50.67 sec. From these findings it can be claimed that the addition of the superdisintegrants has certainly improved the disintegration rate of tablets. Among the three superdisintegrants used, Polyplasdone XL10 showed less disintegrating time followed by Ac-Di-Sol and Primojel. The probable reason may be high gelling tendency of Ac-Di-Sol and Primojel than Polyplasdone XL10 which causes swelling of tablet mass with subsequent retardation of disintegration.13 Besides the type, the concentration of superdisintegrant used also affected the disintegration time. In case of the tablets containing Polyplasdone XL10 and Ac-Di-Sol, an increase in concentration of superdisintegrant resulted in definite decrease in disintegration time. The same result was found for tablets containing Primojel up to 4 %. At 5 % concentration, it resulted in slight increase in disintegration time from 25 sec. to 27 sec. This delay in disintegration time might have occurred due to probable blockade of capillary pores in tablet mass as a result of formation of the viscous plug by Primojel particles,

which subsequently prevented free access of fluid into tablets.13 The disintegration time for all tablet formulations was also evaluated “in vivo” in healthy volunteers. For all formulations “in vivo” disintegration time values were more than corresponding “in vitro” values. This may be due to low volume of saliva available and weak agitation in the mouth as compared to in vitro test.14 ii) Evaluation of effect of concentration of

superdisintegrant on drug release profile from

tablets

Dissolution profile of control tablet formulation that is formulation without addition of any superdisintegrant (M10) showed 100% drug release within 8 min. Three different levels of superdisintegrant concentrations; 3 %w/w, 4 %w/w and 5 %w/w were used to assess the effect of their increasing concentrations on release profiles of drug from tablets (Fig. 5 to 10). The dissolution rate was found to increase linearly with increasing concentrations of superdisintegrant. This was marked by decreased disintegration time values for tablet formulations containing higher proportions of superdisintegrants except for tablet containing 5 % Primojel. Formulations M1, M2, M3, which contained increasing concentrations of Polyplasdone from 3%w/w to 5%w/w, have recorded 100% drug release within 5 min., 4 min. and 2 min. respectively. Formulations M4, M5, M6, which contained increasing concentrations of Ac-Di-Sol from 3 %w/w to 5 %w/w, have recorded 100% drug release within 5 min., 4 min. and 3 min. respectively. Formulations M7, M8, M9 contained increasing concentrations of Primojel i.e. 3 %w/w, 4 %w/w and 5 %w/w respectively. All of them have recorded 100% drug release within 5 min. But T90 values indicated that with the increase in concentration of Primojel from 3 %w/w to 4 %w/w, the rate of dissolution of tablets was slightly enhanced. However further increase in concentration to 5 %w/w did not improve the dissolution rate but infact retarded it. This was probably due to formation of the viscous plugs by Primojel particles. The relative efficiency of different superdisintegrants to improve the dissolution rate of tablets was in order, Polyplasdone >Ac Di Sol > Primojel > Control CONCLUSIONS

1. Both HPβCD and βCD formed 1:1 complexes with Ziprasidone Hydrochloride.


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2. Inclusion complexes of drug with HPβCD exhibited more saturation solubility.

3. Inclusion complexes of Ziprasidone Hydrochloride prepared by different methods showed enhancement in its solubility in the following order,

4. Microwave irradiation method > Kneading method > Coprecipitation method > Physical mixture

5. Results of IR analysis, X- ray diffraction and differential scanning calorimetry suggested formation of inclusion complexes of drug with HPβCD and βCD

6. The inclusion complexes of drug prepared with HPβCD by microwave irradiation method showed highest solubility (246 µg/ ml) and fastest dissolution profile (100% drug release in 5 min.). So it was decided to use this complex for formulation of Ziprasidone Hydrochloride fast disintegrating/ dissolving tablets.

7. Disintegration time decreased with the increase in concentration of superdisintegrants from 3% w/w to 5%w/w except for Primojel where slight decrease in the disintegration time was observed when its concentration was increased from 4% w/w to 5% w/w.

8. For all the formulations in vivo disintegration time values were more than in vitro values.

9. The relative efficiency of these different superdisintegrant to improve the disintegration and dissolution rate of tablets was in the order,

10. Polyplasdone > Ac Di Sol > Primojel. However further in vivo studies are needed to correlate the effect of increasing solubility of Ziprasidone Hydrochloride with its bioavailability.

REFERENCES

1. Seager H. Drug delivery product and Zydis fast dissolving dosage forms. J Pharm and Pharmacol. 1998; 50: 375-382.

2. Indurwade NH, Rajyaguru TH, Nakhat PD. Novel approach- Fast dissolving tablets. Indian Drugs. 2002; 39: 405- 409.

3. Kuchekar BS, Badhan AC, Mahajan HS. Mouth dissolving tablet: A novel drug delivery system. Pharma Times. 2003; 35: 7- 14.

4. Chue P, Jones R, Taylor C. Can J Psychiatry. 2002; 47: 771-774.

5. Chue P, Welch R, Binder C. Can J Psychiatry. 2004; 49: 701-703.

6. Loftsson T, Brewster ME, Masson M. Role of cyclodextrins in improving oral drug delivery. Am J Drug Deliv. 2004; 2: 1-15.

7. Suresh S, Shivkumar HN, Kirankumar G. Effect of β-cyclodextrin complexation on solubility and dissolution rate of carbamazepine from tablet. Indian J Pharm Sci. 2006; 68: 301-307.

8. Loftsson T, Brewster ME. Pharmaceutical application of cyclodextrin in drug solubilization and stabilization. J Pharm Sci. 1996; 85: 1017-1025.

9. Wen X, Tan F, Jing Z, Liu Z. Preparation and study of the 1: 2 inclusion complex of Carvedilol with β-cyclodextrin. J Pharm Biomed Anal. 2004; 34: 517-523.

10. Higuchi T, Connors KA. Phase solubility techniques. Adv Anal Chem Instr. 1965; 4: 117- 212.

11. Arias MJ, Moyano JR, Munoz P, Gines JM, Justo

A. Study of Omeprazole-γ- cyclodextrin complexation in the solid state. Drug Dev Ind Pharm. 2000; 26: 253-259.

12. Bi YX, Sunada H, Yonezawa Y. Evaluation of rapidly disintegrating tablets prepared by direct compression method. Drug Dev Ind Pharm. 1999; 25: 571-581.

13. Bi YX, Yonezawa Y, Sunada H. Rapidly disintegrating tablets prepared by wet compression method: mechanism and optimization. J Pharm Sci. 1999; 88: 1004-1010.

14. Koizumi K, Watanabe Y, Matsumoto M. Int J Pharm. 1997; 152: 127-131.


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Synthesis and Antimicrobial Evaluation of Some New Pyrimidine

Derivatives Shashikanth R. Pattan*1, A.B. Khade2, M.A. Khade2, J.S. Pattan3, S.G. Jadhav2,

N.S.Dighe1, S.A. Nirmal1, S.V. Hiremath4, A.N. Merekar4 1 Dept. of Medicinal Chemistry, Pravara College of Pharmaceutical Sciences, Loni, M.S.

2.P.S.P.S IIP, Sadavati, (Devrukh), Dist. Ratnagiri, 415 804 M.S 3 PVP Science College, Loni, 413 736 MS

4Dept. of Pharmacology, JNMC, Belgaum, 590 010 Karnataka

Author for Correspondence : [email protected]

`

ABSTRACT:

A new series of pyrimidine derivatives have been synthesised by reacting corresponding chalcones with

guanidine carbonate, followed by condensing the active hydrogen atom of 2-amino group of 4-(4-substituted-

phenyl)-6-(furan-2-yl)-2-amino-pyrimidine with isonicotinic acid hydrazine/ p-amino salicylic acid/ 5-amino

tetrazole under microwave irradiation. The structures of these compounds were established by means of IR, 1H-

NMR, and elemental analyses, The title compounds were also evaluated for antibacterial and antifungal

activities. Compound B1, B2, B3, B4, B6, B8, B9 and B10 have shown maximum antibacterial activity against

Norfloxacin at 100mcg/mL, whereas compounds B1, B3, B4, B8, B9, and B10 have shown maximum antifungal

activity against Griseofulvin at 100mcg/mL.

Keywords: Pyrimidine, Antibacterial, Antifungal, and Microwave Assisted Organic Synthesis (MAOS).

INTRODUCTION:

Appreciable number of six membered heterocycles containing nitrogen atom, have turned out to be potential chemotherapeutic and pharmacotherapeutic agents. Various useful synthetic analogs with improved therapeutic properties can be obtained from single lead compound by structural modification. The same principle is applicable to the group of pyrimidines. Many classes of chemotherapeutic agents containing pyrimidine nucleus are in clinical use such as antibacterial agents (sulfadiazines, sulfamerazine & sulpfamethazine), anticancer (5-flurouracil and ftorafur), antiviral agents (iodoxuridine, trifluridine and zidovudine), antifungal (flucytosine) and antimalarial agents. (pyrimethamine)1 Nitrogen containing heterocycles such as pyrimidine is a promising structural moiety for drug designing. Pyrimidine based heterocycles are of interest as potential bioactive molecules and exhibit antimicrobial, antiviral, antiplatelet, antithrombotic,

antihypertensive, antipyretic, anticancer, anticonvulsant, antiallergic, hypoglycaemic, vasodilator, β-adrenoceptro antagonist and also act as enzyme inhibitors1-3. Inspired from these facts, we have attempted to synthesise some pyrimidines derivatives and evaluated for their antibacterial and antifungal activities. MATERIAL AND METHODS

Antimicrobial activity4

The antimicrobial activities of the synthesised compounds were determined by cup-plate method. The organisms selected for antimicrobial activity were Staphylococcus aureus and Escherichia coli. Similarly the antifungal activities were evaluated by using Aspergillus niger and Candida albicans. The concentrations of sample compounds were 100 mcg/mL. Norfloxacin and Griseofulvin were used as standard drug for antibacterial and antifungal activity respectively. Control test with solvents were performed for every assay but showed no inhibition of microbial growth. Indian Journal of Pharmaceutical Education & Research

Received on 9/11/2007 ; Modified on 17/7/2008 Accepted on 11/2/2009 © APTI All rights reserved

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EXPERIMENTAL:

Melting points were determined by open capillary method and are uncorrected. IR(KBr) spectra were recorded on Thermo Nicolet IR 200 spectrophotometer. The 1H-NMR spectra were recorded on multinuclear FT-NMR Spectrometer model Avance-II (Bruker), DMSO-d6 as solvent. The instrument is equipped with a cryomagnet of field strength 9.4 T. Its 1H frequency is 400 MHz. The purity of the compounds was determined on Pre-coated TLC using suitable solvent system. The reactions are carried out in an ONIDA domestic microwave oven. Power consumption: 220V~50Hz, 1350W (Microwave), 1050W (Grill), 2450 MHz (Operational frequency). Preparation of 1-(4-chloro-phenyl)-3-(furan-2-yl)

propen-1-one chalcones (I),5

A mixture of 22 gm of NaOH, 200 mL of water and 100 gm of (122.5 mL) of rectified spirit was taken in a 500 mL flask. The flask was immersed in an ice bath, provided with mechanical stirrer and to this 4 substituted (Cl, CH3, Br, and OCH3) actetophenone was poured (0.43 mol). The mixture was stirred, and then 46 gm (44 mL, 0.43 mol) of pure 2-furaldehyde was added. The mixture temperature was kept at about 25 °C (the limits are 15-30 °C) and stirred vigorously with mechanical stirrer until the mixture is so thick that stirring is no longer effective (2 to 3 hrs.). Then the mechanical stirrer was removed and left the reaction mixture in an ice chest or refrigerator overnight. The product was filtered with suction on a buchner funnel and washed with cold water until the washings are neutral to litmus and then with 20 mL of ice-cold rectified spirit. The crude chalcone was dried in the air and recrystallized from (5 mL per gm, warm 50 °C) rectified spirit. Yield 82%, m.p. 54-57 °C. Preparation of 4-(4-chloro-phenyl)-6-furan-2-yl-

pyrimidine-2-ylamine (II) 1

A mixture of 1-(4-chloro, bromo, methyl, methoxy-phenyl)-3-(furan-2-yl) propen-1-one chalcones I (0.01 mol) and guanidine carbonate (0.04 mol) was heated under reflux in (1 mL) ethanol for 8 hours. The contents were evaporated to dryness and the product so obtained was washed with water repeatedly and then recrystallized from ethanol. Yield 58%, m.p. 192-194 °C.

Synthesis of isonicotinic acid N’-{[4-(4-chloro-

phenyl)-6-furan-2-yl-pyrimidin-2-ylamino]-methyl}-

hydrazide (B1)

A mixture of 4-(4 chloro, bromo, methyl, methoxy -phenyl)-6-furan-2-yl-pyrimidine-2-ylamine II (0.01 mol), isonicotinic acid hydrazide (INH) (1.37 gm, 0.01 mol), formaldehyde (0.5 mL), ethanol (1 mL), NaHCO3 (1 mL of 20 %) were taken in a mortar and made a homogenous paste using a pestle. The paste was transferred to a beaker and exposed to microwave irradiation for 5 min, at intervals of 30 seconds. After the completion of the reaction (By running pre-coated TLC), ice-cold water was added to the reaction mixture and precipitated solid was separated by filtration and recrystallized from ethanol to get isonicotinic acid N’-{[4-(4- chloro, bromo, methyl, methoxy -phenyl)-6-furan-2-yl-pyrimidin-2-ylamino]-methyl}-hydrazide

(B1). Yield 55%, m.p. 154-157 °C. The compounds B2-

B12 were synthesized following a similar procedure. The physical data of the synthesised compounds are shown in Table 1. RESULT AND DISCUSSION:

The Antibacterial activity was carried out by cup-plate method against Staphylococcus aureus and Escherichia

coli. The compounds B1, B2, B3, B4, B6, B8, B9 and B10

have shown significant antibacterial activity when compared against Norfloxacin at 100mcg/mL as standard drug The Antifungal activity was also carried out by cup-plate method against A. niger and C.

albicans. The compounds B1, B3, B4, B8, B9, and B10

have shown promising antifungal activity against Griseofulvin at 100mcg/mL as standard drug. The furfuraldehyde was reacted with substituted acetophenones to obtain chalcones further they were refluxed for 8hrs with guanidine carbonate and alcohol to get 4(-4substituted)-phenyl 6-furan-2yl- pyrimidine-2yl amine. The various 12 derivatives were obtained by mannich’s reaction. Isonicotinic acid hydrazine, p-amino salicylic acid and 5-amino tetrazole were incorporated with the pyrimidine. The compounds have shown promising antibacterial and antifungal activities with suitable molecular modifications the compounds may show the better activities.


310

O CHO

2-furaldehyde

RCH3CO+

Substituted acetophenones

R = Cl, Br, OCH3, CH

3

NaOH

C2H

5OH O C

HCH

C

O

R

Chalcones Ref:5

I

Reflux for 8 HrsGuanidine carbonateC

2H

5OH

O

N

NH

NH2

R

II

MWI 5 MinPower 40%

HCHOC

2H

5OH

NaHCO3R'

20 %

Compd. R R'

B1

B2

B4

B3

B5

B6

B7

B8

B9

B10

Cl

Br

OMe

CH3

Cl

Br

OMe

CH3

Cl

Br

OMe

CH3

B11

B12

N

O

NH

NH

NH

COOH

OH

NN

NN

NH

Ref:1

O

N

NH

NH

R

CH

2

R'

(B1-B

12)

Scheme

Table No: 1 : Physical data of synthesised compounds

Compd. Mol. Formula Mol. Wt. m.p. ° C

Yield %

Elemental analyses Calcd. (Found)

C H N

B1 C21H17ClN6O2 420 154-57 55 59.93

(59.72) 4.07

(4.10) 19.97

(19.57) B2 C21H17BrN6O2 465 165-68 62 54.21 3.68 18.06

B3 C22H20N6O3 416 164-66 68 63.45

(63.62) 4.84

(4.50) 20.18

(20.26)

B4 C22H20N6O2 400 148-50 62 65.99 (65.88)

5.03 (5.01)

20.99 (20.85)

B5 C22H17ClN4O4 436 162-65 63 60.49 3.92 12.83 B6 C22H17BrN4O4 481 178-81 60 54.90 3.56 11.64

B7 C23H20N4O5 432 165-67 58 63.88

(63.75) 4.66

(4.45) 12.96

(12.75) B8 C23H20N4O4 416 155-57 59 66.34 4.84 13.45

B9 C16H15ClN5O 370 161-63 56 51.83 4.08 30.22

B10 C16H15BrN8O 414 171-73 63 46.28 3.64 26.98

B11 C17H18N8O2 366 153-55 60 55.73

(55.65) 4.95

(5.00) 30.58

(30.28)

B12 C17H18N8O 350 173-75 58 58.27

(58.35) 5.18

(5.35) 31.98

(31.80) The CHN analysis of the compounds synthesised is within the limits of permissible errors. (± 0.04) The proto types

of compounds were sent for CHN analysis.


311

Table No:2 : Infra Red/

1H-NMR spectral study of the synthesised compounds.

Compd. IR (cm-1) 1H-NMR (δ, ppm)

B1 3383 (N-H Str.), 2990 (C-H Ar. str.), 2850 (C-H str. ), 1667 (C=O Amide str.), 1592 (C=N str.), 879 (C-H Ar. def.)

---

B2 3384 (N-H str.), 1680(C=O Acid str.), 1580 (C=N str.), 1432 (O-H def.), 1167 (C-O str.), 913 (C-H Ar. def.)

---

B3 3383 (N-H str.), 2990 (C-H Ar. str.), 2850 (C-H str.), 1667 (C=O Amide str.), 1592 (C=N str.), 1254 & 1170 (C-O str.), 879 (C-H Ar. def.)

7.44-7.80 (m, 12 H, Ar-H), 8.40 (3H, NH), 3.89 (s, 3H, OCH3), 3.79 (s, 2H, CH2)

B4 3437 (N-H str.), 2926 (C-H Ar. str.), 2858 (C-H str.), 1663 (C=O Amide str.), 1550 (C=N str.), 1165 (C-O str.), 845 (C-H Ar. def.)

---

B5 3394 (N-H str.), 2840 (C-H Alkane.str.), 1669 (C=O Acid. str.), 1578 (C=N str.), 1498 (O-H def.), 1174 (C-O str.), 870 & 831 (C-H Ar. def.)

---

B6 3387 (N-H str.), 2811 (C-H Alkane. str.), 1666 (C=O Acid str.), 1557 (C=N str.), 1405 (O-H def.), 1173 (C-O str.), 885 (C-H Ar. def.)

7.23-7.66 (m, 11 H, Ar-H), 6.12 s, H, NH), 4.94 (s, 2H, CH2)

B7 3383 (N-H str.), 2990 (C-H Ar. str.), 2850 (C-H str.), 1667 (C=O Amide str.), 1592 (C=N str.), 879 (C-H Ar. def.)

---

B8 3384 (N-H str.), 2800 (C-H Alkane str.), 1680 (C=O Acid str.), 1580 (C=N str.), 1432 (O-H def.), 1167 (C-O str.), 913 & 880 (C-H Ar. def.)

---

B9 3980 (C-H Ar. str.), 3409 (N-H str.), 1676 (C=C Ar. str.), 1581 (C=N str.), 1490 (C-H Alkane str.), 1148 (C-O str.), 819 (C-H Ar. def.)

7.12-7.64 (m, 8 H, Ar-H ), 4.98 (s, 2 H, CH2), 4.50 (s, 1 H, NH)

B10 3392 (N-H str.), 2804 (C-H Ar. str.), 1674 (C=C Ar. str.), 1579 (C=N str.), 1486 (C-H Alkane str.), 1169 (C-O str.), 885 (C-H Ar. def.)

---

B11 3980 (C-H Ar. str.), 3409 (N-H str.), 1676 (C=C Ar. str.), 1581 (C=N str.), 1490 (C-H Alkane str.), 1148 (C-O str.), 819 (C-H Ar. def.)

---

B12 3392 (N-H str.), 2804 (C-H Ar. str.), 1674 (C=C Ar. str.), 1579 (C=N str.), 1486 (C-H Alkane str.), 1169 (C-O str.), 885 (C-H Ar. def.)

---

ACKNOWLEDGMENTS

The authors thank Shri. Balasaheb Vikhe Patil, Honourable Chairman, M.P. PRES and Dr. S. Waluni, Secretary, PRES, Loni for their kind encouragement and support REFERENCES:

1. Ozair A, Mohd I, Khan SA. Synthesis and biological activity of some pyrimidine

derivatives. Indian J Heterocyclic Chem. 2005;14:293-6.

2. Ramesh D, Chandrashenhar, Vaidya VP. Synthesis, analgesic, antiinflammatory and antimicrobial activities of naphtho [2,1-b] furan incorporated substituted pyrimidines. Indian J Heterocyclic Chem. 2006; 15:319-22.

3. Fathalla OA, Radwan HH, Awad SM, Mohamed MS. Synthesis and biological


312

evaluation of new pyrimidine derivatives. Indian Journal of Chemistry 2006; 45B, 980-5.

4. AL Barry. The antimicrobial susceptibility test: principle & practices, edited by IIIus Leu & Febiger (Philadelphia, Pa. USA), Biol. Abstr., 64 (1976), 25783.

5. Furniss BS, Hannaford AJ, Smith PWG, Patchel AR. Vogel’s Textbook of Practical Organic Chemistry. 5th ed., Singapore: Pearson education (Singapore) Pvt. Ltd; 1996.

© APTI, 2009

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