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Vol. 41(4), Oct – Dec, 2007

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.

[email protected]

ASSOCIATE EDITORS

EDITORIAL OFFICE

INDIAN JOURNAL OF PHARMACEUTICAL EDUCATION AND RESEARCH

The Official Publication of Association of Pharmaceutical Teachers of India

H.Q.: Al-Ameen College of Pharmacy,

Opp. Lalbagh Main Gate, Hosur Road, Bangalore 560 027, INDIA

Mobile: 91-9448207428 | 91-9242898028 | 91-9845655732 | 91-9880423041

| 91-9448445612 Fax: 080-22225834; 080-22297368

email: [email protected] | Website: www.ijper.org

Dr. Srinivasa Murthy

[email protected]

Dr. Kulkarni P.K.

[email protected]

Dr. Mallikarjuna Rao C.

[email protected]

Dr. Mueen Ahmed K. K.

[email protected]

ijper Indian Journal of Pharmaceutical

Education & Research

Vol. 41(4), Oct – Dec, 2007

EDITORIAL ADVISORY BOARD

Dr. Betgeri G.V., USA.

Dr. Mrs.Claire Anderson, UK.

Mr. Frank May, USA.

Dr. Gaud R.S., Mumbai.

Dr. Goyal R.K., Ahmedabad.

Dr. Harkishan Singh, Chandigarh.

Dr. Hukkeri V.I., Bangalore.

Dr. Jagdeesh G., USA.

Dr. Katare O.P., Chandigarh.

Dr. Khar R.K., New Delhi.

Dr. Madan A. K., Rohtak.

Dr. Madhusudhan Rao Y., Warangal.

Dr. Manavalan R., Annamalai Nagar.

Dr. Miglani B.D., New Delhi.

Dr. Murthy R.S.R., Vadodara.

Dr. Nagavi B.G., Dubai.

Dr. Pulok K Mukherjee, Kolkata.

Dr. Rao M.N.A., Hyderabad.

Dr. Ravi T.K., Coimbatore.

Prof. Shivananda B.G., Bangalore.

Dr. Shivakumar H.G., Mysore

Dr. Subrahmanyam C.V.S, Hyderabad.

Dr. Suresh B., Ooty.

Dr. Tipnis H.P., Mumbai.

Dr. Udupa N., Manipal.

Dr. Vyas S.P., Sagar.

Publication Committee

• Pharmaceutics - Dr. Paradkar A.R., Dr. Sarasija Suresh,

Dr. Vavia P.R.

• Pharmaceutical Chemistry and

Analysis

- Dr. Gopal Krishna Rao, Dr. Raghurama Rao A.

Dr. Valliappan K.

• Pharmacology - Dr. Krishna D.R., Dr. Kshama Devi,

Dr. Sreenivasan B.P.

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

Dr. Swati S.Patil

• Pharmacy Practice - Dr. Nagappa A.N., Dr. Rajendran S.D,

Dr. Shobha Rani R.H.

• Pharmaceutical Education - Dr. Raman Dang, Dr. Unnikrishnan M.K.,

Dr. Bhise S.B.

• Pharmaceutical Marketing - Dr. Burande M.D., Dr. Gayathri Devi S.,

Dr. Kusum Devi V.

Note : The Editor does not claim any responsibility, liability for statements made and opinions expressed by

authors.

INDIAN JOURNAL OF PHARMACEUTICAL EDUCATION AND RESEARCH

The Official Publication of Association of Pharmaceutical Teachers of India

H.Q.: Al-Ameen College of Pharmacy

Opp. Lalbagh Main Gate, Hosur Main Road, Bangalore - 560027 INDIA

Mobile : 91-9448207428 | 91-9242898028 | 91-9845655732 | 91-9880423041 | 91-9448445612

Fax: 080-22225834; 080-22297368; email: [email protected] | Website : www.ijper.org



Vol. 41 (4), Oct– Dec, 2007

CONTENTS

Editorial

Review Articles

• Huntington’s Disease: A Review

Puneet Kumar, PS Naidu, SSV Padi and Anil Kumar...........................................................................287-294

• Gastric Floating Drug Delivery Systems: A Review

Gangadharappa.H.V., Pramod Kumar T.M and Shiva Kumar H.G…………………..........................295-305

• In vitro-In vivo Correlation: A Ground Discussion

Kalaskar S. G., Yadav A. V and Patil V. B…………………………………………..................................306-318

Research Article

• Formulation and in vitro evaluation of taste masked orodispersible dosage form of Levocetirizine

dihydrochloride

Chaudhari P.D, Chaudhari S.P., Lanke S.D. and Patel Nakul.............................................................319-328

• A study on the effect of different polymers on frusemide loaded calcium alginate micropellets prepared by

ionotropic gelation technique

Ghosh Amitava, Nath L.K, Dey B.K and Roy Partha............................................................................329-336

• Phytochemical investigation and Immunomodulator activity of Amaranthus spinosus linn.

Tatiya A.U., Surana S.J., Khope S.D., Gokhale S.B and Sutar M.P………………..…..........................337-341

• Pharmacodynamic drug interaction of mexiletine with tolbutamide in rats

S. Satyanarayana, M. Nitin and K. Prasad...........................................................................................342-346

• Validated, Reversed Phase High Performance Liquid Chromatography Method for the Estimation of

Etoposide in Bulk and Formulations

Movva Snehalatha, Bende Girish, Kolachina Venugopal and Ranendra N. Saha...............................347-352

• Spectrophotometric Estimation of Bisoprolol Fumarate in Bulk Drug and Tablets

Akmar Sandip, Paramane Sonali, Kothapalli Lata, Thomas Asha, Jangam Sumitra, Mohite Mukesh and

Deshpande Avinash...............................................................................................................................353-357

• Preparation and in Vitro Evaluation of Mucoadhesive Microcapsules of Atenolol

Swamy P.V., Hada Amit, Shirsand S.B., Hiremath S.N and Raju S.A...................................................358-364

• Evaluation of Analgesic activity of root tuber of Curculigo orchioides Gaertn.

V. Madhavan, Joshi Richa, Murali Anita and S.N. Yoganarasimhan...................................................365-368

• Effect of Eclipta alba Linn on learning and memory in rats

G.P.Rajani and KVSRG Prasad............................................................................................................369-372

• Application of Hibiscus Leaves Mucilage as Suspending Agent

Edwin Jarald, Edwin Sheeja, Dosi Shweta, Amal Raj and Gupta Smita..............................................373-375

• The New Patent Regime – Implications for Indian Pharma Industry

K.Madhavi.............................................................................................................................................376-382



• Marketing and Advertising of Prescription and Over the Counter (OTC) Products – Ethical Issues

Manthan D.Janodia and Udupa N……………………………………………………….............................383-387

• Development and Evaluation of Propranolol Hydrochloride Transdermal Patches by using Hydrophilic and

Hydrophobic Polymer

Dey B.K, Nath L.K, Mohanti B and Bhowmik B.B…………………………………….............................388-393

• Free Radical Scavenging Activity of Ficus Racemosa roots

Surendra Kumar Sharma and Vivek Kumar Gupta..............................................................................394-396

• INSTRUCTIONS TO AUTHORS -2008

Editorial

The APTI 12th

Annual National Convention at Chandigarh was an opportunity for the Pharmacy teachers

and educationalists to deliberate upon many issues facing them in the wake of globalization. The

emergence of a globalized world underscoring a framework of competition, and coupled with the pressures

of an exploding knowledge base has given birth to new challenges. It was time for introspection and gave

an occasion to think of the current developments in technological innovations, manufacturing practices and

health trends. Are the recent trends in pharmaceutical sciences a part of our curriculum? If the

pharmaceutical education imparted to our learners is not revised periodically with the change in time, the

consequences are going to be mind boggling.

Concerns were raised on disharmony of curriculum content on global levels. Disparity in the

pharmaceutical knowledge horizon pertaining to biotechnology, genomics, disaster management and

polymer sciences have started creating vacuum in the knowledge base of our students. They are not in a

position to compete with their counterparts at the global level. The challenges are complicated further with

the amendments to Patent laws. More focus now needs to be laid on new drug discovery and R&D

programmes. Have we generated the platforms for our students to work in these areas? Do we have

adequate facilities to promote world-class research, train world-class scientists, regulatory officials and

managers for the drug sector? How many universities and departments in the country have opportunities to

collaborate with foreign research/ teaching institutes and industries? I am sure the situation is not

encouraging.

It is also quite alarming to know further that the number of pharmacy colleges in the country is growing at

an alarming rate. Are these institutions of learning equipped with proper infrastructure? Are we in a

position to supply quality teaching staff for these institutions? Are the teachers appointed in such large

numbers competent enough to facilitate proper training to the students? The knowledge dissemination

consistent with the country’s growth and opportunities should be the top most priority of the teachers.

Nurturing, retaining and hiring such excellent faculty is the back bone for an institution.

The paradigm shift of achieving the objective to produce a ‘seven star pharmacist’ has now made a

beginning in our country after the Vancouver declaration in 1997 (preparing the future pharmacist) with

the introduction of Pharm. D programme. In reality, time is now ripe enough for our graduate course

curriculum to adorn a new look consistent with the global needs.

The new generation pharmacy teachers should now give the best of their time and energy to critically

identify the issues which need to be addressed, in order to enhance effectiveness and efficiency in student

learning. Teachers should take stock of the current curriculum, identify demand-supply gaps in terms of

skill development like information, communication and technology (ICT) and address these issues on a war

footing. It is necessary that we should respond to changing realities. Responsibilities need to be redefined;

teachers become more empowered and should equip themselves to face emerging challenges. As the

country is moving closer towards becoming a knowledge centre, quality education has become determinate

for the well being of one and all. Let us all come together and make India a better place to groom the next

generation pharma professionals. In this era of fiery competitiveness, the catch word is – PERFORM or

PERISH.

Dr. Sanjay Pai P.N. Editor-in-Chief

Indian J.Pharm. Educ. Res. 41(4), Oct – Dec, 2007

287

Huntington’s disease: A Review Puneet Kumar, PS Naidu, SSV Padi and Anil Kumar*

Pharmacology Division, University Institute of Pharmaceutical Sciences,

Panjab University, Chandigarh-160014, India.

Email: [email protected]

Abstract

Huntington's disease (HD), inherited genetic disorder is characterized by abnormal body movements called

chorea, and cognative dysfunction. George Huntington, Ohio physician described it precisely in 1872. HD is a

dominantly inherited disorder characterized by a progressive neurodegeneration of the striatum that also

involves other regions, primarily the cerebral cortex. The mutation responsible for this fatal disease is an

abnormally expanded and unstable CAG repeat within the coding region of the gene encoding huntingtin. The

pathogenic mechanisms by which mutant huntingtin cause neuronal dysfunction and cell death remain

uncertain. HD is considered as important disease, embodying many of the major themes in modern

neuroscience, including molecular genetics, selective neuronal vulnerability, excitotoxicity, mitochondrial

dysfunction, apoptosis, and transcriptional disregulation. A number of recent reports concluded that oxidative

stress plays a key role in the pathogenesis of HD. Although there is no treatment to fully stop the progression of

the disease, there are treatments available to help control the chorea. The present review deals with the

pathophysiology and current drug treatment options and future therapeutic interventions for HD. Present

review focuses on the animal models (behavioural and genetic) emplyoed for unraveling pathogenetic

mechanisms and identification of novel drug targets.

Key words- Excitotoxicity, Gait abnormalities, Huntington’s disease, Memory, Oxidative stress,

INTRODUCTION

Huntington's disease is a genetic, progressive,

neurodegenerative disorder characterized by the gradual

development of involuntary muscle movements

affecting the hands, feet, face, and trunk and

progressive deterioration of cognitive processes and

memory (dementia). Neurologic movement

abnormalities may include uncontrolled, irregular,

rapid, jerky movements (chorea) and athetosis, a

condition characterized by relatively slow, writhing

involuntary movements 1-2 . Dementia is typically

associated with progressive disorientation and

confusion, personality disintegration, impairment of

memory control, restlessness and agitation. In

individuals with the disorder, disease duration may

range from approximately 10 years up to 25 years or

more. Life-threatening complications may result from

pneumonia or other infections, injuries related to falls,

or other associated developments 3-4.

HISTORY

HD has rich historical literature stretching back well

over a century and involves some of the most

prominent figures in medicine and neurology. The

description by George Huntington in 1872 of the

disease that has subsequently borne his name is one of

the most remarkable in the history of medicine 5, 6.

Until recently the history of research on HD has been

one of gradual progress rather than of sudden leaps.

Initially development in this area arose from the illness

of Woody Guthrie, the American folk singer, who

developed HD symptoms around 1952 and died in 1967

at the age of 55. His widow Marjorie devoted the later

part of her life for promoting all aspects of HD. Now a

day’s number of HD research groups is working on this

disease 7.

EPIDEMIOLOGY

Huntington’s disease is currently found in many

different countries and ethnic groups around the world.

There are varying rates of prevalence in different racial

groups 2. HD has a worldwide prevalence of five to

APTI ijper

Indian Journal of Pharmaceutical Education & Research

Received on 06/11/06; Modified on 13/3/2007

Accepted on 4/6/2007 © APTI All rights reserved


288

eight per 100,000 people with no gender preponderance. The highest frequencies of HD are

found in Europe and countries of European origin. The

lowest frequencies are documented in Africa, China,

Japan, and Finland. In the USA Estimates of the

prevalence of HD range from 4.1-8.4 per 100,000

people. In the United States, it is estimated that 25000

individuals have HD with another 125,000 individuals

at risk 8.

In India: A recent study on the distribution of C-A-G

repeats in the normal population suggests a higher

prevalence of HD in India closer to that seen in Western

Europe. Based on the results, haplotype analysis

suggested the presence of a founder mutation in a

subset of families and provide evidences for multiple

and geographically distinct origins for HD mutation in

India 9. One of the studies conducted on 124 (94 male

and 30 female) elderly patients (aged more than 60

years) in a teaching hospital in India reported that there

were 2.4% cases of HD, Parkinson's disease in India9.

NEUROPSYCHOLOGICAL AND

NEUROPSYCHIATRIC ASPECTS OF HD

HD, an inherited neurodegenerative disease, damages

specific areas of the brain resulting in movement

difficulties as well as cognitive and behavioral changes.

The cognitive changes in HD have traditionally been

referred to as dementia. People with HD have specific

and characteristic cognitive difficulties, with other

aspects of cognitive function remaining well preserved.

Behavioral changes are a characteristic feature of HD

and are often the most distressing aspect of the

condition for individuals and families dealing with

HD8.

Behavioral changes associated with HD

Psychomotor function - Early motor signs of HD

typically include the gradual onset of clumsiness,

balance trouble, tremor and brief random, fidgeting

movements. The primary involuntary movement

abnormality and often the earliest symptom, is chorea

or choreoathetosis, continuous and irregular writhing

and jerking movements 10. Many HD patients develop a

distinctive manner of walking (gait) that may be

unsteady, disjoined, or lurching as disease progresses 11,

12.

Frustration, Irritability, Aggression & Anxiety-

People suffering from HD may remain even-tempered;

others may lose the ability to control their emotions.

Emotional volatility may evident in increased

irritability or episodes of explosiveness 10. These

individuals may become irritable, frustrated or

aggressive if demands are not met. Anxiety, a

behavioral symptom of HD, is characterized by

nervousness, restlessness, fidgeting, shallow breathing,

sweating, fear, and panic rapid heart rate 13. For

individuals with HD, continual life changes as HD

progresses can be a source of anxiety. Depression is

often dismissed as an understandable reaction being

diagnosed with HD14.

Altered Sexuality- A very common behavioral

symptom of HD is altered sexuality. Possible cause is

that the delicate balance of hormones in the brain is

disrupted by the progression of HD causing changes in

behaviors regulated by hormone levels. Most

commonly, people with HD suffer from a decreased sex

drive. Increased sex drive and inappropriate sexual

behavior are less common alterations of sexuality

resulting from HD 15

.

Cognitive changes in HD

The term “cognitive” refers to tasks of the brain that

involve knowing, thinking, remembering, organizing

and judging. Cognitive changes in the HD may be due

to the disruption of striatal –frontal circuits 16.

Memory and Visual spatial ability

An individual suffering from the cognitive symptoms of

HD may have memory difficulties. Several

investigators have shown that memory recall is

generally affected more than memory storage in HD 16.

It is important to note that the memory problems that

can occur in people with HD are different from the

memory difficulties that can occur in people with

Alzheimer’s disease (AD)17. Most commonly, the

individual suffering from cognitive symptoms of HD is

aware of his or her visual spatial impairment. Reading

difficulties may also be the result of visual spatial

impairment; however, the inability to maintain attention

may be a contributing factor as well 18.

NEUROPATHOLOGY OF HUNTINGTON’S

DISEASE

The specific symptoms and progression of HD can be

related to its pathology, which is characterized by the

loss of specific neuronal populations in many brain

regions. Motor dysfunction in HD results from the


289

disruption of basal ganglia-thalamocortical pathways

regulating movement control 19-20. The primary site of

neuronal loss and atrophy in HD brain is in the caudate-

putamen21.

Vulnerability in HD

The striatum is composed of a variety of medium to

large neurons that differ in their size and dendritic

profile as well as neurochemical content and output.

Severe loss of medium sized striatal neurons was seen

in the HD brain. They have large dendritic tree and use

GABA as their neurotransmitter 22. As these neurons

degenerate in HD, the neurochemical they contain,

including glutamic acid decarboxylase (GAD),

substance-P, enkephalin, calcineurin, calbindin,

adenosine receptors and dopamine receptors, also

decrease.

Number of theories has been presented, to determine

the exact events involved in the progression of cell

deaths caused by HD. One theory proposes that neurons

die in HD because of an over-accumulation of normal

excitatory chemicals involved in nerve impulses.

Excitatory neurotransmitters (mainly glutamate) are

normally present in the brain, but, if they are released in

excessive amounts or if brain cells are weak, these

excitatory chemicals can cause cell damage and become

chemicals known as “excitotoxins.” Studies show that

when glutamate is injected into the basal ganglion

region of brains of living rats, the rats exhibit symptoms

of HD23. This first theory had to be modified when

high levels of glutamate were not found in the brains of

all HD patients. The mitochondrial dysfunction plays a

role in pathogenesis of HD. The mitochondria of striatal

cells may be damaged with the onset of HD. Scientists

today believe that the damaged mitochondria of people

with HD make striatal cells unable to produce as much

energy as they need, which then makes the cells more

susceptible to normal levels of glutamate 24.

Another theory to explain the death of nerve cells

postulates that the cells actually kill themselves in

response to chemical changes caused by HD. HD

triggers the early death of neurons by accelerating a

normal process called apoptosis 25.3-Nitropropionic

acid and malonate also induce apoptotic profiles and

induce pro-apoptotic proteins 26.

To sum up, the neurobiological effects of HD appear to

be the result of a number of different changes that

ultimately go out of control. Many studies have shown

that neurodegeneration is not confined to the basal

ganglia but also occurs widely in cortical and other sub

cortical regions.

NEUROCHEMISTRY OF HUTINGTON’S

DISEASE

Neurochemical alterations in HD have long attention

from researchers. It is now clear that some of the

earliest pathological changes in HD are indeed

neurochemical. It is conceivable that these

neurochemical alterations not only produce the

characteristic clinical symptoms of HD but also

accelerate the process of cell death, and are thus

essential mediators of disease pathogenesis21

GENETICS OF HD

The disease gene for HD, huntingtin, was identified in

1993 and it encodes a large protein (348kDa) with a

polyglutamine stretch named huntingtin (Htt) 3, 35.

Genetic defect in HD is an expansion of an unstable

CAG repeats encoding polyglutamines at the 5’ end of

a huntingtin [also termed “interesting transcript 15”

(IT15)] gene on chromosome 4 2, 26. The biological

function of the huntingtin protein is still unknown; it is

known that the alteration of this protein ultimately

results in HD 23, 36.

INFLAMMATION AND HUNTINGTON’S

DISEASE

Studies of the HD brain indicate that long-term

inflammation plays a significant role in the progression

of HD. It is suggested that excitotoxic amino acids such

as glutamate induce a direct activation and proliferation

of cells involved in inflammation. Since glutamate

activity is also implicated in the progression of HD, it is

possible that the glutamate molecules in the HD brain

induce an inflammatory response 37. One of the first

steps in excitotoxic neuronal damage involves the

hyperstimulation of N-methyl-D-aspartate (NMDA)

receptors leading to a massive calcium influx that

activates, among other processes, the calcium

dependent phospholipase A2 (PLA2). Further, PLA2

cleaves membrane phospholipids to yield arachidonic

acid (AA), a free fatty acid, which is converted by

cyclooxygenases (COX) into prostaglandins (PGs).

The inflammatory response results in the activation of

various types of cells and the production of different

molecules that can lead to cell death 38. An example of


290

cells activated by the inflammatory response is the

microglia (a type of immune cell), which have been

found to be highly activated in the HD brain. Research

has shown that there is a marked increase in microglia

in the HD brain 37-38 . In the HD brain, an increase in

activated microglia is found along the vicinity of nerve

cells that contain neuronal inclusions (NIs) –

accumulation of the huntingtin protein. This finding

suggests that the huntingtin protein accumulation

influences the activation of reactive microglia. Nerve

cell injury due to excitotoxins such as glutamate also

induces long-term microglial activation in the brain 37-

38.

MANAGEMENT OF HD

Huntington’s disease is a devastating neurological

disorder without effective treatment. There is an urgent

need for developing effective therapies for HD.

TREATMENT OF CHOREA

Dopamine blocking or dopamine depleting

medications

Increased dopamine level plays a major role in the

pathogenesis of HD. On the basis of these reports

dopamine-depleting drug like Tetrabenzine was also

used for the treatment of chorea in clinical trial 40. But

due to lot of side effects the FDA did not approve this

drug.

Glutamate antagonism

Excitotoxicity is the major cause of death of neurons in

the HD. Increase in glutamate release activate the

NMDA receptors and increase the level of Ca2+ and

cause neurotoxicity. The drugs, which block the

NMDA receptors, may be useful to decrease the

symptoms of HD 41.

GABAergic modulation

GABA an inhibitory neurotransmitter is decreased in

the HD brain and cerebrospinal fluid. Indeed the GABA

mimetic drugs and GABA transminase inhibitors are

also be used in the clinical trial for the treatment of

HD42

Cannabinoids receptor agonists

In the brain the cannabinoids and their receptors behave

as neurotransmitters or neuromodulators in a variety of

processes, such as the regulation of motor behaviour,

cognition, learning, memory and antinociception. It is

also reported that the cannabinoid receptors are

destroyed in the basal ganglia 43. Therefore the

treatment with cannabinoids could be beneficial for

HD.

Antioxidants

One component of excitoxicity in HD is oxidative stress

and antioxidants may therefore have therapeutic utility.

A novel antioxidant, BN-82451 improved motor ability

and survival and ameliorated neurodegenration in R6/2

HD mice 35, 40.

DEVELOPMENT OF NOVEL THERAPEUTICS

FOR HD

Agents that inhibit mutant huntingtin aggregation and

Transglutaminase inhibitors

The huntingtin aggregates and inclusions play a major

role in the pathogenesis of HD. Inhibit mutant

huntingtin from aggregation would provide a way to

prevent the progression of the disease44. Congo red

showed protective effect on survival, weight loss and

motor function even after the onset of symptoms of HD

in R6/2 transgenic mice 35. Transglutaminase (TGase)

can use huntingtin as a substrate to cross-link huntingtin

molecules. TGase activity was found to have increased

in HD postmortem brains45. Cystamine is an inhibitor

of TGase and showed a small but significant

neuroprotective effect with improvement of motor

function, survival and loss of bodyweight.

Protease inhibitors

Recent findings showed that huntington could be

cleaved by proteases, including caspases, calpain, and

aspartyl protease. Caspase and calpain-mediated partial

cleavage of mutant huntingtin promotes huntingtin

aggregation and cellular toxicity, inhibitors of

huntingtin partial cleavage might have therapeutic

values. Caspase inhibitors, z-VAD-fmk and z-DEVD-

fmk, can prevent cleavage of huntingtin by caspases

and reduce cytotoxicity caused by expanded

polyglutamine tract 46. Caspase inhibitor minocycline

was able to inhibit huntingtin aggregation, retard

disease progress and prolong the lifespan of HD mice.

Protease inhibitors could reduce N-htt fragments and in

turn, prevent or delay disease progression 47.

Histone deacetylase (HDAC) inhibitors

Inhibitors of histone deacetylase (HDAC) can increase

gene transcription and have been examined as a

potential therapy in both HD Drosophila and transgenic

R6/2 HD mice. Suberoylanilide hydroxamic acid

(SAHA), a selective HDAC inhibitor, reduced

neurodegeneration in HD Drosophila 48.


291

Fig. 1. The basal ganglia of the human brain, showing the impact of HD on brain structure in this region. Note especially

that the brain of a person with HD has bigger openings due to the death of nerve cells in that region

Table.1 Worldwide prevalence of Huntington’s disease

Population Frequency of HD (cases per million people)

South Africa (blacks) 0.6 Japan 1-4 Hong Kong 3.7 Finland 6.0 Europe & countries of European descent 40-100 Northern Ireland 64 South Wales 76.1 Scotland (Grampian Region) 99.4 United States 100

Table.2 Comparison of Huntington’s disease and Alzheimer’s disease

Comparison of Huntington’s Disease and Alzheimer Disease

Ability Huntington’s Disease Alzheimer Disease

Speed of processing Slow, mostly accurate Slow, often inaccurate Speech output Slurred & slow, accurate Normal rate & clarity; often inaccurate Learning new information

Disorganized & slow, can learn Rapid forgetting; cannot store information

Free recall Impaired

• Cannot find the right words

• Cannot recognize with choices

• Benefits from clues

Impaired

• Cannot recall memories

• Cannot recognize with choices

• Does not benefit from clues Motor memory Impaired

• Cannot learn or recall motor memories

Impaired

• Can learn& retain motor memories

Table.3. Level of different neurotransmitters in the HD brain

S.No Neurotransmitter Level in HD brain

1. γ-Amino butyric acid (GABA) and GAD enzyme Decrease 21-29 2. Substance P Decrease 30- 31. 3. Enkephalin Decrease31 4. Dynorphin Decrease31 5. Acetylcholine Decrease32 6. Dopamine Decrease33

7. Glutamate Increase34


292

Other neuroprotective approaches

Gene therapy-

Intracellular antibodies (intrabodies) and RNA

interference (RNAi) are two potential methods that

could be used for gene therapy of HD. Mitochondria

dysfunction has been implicated in HD pathogenesis.

Therefore, compounds enhancing energy metabolism

have been evaluated for treatment of HD. Coenzyme

Q10 and creatine are neuroprotective, putatively via

enhancing cerebral energy metabolism 4, 21. Neural cell

transplantation is also under development for the

treatment of HD 60. Brain derived Neurotrophic factors:

Brain derived neurotropic factor (BDNF) expression is

reduced in the caudate and putamen of patients with

HD. That enhanced expression of neurotropic factors

may mitigate the effects of neurotoxins and thus be a

potential therapeutic strategy was explored in animal

and cell models 49-50.

CONCLUSION

The exact mechanisms underlying neuronal death in

Huntington's disease remain unknown. Over past 10

years, the leading models of neurodegeneration in the

disease have involved mitochondrial dysfunction and

subsequent excitotoxic injury, oxidative stress, and

apoptosis. Recent studies have lent support to these

models, but additional theories involving abnormalities

of protein metabolism and transcriptional dysregulation

have emerged as well. Since the identification of the

Huntington's disease gene in 1993, there have been

great advances in the understanding of the molecular

biology and pathophysiology of the disorder. These

advances have suggested new therapeutic strategies

aimed at slowing progression or forestalling onset of

this devastating neurodegenerative disease. In

preparation for future clinical trials, clinical studies

have begun to provide more quantitative measures of

disease onset and progression. Recent progress in the

basic science and clinical realms raises hopes for

affective therapies in the near future.

REFERENCES

1. Bonelli RM, Hofmann P. A review of the treatment

options for Huntington's disease. Expert Opin

Pharmacother. 2004; 5: 767-776.

2. Kent A. Huntington’s disease. Nursing Standard.

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295

Gastric Floating Drug Delivery Systems: A Review Gangadharappa H. V, Pramod Kumar T. M and Shiva Kumar H. G.

Dept. of Pharmaceutics. JSS College of Pharmacy, Mysore, Karnataka, India

Abstract

In the last three decades various attempts have been made to develop a novel and efficient gastroretentive

dosage forms which can retain in the stomach for an extended period of time in a predetermined manner. This

can be achieved by improving scientific and technological advancement to over come physiological problems

like pH of the stomach, motility, gastric emptying time by altering physiological and formulation variables.

Many approaches are utilized in the development of gastric retention drug delivery systems viz.

hydrodynamically balanced systems, swelling, expanding, high density, super porous hydrogels, bioadhesive,

modified shapes etc. By utilizing one of the above techniques it is possible to deliver drugs, which have narrow

absorption window.

Key words: Gastric floating systems; effervescent, noneffervescent, hydrodynamically balanced system.

INTRODUCTION

The primary aim of oral controlled DDS is to achieve

better bioavailability and release of drug from the

system, which should be predictable and reproducible.

But this is difficult due to number of physiological

problems such as fluctuation in the gastric emptying

process, narrow absorption window and stability

problem in the intestine. This can be over come by

altering the physiological state and designing the

formulations, by which gastric emptying process can be

extended from few minutes to 12 hours. A drug can act

locally in the stomach in case of H. Pylori (tetracycline)

or in the proximal part of the intestine by prolonged

contact with absorbing area1,2. A prolonged gastric

retention increases bioavailability, decreases wastage of

drugs, increases solubility of drugs, which are less

soluble in alkaline pH e.g.verapamil3. It has been

suggested to prepare a suitable dosage forms for the

drugs that have narrow absorption window. These

dosage forms prolongs the gastric residence time

enabling an extended absorption phase for the local

treatment of drugs4. Floating drug delivery systems

provides better bioavailability for the drugs that are

unstable in intestinal or colonic environment5.

Gastric retention can be achieved by the mechanism of

mucoadhesion or bioadhesion systems6, expansion

systems7,8, high density systems9,10,11, magnetic

systems12,13,14, superporous hydrogels15,16, raft forming

systems17,18,19, low density systems20,21,22 and floating

ion exchange resins.23

Based on the mechanism of floatation, delivery systems

can be classified into two types

1. Effervescent floating drug delivery system

(EFDDS).

2. Non- effervescent floating drug delivery system.

This article reviews gastroretentive dosage forms;

technological developments and advantages of delivery

systems.

BASIC PHYSIOLOGY OF GASTROINTESTINAL

TRACT

The anatomy and physiology of GIT should be

understood, while developing floating drug delivery

systems. Factors affecting GI motility like, pH, nature

and volume of gastric secretion and gastric mucus24,25.

Anatomically stomach is mainly divided in to 3 parts:

fundus, body and antrum (pylorus). The proximal part

of the stomach is made up of fundus and body region,

which serves as reservoir of the undigested substances.

Where as distal region (antrum) is major site for mixing

motion and acting as a pump for gastric emptying26.

Gastric emptying occurs based on fed and fasted state

of the stomach. Saliva, mucus and debris are

APTI ijper





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commonly present in the fasted state of the stomach.

The fasted state is characterized by intra gastric series

of cyclic contractions or electrical events takes place,

which is known as interdigestive migrating myloelectric

complex or migrating myloelectrical cycle (MMC).

This activity occurs both through stomach and intestine

every 2-3 hours27, 28. Apparently MMC is further

divided into four consecutive phases as described by

Wilson and Washington29.

Phase I (basal phase): It is a quiescent period which

lasts from 30–60 minutes with rare contractions.

Phase II: It consists of intermittent action potential and

gradually increases in intensity and frequency as the

phase progress and lasts for about 40-60 minutes.

Phase III: This is a shorter period of intense, large

regular distal and proximal gastric contractions (4-5

contractions per minutes) lasting for about 4-6 minutes.

This cycle is also known as “house keeper wave “.

Since it sweeps undigested gastric contents from

stomach to intestine.

Phase IV: A brief transitional phase about 0-5 minutes,

which occurs between last part of phase III and

beginning of phase I.

After feeding this cycle leads to change in the pattern of

contractions, which may last for many minutes. This

frequent feeding of mixed meal may increase gastric

retention time30.

FACTORS AFFECTING GASTRIC RETENTION

There are many factors that affect gastric emptying of

an oral dosage forms, viz. density, size, shape of the

dosage forms, concomitant intake of food, volume of

meals and drugs like anticholinergic, laxatives,

purgatives and biological factors such as gender,

posture, age, sex, race body mass index and diseased

state like diabetes, crohn’s disease31. Most important

factors that influence the gastric emptying rate is caloric

content of the meals. Oily layer formed by fats on other

gastric contents such as fatty substances are emptied

later than the other32. In addition to this, body exercise

may also influence gastric emptying. Stress can

increase the gastric emptying rate while it is decreased

in case of depression33. Men and younger people have

faster gastric emptying rate when compared to women

and old people34, 35.

Density also plays an important role in determining the

location of the delivery systems in the stomach. If

density of the delivery system is higher than the gastric

contents, then it sinks to the bottom of the stomach,

while low-density drug delivery systems float on the

surface. Both positions may isolate the system from

pylorus15. It is observed that, multi particulate

formulations are more reliable as compared to single

unit formulations, which suffers “all or none concept”.

The units of multiparticulate systems are freely

distributed through out the GI tract10. Timmermans36,

carried out a comparative evaluation of gastric transit

floating (F) and non-floating (NF) matrix dosage forms

and results were found that GRT of the non-floating

forms were variable and greatly dependent on their size

which are in the order small<medium<large units.

Floating strength may vary with time and usually

decrease after immersion into the fluid as a result of

hydrodynamic equilibrium development.

Iannuccelli et al.37 described that in the fed state after a

single meal, all the floating units had a floating time up

to 5 hours and gastric residence time prolonged by 2

hours and after succession of meals, most of the

floating units showed a floating time of about 6 hours,

gastric residence time extended about 9 hours over the

control, though a certain variability of data owing to

mix with heavy solid food ingested after the dosing was

observed. It was suggested that floating dosage forms

will be suitable for patients with wide range of eating

habits and concluded that different amount of intake of

food did not affect the duration of gastroretention38.

Curatolo39 studied the gastric retention forms for

controlled release and effect of shapes and size of the

dosage forms on gastric retention time. The size of the

dosage form must be suitable for oral administration.

He showed that for humans, from the practical

standpoint the largest dimension in the expanded form

can be varying from 2.5 to 6.0 cm and preferably 3 to 5

cm. If the systems are other than round or circular,

maximum and minimum dimension is 2.5 and 6 cm,

respectively in the expanded form.

PRACTICAL APPROACHES TO DESIGN FDDS.

The concept of floating drug delivery system was

described in the literature as early as 1968, when David

disclosed a method of overcoming the difficulty

experienced by some persons of gagging or choking

while swallowing medicinal pills. The author suggested

that such difficulty could be overcome by providing

pills having a density less than 1.0 g/ml so that pill will

float on the surface of water. Since then several


297

approaches have been used to develop an ideal floating

drug delivery system. The various buoyant preparations

include hallow miocrospheres (microballoons),

granules, powders, capsules, tablets, pills and laminated

films. Most of the floating systems reported in literature

are single unit systems, such as hydrodynamically

balanced systems and floating tablets. But these

systems are unreliable and nonreproducible in

prolonging gastric residence time in the stomach when

orally administered, owing to their fortuitous (‘all or

nothing’) emptying process40, 41. On the other hand

Rouge and coworkers showed that multiple unit dosage

forms decreases the intersubject variability in

absorption and minimizes probabilities of dose

dumping by uniform distribution within the gastric

content and provides longer duration of action42. In the

designing of FDDS, following rationale should be

sought:

I) Retention in the stomach as per the clinical demand

or need; II) Convenience for patient; III) Ability to load

substantial amount of drug with different

physicochemical properties and release them in a

controlled manner ;IV) Complex matrix integrity of SR

formulation in the stomach, inexpensive optimization

between floatation time and release rate, lag time (time

taken by the system to float) must be less43.

The FDDS are classified based on the mechanism of

buoyancy into effervescent and noneffervescent

systems and these technologies are utilized in their

development.

NON-EFFERVESCENT FDDS

The non-effervescent FDDS works on the mechanism

of polymer swelling, bioadhesion of the polymer to

mucosal layer of GI tract. The most commonly used

excipients for the preparations of non-effervescent

FDDS are gel forming or swellable type hydrocolloids,

polysaccharides and matrix forming polymers like

polymethacrylates, polycarbonates, polyacrylates

polystyrenes and bioadhesion polymers like chitosan

and carbopols. One of the approach in the development

of such floating dosage forms involves thorough mixing

of drug and gel forming hydrocolloids. After oral

administration, the dosage form comes in contact with

gastric fluids and gets swollen, form a gelatinous

barrier at the surface. The swollen dosage forms

maintains a relative integrity of shapes and bulk density

less than 1.0.The air entrapped with in the swollen

polymer matrix imparts buoyancy to the dosage forms.

Apart from this, swollen gel structure acts as a reservoir

for the dosage forms and provides sustained release

effect to the dosage forms. The slow release of drug is

controlled by the formation of gelatinous barrier by

diffusion mechanism44.

El-kamal et al.45 prepared floating micro particles by

solvent emulsion diffusion technique. Four different

ratios of Eudragit S100 (ES) with Eudragit RL were

used. Microparticles of ketoprofen for sustained release

system were designed to increase the residence time in

the stomach without contact with mucosa. The

formulation containing 1:1 ratio of ES100 and ERL

gave best floating ability in all the media. The floating

ability of the particles is due to its low bulk density.

Moreover, it has high packing velocity and its inter void

space is relatively low.

Iannuccelli et al.46 prepared air compartment multiple

unit system for prolonged gastric residence. These units

were composed of a calcium alginate core separated by

an air compartment from membrane of calcium alginate

or calcium alginate/PVA.The porous structure

generated by leaching of PVA, which was employed as

water soluble additive in the coating composition was

found to increase the membrane permeability

preventing the air compartment shrinkage. The ability

of the floatation increases with increase in PVA,

molecular weight, and presence of air compartment. It

was found to give excellent floating PVA 100,000 was

at the concentration of at least 5%.

Sheth and Tossounian47 developed hydrodynamically

balanced capsules containing mixture of drug and

hydrocolloids. Upon contact with gastric fluid, the

capsule shell dissolved in gastric fluid followed by

swelling of mixtures, formation of a gelatinous barrier

and maintains bulk density less than 1.0, which

remained buoyant on the gastric fluid for an extended

period of time (Fig.1).

Harrigan48 developed intragastric floating drug delivery

device. The system composed of a drug reservoir

encapsulated in a microporous compartment having

pores on top and bottom surfaces. The peripheral walls

of the reservoir compartment were completely sealed to


298

Fig.1.Working principle of the hydrodynamically balanced system (HBS).

prevent any physical contact of the undissolved drug

with walls of the stomach (Fig.2).

Fig.2.Intragastric floating drug delivery device

Mitra49 prepared “multilayered, flexible sheet like

medicament device. It was buoyant in the gastric juice

and had sustained release characteristics. The device

composed of at least one dry, self-supporting carrier

film made up of a water insoluble polymer. The drug

was dispersed or dissolved in the polymer layer and the

barrier film overlaid the carrier film. The barrier film

composed of one water insoluble, a water and drug

permeable polymer or copolymer. The peripheral walls

were sealed to prevent direct contact of the drug

reservoir with stomach walls. The buoyancy of

laminated film is due to presence of small air pockets.

Klausner et al.50 described a novel levodopa

gastroretentive dosage form, based on unfolding

polymeric membranes, that combines extended

dimensions with high rigidity. It was folded into a large

size gelatin capsules. In vitro studies showed that

unfolded form reached within 15 min after administrat

-tion and it was confirmed in vivo in beagle dogs. The

unfolded form was maintained for at least 2 hours. It

was concluded that this dosage form could improve

therapy of different narrow absorption window drugs.

However, there are possibilities of the polymeric films

to get stuck in the esophagus causing extreme

discomfort to the patient or drug related injuries and

repeated administration of rigid dosage form may result

in gastric obstruction.

Bolton and Desai30, 51 developed controlled release

floating tablets of theophylline using agar and mineral

oil. Tablets were made by dispersing a drug/mineral oil

mixture in a warm agar gel solution and pouring the

resultant mixture into tablet moulds, which on cooling

and air drying formed floatable tablets. The amount of

agar required to form the floatable tablet was

remarkably low (2% tablet). The light mineral oil

prevents escape of entrapped air in the gel matrix when

placed in gastric fluid due to its inherent hydrophobic

property.

The dosage forms were prepared by using different

drugs, polymer(s) and techniques are given in the table

No.1.

EFFERVESCENT FDDS

These are matrix type systems prepared with the help of

swellable polymers such as hydroxypropyl

methylcellulose or polysaccharides and chitosan68


299

Table No.1 other preparations or developments in floating delivery system.

Sl. No.

Drug(s) Dosage form Polymer(s) used Method of preparation

Ref. No.

1 Melotonin Microspheres Chitosan Ionic interaction 52

2 Repaglinide Microspheres Eudragit S Solvent emulsion

53

3 Flurorescein Sodium

Beads Casein-gelatin Emulsification extraction

54

4 Acetohydroximic Acid

Microspheres Eudragit E

Carbopol

Novel quassi- emulsion

55

5 Verapamil Microparticles Polypropylene foam powder,Eudragit RS,Ethylcellulose, Methylacrylate

Solvent emulsion

56

6 Nifedipine Nicardipine Verapamil Dipyridimol

Hallow microspheres

Cellulose acetate O/W emulsification 57

7 Riboflavin Microballoons HPMC,

EudragitS100

Solvent emulsion diffusion

58

8 Meloxicam Beads Sodium alginate

FloriteRE

Ionotrpoc gelation 59

9 Piroxicam Hallow microspheres

Polycarbonates Solvent evaporation 60

10 Chlorpheniramine maleate, Verapamil, Diltiazem HCl, Theophylline.

Tablet HPMC, Carbopol Na-alginate, Noveon Aal, Guargum,Gum arabica.

Direct compression

61

11 Acetylsalicylic acid Tablet Hydroy propyl methyl cellulose

Direct compression 62

12 Captopril Tablet MethocelK4MCR, HPMC, Carbopol

Wet granulation 63

13 Seratiopeptidase Capsule Glyceryl monooleate,

Gelucire43/01

Conventional 64

14 Misoprostol Capsule HPMC Conventional 65 15 Diltiazem HCl Granules Gelucire 43/01,

HPMC, Ethocel 20 FP Melt granulation 66

16

Amoxycillin Hydrogels Chitosan,Carbopol Ionic interaction 67

and various effervescent components like sodium

bicarbonate, calcium carbonate, citric acid or tartaric

acid. These dosage forms are developed in such a way

that, when they come in contact with gastric juice in the

stomach, CO2 is liberated and is trapped in the swollen

hydrocolloids. This provides buoyancy to the dosage

form. The liberated carbon dioxide may intimately get

mixed within the tablet matrix in case of single layered

tablet.69 The multiparticulate floating reservoir types of

delivery systems may contain double or triple layers.

The triple layered tablets may be prepared, which

contains swellable gas generating layer, sustainable

approach was utilized in the development of floating or

pulsatile drug delivery system based on the coated

effervescent core. The dosage form had two layers, first

layer consisted of drug, cellulose acetate or HPMC (10-

50) as a sustained release core and second layer

consisted of effervescent agents, PEG 4000 (4%based

on the weight of the second layer) and lactose or

microcrystalline cellulose as filler. Sodium bicarbonate

and citric acid were used as an effervescent agent in a

ratio of 1:0.76 in the concentration of 30-50 % of the

w/w of the core. The CO2 is generated upon contact

with the medium and gets entrapped in the polymeric


300

matrix, which provides buoyancy to the dosage form.

They observed that addition of 10-20% w/w of HPMC

significantly retarded drug release compared to the

dosage form without HPMC.The pulsatile release from

the ethyl cellulose coated tablets were highly

reproducible. They concluded that floating or pulsatile

drug delivery systems based on the effervescent cores

can be obtained depending on the choice of the

polymeric coating and core components71.

Farouk sakr72 developed programmable drug delivery

systems for oral administration (Fig.3). It was a new

prototype model device (3 cm long and 0.9 cm internal

diameter) made to comprise of a cylindrical shell in the

form of oral capsule. Drug was placed in a cylindrical

disc made up of slowly eroding polymer and

compressed to zero porosity, a flexible rubber disc,

compressible acid resistant spring and a special acid

impervious non -permeable rubber ballooning system

containing bicarbonate granules. The device in the form

of non-digestible oral capsule containing drug in a

slowly eroding matrix was designed to utilize on

automatically operated geometric obstruction that keeps

the device floating in the stomach and prevents the

system from passing through remainder of GIT.The

different grades of HPMC were used to develop the

eroding matrix. They concluded that duration of action

was dependent on erosion rate of the incorporated

polymer and the in vitro release of drug from developed

device could be maintained upto 20 days.

Fig. 3. Diagrammatic sketch of the device representing

its operation mechanism.(A,B,C,D.) (A) intact device;

(B) device at the beginning of drug release; (C) device

with half drug-polymer compact eroded; and (D) device

after complete drug–polymer erosion and evacuation of

entrapped carbon dioxide (inflated balloon).

Choi et al.73 prepared alginate beads consisting of gas

forming agent. The beads were made up of HPMC and

sodium alginate(9:1w/w) with gas generating agent in

the concentration 0:1 to 1:1(gas forming agent/alginate

w/w). The resultant solution was dropped in to 1%

(w/v) calcium chloride solution containing 10% (v/v)

acetic acid. The suspended beads were stirred with a

magnetic stirrer for 10 minutes. The prepared beads

were evaluated for the effect of CO2 producing agent

on size, floating properties, porosity, morphology and

mechanical strength of beads. It was observed that

amount of gas forming agent had a significant effect on

size, floating ability, porosity, morphology, release rate

and mechanical strength. Calcium carbonate formed

smaller but stronger beads as compared to sodium

bicarbonate. Calcium carbonate was found to be less

effective gas generating agent than sodium bicarbonate.

But it forms superior quality floating beads with

significantly extended drug release.

Atyabi et al74 developed a floating system using ion

exchange resin. The system composed of resin beads,

which were loaded with gas generating agent and

negatively charged drug. The drug-loaded beads were

encapsulated by semipermeable membrane to overcome

sudden loss of CO2 upon arrival in contact with gastric

environment of stomach. An exchange of chlorides and

bicarbonate ions took place. As a result of this reaction,

CO2 was released and trapped in the membrane, there

by carrying beads towards the top of gastric contents.

The in vivo behavior of the coated and uncoated beads

was monitored using a single channel analyzing study

in twelve healthy human volunteers by γ-

radioscintigraphy. They showed that a coated bead

remained in the upper stomach for over 3 hours, which

was superior to the non-coated beads.

Ichikawa et al.76 developed floating capsules composed

a plurality of granules having different residence time

in the stomach and granules were comprised of a core

containing the drug coated by double layer. Inner layer

was further divided into 2 sub layers, inner tartaric acid

and outer sodium bicarbonate. This layer was coated

with expansive polymeric membrane (PVA and

shellac), which allowed gastric juice to pass through it

and expanded by foam produced by the reaction

between gastric juice and foamable layer.

Chen and coworker77 studied effect of formulation

variable on in vitro performance of floating sustained

release capsules of verapamil. The formulations were

comprised of variables like polymer excipients,

polymer content, weight of the filled powder mixture


301

(density of the capsule), and amount of effervescent

agent.

Ichikawa et al.20 developed multiple unit type of

floating pills, composed of inner effervescent layer

containing sodium bicarbonate and tartaric acid and

outer swellable polymeric membrane made up of

polyvinyl acetate and purified shellac (Fig 4). The inner

layer was further divided into two sublayers to avoid

physical contact between sodium bicarbonate and

tartaric acid. When the pill was immersed in buffer

solution at 37 °C, it settled down at the bottom, buffer

solution entered in to the effervescent layer through the

outer swellable membrane. CO2 was generated due to

reaction between sodium bicarbonate and tartaric acid

and formed swollen pills (like balloons) with a density

much lesser than 1.0 g/ml. The system was found to

float completely within 10 minutes and had a good

floating ability independent of pH, viscosity of the

medium and drug release in a sustained manner.

.

Fig.4. Floating pills a) The penetration of water into

effervescent layer leads to a CO2 generation and makes

the system to float (b) Mechanism of floatation

Alza Corporation got two patents for the development

of drug delivery devices for the controlled and

continuous administration of drugs. The osmotically

activated device comprised of a hallow deformable unit

that was convertible from collapsed to expandable form

and returns to original form. The deformable unit

supported by housing that internally divided into first

and second chambers separated by pressure sensitive

permeable bladder. The first chamber having medicinal

agent and second chamber having volatile liquid, like

cyclopentene or ether vaporizes at body temperature

and provides floating ability to the system. The device

contained a bioerodible plug that allowed the vapor to

escape from the system77, 78.

Fig.5. Intragastric floating tablet (US Patent [4, 167,

58, September 11, 1979).Ref.No.84.

Fig.6. Intragastric floating bilayer tablet (US Patent [4,

140, 755, February 20, 1979). Ref.No.80.

PATENTS:

Dosage form US Patent No. Ref

No.

Multilayer flexible sheet

device

4451260 49

Tablet 4814179 51

Tablet 4140755 80

Tablet 4167558 62

Tablet/capsule 3574820 84

Hydrogel/pills 4434153 7

Minicapsule 4101650 79

Capsule 4126672 54

Capsule 4702918 81

Capsule 3976764 82

Intra gastric floating device 4055178 48

Gastric inflatable device 3901232 77

Osmotically controlled

DDS

3786813 78

Granules 4844905 75

Powder(capsule/compressed

into tab)

5169638 83

Multilayered floating

dosage form

Pub.No.US2003232081 85


302

CONCLUSION

To develop an efficient FDDS is a real challenge.

Because several drugs having narrow absorption

window, unstable at higher pH value and drugs have

local effect may benefit from developing a FDDS.

Improvements are needed in all aspects to develop a

perfect system that will be retained in the stomach for a

long time. The research in this area is ongoing until to

develop an ideal dosage form.

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306

In vitro-In vivo Correlation: A Ground Discussion Kalaskar S. G. Yadav A. V

* and Patil V. B.

Dept. of Biopharmaceutics, Govt. College Of Pharmacy, Karad.

* For correspondence: [email protected]

ABSTRACT

Rapid drug development necessitates the research to find out link between dissolution testing and

bioavailability, which result as concept of in vitro in vivo correlation (IVIVC). IVIVC is a mathematical model

that can be used to predict in vivo behavior of drug product from its in vitro performance. Although there are

several levels of correlation level A correlation is most meaningful and acceptable by regulatory committees.

IVIVC is not suitable for all drugs but it is suitable for drugs that have particular properties. This can be

explained by Biopharmaceutical Classification System. Design of dissolution method is crucial step in IVIVC.

Higher the possibility of simulating in vivo condition higher will be the correlation. There are several factors

that should be considered while developing IVIVC like, stereochemistry, first pass effect, effect of food on

bioavailability etc. Now a days, the research is not only focused on IVIVC of oral dosage forms but also it is

extended to novel dosage forms. Applications of IVIVC include drug product development, certain scale up and

post approval changes and even setting dissolution specifications for quality control. Hence the development of

IVIVC should be initiated parallel to drug product development.

Key words: IVIVC, Biopharmaceutical Classification System (BCS), Biowaiver, Dissolution, Novel dosage

form.

INTRODUCTION

Pharmaceutical industries are hungry for rapid drug

development and approval while regulatory agencies

need assurance of product quality and performance.

This necessitates the research to find out link between

dissolution testing and bioavailability1.

In 1980s, attention was focused towards in vitro in vivo

correlation (IVIVC). A workshop sponsored jointly by

the USFDA and industry concluded that the state of

science and technology at that time did not permit

meaningful IVIVC for Extended Release (ER) products

on consistent basis, but encouraged further research in

the area2. Subsequent workshop reports3, 4, showed a

trend towards increasing confidence in IVIVC for ER

oral drug product. Thereafter USP published a stimuli

article5 indicating different levels of correlation, which

was further extended as USP chapter <1088> 6.

Compilation and modification of all these literatures led

to publication of FDA's guidance for industry viz.

Extended Release Oral Dosage form: development,

evaluation and application of in vitro-in vivo

correlations, in 19977. The objective of present review

is to have a ground discussion on IVIVC, dissolution,

factors to be considered while developing IVIVC and

progress in IVIVC.

IVIVC BASIC

IVIVC simply means a mathematical model that can

describe the relationship between in vitro and in vivo

properties of a drug product, so that in vivo properties

can be predicted from its in vitro behavior. Extensive

discussion by several scientists in several workshops

and publications supports the concept of IVIVC7.

According to FDA, “IVIVC is a predictive

mathematical model describing relationship between in

vitro properties of dosage form and relevant in vivo

response. Generally the in vitro property is rate or

extent of drug dissolution or release while in vivo

response is the plasma drug concentration or amount

absorbed7.”

APTI ijper


Received on 15/3/2007; Accepted on 7/8/2007

© APTI All rights reserved


307

Levels of Correlation

Five levels of correlation can be found in FDA

guidelines. Each level denotes its ability to predict in

vivo response of dosage form from its in vitro property.

Higher the level better is the correlation7.

1. Level A correlation.

2. Level B correlation.

3. Level C correlation.

4. Multiple level C correlation.

5. Level D correlation.

1. Level A correlation

It represents the relationship between in vitro

dissolution and in vivo in put rate (in vivo absorption of

drug from dosage form) 7. A hypothetical level A model

showing relationship between fraction absorbed and

fraction dissolved, is shown in Figure No. 1.

For developing correlation between two parameters one

variable should be common between them. Here data

available is in vitro dissolution profile and in vivo

plasma drug concentration profile. As shown in Figure

No. 4, no direct comparison is possible7. To have

comparison between these two data, data

transformation is required. As shown in figure no. 4,

data transformation makes the comparison possible.

The in vitro properties like percent drug dissolved or

fraction of drug dissolved can be used while in vivo

properties like percent drug absorbed or fraction of drug

absorbed can be used respectively. Level A IVIVC is

considered as predictive model for relationship between

the entire in vitro release time course and entire in vivo

response time course8.

Most commonly there should exist a linear correlation

but some times non-linear correlation may be

appropriate. However no formal guidance on non-linear

IVIVC has been established9.

When in vitro curve and in vivo curve are super-

imposable the relationship is called as 1:1 relationship.

But if scaling factor is required to make curve super-

imposable the relationship is called as point-to-point

relationship3.

All data with every point from both in vitro and in vivo

curve is utilized for development of correlation,

therefore it becomes more meaningful than any other

type of correlation and very useful from regulatory

perspective7. It is the highest level of correlation and

most preferred to achieve, since this allows biowaiver

for changes in manufacturing site, raw material

suppliers, and minor changes in formulation3, 7.

2. Level B correlation

In this level of correlation (Figure No. 2), the mean in

vitro dissolution time (MDT in vitro) is compared with

either the mean in vivo residence time (MRT in vivo) or

mean in vivo dissolution time (MDT in vivo) derived by

using principle of statistical moment analysis7.

Even though it utilizes all in vitro and in vivo data, it is

not considered as point-to-point correlation, since

number of in vivo curves can produce similar residence

time value. Hence this correlation becomes least useful

for regulatory purposes.

3. Level C correlation

It is a single point correlation that is established in

between one dissolution parameter like t50% and one of

the pharmacokinetic parameters like tmax, cmax or AUC

(Figure No. 3). It does not reflect the complete shape of

plasma drug concentration time curve, which is the

critical factor that defines the performance of drug

product7.

However it can be helpful in early stages of formulation

development when pilot formulations are being

selected7.

4. Multiple level C correlation

Multiple level C correlation reflects the relationship

between one or several pharmacokinetic parameters of

interest and amount of drug dissolved at several time

point of dissolution profile7. It should be based on at

least three dissolution time points that includes early,

middle and late stage of dissolution profile.

When multiple level C correlation is developed there

are more chances of development of level A

correlation, which should be preferred7.

5. Level D correlation

It is a rank order and semi quantitative correlation and

is not considered useful for regulatory purpose.

Development of level a correlation

As level a correlation is most meaningful and useful,

only level a correlation development and evaluation

will be discussed in detail. Schematic diagram of

development of level a correlation is shown in Figure

No. 4. At least three formulations should be developed

with different release rates that is slow, medium and

fast release rate (at least differ by ±10%) so that


308

comparable difference between in vivo property (tmax,

cmax or AUC) of these formulation is possible7.

In vitro data should be obtained from optimized in vitro

dissolution study, generally by using official dissolution

apparatus. In case unofficial apparatus is used, it should

be permitted by Center for Drug Education and

Research (CDER) office before study 7.

Optimization of dissolution testing should be done to

get best in vivo simulations and higher possible

correlation. Once dissolution testing method is

developed, same method should be used for all the

formulations. The dissolution testing should be

performed on 12 individual dosage forms from each lot

and mean values should be considered7.

To obtain in vivo data, bioavailability (BA) study in

sufficient number of healthy volunteers (6-12) should

be performed. The crossover study design is generally

preferred; if not possible parallel study design is also

acceptable. The drug product should be administered in

fasting state, however if intolerable it can be

administered in fed state and the effect of food should

be considered10.

The data so obtained in in vitro and in vivo studies after

data transformation should be processed to develop

mathematical model that describe relationship between

them. The time scaling factor (if required) for

superimposition of curves should not be different for

different release rates7.

Generally two methods are used for development of

correlations (as shown in Figure No.5)-

1) Two stage deconvolution approach: It involve

estimation of in vivo absorption profile from

plasma drug concentration time profile using

Wagner Nelson or Looe-Riegelman method11, 12,

subsequently the relationship with in vitro data is

evaluated.

2) One stage convolution approach: It computes the in

vivo absorption and simultaneously models the in

vitro – in vivo data.

Two stage methods allows for systematic model

development while one stage method obviates the need

for administration of an intravenous, oral solution or IR

bolus dose8.

Mostly IVIVC models developed are simple linear

equation between in vitro drug released and in vivo

drug absorbed. But some times these data can be better

fitted by using non-linear models like Sigmoid,

Weibull, Higuchi or Hixson-Crowell9.

Evaluation of Predictability of Correlation

It is done by calculating prediction error that is the error

in prediction of in vivo property from in vitro property

of drug product. Depending on therapeutic index of

drug and application of IVIVC evaluation of prediction

error internally and/or externally may be appropriate.

Internal validation serves the purpose of providing basis

for acceptability of model while external validation is

superior and affords greater confidence in model7.

The % Prediction Error (P.E) can be calculated by

following equations13-

% P.E = (Cmax observed - Cmax predicted) X 100/

(Cmax observed) or

% P.E = (AUC observed – AUC predicted) X 100/

(AUC observed)

Internal Predictability

The BA (Cmax/ Tmax/ AUC) of formulation that is

used in development of IVIVC is predicted from its in

vitro property using IVIVC. The predicted BA is

compared with observed BA and % P.E. is calculated.

According to FDA guidelines, the average absolute %

P.E. should not exceed 10% and % P.E. for individual

formulation should not exceed 15%, for establishment

of IVIVC7.

External Predictability

The BA of formulation that was not used in IVIVC

development is predicted from IVIVC model. This

predicted BA is compared with known BA and % P.E is

calculated. The prediction error for external validation

should not exceed 10% whereas prediction error of 10-

20% indicates inconclusive predictability and need of

further study using additional data set. For dugs with

narrow therapeutic index, external validation is required

despite its acceptable internal validation, whereas

internal validation is usually sufficient with non-narrow

therapeutic index7.

BIOPHARMACEUTICS CLASSIFIACTION

SYSTEM AS BASIS FOR BIOWAIWER AND

IVIVC

Development of IVIVC is not suitable for all class of

drugs. It is suitable for only certain classes of drugs that

can be explained by BCS concept. BCS is based on

solubility, intestinal permeability and dissolution rate,

all of which governs the rate and extent of oral drug


309

absorption from immediate release solid oral dosage

forms14. Based on solubility and permeability there are

four classes of BCS as shown in table 1.

A drug is said to be highly soluble when its highest

dose strength is soluble in 250 ml or less of aqueous

media over pH range 1.0 – 7.5, otherwise drug is

considered as low soluble. A drug is considered as

highly permeable when extent of intestinal absorption is

found to be 90 % or higher, otherwise it is considered

as poorly permeable.

Immediate Release (IR) drug product is one which

releases not less than 85 % of labeled drug within 30

min using USP I (100 rpm) or USP II (50 rpm)

apparatus in a volume of 900 ml or less of official

dissolution media. Otherwise product is considered to

be slow dissolution product.

Biowaiver for BCS Class I

Based on FDA guidelines14 sponsor can request

biowaiver for BCS Class I in IR solid oral dosage form,

if drug is stable in gastrointestinal tract (GIT), not a

narrow therapeutic index, excipients in formulation

does not affect absorption and drug product is not

designed to be absorbed in oral cavity. The principle of

applying for biowaiver is that, once drug product enters

in stomach, it gets solubilized in gastric fluid rapidly

before gastric empting. So the rate and extent of

absorption is independent of drug dissolution as in case

of solution15and in vivo bioequivalence is not necessary.

Biowaiver Extension for BCS Class III

It has been contented that there are equally compelling

reasons to grant biowaiver to BCS Class III drugs as

they are for BCS Class I drug16 since drug from both

classes are highly soluble. If excipients used in two

pharmaceutical equivalent solid oral IR product does

not affect the drug absorption and the two products

dissolves very rapidly (>85% in 15 min.) in all relevant

pH ranges, there is no reason to believe that these two

products would not be bioequivalent17.

Biowaiver Extension Potential for BCS Class II

The rate and extent of absorption of BCS Class II drug

(poorly soluble and highly permeable) is dependant on

in vivo dissolution behavior of IR drug product. If in

vivo dissolution can be predicted from in vitro

dissolution studies, in vivo bioequivalence study can be

waived17. In vitro dissolution methods that can mimic

in vivo dissolution behavior of BCS Class II drug are

appealing but experimental methods can be difficult to

design and validate such methods because of numerous

in vivo processes involved18. Here the role of IVIVC

comes to apply for biowaiver. Even though IVIVC is

considered as difficult perspective but it is possible to

achieve and worth the efforts19.

DISSOLUTION AND IVIVC

The dissolution method having higher discriminating

power and is able to detect minor changes in

manufacturing process is useful for quality control.

Such methods can be developed by using USP

dissolution apparatus and USP dissolution media. This

approach of dissolution method development is known

as Quality Control (Q.C) approach. Another approach

that is known as Research and Development (R and D)

approach is directed to develop dissolution method that

is capable of predicting in vivo dissolution behavior of

drug product20. In R and D approach the dissolution

method should be sensitive and reliable predictor of

BA21. Both the USP dissolution media and apparatus

may be less useful and require further physiological

adaptation in them22, 23. For this reason dissolution

apparatus which is able to mimic in vivo hydrodynamic

flow conditions and dissolution medium that can

simulate gastro intestinal environmental conditions

should be used for R and D approach.

Dissolution Apparatus

Even though seven dissolution apparatus are mentioned

and described in USP still there exist scope in

developing new dissolution apparatus. USP Apparatus I

and II are widely used and Apparatus II is most widely

found in literature than Apparatus I. This is because of

simple instrumentation and handling. However, there

are number of limitations associated with them. These

two apparatus have limited volume capacity so become

unsuitable for formulations with poor soluble drugs1, 24.

It is not possible to have automatic changes in pH

ranges during run24,which is important to simulate in

vivo conditions for ER drug product. The major

problem is high variability25 in results, which is mainly

due to variable flow dynamics and poor mixing26. The

last problem was addressed by using crescent spindle

instead of paddle27, which improves the flow dynamics,

and mixing in dissolution vessel. In one study, it was

found that the hydrodynamic flow rate in human GIT is

very low and corresponds to paddle speed of 10 rpm28


310

(as compared to recommended 50 rpm). USP apparatus

III and IV are suitable for poorly soluble drug products

and ER drug products. USP apparatus V, VI and VII are

suitable for transdermal drug products.

Several non-official dissolution apparatus have been

developed till today, for example paddle with beads

apparatus29, crescent spindle instead of paddle27 etc. A

new wave dissolution apparatus30 have been developed

which shows higher level of IVIVC. This apparatus

involve three units named as gastric cell, intestinal cell

and systemic cell so that amount of drug in each cell

represents amount of drug in respective body

compartments. IVIVC developed by using this

apparatus does not require tedious mathematical models

as it can predict the amount of drug entered in systemic

circulation directly30.

Dissolution medium

In vitro dissolution testing that can simulate and predict

invivo dissolution behavior of ER drug product, become

very important in development of IVIVC. For

simulating invivo conditions following parameters

should be considered; pH, buffer composition, ionic

strength, buffer capacity, temperature, volume,

hydrodynamics etc.

Even though compendial dissolution media are listed in

official books, the use of non-compendial media

(Biorelevant Dissolution Media) have shown to

improve IVIVC 31,32 and hence have been proved to

have better discriminating power. 33

Generally speaking pH increases from stomach to large

intestine (pH 1-7-8). Hence, dissolution testing of oral

ER drug product should be carried throughout entire

physiological pH range (1-7-8). The use of dissolution

apparatus that allows the changes in pH during

dissolution testing becomes appropriate. But it is

difficult to determine the time interval, which closely

relate to a particular pH segment of invivo conditions24.

Influence of dissolution media composition on drug

release and IVIVC has been studied. The susceptibility

of dissolution and hence IVIVC to the dissolution

media composition has been reported particularly for

anionic polymeric system34.

Ionic strength of dissolution media also play important

role in dissolution testing35. Dissolution behavior of ER

formulations that use hydrophilic gel forming polymers

(like HPMC) is known to be significantly affected by

any changes in ionic strength24 of dissolution medium.

Ions present in food and food induced secretions in

G.I.T cause changes in ionic strength of G.I.T fluid. It

makes difficult to generalize ionic strength of

dissolution media to be used to test ER dosage form24.

Buffer capacity plays important role in dissolution

behavior of formulation that contains acidic or basic

excipients. It has been shown in one study that,

buffering capacity of medium is an important factor in

design of dissolution media for IVIVC36.

As the human body temperature is about 37 °C,

standard dissolution testing is carried out at 37 ± 0.5 °C

as recommended in most pharmacopoeias. But a report

exists about significant difference in dissolution profiles

of some commercially available extended release solid

dosage forms containing isosorbide dinitrite tested at

various temperatures within this specified range of 36.5

- 37.5 0C 37 .

FACTORS TO BE CONSIDERED WHILE

DEVELOPING IVIVC

Before developing IVIVC some properties like

stereochemistry, first pass effect, effect of food on

bioavailability etc., which may influence the

development and validation of IVIVC should be

considered

1. Stereochemistry

When one of the enantiomer has higher affinity towards

receptors (involved in Pharmacokinetics or

Pharmacodynamics) than other, the phenomenon is

referred as stereo selectivity. This results in difference

in Pharmacokinetics or Pharmacodynamics behavior of

two enatiomers. If such stereoisomers in the form of

racemate are administered orally, one form may have

higher BA than other. Obviously use of in vitro

dissolution data of racemate will not be useful for

development of IVIVC and hence prediction of in vivo

availability of active enantiomer. So consideration of

stereoisomerism in development of IVIVC may provide

more meaningful relationship.

Sirisuth et al. 2000 have studied influence of

stereoselectivity on development and predictability of

IVIVC using Metaprolol Tartrate ER tablet. Study

concludes that Metaprolol Racemate data cannot be

used to accurately predict R-enantiomer drug

concentrations. However, the racemate data was

predictive of active stereoisomer38.


311

2. First Pass Effect

Drug with high hepatic extraction ratio undergo

biotransformation before entering into systemic

circulation. This is known as first pass effect. This

decreases systemic availability of parent drug.

Therefore the amount of drug reaching to systemic

circulation will not match with amount of drug released

in G.I.T. Hence use of plasma concentration data of

parent drug will not be appropriate to calculate in vivo

drug release.

Sirisuth et al have studied the influence of first pass

effect on development and validation of IVIVC. They

developed non-first pass effect IVIVC (NFPE IVIVC)

by using Metaprolol Tartrate ER Tablet as model. The

correlation was developed between the fraction of

Metaprolol dissolved and fraction of total drug

absorbed from various release rate formulations. The

fraction of total drug absorbed can be obtained from

plasma concentration profile of total drug that is

Metaprolol and the major metabolites. The NFPE

IVIVC shows stronger relationship as compared to poor

correlation developed by IVIVC39.

3. Food Effect

Few drug products have to be administered after meal

because of certain reasons. Presence of food may alter

the dissolution behavior of drug and hence it becomes

an important factor that should be considered in IVIVC

development. Presence of food in stomach alters the

gastric environment, which includes alteration in pH,

ionic strength, level of enzymes, gastric emptying time,

etc. Not only presence of food but also nature and its

extent determines the BA. All these facts should be

taken in to consideration while optimizing dissolution

medium for development of IVIVC.

Al-Behaisi et al 2002 studied the in vitro dissolution

profile of Deramciclane containing film coated tablets

under simulated in vivo conditions in both fasting and

fed state. The relevance of food effect on dissolution

profile was studied and a correlation between in vitro

dissolution data and certain pharmacokinetic

parameters was investigated10.

IVIVC OF NOVEL DOSAGE FORMS

1. PARENTERAL CONTROLED OR SUSTAINED

RELEASE (CS/SR) DRUG DELIVERY SYSTEM

Even though the in vitro release testing of CR/SR

parenterals is primarily for quality control purposes but

the ultimate aim of Q.C is to ensure the clinical

performance that is efficacy and safety. So in vitro

release test should be developed with biorelevance40.

Unlike controlled release oral formulations there are no

regulatory standards for parenteral Microparticles

delivery systems41.

In recognition of need for standard in vitro method, a

series of national and international workshops on

quality assurance and performance of CR/SR

parenterals have been conducted in recent years40, 42.

These workshops addressed methodology, apparatus,

out comes, parameters necessary for method

development and IVIVC for SR parenteral dosage form.

The resulting publications included important guideline

for “novel” or “special” dosage forms, including

implants, injectable microparticles, formulations and

liposomes40. Currently research is focused on

shortening the time span of in vitro release experiments

with aim of providing quick and reliable methods for

assessing and predicting drug release43, 44.The use of

animals is considered acceptable to prove that an in

vitro release system is discriminating. However the use

of animals is considered inappropriate to prove an

IVIVC for regulatory purposes.

S.S. D’souza and PP DeLuca have explained three

methods for in vitro drug release study of

Microparticles system for parenteral administration.

These methods include sample and separate, flow

through cell and dialysis technique41.

2. TRANSDERMAL DRUG DELIVERY SYSTEM

(TDDS)

Even though USP 29 gives methods for in vitro drug

release testing of transdermal patches like paddle over

disk, cylinder method and reciprocating disk method,

numerous examples of use of Franz diffusion cell are in

literature21.

Testosterone TDDS patches were studied in vitro for

skin permeation characteristics and in vivo for

pharmacokinetic parameters. In vitro evaluations were

conducted using both human cadaver skin and

reconstructed skin models in order to determine a

model predictive of in vivo delivery. The preliminary

IVIVC developed using human cadaver skin was very

strong45.

Qi X et al have developed IVIVC for 2, 3, 5, 6-

tetramethyl pyrazine (TMP) using convolution


312

technique. In vitro data was obtained from skin

permeation study and in vivo data obtained by

transdermal application of TMP in rabbit. There was

good agreement between predicted and observed drug

absorption profile46.

3. NASAL DRUG DELIVERY SYSTEM

Nasal drug delivery can be assessed by variety of

means, but high reliance is often placed on in vitro

testing methodology like emitted dose, droplet or

particle size distribution, spry pattern and plume

geometry47, 48. Current FDA guidance recommends

these methods as a means of documenting BA and BE

for topically acting solution formulations, because they

can be performed reproducibly and are more

discriminating among products48.

Recent studies have shown a poorer IVIVC for similar

nasal pump spray, where significant in vitro differences

in in vitro parameters were not reflected in differences

in nasal deposition in vivo. It is suggested that

radionuclide imaging data may have important role to

play as adjuvant to in vitro testing in bioavailability

(BA) and bioequivalence (BE) assessment and may

provide a clear understanding of the change in in vitro

parameters that are important for predicting differences

in in vivo performance48.

4. ENTERIC COATED MULTIPLE UNIT

DOSAGE FORM

In case of multiple unit type dosage forms, individual

unit is emptied gradually and separately from stomach

to duodenum. Simulation of these conditions in vitro is

troublesome and may be impossible. Takashi H et al

developed a method to predict dissolution in GIT from

in vitro data in consideration of gastric emptying (GM)

process. Direct prediction of in vivo absorption profile

from in vitro dissolution data in multiple unit system

was very difficult. GE – Convolution method,

overcame this problem. Good correlation (level A) was

obtained for multiple unit type enteric-coated granules

by using GE – Convolution method49.

5. BUCCAL TABLETS

Spiegeleer et al have developed a useful correlation

between in vivo residence time of mucoadhesive tablets

in mouth and in vitro bending point of same. This linear

regression models permits further optimization of

buccal tablets to enhance the adhesion time using in

vitro bending point as selection criteria50.

6. SUPPOSITORIES

It is difficult to establish single standard method for in

vitro evaluation of all types of suppositories42. Modified

basket or paddle method and modified flow through cell

method are recommended for lipophilic suppositories

while conventional basket, paddle or flow through cells

are recommended to be suitable for hydrophilic

suppositories42. However no simulated rectal fluid

exists at the moment to simulate in vivo dissolution of

suppositories21.

IVIVC has been reported by using dialysis rotating

cell51 and reciprocating dialysis tube method52. The

reciprocating dialysis tube method showed highest level

of correlation between in vitro drug release and in vivo

absorption53. The main problem associated with

reciprocating dialysis tube method was the

reproducibility, which was addressed by addition of

periodic tapping to reciprocating dialysis tube method.

It was found that periodic tapping also increases the

predictability of release of drug from suppository

base54.

APPLICATIONS OF IVIVC

The most important application of IVIVC is to use in

vitro dissolution study as surrogate for human

bioequivalence (BE) studies. This will reduce the

number of human bioequivalence studies during initial

approval process as well as certain scale up and post

approval changes (SUPAC) 7. The FDA guidance

explains applications of IVIVC as biowaivers for

changes in manufacturing of a drug product and setting

dissolution specifications.

1. Early Development of Drug Product and

Optimization

In early stages of drug product development drug

products are characterized by some in vitro systems and

some in vivo studies in animal models to address

efficacy and toxicity issue55. At this stage if preliminary

relationship between in vitro and in vivo properties can

be made, it can add better vision in design and

development of drug product55. Latter when valid

IVIVC developed formulation can be optimized based

on in vitro dissolution studies only.

2. Biowaiver for Minor Formulation and Process

Changes

When relationship between critical manufacturing

variables and in vitro dissolution rate have been clearly

defined for CR formulation and IVIVC has been


313

established, it may be possible to use in vitro

dissolution data to justify minor formulation and

process changes. These changes may include minor

changes in colour, shape, size, preservative, flavor,

coating procedure, amount and composition of

materials, source of inactive and active (if adequately

characterized) ingredient, equipment or site of

manufacturing3.


314

Figure No. 4: Development of level A correlation

Slow, medium and fast indicates release rates of formulations


315

Figure No. 5: Two approaches for development of correlation

3. Setting Dissolution Specifications

In absence of IVIVC, the range of dissolution

specification rarely exceeds ± 10 percent of dissolution

of pivotal batch. However, in presence of IVIVC, wider

specifications may be applicable based on the predicted

concentration time profile of test batches being

bioequivalent to reference batch7. The specification

should be optimally established such that all batches

with dissolution profile between fastest and slowest

batch are bioequivalent and less optimally

bioequivalent to reference batch7. The above exercise in

achieving the widest possible dissolution specification,

allow majority of batches to pass. This is possible only

when valid level A model is available. Hundreds of

studies related to IVIVC can be found in literature, few

of them pointed out in the Table number 2.

CONCLUSION

IVIVC is a link between in vitro and in vivo

performance of drug product. It has wide application

right from drug product development to setting

dissolution specifications. A valid IVIVC can allow

biowaiver for certain class of drug. This will definitely

reduce the time and cost requirements in drug product

development without compromising the quality of

same. Extensive research in design of dissolution

method may help in development of more meaningful

and valid IVIVC.

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**********


319

Formulation and in vitro evaluation of taste masked

orodispersible dosage form of Levocetirizine

dihydrochloride Chaudhari P.D

1*, Chaudhari S.P.

1, Lanke S.D.

1 and Patel Nakul

1

1Pad. Dr. D.Y.Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-18, (MS), India.

Email: [email protected] / [email protected].

ABSTRACT

Levocetrizine Dihydrochloride is an active nonsedative antihistamine. Allergic rhinitis is a significant public

health concern in many developed and developing countries. Thus formulating Levocetrizine into an

orodispersible dosage form would provide fast relief. But, it is bitter in taste and taste should be masked to

formulate it in a palatable form. So in the work undertaken, an attempt was made to mask the taste, by

complexation technique using ion-exchange resin, Tulsion 335 (polyacrylic hydrogen with carboxylic

functionality) and to formulate into an orodispersible dosage form. The drug loading onto ion-exchange resin

was optimized for concentration of resin, swelling time of resin, stirring time, pH of resin solution and stirring

temperature. The resinate was evaluated for taste masking and characterized by DSC and IR. Using drug-resin

complex orodispersible tablets were formulated. The tablets were evaluated for drug content, content

uniformity, weight variation, hardness, friability, water absorption ratio, invitro and invivo disintegration time

and invitro drug release. The tablets disintegrated invitro and invivo within 18 and 22 s respectively. Complete

drug was released from tablet within 2 minutes. The results showed that Levocetrizine dihydrochloride was

successfully taste masked and formulated into an orodispersible dosage form as an alternative to conventional

tablets.

Keywords: Levocetrizine dihydrochloride, Tulsion 335 Taste masking, Orodispersible tablet.

INTRODUCTION

Consumer satisfaction is the buzzword of the current

millennium; and moment to achieve it has already

begun in the pharmaceutical industry. An inability or

unwillingness to swallow solid oral dosage forms such

as tablets and poor taste of medicine are some of the

important reasons for consumer dissatisfaction.1, 5

Levocetrizine is a nonsedating antihistamine used in

treatment of allergic diseases. Ion exchange resins are

water-insoluble, cross-linked polymers containing salt

forming groups in repeating position on the polymer

chain. The unique advantage of ion exchange resins for

complexation is due to the fixed positively or

negatively charged functional groups attached to water

insoluble polymer backbones.

These groups have an affinity for oppositely charged

counter ions, thus absorbing the ions into the polymer

matrix. Since most of drugs possess ionic sites in their

molecule, the resins charge provides means to loosely

bind such drugs. The binding is generally an

equilibrium process, resulting in continuous desorption

or elution of drug from the resin as drug is absorbed

into the body. 6, 7

Ion exchange resins are high molecular weight water

insoluble polymers and so are not absorbed by the body

and therefore inert and safe for oral use. The complex

of cationic drug and weak ion exchange resin does not

break at the pH of saliva i.e. 6-7 with cation

concentration of 40 meq/lit. But at high cationic

concentration in stomach and pH 1.2, free drug is

immediately released. This implies that while passing

APTI ijper





320

through mouth, the drug remains in the complex form,

thereby imparting no bitter taste in the mouth. This

property was exploited to formulate the consumer

friendly dosage form i.e. mouth dissolving tablets.4, 8

Mouth dissolving tablets are dosage form, which

disintegrate in patient’s mouth within a few seconds

without the need of water, or chewing, providing best

remedy for the patient suffering from dysphasia. Some

drugs are absorbed from mouth, pharynx and esophagus

as the saliva passes down the stomach. In such cases the

bioavailability is greater than those observed for

conventional dosage form. The advantages of mouth

dissolving dosage form are increasingly being

recognized in both industry and academia. Their

growing importance was underlined recently when

European pharmacopoeia adopted the term

“Orodispersible Tablet” as tablet that is to be placed in

mouth where it disperses rapidly before swallowing.1, 2,

3

MATERIALS AND METHODS

Materials

Levocetrizine dihydrochloride and Tulsion 335 were

provided as gift samples by Emcure Pharmaceuticals

Ltd., Pune, India and Thermax India Ltd., Pune, India

respectively. Starlac and MCC were provided by Signet

Chemicals Ltd., Mumbai, India.

Sensory Test on threshold value of bitter taste for

Levocetrizine dihydrochloride8:

Ten healthy human volunteers (20-25 years) held 10ml

of aqueous solution of Levocetrizine dihydrochloride of

conc. 0, 25, 50, 75, 100, 200, 225 and 250 µg/ml in

water, respectively, in their mouths for 10 seconds and

washed their mouth with 50 ml distilled water. The

bitterness threshold concentration was judged by

considering opinion of volunteers and statistics was

applied for the same.

Preparation of Resinate 9, 10, 11:

Resinate were prepared using batch method. The resins

were first washed with distilled water till neutralization.

100mg of resin was placed in a beaker containing 25 ml

of deionised water and allowed to swell for 60 min.

Accurately weighed 100 mg of Levocetrizine

dihydrochloride was added to the resin solution and

stirred for 360 min. The mixture was filtered through

Whatman filter paper no. 41 and residue was washed

with 75 ml of deionised water. Unbound drug in filtrate

was estimated at 230.5 nm and drug-loading efficiency

was calculated.

Optimization of Levocetrizine-Tulsion 335

Complexation: The drug loading onto resin was

optimized by considering various parameters such as

concentration of resin, swelling time of resin, stirring

time, pH of resin solution and stirring temperature.

These parameters were studied and optimized for the

maximum amount of drug loading.

Optimization of concentration of resin for drug

loading: 10, 11

The resin which showed the highest amount of drug

loading was then optimized for various Drug: Resin

concentrations varying from 1:1 to 1:5. Accurately

weighed Levocetrizine dihydrochloride (100 mg) was

added to the 100, 200, 300, 400 and 500 mg of Tulsion

335 respectively. The best ratio showing maximum

adsorption of drug was then optimized.

Effect of swelling time on drug loading: 11

Separate batches of Tulsion 335 (400 mg) were soaked

in 25 ml of deionised water contained in a beaker for

10, 20, 30, 40, 50, 60 and 120 minutes. The

complexation in batch process was performed and the

loading efficiency with resin swollen at different time

was determined.

Optimization of stirring time, pH, temperature on

maximum drug loading:9,10,11

For Optimization of stirring time on drug loading,

accurately weighed levocetrizine dihydrochloride (100

mg) was added to 400 mg of Tulsion 335 solution and

slurred in 25 ml of deionised water. Six batches with

stirring time of 30, 60, 120, 180, 240, 300, 360 and 420

mins were processed. Amount of bound drug at the end

was estimated at 230.5 nm by UV spectroscopy and the

time required for maximum adsorption of drug was

optimized.

For Optimization of pH on drug loading, accurately

weighed, 100 mg of drug was added to 400 mg of resin

solution in 25 ml of deionized water. The pH of the

solution was maintained at 1.2, 2, 3, 4, 5, 6, 7, 8, and 9

using standard solution of hydrochloric acid and

sodium hydroxide and maintained at 25º C. The drug

loading efficiency at particular pH was estimated.

For Optimization of temperature on drug loading

levocetrizine dihydrochloride- Tulsion 335 complex

formulations were carried out at temperature range


321

from 25 oC to 80 oC and the effect of temperature on

drug loading was studied.

Characterization of Resinate:

Infra Red (IR) Study:

The drug, resin and resinate were subjected to Fourier

Transform Infra Red (FTIR) studies to check the

interaction in the resinate. The KBr disk method was

used for preparation of sample and the spectra were

recorded over the wave number 4000 to 400 cm-1. Then

the spectra were comparatively analyzed for drug

interaction.

Differential Scanning Colorimetery (DSC) Study:

DSC studies were carried out using, Mettler Toledo

DSC 821e instrument equipped with an intracooler

(Mettler-Toledo, Switzerland). Indium/Zinc standards

were used to calibrate the DSC temperature and

enthalpy scale. The samples were hermatically sealed in

aluminium pans and heated at a constant rate of

200C/min over a temperature range of 25-3000C/min.

Inert atmosphere was maintained by purging nitrogen

gas at flow rate of 50 ml/min.

Determination of Drug content:

Resinate prepared by above process was evaluated for

the drug content. Resinate equivalent to 10 mg of drug

was stirred with 100ml of 0.1N HCl for 60 minutes, till

the entire drug leached out, then the solution was

filtered. Further dilutions were made with 0.1 N HCl

and the drug content was noted spectrophotometrically

at 231.5 nm using 0.1 N HCl as blank.

Taste evaluation: 9

Bitterness evaluation test was performed to compare the

bitterness of the drug-resin complex to that of

levocetrizine dihydrochloride. The healthy human

volunteers were used for evaluation of taste masking

and the feedback was obtained from all of them. Taste

evaluation was done by a panel of 10 members using

time intensity method. Sample equivalent to 5 mg i.e.

dose of drug was held in mouth for 10 sec. Bitterness

levels were recorded instantly and then after 10sec, 1, 2,

4, 6 and 8 minutes. Volunteer’s opinion for bitterness

values were rated by giving different score values i.e. 0:

no bitterness, 1: acceptable bitterness, 2: slight

bitterness, 3: moderately bitterness, 4: strong bitterness.

Descriptive statistics mean and standard deviation were

calculated for all variables. Paired t test was applied

using INSTAT software. Value P< 0.05 has been

considered as statistical significant level.

In vitro dissolution:

Resinate equivalent to 5 mg of drug was subjected to

dissolution studies using USP type II dissolution

apparatus at 100 rpm with temperature of 37 ± 0.5º C

and 900 ml 0.1 N HCl used as the dissolution medium.

Aliquot equal to 5 ml was withdrawn at specific time

interval and it was filtered through Whatman filter

paper no.41. Absorption of the filtered solution was

checked by UV spectroscopy at 231.5 nm and quantity

of drug released was determined periodically. The

testing was carried out in triplicate.

Formulation development:

Mouth dissolving tablets of levocetrizine

dihydrochloride were prepared using drug-resin

complex, Crosscarmellose sodium, microcrystalline

cellulose, spray dried mixture of starch and lactose

(Starlac) by direct compression technique. Each

formulation was composed of drug and Excipients in

various proportions as shown in table 1. All ingredients

were passed through mesh no.60. Required quantity of

each was taken for particular formulation (Table 1) and

the blend was mixed by using laboratory mixer. Powder

blend was evaluated for micromeritic properties like

Angle of Repose, Bulk Density, Tapped Density,

Powder Flow Properties and Porosity.13,14 Mixed blend

of drug and excipients was compressed on 16-station

rotary punch tablet machine (Cadmach India). Tablets,

each weighing 150 mg, were prepared.

Evaluation of Tablets:

The prepared tablets were evaluated for various official

and nonofficial specifications.

Weight Variation

Twenty tablets were selected at a random and average

weight was calculated. Then individual tablets were

weighed and the individual weight was compared with

an average weight.

Hardness, Friability and Content Uniformity Test 14, 15,16

Tablets were evaluated for hardness and friability

testing using Monsanto hardness tester and Roche

Friabilator respectively.

Content uniformity test were done as per procedure

given below:


322

One tablet was crushed and 1 ml of dilute hydrochloric

acid and 30 ml of water was added and shaked for 15

minutes. Sufficient water was added to produce 50 ml

and centrifuged. To 5 ml of the clear supernatant liquid

10 ml of 0.1 M hydrochloric acid and sufficient water

was added to produce 100ml and the absorbance of the

resulting solution at the maximum of about 231.5 nm

was recorded. Same procedure was followed for

remaining 9 tablets.

Water Absorption Ratio: 13, 15, 16

A piece of tissue paper folded twice was placed in a

small petri dish containing 6 ml of water. A tablet was

put on the tissue paper and allowed to wet completely.

The wetted tablet was then weighed.

Water absorption ratio, R, was determined using

following equation:

R = 100 x Wa – Wb / Wb

Where Wb = Weight of tablet before water absorption

Wa = Weight of tablet after water absorption.

In-vitro Dispersion Time: 15

Tablet was put into 100 ml distilled water at 37 ± 20C.

Time required for complete dispersion of a tablet was

measured with the help of digital tablet disintegration

test apparatus.

In-Vivo Dispersion Time:

In-Vivo dispersion time of a tablet was checked in

healthy human volunteers by putting a tablet on tongue

and time required for complete dispersion of a tablet

was checked.

Dissolution Study:

Dissolution rate was studied by using USP type II

apparatus (VEEGO Tablet dissolution test apparatus

DA-6D) under following experimental condition:

- 100 rpm

- 900 ml of 0.1 N HCl as dissolution medium.

- 37 ± 2 0C as a temperature of dissolution

medium.

Aliquot equal to 5 ml of dissolution medium was

withdrawn at specific time interval and replaced with

fresh medium for maintaining sink condition. Sample

was filtered and absorbance of filtered solution was

determined by UV spectroscopy at 231.5 nm.

Dissolution rate was studied for all designed

formulations and conventional marketed tablet.

RESULTS AND DISCUSSION

Sensory Test on Threshold Value of Bitter Taste for

levocetrizine dihydrochloride:

Panel of 10 members using time intensity method

determined the threshold bitterness value. From

majority of volunteers it was found that 100µg/ml was

the threshold concentration of bitter taste of

levocetrizine dihydrochloride.

Optimization of Levocetrizine-Tulsion 335

Complexation:

Levocetrizine dihydrochloride was loaded on Tulsion

335 by batch process. Batch process is simpler and

quicker than column process. Complexation between

drug and resin is essentially a process of diffusion of

ions between the resin and the surrounding drug

solution. As reaction is an equilibrium phenomenon,

maximum efficiency is best achieved in batch process.

Also, higher swelling efficiency in the batch process

results in more surface area for ion exchange10. Hence,

the batch process was selected.

The drug loading in various drug: resin concentration

was found to be 77.20 ± 0.12, 81.30 ± 0.22, 86.90 ±

1.05 and 91.08 ± 0.76 respectively for 1:1, 1:2, 1:3 and

1:4 ratio. Swelling time showed the significant effect on

drug loading showing that the swelling of resin

enhances the drug loading capacity of resin. Percent

drug loading with swelling time of 10, 20, 30, 40, 50,

60 and 120 minutes was found to be 67.50 ± 0.363,

72.81 ± 0.758, 79.48 ± 0.942, 83.78 ± 0.169, 89.42 ±

1.03, 94.97 ± 0.827, 95.31 ± 0.495 % w/w respectively.

Thus the 60 minutes swelling time was used as

optimized time for drug loading. The swelling and

hydrating properties of Tulsion 335 affect the rate of

ion exchange, which in turn affects the percentage

loading. In unswollen resin matrix, the exchangeable

groups are latent and coiled toward the backbone, hence

less drug loading efficiency 10.

The equilibrium ion exchange in solution occurs

stoichiometrically and hence it is affected by stirring

time 10, 11. The % drug loading (w/w) with stirring time

of 30, 60, 120, 180, 240, 300, 360 and 420 min was

found to be 52.37 ± 0.311 65.45 ± 0.117%, 73.57 ±

0.754%, 79.27 ± 0.865%, 84.23 ± 0.547%, 89.58 ±

0.784%, 95.79 ± 0.592% and 96.01 ± 0.142%

respectively. These figures reveal that as time increased

percentage drug loading is increased rapidly upto 6 hr.

Increase in stirring time above 360 mins did not further

increase the percentage drug loading. Hence 360 min

contact time between drug and resin could be optimized


323

Table 1: Formulation Design

Tablet Ingredients (mg) A B C D E F G H I J

L-Cetirizine: Tulsion 335 Complex (1:4)

25 25 25 25 25 25 25 25 25 25

Ac-Di-Sol - - 3 4.5 6 7.5 3 4.5 6 7.5 Starlac 101 - 98 96.5 95 93.5 - - - - Avicel PH 102) - 101 - - - - 98 96.5 95 93.5 Aspartame 1 1 1 1 1 1 1 1 1 1 Mannitol 20 20 20 20 20 20 20 20 20 20 Tutti-Frutti 1 1 1 1 1 1 1 1 1 1 Magnesium stearate 0.75 0.75 0.75 1 1 1 0.75 0.75 0.75 0.75 Aerosil 1.25 1.25 1.25 1 1 1 1.25 1.25 1.25 1.25 Total 150 150 150 150 150 150 150 150 150 150

Table 2: Volunteers Opinion Test for Levocetrizine dihydrochloride before and after Taste Masking (n=10)

p < 0.001***

Table 3: Micromeritic properties of powder blend:

Evaluation Parameters Formulations Angle of

Repose Bulk

Density (g/cm3)

Tapped Density (g/cm3)

Percentage Compressibility

Porosity (%) Flowability

A 6.96

±0.270 0.5050 ±1.018

0.5464 ±1.109

8.57 ±0.883

12.24 ±0.391

Excellent

B 6.37

±0.423 0.5076 ±1.331

0.5551 ±1.760

8.62 ±0.943

9.61 ±1.130

Excellent

C 7.54

±1.403 0.4830 ±0.751

0.5347 ±1.368

9.66 ±1.759

13.19 ±1.219

Excellent

D 8.06

±0.498 0.4629 ±1.283

0.5208 ±0.182

11.31 ±0.730

15.13 ±0.628

Excellent

E 9.17

±0.813 0.4366 ±0.891

0.4975 ±0.759

12.22 ±1.208

15.85 ±1.313

Excellent

F 9.98

±0.257 0.4201 ±0.519

0.4830 ±1.020

13.02 ±0.402

15.94 ±1.268

Excellent

G 9.38

±0.575 0.4854 ±1.645

0.5464 ±0.480

11.16 ±2.07

12.06 ±0.759

Excellent

H 9.16

±1.207 0.4672 ±1.295

0.5291 ±0.495

11.68 ±0.421

13.0 ±0.437

Excellent

I 9.45

±0.915 0.4424 ±0.974

0.5102 ±0.883

13.27 ±1.04

14.75 ±1.004

Excellent

J 9.78

±1.573 0.4255 ±0.817

0.4926 ±0.810

13.61 ±0.915

15.02 ±1.172

Excellent

Time (seconds) Before taste masking Mean ± SD

After taste masking Mean ± SD

10 4.0 ± 0.48*** 0.2 ±0.63***

60 3.8 ± 0.43*** 0.1± 0.84***

120 3.4 ±0.51*** 0

240 3.0 ±0.42*** 0

360 2.5 ±0.42*** 0

480 1.9 ± 0.38*** 0

600 1.6 ± 0.08*** 0


324

Table 4: Evaluation of Tablet

Evaluation Parameters

Disintegration Time (sec)

Formulations Weight

Variation

(±%)

Hardness (Kg/cm2)

Friability (%)

Uniformity of Content (%)

Water Absorption

Ratio Invitro Invivo

A Passes 3.1

± 0.985

0.65

± 0.894

101.15

± 1.098

88.91

± 0.96

29

± 1.08

32

± 0.286

B Passes

3.2

± 0.199

0.62

± 0.172

99.76

± 1.045

52.03

± 0.392

56

± 1.034

68

± 0.859

C Passes 3.2

± 1.163

0.76

± 1.047

98.99

± 1.29 92.48 ± 0.195

24

± 0.863

28

± 0.534

D Passes 3.0

± 0.603

0.70

± 0.197

99.89

± 0.928 96.29 ± 1.09

22

± 0.682

25

± 0.146

E Passes 2.8

± 0.682

0.71

± 0.749

100.06

± 1.045

104.04 ± 0.946

21

± 0.516

24

± 1.016

F Passes 2.7

± 0.263

0.72

± 0.992

99.37

± 0.586

115.95 ± 0.503

19

± 0.938

22

± 0.638

G Passes 3.0

± 0.305

0.74

± 0.376

99.62

± 1.613

93.17 ± 0.52

33

± 0.123

38

± 0.659

H Passes

2.9

±0.756

0.75

±0.358

100.02

±1.419 95.97 ± 0.83

27

±0.256

32

± 0.364

I Passes 3.2

± 0.648

0.68

± 0. 982

101.05

± 2.195

100.02 ± 0.381

25

± 0.156

29

± 0.769

J Passes 2.9

± 0.733

0.64

± 0.594

99.73

± 0.914 113.5 ± 0.183

22

± 0.365

25

± 0.804

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9

pH

% D

rug

Lo

ad

ing

Drug:Resin (1:4)

Figure 1: Effect of pH on drug adsorption Figure 2: IR spectra of A. Levocetrizine dihydrochloride, B.

Tulsion 335 and C. Levocetrizine dihydrochloride: Tulsion

335 complex.


325

0

20

40

60

80

100

120

0 1 2 3 4

Time ( min.)

Cumulative % Drug Release

A

C

D

E

F

Figure 3: DSC curves for A. Levocetrizine dihydrochloride,

B. Tulsion 335 and C. Levocetrizine dihydrochloride: Tulsion

335 complex.

Figure 4: Dissolution profile of control tablet and tablets

consisting Ac-Di-Sol containing Starlac as filler binder

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Time ( min.)

Cumulative %

Drug Release

B

G

H

I

J

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16

Time (min)

Cu

mu

lati

ve %

dru

g r

ele

ase

Figure5: Dissolution profile of control tablet and tablets

consisting Ac-Di-Sol containing MCC as filler binder

Figure 6: Dissolution profile of conventional marketd tablet

to equilibrate ion exchange process to achieve

maximum drug loading.

Resin: drug complexation involved exchange of

ionisable drug and metal ion in resin. Such a mode of

complexation between drug and resin can be affected

by pH of media. Complexation was enhanced between

pH 2.5 to 4; a maximum of 95.25% w/w, drug loading

was obtained at pH 3. As pH increases above pH 5

percentage of drug loading decrease (Figure 1). pH of

the solution affects both solubility and degree of

ionization of drug and resin. Results can be attributed to

the fact that a cationic drug is ionized at lower pH value

and hence demonstrate high binding capacity while at

higher pH protonated fraction of cationic drug

decreases and hence interaction with resin also

decreases.10,11,17 Hence levocetrizine dihydrochloride as

a cationic drug will have maximum solubility and

complete ionization in this range. Decreased

complexation at lower pH i.e. below 2 is due to excess

H+ ions in solution which have more binding affinity to

the –COO- group of resin and compete with drug for

binding.

Efficient drug loading on Tulsion 335 occurred

uniformly (95.15% ± 0.80 w/w) in the experimental

temperature range of 250C to 800C. Increased

temperature during complexation increases the

ionization of drug and resin. The effect is more

pronounced for poorly water soluble and unionized

drugs. Higher temperatures tend to increase the

diffusion rate of ions by decreasing the thickness of

exhaustive exchange zone.10 As levocetrizine

dihydrochloride is water soluble drug and ionisable

drug temperature does not show any significant effect

on drug adsorption and also cation exchange resins are

not significantly affected by temperature changes.

The drug content in the resinate was found to be 99.1%.

The dissolution profile of drug showed complete drug

release within 2 minutes. Results of evaluation of taste

indicated complete masking of bitter taste as no

bitterness was felt in the drug-resin complex. (Table 2)


326

Characterization of levocetrizine dihydrochloride

and Tulsion 335:

Infrared (IR) study:

The interaction between the drug and the resin often

lead to identifiable changes in the IR profile of drug

dispersion. So levocetrizine dihydrochloride:Tulsion

335 (1:4) were subjected to IR analysis in order to

evaluate possible interaction between drug and Tulsion

335. From IR data, pure drug shows C=O, aromatic C-

H, C-O, C-Cl, and C-N streching, at 1720-1740 cm-1,

3000-3020 cm-1, 1130-1140 cm-1, 800-810 cm-1, 1300-

1330 cm-1, 1160-1190 cm-1 respectively. Tulsion 335

shows C=O, aromatic C-H steching at 1720-1740 cm-1,

3100-3150 cm-1 respectively , and C-H bending at

1200-1250 cm-1. While the drug resin-complex show

similar peak as that of pure drug therefore it indicates

there was no interaction between ionizable drug and

cations in Tulsion 335. (Figure 2.)

Differential Scanning Calorimetry (DSC) study:

DSC studies revealed that endothermic peaks for pure

levocetrizine dihydrochloride and Tulsion 335 (figure

3) were obtained at 221.94 ° C and 230.5 ° C

respectively. Endothermic peak of levocetrizine

dihydrochloride indicates its amorphous nature.

Thermogram of Drug: Tulsion 335 solid dispersion

showed decrease in height and sharpness of peak of

levocetrizine dihydrochloride indicating its successful

masking by Tulsion 335 (Figure 3). As the entrapped

drug is dispersed monomolecularly in the resin bead

there is decrease in the sharpness and height of peak.

Thus, DSC studies confirm amorphous state of drug,

interaction between ionizable drug and cations of resins

and succesful masking taste of drug by resins.

Formulation of Orodispersible tablets using

Resinate:

The batches of controlled formulations and

formulations containing superdisintegrant were

designed, using higher and lower concentrations of Ac-

Di-Sol and employing different filler, binders, MCC

PH-102 and Starlac and other excipients and

compressed on tableting machine.

For each designed formulation, blend of drug and

excipients was prepared and evaluated for micromeritic

properties (Table 3). Bulk density was found to be

between 0.42 - 0.50 gm/cm3 and tapped density

between 0.48 - 0.55 gm/cm3 for all formulations. From

density data, % compressibility was calculated and was

found to be between 8.57% - 13.61%. Flowability of

the material was found to be excellent. Porosity was

found to be between 9.64% - 15.85%. Angle of repose

was found to be in the range of 6.370- 9.980. As it is

below 300, it indicates good flow properties of blend.

Tablets were prepared by direct compression technique

and evaluated for various official and nonofficial

parameters. As the material was free flowing, tablets

were obtained of uniform weight due to uniform die fill,

with acceptable variations as per I.P. specifications, i.e.

below 7.5%. The hardness, friability and uniformity of

content are given in table 5. Hardness of the tablets for

each formulation was between 2.7 - 3.2 kg/cm2.

Friability below 1.0% was an indication of good

mechanical resistance of the tablets. The uniformity of

content was found to be 99%-101% which was within

acceptable limits.

Ac-Di-Sol is one of the superdisintegrant having

excellent disintegrating ability. Ac-Di-Sol is made by

cross-linking (etherification) reaction of Sodium CMC.

This cross linking greatly reduced water solubility of

Sodium CMC while permitting material to swell and

absorbs water many times its weight without loosing

fiber integrity. It swells to large extent to disintegrate

tablets and has fibrous nature that allows

interparticulate as well as extraparticulate, wicking of

water even at low concentration.18,19 The water

absorption ratio, invivo and in vitro dispersion time are

given in Table 4. It was observed that as concentration

of Ac-Di-Sol increases water absorption ratio increases

and invivo and invitro dispersion time decreases. But in

the formulations consisting Starlac as filler and binder

it was observed that Ac-Di-Sol does not seem to have

much significance effect on the water absorption ratio

and dispersion time of tablets. This may be due to the

fact that Starlac consists of 15% starch and 85%

lactose.21 Starch has good disintegrating ability and

thus enhances water uptake and dispersion ability. As

both starch and lactose are water soluble, the pores

along the Ac-Di-Sol fiber will be enlarged by the

dissolution of starch and lactose, so the swelling of Ac-

Di-Sol will have less effect on destruction of the tablet

matrix 19 compared with the tablets consisting MCC as

filler binder. In tablets consisting MCC as filler binder

Ac-Di-Sol shows significant effect on water absorption

and dispersion time. This may be attributed to the fact

that MCC is a swellable material and its disintegration


327

characteristics in water have been attributed to capillary

action or swelling 18, 19. Thus as Ac-Di-Sol which has

excellent swelling ability enhances the dispersion and

water uptake ability consisting MCC.

Dissolution rate was studied for all the tablet

formulations and conventional marketed tablet. For, all

designed formulations the tablets show about 100%

drug release within 6 minutes, while conventional

marketed tablet required 16 minutes for complete

release of drug (figures 4, 5 and 6). The formulations E

and F consisting Starlac as filler binder and Ac-Di-Sol

showed complete drug release drug within 2 minutes

(figure 4) and formulations I and J consisting MCC as

filler binder and Ac-Di-Sol showed complete drug

release within 3 minutes (figure 5). The drug release

from all formulations was too fast as compared to the

conventional marketed tablet.

CONCLUSION

Levocetrizine dihydrochloride, a bitter drug could be

successfully taste masked using suitable ion exchange

resin. The process of taste masking was optimized with

respect to parameters like time required for

complexation, pH effect, swelling time of resin and

temperature. The taste masked complex was

incorporated into patient compliant and palatable

orodispersible tablets. Tablets formulated with Starlac

as filler binder showed faster disintegration and drug

release.

ACKNOWLEDGEMENTS

The authors wish to thank Emcure Pharmaceuticals

Ltd., Pune, India and Thermax India Ltd., Pune, India

for providing drug and Tulsion 335 respectively. The

authors thank Signet Chemical Corporation, Ltd.,

Mumbai, India for providing excipients and Torrent

pharmaceuticals Ltd., Gandhinagar, India for providing

DSC facility.

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4. Joshi A A, Duriez X. Added functionality

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5. Gowthamarajan K, Kulkarni G T, Kumar N M. Pop

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Kadam S. Molecular properties of ciprofloxacin–

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62, 5(4): 1-8.

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Dosage Form Design, Second Edition, Churchill

Livingstone, 2002, 200-206.

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dissolving tablets of sumatriptan succinate. Indian

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Delhi, 1996, 736.

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dissolving tablets of salbutamol sulphate : A novel

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Dev.Ind.Pharm. 1999; 25(5): 571-581.

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***********


329

A study on the effect of different polymers on frusemide

loaded calcium alginate micropellets prepared by ionotropic

gelation technique Ghosh Amitava *, Nath L.K

1, Dey B.K and Roy Partha

Himalayan Pharmacy Institute, Majitar, East Sikkim- 737136 1 Dept. Of Pharmaceutical Sciences, Dibrugarh University, Assam-

E- Mail: [email protected]

Abstract

The objective of this study was to encapsulate drugs in polymers of varying solubility in an absolute aqueous

environment. The micropellets were prepared using ionotropic gelation technique, where gelation of anionic

sodium alginate, the primary polymer , was achieved with oppositely charged counterion to form microparticles

which were further made sustained by using different polymers namely Methocel K- 15M (Hydroxy propyl

methyl cellulose), Surelease (Ethyl Cellulose) and Acrycoat E30D (poly [ethyl acrylate methyl methacrylate]).

The effect of these polymers on the release profile of the drug has been reported in this paper. Frusemide, a

potent diuretic, was selected as the model drug for the experiments. Nine set of formulations were prepared

using Acrycoat E30D (E1, E2, E4); Methocel K-15M (H1, H2, H4) and Surelease (S1, S2, S4) at concentration

(1%, 2%, 4%w/w). The final formulations were subjected to several characterization studies. Batches with

Acrycoat E30D and Surelease shown zero-order release whereas batches with Methocel K-15M followed

Higuchi model. All the batches sustained the release of the drug for more than 8 hours. Both water soluble and

insoluble copolymers were tested and among them acrylic colloidal polymer dispersion (Acrycoat E30D)

showed high encapsulation efficiencies and maximum prolongation of drug release.

Key words- Micropellets, ionotropic gelation, Frusemide, acrylic polymers

INTRODUCTION

One of the common methods of controlling the rate of

drug release is microencapsulation. The encapsulation

techniques (e.g., solvent evaporation or coacervation–

phase separation) normally involves water insoluble

polymers as carriers which require large quantity of

organic solvents for their solubilization1, 2. As a result

the processes become vulnerable to safety hazards,

toxicity and increases the cost of production making the

techniques non reproducible, economically and

ecologically at an industrial scale. These concerns

demand a technique free from any organic solvent.

Thus, the objective of this study was to encapsulate

drugs of varying solubility within water insoluble

polymers in an absolute aqueous environment.

Recently, aqueous polymeric dispersions have played a

great role in replacing organic solvents in the coating of

solid dosage forms with water soluble polymers3-5.

These polymeric dispersions forms a homogenous film6

on drying and provides a diffusion controlled release of

the drug from the polymer matrix.

The micropellets were prepared using ionotropic

gelation technique7-9 where gelation of anionic

polysaccharide sodium alginate, the primary polymer of

natural origin, was achieved with oppositely charged

calcium ions (counterion)10 to form instantaneous

microparticles. The micropellets thus produced were

further made sustained by using different polymers

namely Methocel K- 15M (Hydroxy propyl methyl

cellulose -HPMC) - a water soluble polymer; Surelease

(Ethyl Cellulose) – a semi synthetic water insoluble

aqueous colloidal polymer; and Acrycoat E30D (poly

APTI ijper





330

[ethyl acrylate methylmethacrylate]) – a synthetic water

insoluble aqueous polymeric dispersion. The effect of

these polymers of varying solubility and other

physicochemical properties, on the release profile of the

drug has been studied and reported in this paper.

Frusemide11 was selected as the model drug for the

experiments. It is a potent high ceiling loop diuretic

agent commonly indicated for the treatment of edema

of hepatic, cardiac and pulmonary systems during acute

or chronic renal failure. In low dose it is a drug of

choice for the treatment of chronic hypertension12. It

shows a prompt onset of action and produces a peak

diuresis far greater than that observed with other

diuretic agents12. This intense diuresis from a

conventional tablet provokes major side effects like

electrolytic imbalance manifested in the form of

tiredness, dehydration and muscular cramps13. The drug

is practically insoluble in water and has a biological

half life of 2 hr in patients with renal insufficiency11.

The aim of the experiment was to produce sustained

release micropellets of Frusemide, that can be tabletted

or capsulated, exhibiting the same diuretic effect as that

of a conventional tablet, but eliminating the toxicity,

patient discomfort and non compliances.


Frusemide was received as a gift sample from Aventis

Pharma Ltd., Ankleshwar. Sodium alginate (viscosity

of 2% aqueous solution at 25°C was 3500cps) was

obtained from Loba Chemie, Mumbai. Calcium

chloride dihydrate (A.R. Grade, E.Merck, Germany);

Acrycoat E30D, aqueous dispersion (solid content-

28.7%w/w) (Corel pharmaceuticals, Ahmedabad);

Surelease (Ethyl Cellulose) (Colorcon Ltd.) Hydroxy

propyl methyl cellulose (HPMC) (Methocel K- 15M)

(Dow Chemical Co.). All other chemicals were

purchased from local supplier in A.R. and L.R. Grade

as required.

Preparations of micropellets

The drug (30% w/w) was dispersed uniformly in

aqueous mucilage of sodium alginate (2% w/v) using

mechanical stirrer maintaining the speed at 500-600

rpm. To this dispersion the desired polymer was mixed

in suitable proportions and the entire mixture was

stirred for 30 min. The pellets were formed by dropping

the bubble free dispersions through a glass syringe into

a gently agitated calcium chloride (5% w/v) solution

100 ml. The gelled pellets were cured for 30 min before

being filtered and washed thoroughly with distilled

water. They are then oven dried for 6 hr at 60°C. The

#22 I.P. standard sieve size fractions were used for

further studies.

Process variables and Process optimization

The following process variables were investigated

(concentration of sodium alginate; concentration of

calcium chloride; curing time; height of dropping;

variation of drug loading; stirring speed and stirring

time) and the different batches thus produced were

analyzed for size, shape, ease of preparation, drug

content and drug release. On the basis of the result

obtained the process parameters were optimized as

follows:-

Sodium alginate concentration – 2% w/v

Calcium chloride concentration – 5% w/v

Drug load – 30% w/w

Curing time – 30 min

Height of dropping – 2 cm from the level of CaCl2

solution

Stirring time and speed – 30 min & 500 rpm

Drying condition – oven drying for 6 hr at 60°C

Different batches of micropellets were then prepared by

using the optimized process variables and the only

variation followed was use of different polymers. Nine

set of formulations were prepared using Acrycoat E30D

(E1, E2, E4); Methocel K-15M (H1, H2, H4) and

Surelease (S1, S2, S4) at concentration (1%, 2%,

4%w/w). The final formulations were subjected to

several characterization studies.

Characterization of micropellets

Particle size determination

Particle size analysis14 of the micropellets was done by

sieving method using Indian Standard Sieves # 16, #22

and #30. Average particle size was calculated using the

formula: - davg = ∑ dn / ∑ n, where n=frequency weight,

d= mean diameter. (Table-1)

Scanning electron microscopy

Morphological characterization of the micropellets was

done by taking scanning electron micrograph in (JEOL

JSM Model 5200, Japan). Cross sectional view were

obtained by cutting the micropellets with a razor blade.

The samples were coated to 200 A° thickness with

gold-palladium using (Pelco model 3 sputter coater)

prior to microscopy. A working distance of 20 nm, a tilt

of 0° and accelerating voltage of 15 kv were the

operating parameters. Micropellets before dissolution


331

were only subjected to SEM study since, after

dissolution the pellets become swollen palpable mass.

Photographs were taken within a range of 50 - 500

magnifications. (Fig 1-5)

Micromeretic study14

To determine the rheological properties of the

micropellets, the angle of repose of all the samples were

measured using funnel method. Bulk density was

determined by taking known quantity of micropellets in

100 ml measuring cylinder and tapping it 3 times from

a height of 1 inch at 2 seconds interval. The bulk

density was calculated by dividing sample weight by

final bulk volume. (Table-1)

Determination of Moisture content14

The formulations were subjected to moisture content

study, by placing the micropellets at 60° C for 10

minutes in an IR moisture balance. (Table-1)

Surface Accumulation study (SA) 15

This study was conducted to estimate the amount of

drug present on the surface of the micropellets which

may show immediate release in the dissolution media.

100 mg of micropellets (# 22 sizes) were suspended in

100 ml of phosphate buffer (pH 6.8), simulating the

dissolution media. The samples were shaken vigorously

for 15 min in a mechanical shaker. The amount of drug

leached out from the surface was analyzed

spectrophotometrically at 277.5 nm. Percentage of drug

released with respect to entrapped drug in the sample

was recorded. (Table-1)

Determination of Drug Entrapment Efficiency

About 100 mg of micropellets (# 22 sizes) were

accurately weighed and dissolved in 25ml of Phosphate

buffer (pH 7.4) for overnight and an aliquot from the

filtrate was analyzed spectrophotometrically, after

suitable dilution, using SHIMADZU UV-VIS, at

277.5 nm. Reliability of the method was judged by

conducting recovery analysis using known amount of

drug with or without polymer. Recovery averaged

100±0.89 %. Drug content of every batch was

determined for every size range of micropellets and the

mean± S.D.was calculated. Drug Entrapment Efficiency

(DEE) was calculated according to the formula %

DEE= (Actual drug content/ Theoretical drug content) x

100. (Table-2)

Disintegration studies9

Disintegration studies were performed in 0.1N HCl and

simulated intestinal fluid (USP XXI) in a rotating bottle

apparatus. 5 pellets per vial were kept in 50 ml medium

at 37° C and the vials were rotated at 25 rpm. The

measured disintegration time was the time taken by the

pellets to disintegrate into crystals, the polysaccharide

being soluble and the drug insoluble in the

disintegrating fluid. (Table-2)

In vitro dissolution study

The USP rotating – paddle Dissolution Rate apparatus

(Veego, Mumbai) was used to study drug release from

the micropellets. The dissolution parameters [ 100 mg

pellets ; 37± 2°C ; 50 rpm ; 500 ml of USP Phosphate

buffer (pH 6.8); n=3; coefficient of variation< 0.05]

were maintained for all the nine formulations. 2 ml of

aliquot were withdrawn at specified intervals and after

suitable dilution assayed by SHIMADZU UV-VIS

PharmSpec 1700 spectrophotometer at 277.5 nm. The

data for percent drug release was fitted for zero order

and Higuchi matrix equation. The polysaccharide did

not interfere with the assay as confirmed from

conducting a dissolution study of blank alginate beads.

(Table-2)

Determination of stability of the micropellets

The formulations showing the best performance, with

respect to in vitro release, from each polymer

composition were stored at 4°C, room temperature and

45°C for a month. Every week samples were withdrawn

and were assayed spectrophotometrically at 277.5 nm

using Phosphate buffer (pH 6.8) as blank. (Table- 3)

Study on Drug – Polymer interaction using Infra

Red Spectroscopy14

The disc method was employed to study the possible

interactions between the drug and the selected polymer

Acrycoat E30D and sodium alginate. Pure drug, blank

alginate pellets and drug loaded pellets with Acrycoat

E30D pellets were separately analysed. KBr (IR Grade)

discs in a proportion of 1: 100 :: Sample : KBr, were

prepared from the samples and are analyzed in FTIR

spectrophotometer (SHIMADZU FTIR - 8400S, Japan)

over a range of 400- 4000 cm-1. Transmittance (T)

spectra were recorded and displayed in an overlay mode

in Figure 10.


The micropellets were prepared in an environment free

from organic solvents, by dropping a mixture of

colloidal copolymer dispersion, the dispersed drug


332

Frusemide, and mucilage of sodium alginate in calcium

chloride solution, which acted as a counterion. The

droplets instantaneously formed gelled spherical beads

due to cross linking of calcium ion with the sodium ion

which remained ionized in the solution. Smaller particle

can be prepared by adjusting the height of the syringe

from the level of counterion solution, compression

force on the plunger of the syringe. The gelled particles

were cured to get sufficiently hardened and then filtered

and dried. The colloidal polymer particles fused into the

polymer matrix during drying with the drug being

dispersed in the latex. The micropellets thus formed

using three different polymers did show significant

results on evaluation.

The size of the micropellets ranged between 600 µm to

800 µm and increased significantly with the

concentration of the copolymers. The average particle

size was on the highest side with Acrycoat polymers

followed by Surelease. The particle size distribution

was uniform and narrow. It can be estimated that with

further increment in the copolymer concentration the

particles would change from micro to granular level.

The scanning electron micrograph (Figure 6-8) shows

the pellets being discoid in shape. Surface depression

was noticed at the point of contact on the drying paper.

On comparison of the pellets prepared from three

polymers in highest concentration, it was evident from

the photograph that more roughness with Acrycoat

copolymers was achieved than that of the other two. It

can be concluded that the roughness is due to the

density of the matrix which in turn justifies its sustained

release. The dense network of drug-polymer-copolymer

increases the tortuisity, as evident from Figure-9, thus

delaying the release of the drug and retarding the

penetration of water required to make the pellets swell

for disintegration. The micrograph of the blank pellets

(Figure-5) act as a control and suggests that increase in

total weight of the pellets makes it more spherical.

The rheological parameters like angle of repose and

bulk density of all the pellets (Table-1) confirms better

flow and packing properties. Thus, the micropellets if

tabletted or encapsulated, requires less amount of

lubricants and ensures low production cost leading to its

feasibility for large scale production.

Surface Accumulation (SA) study was an important

parameter giving an indication of the amount of drug on

the surface of the micropellets without proper

entrapment. With the increase in the copolymer

concentration % SA decreased significantly owing to

high entrapment of drug in the dense network of

polymers.

Low moisture content in all the micropellets indicates

the effectiveness of the optimized drying condition.

Low moisture level ensures better stability of the drug

in the micropellets.

Significantly high entrapment efficiency of drug with

Acrycoat based formulations (Table-2) over other

polymers confirms it being more rigid among the three.

As described during the discussion on the

photomicrograph, the Acrycoat based formulations

showed highest disintegration time which may be due

to its stronger latex network structure. The micropellets

being less porous among the three, delays the

penetration of water needed for swelling and eventual

disintegration. No disintegration was observed in 0.1N

HCl, even when the samples were kept for overnight in

the medium. The ionic character of the polysaccharide

resulted in pH dependant disintegration of the

micropellets.

The in vitro release data of all the formulations were

fitted in Zero order and Higuchi matrix model and the

rate constants and correlation coefficient were

compared to get a trend in the release pattern of the

drug from the formulations. From Table -2 it is evident

that the batches with Acrycoat E30D (E1-E4) and

Surelease (S1-S4) predominantly shown zero-order

release whereas micropellets prepared with Methocel

K-15M followed Higuchi model. All the batches

sustained the release of the drug for more than 8 hours

(Figure 1-3) as compared with the conventional tablet

dosage form (Figure-4). Predominantly, the drug

release followed passive diffusion technique. On

releasing, the drug present on the surfaces leaves

behind pores or channels, through which diffusion of

the drug present in the inner matrix of the micropellets

occurred. Due to loose drug present on the surface of

the micropellets (Surface Accumulation) the in vitro

release profile obtained indicated a biphasic pattern i.e.

initial fast release followed by a sustained pattern.

Batches of Acrycoat micropellets showed more

prolonged action as evident from its t50 values when

compared with other two polymers. Increase in the

polymer concentration increased the crosslink density

thereby creating barrier for drug diffusion.


333

Table- 1 -Comparative study of various physical parameters for alginate micropellets containing frusemide and

release retarded with Acrycoat E30D, Hpmc and Surelease respectively

Formulation Composition (w/w)

Moisture content (% ± S.D.)

SA with respect to Entrapped Drug (%)

Mean Diameter (µm ± S.D.)

Angle of repose (θ ± S.D.)

Bulk density (gm/cc ± S.D.)

E1 Acrycoat E30D-1% 1.48 ± 0.48 3.549 608.16 ± 0.59 18.32 ± 0.79 0.658 ± 0.68 E2 Acrycoat E30D-2% 1.44 ± 0.56 2.369 760.89 ± 0.51 20.56 ± 1.03 0.674 ± 1.52 E4 Acrycoat E30D-4% 2.23 ± 0.68 1.567 782.78 ± 0.36 21.24 ± 1.97 0.682 ± 1.96 H1 HPMC – 1% 2.43 ± 0.74 3.728 589.25 ± 0.62 20.68 ± 1.05 0.629 ± 0.89 H2 HPMC – 2% 1.74 ± 0.81 2.988 667.58 ± 0.56 22.01 ± 1.88 0.641 ± 1.59 H4 HPMC – 4% 2.32 ± 0.91 2.142 759.26 ± 0.42 23.09 ± 2.57 0.652 ± 2.49 S1 Surelease- 1% 1.21 ± 0.77 3.589 601.69 ± 0.68 19.26 ± 1.36 0.657 ± 1.23 S2 Surelease- 2% 0.91 ± 0.68 3.008 728.14 ± 0.48 21.28 ± 1.98 0.669 ± 1.87 S4 Surelease- 4% 1.76 ± 0.59 2.459 776.19 ± 0.41 22.58 ± 2.89 0.678 ± 2.88

* n= 3, SA = Surface accumulation

Table- 2 - Comparative study of various pharmaceutical factors for alginate micropellets containing frusemide

and release retarded with Acrycoat E30D, HPMC and Surelease respectively

Formulation Composition

(w/w)

Drug

Entrapment

Efficiency

(% ± S.D.)

Disintegration

Time (min)

t50

( min )

Zero order Higuchi SQRT

K0 R2 KH R

2

E1 Acrycoat

E30D-1%

94.48 ± 0.48 42 272 11.628 0.9918 25.479 0.7939

E2 Acrycoat

E30D-2%

93.44 ± 0.56 64 290 11.175 0.9928 23.909 0.7857

E4 Acrycoat

E30D-4%

91.23 ± 0.68 97 341 11.131 0.9397 20.151 0.6492

H1 HPMC – 1% 82.43 ± 0.74 29 117 12.331 0.7806 29.928 0.9531

H2 HPMC – 2% 83.74 ± 0.81 38 120 11.948 0.7971 29.432 0.9464

H4 HPMC – 4% 89.32 ± 0.91 59 129 11.571 0.8841 30.356 0.9452

S1 Surelease- 1% 77.21 ± 0.77 28 158 11.899 0.8853 26.781 0.9057

S2 Surelease- 2% 80.91 ± 0.68 41 187 10.718 0.9576 25.102 0.9131

S4 Surelease- 4% 84.76 ± 0.59 77 316 08.883 0.9746 21.482 0.8386

* n= 3

K0 , KH – Release Rate Constants for Zero Order and Higuchi release Kinetic Model respectively

R2 – Correlation coefficient.

Table- 3 - Accelerated stability studies of frusemide micropellets prepared with different polymers

TIME S4 H4 E4

(WEEK) 4°C RT 45°C 4°C RT 45°C 4°C RT 45°C

0 100 100 100 100 100 100 100 100 100

1 98.28 97.31 84.39 98.83 97.75 86.41 99.28 98.52 89.28

2 96.93 96.62 81.11 95.97 96.13 85.13 97.57 96.93 88.42

3 93.11 94.26 78.44 94.33 95.07 84.21 96.62 95.89 86.74

4 91.37 92.57 76.41 93.78 93.89 83.37 95.09 95.37 85.33

RT- ROOM TEMPERATURE


334

INVITRO RELEASE OF SURELEASE RETARDED

ALGINATE MICROSPHERES

0

20

40

60

80

100

120

0.5 1 2 3 4 5 6 7 8 9

TIME (hr)

CU

MU

LA

TIV

E P

ER

CE

NT

RE

LE

AS

E

S1

S2

S4

INVITRO RELEASE FROM HPMC RETARDED

ALGINATE MICROSPHERES

0

20

40

60

80

100

120

0.5 1 2 3 4 5 6 7 8 9

TIME (hr)

CU

MU

LA

TIV

E P

ER

CE

NT

RE

LE

AS

E

H1

H2

H4

Figure 1 Figure 2

IN VITRO RELEASE OF ACRYCOAT E30D

RETARDED ALGINATE MICROSPHERES

0

20

40

60

80

100

120

0.5 1 2 3 4 5 6 7 8 9

TIME (hr)

CU

MU

LA

TIV

E P

ER

CE

NT

RE

LE

AS

E

E-1

E-2

E-4

IN VITRO RELEASE OF FRUSEMIDE FROM

CONVENTIONAL TABLET (40 mg)

y = 12.507x

R2 = 0.9034

0

20

40

60

80

100

120

5 10 15 20 25 30 35 40 50

TIME (min)

%C

um

ula

tiv

e r

ele

as

e

Figure 3 Figure 4

Figure 5- SEM Photograph of blank alginate micropellets

(50X)

Figure 6- SEM Photograph of Frusemide loaded alginate

micropellets with Acrycoat E30D (4%w/w) (50X) Formulation

# E4


335

Figure 7 - SEM Photograph of Frusemide loaded alginate

micropellets with Surelease (4%w/w) (50X) Formulation # S4

Figure 8 - SEM Photograph of Frusemide loaded alginate

micropellets with Methocel K-15M (4%w/w) (50X)

Formulation # H4

Figure 9- SEM Photograph of Frusemide loaded alginate

micropellets with Acrycoat E30D (4%w/w) (350X) Formulation

# E4

FIGURE 10 - IR Spectra overlapped (Frusemide + Blank

Alginate pellets + Final pellets with Acrycoat E30D)

When studied for stability at 4°C, room temperature

and 45°C for a month, the drug was found to be stable

at 4°C and room temperature for all the formulations

but showed gradual degradation at high temperatures.

From the infrared spectra (Figure 10) it is clearly

evident that there were no interactions of the drug. The

main peaks in the spectrum of the drug Frusemide like

1143.83 and 1323.21 /cm for S=O bond; 1674.27 /cm

for C=O bond; 3487.42 /cm for N-S bond and 582 for

C-Cl bond remained undisturbed in the final

formulation. This proves the fact that there is no

potential incompatibility of the drug with the polymers

(alginate and Acrycoat E30D) used in the formulations.

Hence, the formula for preparing Frusemide loaded

Calcium alginate microspheres can be reproduced in the

industrial scale without any apprehension of possible

drug- polymer interactions.

In conclusion, sustained release micropellets containing

water insoluble drug were successfully prepared

employing ionotropic gelation technique entirely

avoiding the use of organic solvents. Apart from the

natural water soluble polymer, the use of copolymer

further prolongs the release of the drug. Both water

soluble and water insoluble copolymers were tested and

among them acrylic based colloidal polymer

dispersions (Acrycoat E30D) showed high

encapsulation efficiencies and maximum prolongation

of drug release. A further study using different acrylic

polymers are on progress for new revelations.

Considering the end product, the micropellets could be


336

administered as prepared or could be compressed into

tablet or filled in capsule shell. The entire process is

feasible in an industrial scale and demands pilot study.

ACKNOWLEDGEMENTS

Authors wish to thank Aventis Pharmaceuticals

(Ankleshwar, India) for providing gift sample of

Frusemide and Corel Pharmaceuticals (Ahmedabad,

India) for providing gift sample of Acrycoat E30D. We

are also thankful to the staff of University Science

Instrumentation centre (USIC), Jadavpur

University,Kolkata for their relentless cooperation in

SEM study.

REFERENCES 1. Bodmeier R, Chen H. Pseudoephedrine HCl

microspheres formulated into an oral suspension

dosage form.J.Controlled release. 1991; 15: 65-77

2. Baken JA.Microencapsulation.In: Lachman and

Lieberman (Eds) The Theory and Practice of

Industrial Pharmacy. 3rd ed. Varghese Publishing

House, Mumbai; 1987; 412-429

3. Lehmann KOR. In. Aqueous Polymeric Coatings

for Pharmaceutical Applications.McGinity JW. eds.

New York: Marcel Dekker 1989;153-245

4. Steuernagel CR. In. Aqueous Polymeric Coatings

for Pharmaceutical Applications.McGinity JW. eds.

New York: Marcel Dekker 1989; 1-61

5. Bodmeier R, Wang J.Microencapsulation of Drugs

with Aqueous Colloidal dispersion.

J.Pharm.Sci.1993;82(2):191-194

6. JamesW.McGinity, Aqueous polymeric coatings

for pharmaceutical dosage forms, 2nd ed. 1997;

355,381

7. Lim F, Sunn AM. Microencapsulated islets as

bioartificial endocrine pancreas. Science.1980; 210:

908-910.

8. Segi N, Yotsuyanagi T. Ikeda K. Interaction of

Calcium- induced Alginate Gel beads with

Propranolol. Chem Pharm Bull, 37 (11), 1989,

3092-3095.

9. Bodmeier R, Paeratakul O. Spherical Agglomerates

of water insoluble drugs. J.Pharm. Sci. 1989; 78:

964-967.

10. Lim LY, Wan SC. Propranolol hydrochloride

binding in calcium alginate bead. Drug Dev Ind

Pharm. 1997; 23 (10): 973-980.

11. Abdurrahman MA, Fahad JA, Khalid AMA.

Frusemide. Analytical Profiles of Drug Substances

and Excipients. Academic Press Inc. ed Florey

K.1992; 18: 153-193

12. Gilbert HM. The Pharmacological basis of

Therapeutics. 6th ed. New York: McMillan Co.

Inc.;1975; 903

13. Martindale. The Extra Pharmacopoeias. 31st ed:

870-874

14. Indian Pharmacopoiea. 5th ed.Controller of

Publication.1996; 328-329,393-395

15. Abu IK, Gracia CL, Robert LD. Preparation and

evaluation of zidovudine-loaded sustained-release

microspheres. J Pharm Sci. 1996; 85(6): 575-576

*************


337

Phytochemical investigation and Immunomodulator activity

of Amaranthus spinosus linn. Tatiya A.U,* Surana S.J, Khope S.D, Gokhale S.B, and Sutar M.P.

*Dept. of Pharmacognosy, R. .C. Patel College of Pharmacy, Shirpur- 425 405, India.

E-mail: [email protected]

Abstract

Amaranthus spinosus Linn. is a herbaceous plant studied for its immunomodulatory activity by cell-mediated

immune response (CMIR) measured by delayed type of hypersensitivity reaction to SRBC and Humoral immune

response (HIR) measured by hemagglutination antibody titre. Among the various leaf extracts the aqueous and

alcoholic extracts showed a significant elevation in humoral as well as cell-mediated response and Pet. ether

extract significantly reduced humoral as well as cell-mediated response. The results indicate the potential of

Amaranthus spinosus Linn. as an immunomodulatory agent.

Key wards- Amaranthus spinosus , Phytochemical, Cell mediated, Humoral .

INTRODUCTION

Immunomodulation is a process that can alter the

immune system of an organism by interfering with its

function. If it results in an enhancement of immune

reactions named as immunomodulatory. While

immunosuppressant implies mainly to reduce resistance

against infection, stress and may occur on account of

environmental or chemotherapeutic factors 1,2.

Immunomodulatory agents may selectively activate

either cell mediated or humoral immunity. The primary

target of the immunomodulatory compounds is believed

to be the macrophages, which play a key role in the

generation of an immune response. Traditional Indian

System of medicine (Ayurveda, Siddha, and Unani)

suggests means to increase the body’s natural resistance

to disease. A number of Indian medicinal plants and

various ‘Rasayanas’ (Charak Samhita, 1000B.C) have

been claimed to possess immunomodulatory activity.

Rasayanas are a group of non-toxic herbal drug

preparations that stimulates the immune response of the

human.

Amaranthus spinosus Linn. is a herbaceous plant and

is a constituent of herbal formulation LEUCOSOL-H

which is found to be effective in leucorrhoea.3 The

fresh juice of leaves was used for snakebite, rat bite and

insect bite poisoning 4. Fresh young leaves are used as

laxative 5. Leaf paste is used as antiseptic 6. The boiled

leaves & root gives an emollient effect also helpful in

case of abscesses, boils and burns. Water extract of

Amaranthus spinosus directly stimulates the

proliferation of B lymphocytes in vitro2 ,it suggest that

the immuno-stimulamnt effects of amaranthus might

be lead to B-lymphocytes activation and subsequent T

cell proliferation in vitro. These results are potentially

valuable for future Nutraceuticals and immuno-

pharmacological application of amaranthus. The present

study was undertaken to investigate in vivo

immunomodulatory potential of Amaranthus spinosus

Linn. and estimation of total cardiac, anthraquinone and

saponin glycosides present in it .


The plant was collected from the local areas of Shirpur

and authenticated by Dr. D.A Patil (Dept. of Botany)

Taxonomist. at S.S.V.P.S College, Dhulia (M.S) .The

voucher specimen no RCP /11 was preserve in the

Department of Pharmacognosy.

Preparation of extract

Amaranthus spinosus powdered material was subjected

to extraction using 95% ethyl alcohol. The extract was

concentrated under reduced pressure. According to

qualitative chemical tests extract was further

APTI ijper


Received on 21/11/06 ; Modified on 21/5/2007



338

fractionated into pet ether (60-800C) and chloroform

extract for separation of non-polar

components.Remaining marc was subjected to

maceration in aqueous to obtained polar components.

All the extracts were concentrated under vaccum

evaporator (Roteva, equitran). Percentage yield of

alcoholic (9.6 %), petroleum ether (2.5 %), chloroform

(0.4 %) and aqueous extract (30.4 %) was reported.

Phytochemical investigation

The qualitative chemical tests of aqueous, alcoholic,

pet.ether and chloroform extract of Amaranthus

spinosus was carried out using standard procedure to

determine the presence of various phytochemicals.

Aqueous extract was subjected to quantitative

estimation of cardiac glycoside, anthraquinone

glycoside and saponin glycoside .7, 8

Experimental protocols

Requirements

Phosphate buffered saline, Sheep red blood cells

(SRBC), Alsevier’s solution, micro titer plate,

compound microscope (Metzer), Digital

plethysmometer (UGO Basile Italy), Gum acacia

solution and micropipette.

Animals

Male Swiss albino mice (25-30gm) were used for the

activity. They were housed under controlled conditions

of light and dark cycle (12:12) and temp (25±20C). All

animals had access to standard

pelleted food and water. Working protocol was

approved by Ethical committee No 651/02/BC/

CPCSEA.

Acute toxicity studies

The acute toxicity of all extracts was evaluated in mice.

The animals were fasted overnight prior to the acute

toxicity study. Different groups containing 2 mice in

each were orally administered with

aqueous, alcoholic, chloroform and pet ether extract at

0.5, 1, 1.5, 2 and 3gm/kg, p.o. respectively.Mortality

and general behavior of the animals were observed

continuously for initial four hour and then at 24 hours

and 48 hours after dosing.The parameter observed and

recored were sedation, grooming, loss of righting

reflex, respiratory rate and convulsion.1/10th of lethal

dose was taken as the screening dose.9

Antigen

SRBC – The blood was withdrawn from the external

jugular vein of sheep. It was mixed with Alsevier’s

solution in 1:1 proportion and was stored at 40C.

Cell-mediated immune response to SRBC (Delayed

Type Hypersensitivity) 10,11

Animals were divided into 5 groups, each group

containing 6 animals immunized on day 0 by i.p.

administration of 0.5 X 109 SRBC/ml. The suspensions

of solvent dried extracts of aqueous, alcoholic, pet.

ether and chloroform were prepared in gum acacia

solution( 0.5%) and administered orally to mice from

day 1 to day 13 at a dose of 200 mg/ kg bw. At the

same time control group received 0.5% gum acacia

solution. Animals from all groups were antigenically

challenged by subcutaneous administration of 0.25 X

109 SRBC/ml into the right hind foot pad of the mice on

day 13. DTH response was measured after 48 hrs. with

respect to increase in paw volume by plethysmometer

(UGO Basile,Italy). The change in the volume of the

left hind paw, injected similarly with phosphate

buffered saline served as control. The percentage

increase in the volume of the paw in various extract

treated groups was considered as an index of cell

mediated immune response.12

Humoral immune response (haemagglutination

antibody titer) 13,14

The humoral immune response was measured by

hemagglutination-antibody titre method on days 13 and

20. Animals were divided into 5 groups, each group

containing 6 animals; Group I served as control and

Groups II, III, IV, V were treated with aqueous,

alcoholic, Pet. ether and chloroform extract respectively

at a dose of 200mg/kg for 13 days and each animal was

immunized with 0.5 X 109 SRBC/ml. by i.p. route

including control group on day 0. On day 13 and 20,

blood samples were collected from the retro-orbital

plexus of mice of all the groups and serum was

separated. In the wells of micro-titrating plate, 25 µl of

serum was serially diluted with same amount of

phosphate buffer saline (pH 7.4) to get successive two-

fold dilutions so that the antibody concentration in any

of the wells was half that of the previous. The minimum

dilution of serum (1/2) was ranked as 1.Each dilution

with 25 µl of SRBC suspension (0.025 x 109 cells/ml)

was incubated at 37o C, for one hour. The highest rank

number of the serum dilution, that exhibited

agglutination, was considered as an antibody titre. The


339

Table no. 1 - Results of Preliminary Phytochemical Investigation of Amaranthus spinosus L.

Sr.No. Phytoconstituents Aqueous

Extract Alcoholic Extract

Pet. Ether Extract

Chloroform Extract

1. Alkaloids - - - -

2. Amino acids + + - -

3. Carbohydrates + + - +

4. Flavonoids - + + -

Glycosides

i. Anthraquinone + + - +

ii. Cardiac + + - +

iii. Coumarin + + - -

iv. Cyanogenetic - - - -

5.

v. Saponin + - - -

6. Tannins + - - -

7. Proteins + + - -

8. Steroids - + + -

9. Terpenoids - + - +

Table No. 2 -Quantitative estimation of Phytoconstituents of Amaranthus spinosus Linn.

Phytoconstituents % yield

Cardiac glycoside 0.0025 %

Anthraquinonoe glycoside 0.0139 %

Saponin glycoside 0.40 %

Table No. 3- Effect of the various extracts of Amaranthus spinosus Linn. on Delayed Type Hypersensitivity

reaction in mice.

Groups

Mean Increase in paw Volume

(%)±SEM

Control 39.11± 0.22

Aqueous Extract 46.40± 0.20*

Alcoholic Extract 45.23 ±0.18*

Pet Ether Extract 26.27± 0.17

Chloroform Extract 37.50± 0.16

The values are mean ± SEM, P< 0.01*, when compared with control group

(One way ANOVA followed by Dunnett’s test).


340

Table No.4 -Effect of various extracts of Amaranthus spinosus Linn. on Haemagglutination titer in mice

Humoral Immune Response Mean Antibody Titer Groups

Primary Secondary

Control 07.60±0.09 11.08± 0.17

Aqueous Extract 10.64±0.09* 14.49±0.16*

Alcoholic Extract 11.33±0.22* 15.52±0.29*

Pet Ether Extract 03.27±0.20 06.25±0.18

Chloroform Extract 07.52±0.18 11.38±0.18

The values are mean ± SEM, P< 0.01*, when compared with control group

(One way ANOVA followed by Dunnett’s test).

Fig. No 1 - Graphical representation of various

extract of Amaranthus spinosus Linn. on DTH

reaction in mice

Fig No.2 - Graphical representation of various extract of

Amaranthus spinosus Linn. on Haemagglutination titer

in mice.

initially observed antibody titre was considered as

primary humoral immune response while the

observation on second challenge considered as

secondary humoral immune response.

Statistical Analysis

The data expressed as mean ±SEM using one way

ANOVA, followed by Dunnet’s post –hoc test.

RESULTS

Phytochemical investigation of aqueous, alcoholic, pet

ether and chloroform extract of Amaranthus spinosus

Linn. leaves revealed the presence of flavonoid,

glycoside, steroids, terpenoids and tannins as shown in

Table No.1. According to Quantitative estimation of

anthraquinone, cardiac and saponin glycoside as

shown in Table No. 2.

Aqueous, alcoholic, pet ether and chloroform extract of

Amaranthus spinosus Linn. were evaluated at the dose

of 200mg/Kg of body weight for immunomodulatory

activity. The DTH response(%) to SRBC which

corresponds to cell mediated immunity for aqueous,

alcoholic, pet. ether and chloroform extract having

values 46.40± 0.20, 45.23 ± 0.18, 26.27± 0.17and 37.50

± 0.16 respectively,

in comparison to corresponding values 39.11± 0.20 for

untreated control group as shown in Table No.3. while

in Humoral immunity response,haemagglutination

antibody titer values for the aqueous, alcoholic, pet.

ether and chloroform extracts were 10.64 ± 0.09, 11.33

± 0.22, 3.27± 0.20 and 7.52 ± 0.18 in primary response

and 14.59 ± 0.16, 15.52 ± 0.29, 6.25 ± 0.18 and 11.38 ±

0.18 respectively in secondary response as shown in

Table No.4 .

DISCUSSION

The result indicates that daily treatment of mice with


341

alcoholic and aqueous extracts showed a significant

elevation in the humoral as well as cell mediated

immunity. The effect offered by chloroform extract was

not significant. On the other hand, the petroleum ether

extract treatment has significantly reduced the humoral

as well as cellular immune response. Aqueous and

alcoholic extracts suggest the profound

immunostimulatory effect in both responses while

petroleum ether extract produce immunosuppressant

effect.

The humoral immune response has been assessed by

estimating the antibody levels in the mice. The humoral

immunity involves interaction of B cells with antigen

and their subsequent proliferation and differentiation

into antibody-secreting plasma cells. SRBC have been

used as antigenic material for initiating the antibody

formation and subsequently the same was titrated with

SRBC, by haemagglutination method.

In the present study, SRBC were used as antigenic

material to elicit a contact hypersensitivity reaction in

earlier SRBC- sensitized mice. The reaction resulted

into an increase in the paw volume, which was

considered as an index of DTH or cell mediated

immune response. Like humoral response, alcoholic

treated group of animal produced significant decrease

in the paw swelling which suggests suppressed cell

mediated immune response. DTH reaction is always in

response to thymus-dependent antigen.

CONCLUSION

It is interesting to note that Amaranthus spinosus may

be having both immunostimulant and

immunosuppressant activity due to presence of various

glycosides, steroids and other Phytochemicals.

Therefore present investigation may demonstrate

immunomodulatory activity of aqueous and alcoholic

extract of Amaranthus spinosus thus have tremendous

future potential for developing new pharmaceutical

product and also correlation of botanical and

phytochemicals properties are having specific

pharmacological activity.

ACKNOWLEDGEMENTS

Authors are greatly thankful to the Department of

Pharmacology for providing free access to their

facilities to carry out research work. We also wish to

thank Dr. D. A Patil for identification of plant material.

REFERENCES

1. Hennessey L R, Baker J R. Clinical Immunology.

8th ed. New Jersey: Lange ;1994: 781

2. Lin Bi-Fong , Chiang Bor-Luen , Lin Jin-Yuarn. Int

Immunopharmacol. 2005;l 5(4): 711-22.

3. Anonymous wealth of India : Raw Materials.

(New Delhi ) :1998: 53.

4. Kirtikar K R, Basu B D. Indian Medicinal Plants

.2nd ed. 1996;3: 2056-58.

5. Pal D C , Jain S K. Tribal Medicine. 1st ed. Naya

Prakashan;1989:57.

6. Varier V P S. Indian Medicinal Plants .Orient

Longman;1994; 1:121.

7. Gupta A K. Quality Standards of Indian Medicinal

Plants. ICMR; New Delhi: 2003 ;62 .

8. Rajpal V, Standardisation of Botanicals. Eastern

Publisher : New Delhi:2004; 41-42

9. Ghosh M N, Fundamental of Experimental

Pharmacology. 2nd ed. Scientific Book Agency,

Kolkata: 1994; 153-58.

10. Joharapurkar A A , Zamad S P, Wanjari M M ,

Umathe S N .Indian J Pharmcol. 2003; 35: 232-

36.

11. Mediratta P K, Sharma K K ,Singh S. J

Ethnopharmacol. 2002; 80: 15-20.

12. Bhattacharya S K, Bhattacharya A , Chakrabarti A.

Indian J Exp Bio .2000; 38:119-28.

13. Mitra S K, Gupta M, Sharma D N K. Phytotherapy

Res .1999;13(4): 341-43.

14. Fulzele S V, Saturwar P M, Joshai S B , Dorle A K.

Indian J Pharmacol.2003;-35: 51-54.

**********


342

Pharmacodynamic drug interaction of mexiletine with

tolbutamide in rats S. Satyanarayana*, M. Nitin**, and K. Prasad.**

*Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam-530 003

**H.K.E.S.’s College of Pharmacy, Gulbarga-585 105

E mail. [email protected]

Abstract

The study was intended to find the pharmacodynamic drug interaction of class I b antiarrhythmic drug

mexiletine with tolbutamide in normal and diabetic rats. The doses of mexiletine selected were half the

therapeutic dose 1/2TD, TD, 2TD and in combination group, TD of mexiletine and TD of tolbutamide were

administered. The dosage calculations were based on therapeutic dose of humans extended to animals3. The

diabetes was induced by alloxan (150 mg/kg, i.p.). The blood glucose was measured by GOD/POD method

using kits manufactured by Dr. Reddy’s laboratories, Hyderabad. Single dose studies of interaction of

mexiletine with tolbutamide induced hypoglycemia indicated enhancement of hypoglycemia of tolbutamide in

normal rats. The studies when extended in diabetic rats also produced prolonged antihyperglycemic effect of

tolbutamide.

Key words: Mexilitine, Tolbutamide, Drug interaction, GOD/POD Method

INTRODUCTION

Diabetes is a group of syndrome characterized by

hyperglycemia; altered metabolism of lipids,

carbohydrates, and proteins; and an increased risk of

complications from vascular diseases. Among diabetics,

approximately 95% of patients have type II diabetes

mellitus (DM), whereas about 5% of patients have type

I diabetes mellitus. Patients with DM are at risk for

microvascular complications like retinopathy,

nephropathy and neuropathy and macrovascular

complications like, myocardial infarction that increase

morbidity and mortality1.

Polypharmacy and multiple drug therapy assume

importance in present day clinical practice, since newer

molecules are invented everyday and newer challenges

face clinicians in managing either a single disease or

simultaneously occurring different diseases. The

clinical observations are very vital in noting the

interactions of drugs, but to study the mechanisms of

such interactions, clinical studies cannot be carried out

using human models.

Hence animal model studies help in understanding the

underlying mechanisms in both healthy animal models

as well as disease induced animal models. This

knowledge would help in designing a rational therapy

with optimum benefits. The present study was intended

for studying such interactions between tolbutamide,

representing a prototype of sulphonylureas and

mexiletine since cardiovascular problems are more

common in diabetics and the possibility for the

simultaneous use of such combination is more.

The drug tolbutamide was selected as model drug

among sulphonylureas since it is the oldest, least

expensive and least fashionable and equally effective

drug to second-generation sulphonylureas2 in the

management of diabetes. Moreover it is the preferred

drug for geriatric patients because it possesses

relatively short half-life and is devoid of nocturnal

hypoglycemia.

The pharmacodynamic data (blood glucose) from blood

samples collected before and after administering drugs

to different group of rats served as parameter to identify

the interaction with the intention of getting results

quickly. Based on this data, in normal rats the study

was extended in diabetic rats, and data was collected

APTI ijper


Received on 1/8/2006; Modified on 10.1.2007



343

from this model for 6 hrs to find out whether the same

interaction occurs in both normal animals as well as in

disease induced model, which represents the condition

of actual use of drugs in humans.


Inbred adult albino rats of either sex were procured

from central animal house facility of M.R. Medical

College, Gulbarga. Prior approval by institutional

ethics committee was obtained for conduction of

experiments.

Glucose kits (GOD/POD method) manufactured by Dr.

Reddy’s Laboratory, Hyderabad were purchased from a

local pharmacy.

Alloxan monohydrate manufactured by Aldrich

Chemical Company Ltd., was purchased from a

chemical dealer.

The drugs were procured as gift samples from the

manufacturers listed below

Tolbutamide - Hoechst Marion Roussel Ltd. Mumbai.

Mexiletine hydrochloride-German Remedies Ltd.,

Mumbai.

Weight of rats selected- 180-220 g of either sex

Rats were fasted for 18 h prior to experiment with water

ad libitum as described previously. They were divided

into 5 groups of 6 each. Tolbutamide 10 mg/ml solution

in distilled water was prepared by solubilizing with few

drops of N/10 NaOH solution. Mexiletine 7.2 mg/ml

solution in distilled water was prepared. Group I served

as control. Group II was administered with, ½ the

therapeutic dose of mexiletine i.e. 3.6 mg/200g body

weight. Group III and group IV were treated with

therapeutic dose and double the therapeutic dose of

mexiletine i.e. 7.2 mg and 14.4 mg/200 g body weight

respectively. Group V was tolbutamide-matching

control. Group VI was treated with combination of

both drugs for studying interaction. In combination

group, therapeutic dose of mexiletine was administered

first, followed by therapeutic dose of tolbutamide after

an interval of 30 min. The blood samples were

collected and glucose was estimated in all the groups at

0, 1, 2, 4, 6 and 8 h intervals by GOD/POD method.

The animals were maintained on uniform diet and

temperature with 12 hrs, light and dark cycle housed in

polypropylene cages. Standard animal pellet food

manufactured by Hindustan Lever Ltd., procured from

local dealer was provided in adequate quantity, with

drinking water ad libitum. The therapeutic dose of

drugs administered to animals was calculated from

human dose as suggested by Laurence and Bacharach3,

1964. The drugs were administered orally. In case of

combination, therapeutic dose of mexiletine drug was

administered first followed by therapeutic dose of

tolbutamide after an interval of 30 min. First the studies

were conducted in normal rats and after collecting the

initial data, the studies were extended to diabetic rats in

alloxan induced diabetic model.

The blood samples were drawn from the tail vein in

small plastic disposable centrifuge tubes containing

anticoagulant (sodium fluoride and potassium oxalate,

1:3). Sodium fluoride was added to prevent in vitro

glycolysis in the blood samples collected. The above

samples were centrifuged immediately after collecting

blood from all the groups of animals at 0, 1, 2, 4 and 6

hrs, intervals. The blood glucose was estimated in the

plasma by GOD/POD method immediately.

INDUCTION OF DIABETES

Alloxan 40 mg/ml solution in distilled water was

prepared. After 3 days of acclimatization in the

laboratory the rats were administered with 100 mg/kg

body weight of alloxan monohydrate by intraperitoneal

route4. After the injection, they were provided with

10% dextrose solution through feeding bottles to

prevent sudden hypoglycemic shock, due to the sudden

release of stored insulin from the destroyed cells.

Standard rat pellet food manufactured by Hindustan

Lever Ltd., procured from a local dealer was provided

in adequate quantity during induction of diabetes. After

4 days, the blood glucose was estimated for verifying

the induction of diabetes. Later, an additional dose of

alloxan 50 mg/kg body weight was administered by i.p.

route if rise of blood glucose was not seen. Standard

food and dextrose solution was provided as described

above. After 2 days, the blood glucose was estimated

and rats with blood glucose above 250 mg/100 ml were

considered suitable for experimentation.

Initial blood glucose was determined after inducing

hyperglycemia. At first stage, therapeutic dose of

mexiletine drug was administered and the blood

glucose of the collected blood samples was determined

at intervals of 0, 1, 2, 4, 6 and 8 hrs, by GOD/POD

method. The rats were kept for a washout period to

ensure complete elimination of the drug exceeding 5-6


344

Table 1. Percent blood glucose reduction by different doses of mexiletine, tolbutamide and their combination on

blood glucose in normal rats.

Mean Percent blood glucose reduction ±SEM

Time (h)

Group Treatment

Dosage mg/200g p.o. 0 1 2 4 6 8

I Control

Distilled water 1 ml

0.0 2.35±0.63 4.85±1.42 7.60±1.59 10.14±1.58 11.95±1.67

II Mexiletine

1/2TD 3.6

0.0 8.51±1.09 18.07±2.04 21.79±2.06 25.02±1.89 7.60±1.02

III Mexiletine

TD 7.2

0.0 10.27±1.32 21.83±1.77 31.80±2.42 32.05±0.82 10.96±1.54

IV Mexiletine

2TD 14.4

0.0 11.07±2.46 26.91±1.03 36.62±1.93 42.96±1. 65 13.91±2. 06

V Tolbutamide

Tolbuta- -mide

10 0.0 22.14±1.27 44.38±1.12 54.60±1.06 26.66±1.10 14.79±1. 52

VI Mexiletine

+ Tolbutamide

7.2 + 10

0.0 24.51±2.06 48.29±1.72 57.53±0.87 51.27±

1. 47*** 21.34± 2. 81

N = 6, p***< 0.001

Table 2. Percent blood glucose reduction by mexiletine, tolbutamide and their combination in diabetic rats.

Percent blood glucose reduction ±SEM

Time (h) Treatment

0 1 2 4 6 8

Mexiletine TD

7.2mg/200g 00 6.81±1.39 13.04±1.91 20.34±2.20 26.23±3.91 8.99±1.13

Tolbutamide TD

10mg/200g

00

21.60±1.53 33.73±2.38 46.25±2.96 30.47±2.59 10.86±1.70

Mexiletine +

Tolbutamide 7.2+10mg/200g

00

27.43±2.35 41.82±2.34 51.96±2.76 37.58±3.04 19.40±0.54***

Number of animals n = 6; p***< 0.01

half-life periods of drug. At 2nd stage, after the above

washout period, therapeutic dose of tolbutamide was

administered and blood glucose of the samples was

estimated as above. At 3rd stage after a further washout

period of 4 days, the therapeutic dose of mexiletine

drug was administered first followed by therapeutic

dose of tolbutamide after an interval of 30 min. and the

blood glucose of samples was estimated at

predetermined intervals by GOD/POD method.

RESULTS

Mexiletine has been found to produce hypoglycemic

effect when administered alone. The maximum

reduction of blood glucose was 25.02%, 32.05% and

42.96% observed at 6 hrs, with different doses selected

for the study. The blood glucose appears to return to its


345

normal level after 8 hrs. In case of combination the

maximum fall in blood glucose was 57.53% at 4 hrs,

and 51.27% at 6 hrs, while tolbutamide alone produced

54.60% fall at 4 hrs, and 26.66% at 6 hrs. Similar

results were obtained from diabetic rats. The maximum

fall in blood glucose in diabetic rats was 46.25% with

tolbutamide and 51.96% in combination group.

The student’s t-test for unpaired observations was

applied between tolbutamide and the combination, and

it was found that the increase in tolbutamide-induced

hypoglycemia by mexiletine was statistically significant

at 6 hrs, compared to tolbutamide matching control.

The results of the study are shown in the table 1and 2

DISCUSSION

The human loading therapeutic oral dose of mexiletine

is 400 mg/day and maintenance dose of 200 mg/8 h5.

In the present study, 400mg of human dose was

considered for extending to the rat dose. Preliminary

studies designed in normal rats provided data about

pharmacodynamic interaction. Based on this data,

further studies were planned in diabetic rats.

The interaction of mexiletine and tolbutamide in normal

rats indicate that, mexiletine produced hypoglycemic

effect when administered alone and also enhanced the

tolbutamide induced hypoglycemic effect. The

interaction studies indicated a similar fall of blood

glucose even in diabetic rats and also enhancement of

tolbutamide effect.

The studies of interaction of mexiletine with

tolbutamide produced statistically significant

pharmacodynamic interaction in normal rats.

Mexiletine produced per se hypoglycemia when

administered alone and enhanced the effect of

tolbutamide. The interaction studies of mexiletine in

diabetic rats produced similar results as observed in

normal rats.

Type II diabetes mellitus is a more common disorder

than Type I and sulphonylureas are the preferred drugs

for its treatment. Among sulphonylureas, tolbutamide

was selected as a model drug, since it is highly protein

bound extensively metabolized by hepatic microsomal

enzymes and excreted in urine completely6. As a result

it may be affected more by other drugs at distribution,

metabolism and excretion pathway apart from the

interference at absorption7. The interacting drugs may

also alter the tolbutamide response at receptors. Since

cardiac disorders are more common in diabetes,

arrhythmias and angina are likely to occur in diabetics

as a result the use with antiarrhythmic/antianginal drugs

along with antidiabetic drugs is also more8,9. For

finding the information about drug interaction between

tolbutamide and mexiletine quickly it was planned to

find the influence of the selected drugs on tolbutamide

induced hypoglycemia in normal rats. Tolbutamide was

used as a prototype of sulphonylureas for studying the

drug interaction in the present investigation.

It is well established that tolbutamide produces

hypoglycemia mainly by pancreatic (insulin release)

mechanism. Additionally it is also known to produce

hypoglycemia by extrapancreatic mechanism (tissue

uptake of glucose) mechanism10,11. It also enhanced the

tolbutamide-induced hypoglycemia, which may be due

to their added pharmacological response. The results

indicate involvement of pharmacodynamic mechanism.

The extension of the drug interaction studies from

normal rat model to diabetic rat model have shown

similar and significant interaction. These studies

indicate that the drug interaction exists between

mexiletine and tolbutamide in normal as well as

diabetic rats representing the actual condition of drug

use.

The studies involving interaction between mexiletine

and tolbutamide indicated that simultaneous use of

mexiletine and tolbutamide need patient counseling and

monitoring of blood glucose to prevent the untoward

effects of hypoglycemia.

ACKNOWLEDGEMENTS

The authors thank M/s. Hoechst Marion Roussel and

German Remedies Ltd., Mumbai, for providing the gift

sample of tolbutamide and mexiletine respectively.

REFERENCES

1. Cerveny JD, Leder RD, Weart CW. Issues

surrounding tight glycemic control in people with

type 2 diabetes mellitus. Ann Pharmacother 1998;

32: 896-905.

2. Rang HP, Dale MM, Ritter JM. Pharmacology.

5thed. Edinburgh:Churchill Livingstone;2003.

3. Laurence DR, Bacharach AL. Evaluation of drug

activities and pharmacometrics. London and New

York: Academic press; 1964.


346

4. Kulkarni SK. Handbook of experimental

pharmacology. 2nd ed. New Delhi: Vallabh

Prakashan; 1993.

5. Dhawan J, Coronary heart disease risks in Asian

Indians. Curr Opin Lipidol

1996;7:196-198.

6. Product information Tolbutamide sodium. PDR

Generics, Ist ed Medical EconomicsData Production

Company. Montvale NJ: 1997.

7. Nakajima M, Kobayashi K, Shimada N et al.

Involvement of CYP 1A2 in mexiletine

metabolism. Br J Clin Pharmacol 1998; 46:55-62.

8. Kleinman JC, Donahue RP, Harris MI, Finucane

FF, Madans JH, Brock DB. Mortality among

diabetics in a national sample. Am J Epidemiol

1988; 128: 389-401.

9. Stamler J, Vaccaro O, Neaton JD, Wentworth D.

Diabetes other risk factors and 12 year

cardiovascular mortality for men screened in the

Multiple Risk factor Intervention trial. Diabetes

care 1993; 16: 434-444.

10. Goodman LS, Gilman A. The pharmacological

basis of therapeutics. 10th ed. McGraw-Hill: New

York; 2001.

11. Ronald C, Kahn, Gordon C, Weir, BI. Joslin’s.

Diabetes Mellitus. 13th ed. New Delhi: Waverly Pvt

Ltd; 1998.

**********


347

Validated, Reversed Phase High Performance Liquid

Chromatography Method for the Estimation of Etoposide in

Bulk and Formulations Movva Snehalatha, Bende Girish, Kolachina Venugopal and Ranendra N. Saha*

Pharmacy Group, Faculty Division III, Birla Institute of Technology and Science (BITS), Pilani - 333031,

Rajasthan, India. Phone: +91-1596-245073-284, Fax: +91-1596-244183,

E-mail: [email protected] (R.N. Saha) and [email protected] (M. Snehalatha)

Abstract

Reversed phase high performance liquid chromatographic method was developed and validated for the

estimation of etoposide in bulk and formulations using UV detector. Selected mobile phase was a combination

of 50 mM ammonium acetate buffer (pH 6.0) and methanol (50:50 %v/v) and wavelength selected was 240 nm.

Retention time of etoposide was 6.7 min. Linearity of the method was found to be 50 to 1000 ng/ml, with the

regression coefficient of 0.9998. This method was validated according to ICH guidelines. Quantification was

done by calculating area of the peak and the detection limit and quantitation limits were 10.34 ng/ml and 33.62

ng/ml respectively. There was no significant difference in the intra day and inter day analysis of etoposide

determined for three different concentrations using this method. Present method can be applied for the

determination of etoposide in quality control samples, formulations without interference of the excipients

present and in the dissolution studies of the drug.

Key words: Etoposide, Liquid chromatography, Validation

INTRODUCTION

Etoposide is a semi synthetic derivative of

epipodophyllotoxin and believed to act by the inhibition

of topoisomerase enzyme and/or induction of direct

DNA breaks 1, 2. It is preferably used for the treatment

of patients with small cell lung cancer, testicular

tumors, Kaposi’s sarcoma and lymphomas. Several

HPLC methods were reported for etoposide with UV 3,

4, fluorescence 5 and electrochemical 6, 7 detectors with

complicated extraction procedures. Most of these

methods were for the estimation of etoposide in

biological matrix like blood 8, plasma 5, 9-11, urine 12,

CSF 12 and leukemic cells 13. Very few HPLC methods

are reported for the estimation of etoposide in injectable

formulations 14, 15. These methods were developed using

phenyl and cyano columns; require internal standard for

estimation of etoposide and retention times were high.

There is a need for new method for the estimation of

etoposide in bulk and dosage forms like injections and

soft gelatin capsules that can estimate etoposide

accurately and can be used for routine analysis and

dissolution studies.

The main purpose of this investigation is to develop and

validate reversed phase HPLC method which is simple,

precise, sensitive and selective for quantitation of

etoposide in bulk and dosage forms. The developed

method can be easily used in routine quality control and

for dissolution studies at very low level of etoposide

concentration. Suitable statistical tests were performed

on validation data.


Materials

Pure Etoposide was obtained as gift sample from Dabur

Research Foundation, Sahibabad, India. Methanol was

of HPLC grade and purchased from Spectrochem,

Mumbai and other buffer salts were of analytical grade.

APTI ijper





348

Triple Distilled water was used for preparation of

buffer. Ammonium acetate buffer solution was

prepared and was filtered through 0.22 µ filters

(Millipore, USA). Formulations of etoposide used for

the study were soft gelatin capsules (Etosid, Cipla LTD,

India) containing 50 mg etoposide, U.S.P. each capsule

and injections (Etosid, Cipla LTD, India and Fytosid,

Dabur India LTD, India) containing etoposide U.S.P.

20 mg/ml and were procured from Indian market.

Etosid capsules contain excipients like ferric oxide red

and titanium dioxide. Etosid and Fytosid injections

contain excipients like benzyl alcohol I.P., ethyl alcohol

I.P. in a sterile non-aqueous vehicle.

Equipment

HPLC equipment (Jasco, Japan) consisted of model

PU-1580 intelligent HPLC pumps, UV-1575 model

intelligent UV/Visible detector and AS-1559 model

intelligent sampler. Chromatograms were analysed

using Borwin software provided with the system.

Chromatographic condition

Separation was performed on a reverse phase

LiChrocart RP-18 (250 x 4.6 mm, 5 µm particle size)

column. Mobile phase consisted of a mixture of 50 mM

ammonium acetate: methanol (50:50) (pH 6.0).

Injection, volume of 50 µl was used. Flow rate was

adjusted to 1 ml/min and the wavelength was set to 240

nm.

Calibration curve:

Primary stock of etoposide was prepared dissolving 2

mg of etoposide in 10 ml of methanol. Secondary stock

of 20 µg/ml was prepared by diluting primary stock

with mobile phase. Stock solution was diluted suitably

with mobile phase to get calibration standards in a

concentration range of 50 to 1000 ng/ml. Calibration

curve was plotted between peak areas of etoposide

against concentration of drug

Analytical validation:

Method was validated according to ICH guidelines 16.

Specificity and selectivity of the method was assessed

by preparing a drug concentration of 500 ng/ml from

pure drug stock and commercial sample stock in

selected mobile phase and analysed. To determine

accuracy of the proposed method, different levels of

drug concentrations (Lower Quality Control – 100

ng/ml, Medium Quality Control – 500 ng/ml and

Higher Quality Control – 800 ng/ml) were prepared

independently from stock solution and analysed. To

give additional support to determine accuracy of the

developed method, standard addition method was done.

Different concentrations of pure drug solutions (100,

300 and 600 ng/ml) were added to a known preanalysed

formulation sample (400 ng/ml) and the total

concentration was analysed. The recovery of the added

pure drug was calculated.

As a part of precision, repeatability was calculated by

taking different levels of drug concentrations (same as

accuracy) prepared from fresh stock solution and were

analysed. Inter day and intra day variation and inter-

analyst variation studies were carried out to determine

intermediate precision of the method. Different levels

of drug concentrations were prepared two different

times in a day and studied for intra-day variation. Same

protocol was followed for three different days to study

inter-day variation. To establish linearity of the

proposed method, six separate series of solutions of the

etoposide were prepared from the stock solution and

analysed. Least square regression analysis was done for

the obtained data to develop calibration equation.

ANOVA test (one-way) was performed to determine

the variation between the replicates and it was based on

the area of the peak observed for each pure drug

concentration during the replicate measurement of the

standard solutions. Signal-to-noise ratio of 3 was taken

as limit of detection (LOD) and signal-to-noise ratio of

10 was taken as limit of quantitation (LOQ). The LOQ

samples were prepared in replicates (n =5) using same

procedure followed for calibration standards and

analysed. Robustness of the method was determined by

changing composition of mobile phase by ± 1%, by

changing the pH of ammonium acetate buffer by ± 0.1

units and by establishing the bench-top, stock solution

stability of etoposide at room temperature.

Analysis of formulations:

Contents of 10 soft gelatin capsules of etoposide were

emptied and material equivalent to 2 mg of drug was

taken to prepare a solution of 20 µg/ml in methanol.

Final dilution was made with mobile phase to obtain

concentrations within the linearity range. For injections,

volume equivalent to 2 mg drug was taken and same

steps were performed to get final concentration in

linearity range. Six replicates were prepared for all

formulations.


For mobile phase optimization various buffers of


349

Table 1: Etoposide calibration curve data.

Concn. (ng/ml) Area a (mV-sec)

(± SD) % RSD Predicted Con. (ng/ml)

50 3168.42 ± 32.62 1.029 49.53

200 11592.33 ± 149.27 1.288 201.91

400 22441.96 ± 302.96 1.350 398.17

600 33600.25 ± 149.08 0.444 600.01

800 45413.97 ± 134.97 0.297 813.70

1000 55145.41 ± 584.23 1.059 989.73 aEach value is average of six determinations.

Table 2: Accuracy and precision data for the developed method.

Predicted con. (ng/ml) a Level

Range Mean (± SD) % RSD Mean % Recovery (± SD)

Accuracy

(%) b

LQC 96.69 - 106.61 102.27 ± 2.496 2.440 102.27 ± 2.496 2.270

MQC 484.61 - 518.19 496.57 ± 9.834 1.980 99.31 ± 1.967 -0.685

HQC 776.0 - 848.16 806.35 ± 18.340 2.274 100.79 ± 2.293 0.794 a - predicted concentration of etoposide was calculated by linear regression equation b - accuracy is given in relative error % (= 100 × [(predicted concentration – nominal concentration)/nominal concentration)]. Each

determination is result of six separate determinations.

Table 3: Intra-day and inter-day precision of the developed method

Intra-day repeatability % RSD (n = 3) Level

Day 1 Day 2 Day 3

Inter-day repeatability % RSD (n = 18)

LQC 2.026 2.789 0.172 1.932 2.393 0.489 0.347

MQC 1.475 1.991 2.138 1.368 1.929 1.589 0.147

HQC 0.730 0.526 2.521 1.431 0.361 1.484 2.170

Table 4: Results of Estimation of etoposide in pharmaceutical preparations.

Commercial products Amount found % Assay

Etosid soft gelatin capsules - 50 mg

Mean ± SD (mg) 50.78 ± 0.81 101.56 ± 1.39

Etosid injection - 20 mg/ml

Mean ± SD (mg/ml) 20.24 ± 0.28 101.18 ± 2.05

Fytosid injection - 20 mg/ml

Mean ± SD (mg/ml) 20.09 ± 0.59 100.46 ± 1.63

Each determination is result of six separate determinations.


350

Figure 1

Figure 1: Chromatograms of blank, 50, 400 and 1000 ng/ml etoposide respectively.

different pH like phosphate buffers, acetate buffers (pH

3.5 to 6.0) (20 mM to 50 mM), and different

combinations of organic phase (acetonitrile and

methanol) along with buffers were investigated.

Changing the buffers, their molarities, pH and addition

of organic solvent in various proportions changed the

retention time of the drug and symmetry of the peak.

The final decision of using 50 mM ammonium acetate

buffer (pH 6.0) with methanol in 50:50 % v/v as a

mobile phase was based on certain criteria like,

asymmetric factor, retention time of the drug,

sensitivity of the method and cost. Sensitivity of the

method was less when detected at λmax 284 nm, whereas

at 240 nm sensitivity was increased considerably.

Chromatograms of blank and three different

concentrations of etoposide prepared in mobile phase

are shown in Figure 1. Retention time of etoposide was

6.74 min in selected mobile phase. Peak was having

good resolution with asymmetric factor of 1.21.

Calibration curve

Different concentrations (50, 200, 400, 600, 800 and

1000 ng/ml) and their peak areas were shown in the

Table 1. At all the concentration levels the standard

deviation was low and the relative standard deviation

(RSD) did not exceed 1.5 %. The predicted

concentrations were nearly matching with the nominal

concentration. In selected mobile phase the linearity

range was found to be 50 - 1000 ng/ml. According to

linear regression analysis, the slope (± standard error)

and intercept (± standard error) were found to be 61.01

(± 0.324) and -477.3 (± 181.41) respectively. These

mean values were found to be within the 95%

confidence limits (confidence limits of slope: 60.36 to

61.66; confidence limits of intercept: -840.5 to -114.1).

Goodness of fit of regression equation was supported

by high regression coefficient value (0.9998), low

calculated F-value (Calculated F-value – 4.49 × 10-4

and critical F (5,24) – 2.621 at P = 0.05 level of

significance) and low standard deviation of response

(Sy,x – 833.8). Lower values of parameters like standard

error of slope, intercept and estimate indicated high

precision of the proposed method.

Analytical validation

Retention time, asymmetric factor and area of the peak

obtained with marketed formulations were not affected

with excipients present in formulations of etoposide.

Selected mobile phase does not have significant signal

at retention time of etoposide. This indicates this

method is selective and sensitive to etoposide.

Accuracy for three different concentrations ranged from

0.794 to 2.270 (Table 2). In standard addition studies,

mean % recoveries (± SD) were found to be 99.01 (±

0.408), 102.59 (± 0.147) and 100.33 ± 0.054) for 100,

300 and 600 ng/ml concentrations respectively. High

recoveries by standard addition method for respective

pure drug concentration and low RSD (< 0.5 %) show

that the method can estimate etoposide accurately.

In repeatability study % RSD was ranged from 1.98 to

2.44. At all three concentration levels, precision showed

satisfactory levels. Intermediate precision expresses

within-laboratory variations in different days and by

different analysts. Results of intermediate precision


351

study, % RSD values for each set (all three levels) were

given in Table 3. In all the cases the RSD values were <

3 %, indicating that these methods have excellent

repeatability and intermediate precision. DL and QL

were found to be 10.34 ng/ml and 33.62 ng/ml

respectively. The mean % recovery (± SD) of the

estimated QL was found to be 101.66 (± 2.79). When

drug was estimated under varying pH and varying

composition of mobile phase conditions the mean %

recovery value was found to be 100.29 with low %

RSD of 1.195, indicating the robustness of the method.

The retention time and area of the bench-top stability

and stock solution stability samples were not changed

till 7 days and there were no extra peaks observed in the

chromatograms.

Analysis of formulations

The method was evaluated by estimation of etoposide

in pharmaceutical formulations and the results were

shown in Table 4. For different formulations % ranged

from 100.18 to 101.56 with standard deviation not more

than 2.05. The estimated drug content with low values

of standard deviation established the precision of the

proposed method. Assay values of formulations were

very close to the label claim. This indicated that the

interference of excipient matrix is insignificant in

estimation of etoposide by the proposed method (Table

4).

In conclusion, the developed method is accurate,

precise and easy to apply for the estimation of

etoposide in bulk, and pharmaceutical formulations.

Etoposide from routine dissolution samples can also be

estimated by this method. This method is simple and

sensitive without the use of any complicated

instruments or sample preparation. Detection limit of

the proposed method is lower than many reported

HPLC methods. The sample recoveries in all

formulations were in good agreement with their

respective label claims, indicating non-interference of

excipients in the estimation.

ACKNOWLEDGEMENTS

The authors are grateful to Centre for Scientific and

Industrial Research (CSIR), New Delhi for financial

support. The authors gratefully acknowledge Dabur

Research Foundation (Sahibabad) for providing

etoposide as gift sample.

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16. International Conference on Harmonisation (ICH),

ICH Harmonised tripartite guideline, Topic Q2B,

Note for Guidelines on Validation of Analytical

Procedures: Methodology, 1996.

***********


353

Spectrophotometric Estimation of Bisoprolol Fumarate in

Bulk Drug and Tablets Akmar Sandip, Paramane Sonali, Kothapalli Lata *, Thomas Asha, Jangam Sumitra,

Mohite Mukesh and Deshpande Avinash.

Department of Pharmaceutical Chemistry, Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research,

Pimpri, Pune-411018, Maharashtra, India.

E-mail: - [email protected]

Abstract

Two simple, precise and economical spectrophotometric methods have been developed for the estimation of

Bisoprolol Fumarate in bulk and pharmaceutical formulations. Bisoprolol Fumarate shows a sharp peak at

220.0 nm in first order derivative spectrum with n = 1 (Method A). Method B applied is based on calculation of

Area under Curve (AUC) for analysis of Bisoprolol Fumarate in the wavelength range of 218-228 nm. The drug

follows the Beer-Lambert’s law in the concentration range of 5-50 µg/ml in both the methods. Results of the

analysis were validated statistically and by recovery studies and were found to be satisfactory.

Key words: - Bisoprolol Fumarate, UV spectrophotometry, Derivative spectroscopy, Area under Curve.

INTRODUCTION

Bisoprolol Fumarate is a synthetic β1-selective

(cardioselective) adrenergic receptor blocking agent.

Chemically Bisoprolol Fumarate is (±)-1-[4-[[2-(1-

Methylethoxy)ethoxy]methyl]phenoxyl-3-[(1-

methylethyl)amino]-2-propanol(E)-2-utenedloate (2:1)

(salt)1. It is clinically useful in the treatment of

hypertension.

It is not official in any of the pharmacopoeias. It is

listed in The Merck Index1 and Martindale, The

Complete Drug Reference2. Literature survey reveals

that only few RP-HPLC methods3-9 are reported for the

determination of Bisoprolol Fumarate in biological

fluids using fluorescence detector. Hence the objective

of the work is to develop new spectrophotometric

methods for its estimation in bulk and pharmaceutical

formulations with good accuracy, simplicity, precision

and economy.


Pure sample of Bisoprolol Fumarate was obtained from

Atra Pharmaceuticals Ltd., Aurangabad as a gift

sample. Distilled water was used as solvent. Shimadzu

UV-1601 UV/VIS spectrophotometer was used with 1

cm matched quartz cells. Tablets of 5 mg strength were

procured from local pharmacy of two commercial

brands i.e. Concor (MERCK) and Corbis

(UNISEARCH).

Accurately about 10 mg of the pure drug was weighed

and dissolved in sufficient quantity of distilled water

and the volume was made up to 100 ml with distilled

water to give standard stock solution (100 µg/ml).

Aliquots of standard stock solution were pipetted out

and suitably diluted with distilled water to get the final

concentration of 5, 10, 15, 20, 25……….50 µg/ml of

standard solutions. The solutions were scanned in the

spectrum mode from 400 nm to 200 nm wavelength

range and the first order derivative spectra were

obtained at n = 1(Method A)10 a sharp peak was

obtained at 220.0 nm (Figure 1). The absorbance

difference at n=1(dA/dλ) was calculated by the inbuilt

software of the instrument which is directly

proportional to the concentration of the standard

solution. A calibration curve was plotted taking the

absorbance difference (dA/dλ) against the concentration

of the standard solutions (Table 1, Graph 1). The

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354

method was applied for the sample solution of known

concentration and was found to be satisfactory for the

analysis of tablet formulations.

Method B, the AUC11 (area under curve) method is

applicable when there is no sharp peak or when broad

spectra are obtained. It involves the calculation of

integrated value of absorbance with respect to the

wavelength between two selected wavelengths λ1 and

λ2. Area calculation processing item calculates the area

bound by the curve and the horizontal axis. The

horizontal axis is selected by entering the wavelength

range over which the area has to be calculated. This

wavelength range is selected on the basis of repeated

observations so as to get the linearity between area

under curve and concentration. Suitable dilutions of

standard stock solution (100 µg / ml) of the drug were

prepared and scanned in the spectrum mode from the

wavelength range 400–200 nm (Figure 2) and the

calibration curve was plotted as concentration against

AUC (Table 2, Graph 2).

The method was checked by analyzing the samples with

known concentration for the AUC. As the results

obtained were satisfactory, the method was applied for

pharmaceutical formulations. For estimation of

Bisoprolol Fumarate in tablet formulations by both the

methods, twenty tablets of each brand were weighed

and triturated to fine powder.

Tablet powder equivalent to 5 mg of Bisoprolol

Fumarate was weighed and dissolved and further

diluted with quantity sufficient of distilled water. It was

kept for ultrasonication for 45 min; this was then

filtered through Whatman filter paper no. 41 to get

stock solution of concentration 100 µg/ml. For method

A, analysis of Bisoprolol Fumarate in both tablet

formulation T1 and T2 was done using various sample

solutions at 220.0 nm against reagent blank in

quantitation mode for six times.

For method B the sample solutions were scanned in the

spectrum mode and AUC calculations were done in the

wavelength range of 218.0–228.0 nm for both the

tablet formulation T1 and T2 for six times and

concentrations calculated using the calibration curve.

Both the methods A and B were validated for linearity,

accuracy, specificity.

Table 1: Standard Calibration Table For Bisoprolol Fumarate(Method A)

Sr. No. Concentration

(µg/ml)

Absorbance difference* (dA/d λ)

1 5 0.003 2 10 0.006 3 15 0.009 4 20 0.012 5 25 0.015 6 30 0.018 7 35 0.020 8 40 0.024 9 45 0.027

10 50 0.031

* mean of six readings

Table 2: Standard Calibration Table For Bisoprolol Fumarate( Method B)

Sr. No. Concentration (µg/ml) AUC*

1 5 1.6034

2 10 3.1287

3 15 4.6985

4 20 6.1876

5 25 7.9528

6 30 9.4823

7 35 10.9712

8 40 12.5794

9 45 14.1094

10 50 15.7988

* mean of six readings


355

AUC Method of Bisoprolol Fumarate

0

5

10

15

20

5 10 15 20 25 30 35 40 45 50

Concentration

AU

C

Table 3: Optical Characteristics And Other Parameters

Parameters Method A Method B

λMax (nm) / wavelength range (nm)

Beer-Lambert’s range (µg/ml) Coefficient of correlation (r) Regression equation Y = mx + c a. Slope (m) b. Intercept (c)

220.0 5-50

0.9987

0.006 0.0000

218-228 5-50

0.9999

1.5734 -0.0124

Where, x is concentration in µg/ml & Y is absorbance unit. ; A is First Order Derivative spectrum method with n = 1. ;B is the AUC method.

Table 4: Estimation Of Bisoprolol Fumarate In Tablet Formulation.

Method Tablet

Label Claim

(mg)

Amount Found

(mg) *

%*

S.D.*

R.S.D* S.E.*

A

B

T1

T2

T1

T2

5

5

5

5

4.986

4.989

4.983

4.991

99.732

99.786

99.656

99.825

0.0980

0.2033

0.1404

0.2641

0.0983

0.2037

0.1409

0.2645

0.0400

0.0830

0.0573

0.1078

Where, A is first order derivative spectrum method with n = 1.; B is the AUC method. T1 (Concor) and T2 (Corbis) are two brands of tablet

formulations ;* The results are the mean of six readings (n = 6).

Table 5: Recovery Study Data

Method Tablet

Level of Recovery

(% )

Label Claim (mg)

Amount Recovered

(mg) *

Recovery* %

S.D.* (±)

R.S.D*

S.E.*

A

B

T1

T2

T1

T2

80 100 120 80

100 120 80

100 120 80

100 120

5 5 5 5 5 5 5 5 5 5 5 5

4.946 4.959 4.925 4.932 4.977 4.968 4.963 4.982 4.963 4.983 4.995 4.990

98.930 99.187 98.513 98.657 99.543 99.373 99.290 99.653 99.667 99.673 99.900 99.813

0.1212 0.0503 0.0971 0.1209 0.2346 0.2774 0.1345 0.0231 0.0416 0.3900 0.1249 0.0757

0.1226 0.0507 0.0986 0.1226 0.2357 0.2791 0.1355 0.0232 0.0418 0.3913 0.1250 0.0759

0.0495 0.0205 0.0396 0.0494 0.0958 0.1132 0.0549 0.0094 0.0170 0.1592 0.0510 0.0309

Where, A is first order derivative spectrum method with n = 1. ; B is the AUC method.

T1 (Concor)and T2(Corbis) are two brands of tablet formulations. ; * The results are the mean of six readings at each level of recovery.

Graph 1: Calibration Curve Of Bisoprolol Fumarate

(Method A)

Graph 2: Calibration Curve Of Bisoprolol Fumarate

( Method B)


356

Figure 1. First order derivative spectrum of Bisoprolol

Fumarate with n = 1

Figure 2. Wavelength range selected for AUC method of

Bisoprolol Fumarate

Linear regression of absorbance and correlation

coefficient (r) of both the methods are given in (Table

3). Relative standard deviation for analysis of six

replicate samples of two brands T1 and T2 in both

method A and B are given in (Table 4).

Recovery studies were carried out at three different

levels i.e. 80 %, 100 % and 120 % by adding the pure

drug (4, 5 and 6 mg respectively) to previously

analyzed tablet powder sample (5 mg) as per ICH

guidelines12 for three times (n = 3). From the amount of

drug found, percentage recovery was calculated (Table

5).


Both the methods A and B for the estimation of

Bisoprolol Fumarate in tablet dosage form were found

to be simple accurate and reproducible. Beer-Lambert’s

law was obeyed in the concentration range of 5-50

µg/ml in both the methods. The values of standard

deviation were satisfactory and the recovery studies

were close to 100 %. As the drug Bisoprolol fumarate

showed a broad spectrum, the derivative spectroscopy

method applied has the advantage that it locates the

hidden peaks in the normal spectrum when the

spectrum is not sharp and it also eliminates the

interference caused by the excipients and the

degradation products present, if any, in the formulation.

The AUC method is also advantageous as it is

applicable to the drugs which show the broad spectra

without a sharp peak. Hence these methods can be

useful in the routine analysis of Bisoprolol Fumarate in

bulk drug and formulations.

ACKNOWLEDGEMENT - We are highly thankful

to Atra Pharmaceuticals Ltd., Aurangabad for providing

us the gift sample of the pure drug and to our Principal,

Dr. D. Y. Patil Institute of Pharmaceutical Sciences and

Research, Pimpri, Pune for providing excellent research

facilities.

REFERENCES

1. Budavari, S., Eds: In; The Merck Index, 13th Edn,

Merck & Co., Inc., White House Station., NJ,

2001;218.

2. Sean, C., Sweetman, Eds: In., Martindale, The

Complete Drug Reference, 34th Edn., The

Pharmaceutical Press; London.,2002;875.

3. Caudron, E.; Laurent, S.; Billaud, E.M.; Prognon.

P.; J.Chromatogr.B.Analyt. Technol. Biomed.

Life.Sci.,2004;Mar5;801(2);339-45.

4. Modamio, P.; Lastra, C. F.; Marino, E. L.; J.

Pharm.Biomed.Anal.,1996;Feb; 4(4); 401-8.

5. Braza, A. J.; Modamio, P.; Lastra, C. F.; Marino, E.

L.; Biomed.Chromatogr., 2002;Dec;16(8); 517-22.

6. Eastwood, R. J.; Jerman, J. C.; Bhamra, R. K.;

Holt, D. W.; Biomed.Chromatogr., 1990 ; Jul;

4(4);178-80.

7. Suzuki, T.; Horikiri, Y.; Mizobe, M.; Noda, K.;

J.Chromatogr., 1993;Sep22; 619(2);267-73.

8. Buhring, K. U.; Garbe, A.;

J.Chromatogr.,1986;Oct31;382;215-24.

9. Clarice.; Journal of Liquid Chromatography &

Related Technologies, Taylor & Francis,

2005;Vol.28;3; 477 – 486.

10. Beckett A.H., Stenlake J.B., “Practical

Pharmaceutical Chemistry”,Part-II, 4th Edn.,


357

2002, CBS Publishers and Distributors, New Delhi,

275-280, 293-300..

11. Lande, N. R.; Shetkar, B. M.; Kadam, S. S.;

Dhaneshwar, S. R.; Indian J. Pharm. Sci., 2001,

63-66.

12. International Conference on Harmonisation, ICH

Harmonised Tripartite Guideline — Validation of

Analytical Procedures: Methodology, Fed. Regist.,

1997;62;27463

**********


358

Preparation and In Vitro Evaluation of Mucoadhesive

Microcapsules of Atenolol Swamy P.V.*, Hada Amit, Shirsand S.B., Hiremath S.N and Raju S.A

Department of Pharmaceutical Technology, HKE Society’s College of Pharmacy, Sedam Road

Gulbarga-585 105 Karnataka-India

*E-mail : [email protected]

ABSTRACT

Sustained release alginate microcapsules of atenolol were prepared by orifice-ionic gelation method using

hydroxypropyl methylcellulose (viz., 50 cps and K4M) as mucoadhesive polymer. Microcapsules were discrete,

spherical and free flowing. Encapsulation efficiency varied from 49% - 66%. Microcapsules were evaluated

for % yield, drug content uniformity, particle size distribution, surface morphology (scanning electron

microscopy), short-term stability (at 40 ± 1º for 3 weeks) and drug-excipient interactions (IR Spectroscopy).

The formulation prepared by using alginate-hydroxypropyl methylcellulose (K4M) in a ratio of 5:1 along with

4% magnesium stearate, emerged as the overall best formulation (t50%=2.25 h, t70%=4.35 h, t90%=7.60 h), based

on drug release characteristics (in pH 6.2 phosphate buffer). This formulation shows slow release upto 8h. In

vitro drug release followed first-order kinetics and fickian-diffusion mechanism (Higuchi’s model). All the

microcapsules exhibited good mucoadhesive property in the in-vitro wash-off test. Short-term stability studies

on the promising formulations (40 ± 1º for 3 weeks) indicated that there are no significant changes in drug

content and dissolution parameters (p<0.05). These mucoadhesive microcapsules are thus suitable for oral

controlled release of atenolol.

Keywords: Atenolol; mucoadhesive microcapsules; orifice-ionic gelation method; hydroxypropyl

methylcellulose (50 cps, K4M); In vitro wash off test.

INTRODUCTION

The novel design of an oral controlled drug delivery

system should primarily be aimed at achieving more

predictable and increased bioavailability of drugs. But

there are several physiological difficulties, which

include restraining / localizing the drug delivery system

within the regions of the gastrointestinal tract and the

highly variable nature of gastric emptying process (a

few minutes to 12 h) 1.

The major absorption zone (stomach or upper part of

the intestine), can result in incomplete drug release

from the drug delivery system leading to diminished

efficacy of the administered dose. Therefore,

restraining a drug delivery system in a specific region

of the gastrointestinal tract due to its mucoadhesiveness

increases the intimacy and duration of contact between

a drug containing polymer and a mucous surface. Such

a drug delivery system offers numerous advantages,

especially for drugs exhibiting an absorption window or

for drugs with a stability problem. Overall, the intimate

and prolonged contact of the drug delivery system with

the absorbing membrane has the potential to maximize

drug absorption and may also influence the rate of drug

absorption2,3. These considerations have led to the

development of oral controlled-release microcapsules /

microspheres possessing mucoadhesive properties.

Alginate is easily gelled by the addition of Ca2+ to an

aqueous solution of sodium alginate, since insoluble

calcium alginate is formed by cation exchange between

Na+ and Ca2+. The mechanism of gelation has been

intensively investigated by circular dichroism (CD) and

nuclear magnetic resonance (NMR) studies. The

gelation and cross-linking are due to the stacking of the

glucoronic acid (G) blocks of alginate chains with the

formation of the egg-box junction.

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Received on 13..2.2007; Accepted on 7/8/2007



359

When an aqueous solution of sodium alginate was

added dropwise to an aqueous solution of calcium

chloride, a spherical gel with regular shape and size was

obtained. The spherical gel is termed ‘Alginate Bead’.

Alginate beads have following advantages: (i) non-toxic

(ii) have a protective effect on mucous membrane of

upper GIT. (iii) since dried alginate beads have

property of reswelling they can act as a controlled-

release system. (iv) porosity of alginate beads gives a

fast release pattern of incorporated drugs and also a

very low efficiency of incorporation of low molecular

weight drugs, except for sparingly soluble drugs4.

Atenolol is a β-adrenoreceptor antagonist used in the

treatment of hypertension and angina-pectoris. It has

an oral bioavailability of only 50%, because of its poor

absorption in lower gastrointestinal tract. It undergoes

little or no hepatic metabolism and its elimination half

life is 6 to 7 h5. Therefore, it is selected as a suitable

drug for the design of mucoadhesive microcapsules

with a view to improve its oral bioavailability.

The main objective of the present study is to develop

alginate mucoadhesive microcapsules of atenolol by

orifice-ionic gelation process using hydroxypropyl

methylcellulose (HPMC 50 cps and HPMC K4M) as

mucoadhesive polymer.


Materials

Atenolol I.P was a generous gift sample from Vapi Care

Pharma Ltd., Vapi. HPMC (50 cps and K4M) was

procured from Colorcon Asia Limited, Goa. Sodium

alginate and magnesium stearate were obtained from Sd

Fine Chemicals, Mumbai. All other chemicals were of

analytical reagent grade.

Preparation of microcapsules4,6

Coating material (sodium alginate) and mucoadhesive

polymer were dissolved in distilled water (16 ml) to

form a homogeneous polymer solution. The core

material, atenolol (1 g) was added to the polymer

solution and mixed thoroughly to form a viscous

dispersion. The resulting dispersion was added drop

wise into calcium chloride solution (10 ml; 10% w/v)

through a syringe fitted with a needle of 18 gauge. The

added droplets were retained in the calcium chloride

solution for 3 h to complete the curing reaction and to

produce spherical rigid microcapsules. The

microcapsules were collected by decantation and the

product thus separated was washed repeatedly with

water and dried at 45º for 12 h.

Since the microcapsules prepared by the above method

could sustain the drug release upto 5h only, further

modifications were made in the method. These

modifications include: (i) increasing the cross-linking

time from 3 to 6 h (Formulations MC-5 to MC-8). (ii)

Addition of magnesium stearate (#200 mesh) in 2 to 4%

w/w concentration (Formulations MC-7 and MC-8),

after thoroughly mixing with the drug atenolol by

spatulation (on a butter paper). The prepared

microcapsules were stored in a desiccator until further

use. (Table-1)

Evaluation of microcapsules

% Yield

The total amounts of microcapsules obtained were

weighed and the percentage yield calculated taking into

consideration the weight of drug and polymer.

100Pr

% xYieldlTheoretica

YieldacticalYield =

Size analysis2

For size distribution analysis, different sizes in a batch

were separated by sieving, using a set of standard sieves

(IP). The amounts retained on different sieves were

weighed.

Drug content estimation7

Atenolol microcapsules (25 mg) from each batch were

initially stirred in 3 ml of sodium citrate solution (1%

w/v) until complete dissolution. Methanol (7 ml) was

added to above solution to gel the solubilized calcium

alginate and further solubilize atenolol. This solution

was filtered through Whatman No.1 filter paper. The

filtrate was assayed for drug content by measuring the

absorbance at 224.6 nm after suitable dilution.

Encapsulation efficiency6,8

Encapsulation efficiency was calculated using the

equation :

= 100

%

%x

lesmicrocapsuincontentdruglTheoretica

lesmicrocapsuincontentdrugEstimated

efficiency

ionEncapsulat

Scanning electron microscopy (SEM) 6

The microcapsules were observed under a scanning

electron microscope (SEM-JEOL, JSM-840A, Japan).

They were mounted directly onto the SEM sample stub

using double-sided sticking tape and coated with gold

film (thickness 200 nm) (FINE COAT, JFC – 100E;


360

Ion-sputtering device, Japan), under reduced pressure

(0.001 mm of Hg).

In vitro wash off test for mucoadhesion2,3,6

The mucoadhesive property of the microcapsules was

evaluated by an in vitro adhesion testing method known

as the wash-off method. Freshly excised pieces of

intestinal mucosa (4x5cm) from sheep were mounted

onto glass slides (3x1 inch) with cyanoacrylate glue.

Two glass slides were connected with a suitable

support. About 50 microcapsules were spread onto

each wet rinsed tissue specimen, and immediately

thereafter the support was hung onto the arm of a USP

tablet disintegrating test machine. When the

disintegrating test machine was operated, the tissue

specimen was given a slow, regular up-and-down

movement in the test fluid (400 ml) at 37 ºC contained

in a 1000 ml vessel of the machine. At the end of 1 h,

and at hourly intervals upto 12 h, the machine was

stopped and the number of microcapsules still adhering

to the tissue was counted. The test was performed both

in simulated gastric fluid (pH 1.2) and simulated

intestinal fluid (pH 6.2 phosphate buffer). The data of

in vitro wash off test are shown in Table-3.

In vitro drug release studies

Drug release study was carried out in USP paddle type

dissolution test apparatus (Electrolab TDT-06 N). A

quantity of microcapsules equivalent to 100 mg of

atenolol was used for the test. Dissolution medium was

phosphate buffer having a pH 6.2. Volume of

dissolution medium was 900 ml, and bath temperature

was maintained at 37± 0.5ºC throughout the study. At

specified time intervals, 5 ml samples were withdrawn

by means of a syringe fitted with prefilter and replaced

immediately with 5 ml of fresh medium. The

absorbance of sample was measured at 224.6 nm after

suitable dilution with the medium. All the studies were

conducted in triplicate (n=3).

Drug-polymer interaction studies

There is always a possibility of drug polymer

interaction in any formulation due to their intimate

contact. The technique employed in the present study

for this purpose is IR spectroscopy.

The IR spectra of atenolol, sodium alginate, HPMC,

magnesium stearate and formulation MC-8 were

obtained by KBr pellet method employing Perkin –

Elmer FTIR 1516 series.

Short-term stability

Short-term stability studies were performed over a

period of 3 weeks (21 days) on the promising

formulation (MC-8). The microcapsules were packed in

screw capped amber colored glass container and kept in

hot air oven maintained at 40º±1ºC at ambient humidity

conditions. Samples were withdrawn at weekly

intervals and were examined for physical changes such

as color and texture, and assayed for drug content. At

the end of 3 weeks period, dissolution test was also

performed to determine the drug release profile.

Data analysis

The in vitro drug release data was fitted into four

popular models of data treatment for the matrix

formulations as follows: (1) zero order kinetics; (2) first

order kinetics; (3) Higuchi’s square root model9; (4)

Peppas model10. The data obtained from the stability

studies was subjected to statistical analysis (student ‘t’

test) in order to find out any significant changes in the

drug content and dissolution parameters (t50% and t70%)

of the promising formulation (MC-8) after storage for 3

weeks at a temperature of 40º±1ºC and ambient

humidity conditions.


All the prepared microcapsules were found to be

spherical, discrete and free-flowing with yellowish

white color. The SEM studies of microcapsules (MC-8)

reveals that the microcapsules were perfectly spherical

in shape and show a rough and porous surface with free

drug crystals. (Fig.1).

Percent yield was in the range of 69 – 83%. The mean

diameter of microcapsules was found to be in the range

of 855 – 1200 µm with only 2.8 to 17.3% being of the

smaller mean diameter 855 µm (16/22 mesh). Overall,

more than 82% of the microcapsules prepared were of

1200 µm mean diameter (12/16 mesh). The mean

percent drug content of the microcapsules ranged from

24.93 – 32.51%. The low values of standard deviations

indicate uniform distribution of the drug within the

various batches of microcapsules prepared.

Encapsulation efficiency ranges approximately from 49

– 66%. Results reveal that the encapsulation efficiency

of microcapsules increases with an increase in alginate


361

Table-1: Composition of Microcapsules

Quantity of Mucoadhesive polymers (mg)

Formulation Code Sodium alginate :

HPMC ratio Atenolol

(mg)

Sodium alginate

(mg)

Magnesium stearate (mg) HPMC

(50 cps) HPMC (K4M)

MC 1 1:1 1000 500 -- 500 -- MC 2 3:1 1000 750 -- 250 -- MC 3 5:1 1000 833 -- 167 -- MC 4 9:1 1000 900 -- 100 -- MC 5 5:1 1000 833 -- -- 167 MC 6 9:1 1000 900 -- -- 100 MC 7 5:1 1000 833 40 -- 167 MC 8 5:1 1000 833 80 -- 167

Table-2: Evaluation of Microcapsules

% Microcapsules with mean diameter in microns

Sl. No.

Formulation Code

% Yield

1200 (12/16 mesh)

855 (16/22 mesh)

Mean % drug content ±SD*

Encapsulation efficiency (%)

1. MC 1 72.40 82.70 17.30 24.93±0.567 49.86 2. MC 2 76.80 86.13 13.87 25.25±0.440 50.51 3. MC 3 80.00 88.47 11.53 26.97±0.711 53.92 4. MC 4 82.80 89.13 10.87 31.60±0.603 63.20 5. MC 5 69.50 91.47 8.53 28.40±0.366 56.8 6. MC 6 73.15 94.47 5.53 32.51±1.270 65.97 7. MC 7 71.30 96.37 3.63 30.34±0.821 60.69 8. MC 8 73.00 97.20 2.80 31.62±0.468 63.25

* Average of three determinations.

Table-3: Results of In vitro wash off test to Assess Mucoadhesive Properties of Microcapsules

% of Microcapsules adhering to tissue at various time intervals* in h

in simulated gastric fluid (pH 1.2) in phosphate buffer (pH 6.2)

Microcapsules 1 2 4 6 8 1 2 4 6 8

MC 1 100 (0) 100 (0) 99.3 (1.2)

98 (2.0)

98 (2.0)

97.3 (1.2)

91.3 (1.2)

78 (2.0)

72.7 (1.2)

68.7 (1.2)

MC 2 100 (0) 99.3 (1.2)

96 (2.0)

94.7 (1.2)

92.7 (1.2)

97.3 (1.2)

92.0 (2.0)

84.7 (2.3)

78 (2.0)

72.7 (1.2)

MC 3 98 (0) 96.7 (1.2)

93.3 (1.2)

91.3 (1.2)

90 (2.0)

98 (2.0)

93.3 (1.2)

88 (2.0)

80.7 (3.0)

76 (2.0)

MC 4 99.3 (1.2)

96.7 (1.2)

92 (2.0)

89.3 (1.2)

84.7 (2.3)

98.7 (1.2)

94 (2.0)

92 (2.0)

81.3 (1.2)

78 (2.0)

MC 5 100 (0) 98 (0)

96.7 (1.2)

95.3 (1.2)

93.3 (1.2)

98 (2.0)

94 (2.0)

93.3 (1.2)

88 (2.0)

80.7 (3.0)

MC 6 99.3 (1.2)

96.7 (1.2)

93.3 (1.2)

90 (2.0)

89.3 (1.2)

99.3 (1.2)

96.7 (1.2)

93.3 (1.2)

90 (2.0)

84.7 (2.3)

MC 7 100 (0) 99.3 (1.2)

97.3 (1.2)

96 (2.0)

94 (2.0)

99.3 (1.2)

96.7 (1.2)

92 (2.0)

89.3 (1.2)

84.7 (3.1)

MC 8 100 (0) 99.3 (1.2)

98.0 (2.0)

96.7 (1.2)

96 (2.0)

100 (0)

98 (0)

95.3 (1.2)

93.3 (1.2)

90.7 (1.2)

* Average of three determinations (SD).


362

Table-4: In vitro Drug Release Data of Microcapsules

Sl. No.

Formulation Code

t50% (h)

t70% (h)

t90% (h)

Cumulative % drug release in

8 h*

‘r’ value (Higuchi’s equation)

‘n’ value (Peppas equation)

1. MC 1 0.45 1.75 3.75 97.26±0.549a 0.9973 0.2616 2. MC 2 0.45 2.00 4.15 96.42±0.207a 0.9952 0.2699 3. MC 3 0.50 2.10 4.00 95.70±1.362a 0.9976 0.2730 4. MC 4 0.60 2.25 4.75 92.52±1.717a 0.9967 0.2802 5. MC 5 0.85 2.45 4.90 96.90±0.749b 0.9987 0.3209 6. MC 6 1.40 3.25 5.60 93.72±1.748b 0.9966 0.3558 7. MC 7 1.50 3.90 7.00 96.36±2.443 0.9974 0.3318 8. MC 8 2.25 4.35 7.60 92.16±2.298 0.9977 0.3833

* Average of three determinations ; a Cumulative % drug released in 5 h ; Cumulative % drug released in 6 h

Table- 5: Statistical Analysis of Drug-Content Data for the Stability Formulation (MC-8)

Sl. No. Trial No. A 1st Day

B 21st Day

A – B

1. I 31.44 31.48 -0.04

2. II 31.28 31.04 0.24 3. III 32.16 31.04 1.12 4.

Mean ( X ) 31.62 31.19 0.44

5. S.D ±0.468 ±0.254 ±0.605

‘t’ = 1.26 (p<0.05)

Table-6: Statistical Analysis of Dissolution Parameters (t50%, t70%) of Stability Formulation (MC-8)

t50% values t70% values Trial No.

1st Day (A) 21st Day (B)

A – B

1st Day (A) 21st Day (B)

A – B

I 2.45 2.50 -0.05 4.75 4.65 0.10 II 2.00 2.30 -0.30 4.15 4.35 -0.20 III 2.15 2.65 -0.50 4.30 4.80 -0.50

Mean ( X ) 2.20 2.48 -0.28 4.40 4.60 0.20

S.D ±0.229 ±0.175 ±0.225 ±0.312 ±0.229 ±0.300

‘t’ = 2.15; (p<0.05) ‘t’=1.16; (p<0.05)

Figure -1: Scanning Electron Photomicrographs of

Formulation MC-8


Formulation MC-8


363


Formulation MC-8

Figure-2: Cumulative % drug released versus time plot of

Microcapsules

concentration. (Table-2).

Microcapsules with a coat consisting of alginate and a

mucoadhesive polymer exhibited good mucoadhesive

properties in the in vitro wash off test. The wash off

was faster at intestinal pH than at gastric pH. It was

observed that the pH of the medium was critical for the

degree of hydration, solubility and mucoadhesion of the

polymers. The rapid wash off observed at intestinal pH

is due to ionization of carboxyl and other functional

groups in the polymers at this pH, which increases their

solubility and reduces adhesive strength. The results of

the wash off test indicated that the microcapsules had

very good mucoadhesive properties with more than 75

% retention after 6 h. (Table-3).

Atenolol release from the microcapsules was studied in

phosphate buffer (pH 6.2). Due to slight solubility of

drug in water, alginate-HPMC (50 cps) formulation

demonstrated a drug release of 92 – 98% in first 5 hr

with an initial burst release of 49-55 % within first 30

min. Increasing the cross-linking time from 3 to 6 h and

addition of magnesium stearate (2-4% w/w) sustained

the drug release upto 8 hr, with an initial burst release

of 33 – 44 % within first 30 min and a cumulative

release of over 92 – 97 % within first 8 h.(Table-4)

Atenolol release from the microcapsules was slow and

depended on the composition of coat (Fig. 2). Release

followed first order kinetics (r > 0.95). Microcapsules

of alginate-HPMC (50 cps) gave relatively fast release

when compared to alginate-HPMC (K4M). The drug

release from the microcapsules was diffusion

controlled, as cumulative percent drug released versus

the square root of time plots were found to be linear (r

> 0.99). From the Higuchi’s and Peppas data (Table–

4), it is evident that the drug is released by fickian

diffusion mechanism (n < 0.45). The formulation

prepared by alginate-HPMC (K4M) in a ratio of 5:1

along with 4% w/w magnesium stearate, emerged as the

overall best formulation (t50%=2.25 h, t70% = 4.35 h, t90%

= 7.60 h), based on drug release characteristics (in pH

6.2 phosphate buffer). This formulation shows slow and

extended release upto 8 h.

Short-term stability studies indicated that the drug

content and dissolution parameter of promising

formulation (MC-8) was not significantly affected by

storage at 40º±1ºC for 3 weeks. The ‘t’ value for the

drug content was found to be 1.26 (Table-5), whereas

the ‘t’ values for t50% and t70% were found to be 2.15 and

1.16 respectively (Table-6) against the table value of

4.30 (p < 0.05).

Drug-excipient interactions were ruled out by IR

spectroscopic studies on the promising formulation

(MC-8) stored for 3 weeks at 40º±1ºC. IR spectrum of

the pure drug shows the characteristic peaks at 3356.12

cm-1 and 1583.53 cm-1 due to N-H stretching and C=O

stretching of amide group respectively. The peak at

2963.87 cm-1 is due to alcoholic – OH group. The IR

spectrum of MC-8 formulation exhibited peaks at

3432.46 cm-1 and 1575.13 cm-1 due to N-H stretching

and peak at 2917.98cm-1 due to alcoholic – OH group.

F ig u r e -2 : C u m u la t iv e % d r u g r e le a s e d v e r s u s t im e p lo t o f

M ic r o ca p s u le s

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

0 1 2 3 4 5 6 7 8 9

T im e (h )

Cu

mu

lati

ve %

Dru

g R

ele

ased

M C - 1 M C -2 M C - 3 M C - 4 M C - 5 M C - 6 M C -7 M C - 8


364

This confirms the undisturbed structure of the drug in

the formulation.

The present study conclusively proves that MC-8

formulation with an alginate – HPMC (K4M) ratio 5:1

containing 4% w/w magnesium stearate has shown

promising results (released 92.16% drug in 8 h) as

sustained released mucoadhesive microcapsules of the

drug atenolol.

ACKNOWLEDGEMENTS

The authors are thankful to M/s. Vapi Care Pharma

Ltd., Vapi, for providing gift sample of Atenolol, Sipra

Labs, Hyderabad and IISC Bangalore for providing

facilities to carry out part of the work. The authors also

wish to thank the Principal, H.K.E Society’s College of

Pharmacy, Gulbarga, for providing facilities to carry

out the present work.

REFERENCES

1. Rouge N, Buri P, Doelker E. Drug absorption

sites in the gastrointestinal tract and dosage

forms for site-specific delivery. Int. J. Pharm.

1996; 136: 117-139.

2. Chowdary KPR, Srinivasa Rao Y. Preparation

and Evaluation of Mucoadhesive Microcapsules

of Indomethacin. Indian. J. Pharm. Sci. 2003;

65(1): 49-52.

3. Pothal RK, Sahoo SK, Chatterjee S, Sahoo D,

Barik BB. Preparation and Evaluation of

Mucoadhesive Microcapsules of Theophylline.

The Indian Pharmacist. 2004; 3(28): 74-79.

4. Kim CK and Lee EJ. The Controlled release of

blue dextran from alginate beads. Int. J. Pharm.

1992; 79: 11-19.

5. Martindale: The Complete Drug Reference,

Thirty-third edn. London: Pharmaceutical Press;

841: 2002.

6. Chowdary KPR and Srinivasa Rao Y. Design

and in-vitro and in-vivo evaluation of

Mucoadhesive microcapsules for oral controlled

release: A Technical Note. AAPS Pharm.

SciTech. 2003; 4(3): Article 39.

7. El-Kamel AH, Al-Gohary OMN, Hosny EA.

Alginate-Diltiazem hydrochloride beads:

Optimization of formulation factors, in-vitro and

in-vivo availability. J. Microencapsulation. 2003;

20(2): 211-225.

8. Chowdary KPR, Sambasiva Rao KRS,

Koteswara Rao.N. Design of EVA Microcapsules

of Glipizide for controlled release: Influence of

solvents used. Int.J.Chem.Sci. 2006; 4(1): 23-30.

9. Higuchi T. Mechanism of sustained-action

medication. Theoretical Analysis of rate of

release of solid drugs dispersed in solid matrices,

J. Pharm. Sci., 1963, 51, 1145-1149.

10. Peppas N.A. Analysis of Fickian and Non-

Fickian drug release from polymers. Pharm.

Acta. Helv, 1985, 60, 110-111

*********


365

Evaluation of Analgesic activity of root tuber of Curculigo

orchioides Gaertn.

V. Madhavan1, Joshi Richa

1, Murali Anita

2, S.N. Yoganarasimhan

1.

1Department of Pharmacognosy, M.S. Ramaiah College of Pharmacy, Bangalore - 560054, India 2Department of Pharmacology, M.S. Ramaiah College of Pharmacy, Bangalore - 560054, India

e-mail : [email protected]

Abstract

The aqueous and alcoholic extracts of the roots of Curculigo orchioides were evaluated for analgesic activity

using Eddy’s Hot Plate method and Heat conduction method on Swiss albino mice. The aqueous extract showed

significant analgesic activity. The findings support the use of this drug, C. orchioides in the treatment of pain.

Both the extracts were not toxic up to 3000 mg/kg body weight.

Key words: Curculigo orchioides, Analgesic activity, Eddy’s Hot Plate Method, Heat Conduction method.

INTRODUCTION

Curculigo orchioides Gaertn (Hypoxidaceae) is known

as Musali or Talamuli in Ayurveda and Nilapanai in

Siddha system of medicine1,2. The genus Curculigo

Gaertn. consists of 10 species, out of which three

species are found in India3. Curculigo orchioides is an

acaluescent herb found in the subtropical Himalayas

from Kumaon eastwards to Khasia Hills, Manipur,

Bihar, Chota Nagpur, West Bengal, Western Ghats4.

The root tuber is used in Siddha system for the

treatment of diseases like diabetes, leucoderma, pain

and as an aphrodisiac2. It is used in Ayurveda for

treatment of diseases like sprue, piles, disorders of

blood and also as an aphrodisiac and rejuvenator2.

Further, they are also used in skin diseases, as a

demulcent, diuretic, tonic, in diarrhoea, piles, jaundice

and asthma in combination with aromatics and bitters2.

Earlier corchioside A, 25- hydroxy-33-

methylpentatriconta-6-one, 21- hydroxy- tetracontan-

20-one, 27- hydroxy- tricontan- 6- one, 2- methoxy- 4

acetyl- 5methyltricontane, linoleic, linolenic, arachidic,

4- methylheptadecanoic acid, oleic, palmitic acids,

curculigol, curculigenin A, curculigosaponin AF,

cycloartenol, sitosterol and stigmasterol5, were

separated from C. orchioides. The present work was

aimed to study the analgesic activity of Curculigo

orchioides in mice.


Plant Material

Curculigo orchioides was collected from the Botanical

garden of the University of Agricultural Sciences,

GKVK Campus, Bangalore on 20th June, 2005. The

plant material collected was identified at the herbarium

of Regional Research Institute, Bangalore and

authenticated by Dr. Yoganarasimhan (Taxonomist and

Research Co-ordinator, Department of Pharmacognosy,

MSRCP, Bangalore) and a voucher herbarium

specimen Richa Joshi 001 is deposited in the herbarium

of PG department of Pharmacognosy, MSRCP,

Bangalore. About 3 kg of fresh root tuber was collected

from forests of Tirunelveli, Tamil Nadu on 9th

November 2004. The cleaned tubers were cut into small

pieces of 1- 2 cm, washed, dried at room temperature,

powdered and sieved through 60 mesh and stored in air

tight containers.

Preparation of Plant extracts

Alcoholic extract

A weighed quantity (500 g) of the air-dried powdered

APTI ijper


Received on 10.3.2006 ; Modified on 3.1.2007

Accepted on : 7/8/2007 © APTI All rights reserved


366

drug was taken and extracted with ethanol (90 %) in a

soxhlet extractor. The extract was concentrated in a

rotary flash evaporator at a temperature not exceeding

50°C. The alcoholic extract (5.6% w/w) was dissolved

in distilled water containing 2 % v/v tween 80 (as a

suspending agent).

Aqueous extract

A weighed quantity (500 g) of the air-dried powdered

drug was taken, macerated with hot water at 80°C. The

maceration process was carried out for 24 h. The

macerate was filtered through Whatman No. 1 filter

paper. The filtrate was concentrated in a rotary flash

evaporator, which yielded 7.03% w/w of aqueous

extract.

Animals

Swiss albino mice weighing 20-30 g of either sex were

maintained under controlled conditions of light (12 hr)

and temperature 25±1°C in the animal house of M. S.

Ramaiah college of Pharmacy, Bangalore, two weeks

prior to the experiment for acclimatization. Animals

had access to food and water ad libitum. All

pharmacological activities were carried out as per

CPCSEA (Committee for the Purpose of Control and

Supervision of Experiments on Animals) norms, after

obtaining the approval from the Institutional Animal

Ethical Committee of M. S. Ramaiah College of

Pharmacy, Bangalore.

Acute Toxicity Studies

Acute toxicity studies were carried out on Swiss albino

mice according to method proposed by Ghosh6.

Alcoholic and aqueous extracts at doses of 30, 100,

300, 1000 and 3000 mg/kg body weight were

administered to separate groups of mice (n=6), after

overnight fasting. Subsequent to administration of drug

extracts, the animals were observed closely for the first

three hours, for any toxic manifestations, like increased

motor activity, salivation, clonic convulsions, coma and

death. Subsequent observations were made at regular

intervals for 24 h. The animals were observed for

further one week.

Analgesic activity

Eddy’ s Hot Plate Method7

The animals were grouped into 6 groups of six animals

each. Group1 received distilled water, which served as

control. Group 2 received Morphine sulfate (5 mg/kg,

i.p.) (Astra Zeneca Pharma India Ltd., Bangalore), and

served as the standard. Groups 3 and 4 received

aqueous extracts at doses of 500 and 1000 mg/kg

respectively. Groups 5 and 6 received alcoholic extracts

at doses of 500 and 1000 mg/kg respectively. All the

extracts were administered orally.

Sixty min after oral administration of extracts and 30

min after i.p. injection of morphine sulfate, animals

were individually placed on the Hot plate (maintained

at 55 °C) and the responses such as paw licking or jump

response, whichever appeared first were noted. Cut off

period of 15 sec was maintained.

Heat conduction method8

The animals were grouped into 6 groups of six animals

each. Group1 received distilled water, which served as

control group. Group 2 received Morphine sulfate (5

mg/kg, i.p.) and served as the standard group. Groups 3

and 4 received aqueous extracts at doses of 500 and

1000 mg/kg respectively. Groups 5 and 6 received

alcoholic extracts at doses of 500 and 1000 mg/kg

respectively. All the extracts were administered orally.

Sixty minutes after oral administration of extracts and

30 min after i.p. injection of morphine sulfate, the tail

tip of individual animals was dipped up to 5 cm into hot

water (maintained at 58 °C) and the response time was

noted as the sudden withdrawal of the tail from the hot

water. Cut off period of 10 sec was maintained.

Statistical analysis

Statistical analysis was performed, using one way

analysis of variance (ANOVA) followed by Tukey-

Kramer Multiple Comparison Test. All values were

expressed as mean ± SEM.


Acute toxicity studies did not reveal any toxic

symptoms or death in any of the animals up to the dose

of 3000 mg/kg body weight, with either extract.

Aqueous extract of C. orchioides showed significant

analgesic activity as evidenced by the increase in

reaction time to the pain stimulus. The results were

significant at p <0.001 for both Eddy’s Hot Plate

method and Heat conduction method. The analgesic

activity is presented in Tables 1 and 2 and in

Histograms 1 and 2. Analgesic activity of the drug in

aqueous extract was comparable to the standard drug

Morphine sulfate.

From this study, it can be concluded that aqueous

extract of root tuber of C. orchioides possesses marked


367

02468

1012

1 2 3 4 5 6Groups

Tim

e i

n S

ec

on

ds

SEM

MEAN

0

2

4

6

8

10

1 2 3 4 5 6

Groups

Tim

e in

Seco

nd

s

SEM

MEAN

Histogram 1: Analgesic activity by Eddy’s Hot Plate method

1-Control; 2- Morphine sulphate (5mg/kg); 3- Aqueous

extract (500 mg/kg); 4- Aqueous extract (1000 mg/kg); 5-

Alcoholic extract (500 mg/kg); 6- Alcoholic extract

(1000 mg/kg)

Histogram 2: Analgesic activity by Heat Conduction Method

1-Control; 2- Morphine sulphate (5 mg/kg); 3- Aqueous

extract (500 mg/kg); 4-Aqueous extract (1000 mg/kg); 5-

Alcoholic extract (500 mg/kg); 6- Alcoholic extract

(1000 mg/kg)

Table 1: Analgesic activity of aqueous and alcoholic extracts of root tuber of C. orchioides by Eddy’s Hot Plate

method

Groups Reaction Time (seconds)

Control 2 ± 0.2582 Morphine sulfate (5 mg/kg) 9.3 ± 0.3333 * * * Aqueous extract (500 mg/kg) 4.1 ± 0.1667 * * * Aqueous extract (1000 mg/kg) 4.6 ± 0.3333 * * * Alcoholic extract (500 mg/kg) 2.5 ± 0.2236 Alcoholic extract (1000 mg/kg) 2.8 ± 0.1667

Values are expressed as mean ± SEM; n=6; * * * p< 0.001.

Table 2: Analgesic activity of aqueous and alcoholic extracts of root tuber of C. orchioides by Heat conduction

method

Groups Reaction Time (sec)

Control 2 ± 0.2582

Morphine sulfate (5 mg/kg) 8.6 ± 0.2108* * *

Aqueous extract (500 mg/kg) 4.3 ± 0.2108 * * *

Aqueous extract (1000 mg/kg) 4.5± 0.4282 * * *

Alcoholic extract (500 mg/kg) 2.3 ± 0.2108

Alcoholic extract (1000 mg/kg) 2.6 ± 0.3333

Values are expressed as mean ± SEM; n=6; * * * p< 0.001.

analgesic activity and is equipotent to standard

analgesic drugs. The present study establishes the

effectiveness and pharmacological rationale for use of

C. orchioides as an analgesic drug. The drug may be

further explored for its phytochemical profile to

identify the active constituents responsible for the

analgesic activity. Also, the medical application of the

drug for use in the treatment of pain in traditional

systems of medicine is substantiated.

ACKNOWLEDGEMENTS

The authors are thankful to Gokula Education

Foundation for providing financial support and facilities


368

to carry out this work.

REFERENCES

1. Narayana Aiyer MA, Kolammal M.

Pharmacognosy of Ayurvedic Drugs, Trivandrum:

Department of Pharmacognosy, University of

Kerala; 1963.

2. Yoganarasimhan SN. Medicinal Plants of India.

Vol 2. Tamil Nadu. Bangalore: Cyber media; 2000.

3. Santapau H and Henry AN. A Dictionary of the

flowering Plants in India. New Delhi: CSIR; 1976

(repr. ed.).

4. Sharma PC, Yelne MB, Dennis TJ. Database on

Medicinal plants used in Ayurveda. Vol. 4. New

Delhi: Ministry of Health And Family Welfare;

2002.

5. Chaterjee A, Pakrashi SC. The Treatise on Indian

medicinal plants. Vol. 6. New Delhi: CSIR; 2001.

6. Ghosh MN. Fundamentals of Experimental

Pharmacology. 3rd ed. Kolkata: Hilton and

company; 2005.

7. Eddy NB and Leimbach DJ. Synthetic analgesics:

II Dithienyl butenyl and Dithenyl butylamines

(Retracted by Turner RA. Screening methods in

Pharmacology I, I ed. New York, London:

Academic Press; 1965; 105-109) J Pharmacol Exp

Therap 1953; 107(3): 385-93.

8. Gawade SP. Experimental Pharmacology. How’s

and What’s of Pharmacology. I st ed. Pune: Nirali

Prakashan;1995.

*******


369

Effect of Eclipta alba Linn on learning and memory in rats G.P.Rajani ∗∗∗∗, KVSRG Prasad ∗∗∗∗∗∗∗∗

∗ K.L.E.Societys College of Pharmacy, Bangalore., Karnataka, India

∗∗ Sri Padmavathi Mahila VisvaVidyalayam,Tirupathi, India

E [email protected]

Abstract

The present study was taken up to evaluate the effect of ethanolic extract of Eclipta alba Linn (EEA) on

learning and memory. Passive avoidance response and elevated plus maze were the two models used for

evaluating the effect of the drug on learning and memory. In the passive avoidance model, EEA treated animals

showed a significant decrease in the latency to reach the safe zone with reduced occurrence of mistakes. In the

elevated plus maze model the transfer latency was reduced in the treated animals. The drug was found to have

significant memory enhancing activity on acute as well as chronic treatment.

Key words: Memory enhancers, Eclipta alba Linn, Elevated plus maze, Passive avoidance response.

INTRODUCTION

Eclipta alba Linn belongs to the family Compositae. It

is an annual herb, erect, branched often rooting at

nodes. Stem and branches have appressed white hairs,

flowers are small and white. It is commonly known as

Bringharaj. The main chemical constituents are

wedelactone, demethyl wedelactone, ecliptal,

triterpenoids like 4 heptaeosanol and flavanoids like

luteolin-7-o glucoside, ursolic acid, oleanolic acid

ecliptasaponin and daucosterol. It is found to have

antiviral, antinociceptive, anti-inflammatory,

bronchodilator, antibacterial, antipyretic, tonic,

expectorant and hepatoprotective activity1,2. The plant

has been extensively studied for its hepatoprotective

activity and a number of herbal preparations comprising

of Eclipta alba are available for treatment of jaundice

and viral hepatitis The aqueous and alcoholic extracts

of the plant are proved to confer protection against the

myotoxic effects of snake venom 3.

It is reported in traditional medicine that Eclipta alba

may be having beneficial effect on learning and

memory4, 5. Eclipta alba has not been studied for its

activity on learning and memory in particular and CNS

activity in general. The present study was taken up to

investigate scientifically if the herb infact can affect

learning and memory.

Cognitive deficits is recognized as a neurological

disorder and is associated with neurodegenerative

diseases like Alzheimers disease, senile dementia,

parkinsonism etc. Learning is defined as the acquisition

of information and skills. Memory is the subsequent

retention of that information6. Cholinergic neurons in

forebrain and brainstem send diffuse projections to

hippocampus and cortex. Degeneration of the above,

nucleus basalis of meynert and septohippocampal

nucleus is involved in Alzheimers disease and in

learning and short term memory. Glutamate and

NMDA agonists are involved in long term potentiation

and formation of synaptic contacts, which form an

important part of learning and memory. Cholinergic

agonists, Glutamate and NMDA agonists help in

improving learning and memory7.


Animals.

Male albino Wistar rats weighing between 200-250 g

were selected for the study. Animals were housed under

standard laboratory conditions in colony cages.

Ambient temparature of 25±2 oC, 45-55% relative

humidity and 12h dark and light cycle was maintained.

The animals had free access to food and water. All the

experiments and observations were carried out under

these ambient conditions between 9.00 to 17.00 h. Prior

APTI ijper


Received on 7/12/2006 ; Accepted on 7/8/2007



370

IAEC approval was obtained to carry out the research

work.

Preparation of the plant extract

The authenticated aerial parts of EEA used for the study

was obtained from Hill green company, Vasanthnagar,

Bangalore. One kilogram of the aerial parts were air

dried and macerated with 90% ethanol for 24 h. The

filtrate was concentrated to dryness in a rotary

evaporator in vaccum at 50 0 C. (Yield –10.8%)

Acute toxicity studies

Acute toxicity studies of the alcoholic extract was

conducted as per OECD guide lines8. Each animal was

administered the aqueous solution of the alcoholic

extract by oral route and the animals were observed for

any changes continuously for two hours and for 24 h for

mortality. The animals were observed for one more

week there after. The extract was found to be safe upto

2000 mg /Kg body wt.

Chemicals

Piracetam –Cetam, Tauras Labs,Ahmedabad,Gujarat.

Apparatus

Both the apparatus used for testing passive avoidance

response and elevated plus maze were fabricated 6.

Passive avoidance response

The apparatus consisted of an electric grid (20x30 cm)

and a shock free zone (2x3x1 cm) in the center, which

was enclosed in a perspex chamber. 20V stimulus could

be applied to the electric grid 6,7.

Elevated plus maze

It consisted of 2 opposite open arms 50x10 cm crossed

with 2 closed arms of same dimensions with walls 40

cm high. The arms were connected with a central

square 10x10 cm to give the apparatus a plus sign

appearance. The maze was elevated 70 cm above the

floor in a dimly lit room 6, 9.

Experimental procedure

Passive avoidance response9

The animals were randomly divided into seven groups.

The group I animals were given sham electric shock.

The group II animals were used for administering acute

electro convulsive shock (ECS) 10mA for 0.2 msec by

applying crocodile clips to the ears. The same animals

were used for chronic studies by continuing the ECS for

7 days. The group III animals were administered EEA

200 mg/kg body weight (acute treatment); the same

animals were used for chronic studies by continuing the

treatment for 7 days. The group IV, V and VI animals

were treated similarly by administering EEA 300

mg/kg, EEA 400 mg/kg and Piracetam 250 mg/kg

respectively EEA was administered by oral route and

Piracetam by intraperitoneal route.

The animals were placed individually on the electric

grid in the passive avoidance chamber to explore for 1

min. The stimulus of 6 mA was applied to the electric

grid and latency to reach the shock free zone (SFZ) was

recorded 3 times as the basal reading. The animals were

administered the drug as specified for the respective

groups. One hour after the administration of the drug,

ECS 10 mA for 0.2 sec was applied. Thirty min after

ECS, the animal was placed on the electric grid to

which stimulus of 6mA was applied and the time taken

to reach the SFZ and the number of mistakes (descents)

made in 15 min was also noted down.

Elevated plus maze8-12

The animals were divided into 6 groups. The group I

animals were used for sham electro convulsive shock.

The group II animals were applied ECS 10mA for 0.2

sec. The group III, IV, V and VI animals were treated

with EEA 200 mg/kG, 300 mg/kg and 400 mg/kg and

piracetam 250 mg/kg body weight respectively.

The drugs were administered by oral route. One hr after

drug administration, ECS 10mA for 0.2 sec was applied

through crocodile clips applied to the ear. The animals

were placed on the open arm of elevated plus maze 30

min after ECS and transfer latency (time taken to enter

enclosed arm) was noted down. After recording transfer

latency (TL) the animals were left for 30 sec more to

explore the maze. The TL was also measured after 24

hr.

Statistical analysis

The results are expressed as mean ± SEM.The data are

analysed by one way analysis of variance (ANOVA)

followed by Neumen Keuls multiple comparision test.


In the present study 2 models were used for studying

the effect of eclipta alba on learning and memory. In

the first model (passive avoidance response), latency to

reach SFZ (acquisition) and the number of mistakes

(descents) made by the animal in 15 min was studied

(retention). In the second model (elevated plus maze),

the transfer latency of the animal when placed on the

open arm was recorded on day 1 and 2.

Generally rats show natural aversion for open arms and

tend to reach enclosed arms and spend more time there.


371

Table I Effect of various doses of Eclipta alba L (acute) on latency to reach SFZ and number of mistakes in 15 min in passive

avoidance response model

Group Treatment Latency to reach SFZ in sec

No of mistakes in 15 min

I Sham electroshock 6.5±0.34 7±0.36 II ECS(vehicle) 7±0.57 7±0.57 III EEA 200mg/kg 4±0.51** 6±0.25**

IV EEA 300mg/kg 3±0.44*** 5±0.44***

V EEA 400mg/kg 3±0.36*** 4±0.36***

VI Piracetam 250mg/kg 2±0.35*** 2±0.36***

Values are mean ± SEM (n = 6); **P< 0.01, ***P< 0.001 compared to ECS group

Table II Effect of various doses of Eclipta alba L (chronic) on latency to reach SFZ and number of mistakes in 15 min in

passive avoidance response model

Group Treatment Latency to reach SFZ in sec No of mistakes in 15 min

I Sham electroshock 6.5±0.34 7±0.36 II ECS(vehicle) 10±0.73 10±0.51 III EEA 200mg/kg 3±0.57** 4±0.25***

IV EEA 300mg/kg 2±0.25*** 3±0.44***

V EEA 400mg/kg 2±0.36*** 2±0.30***

VI Piracetam 250mg/kg 1±0*** 1±0***

Values are mean ± SEM (n = 6); **P< 0.01, ***P< 0.001 compared to ECS group

Table III Effect of Eclipta alba L on transfer latency on elevated plus maze in electroconvulsive shock treated rats

Transfer latency (TL) in sec

Transfer latency (TL) in sec

Group Treatment

1st day 2nd day

I Sham electroshock 41.66±2.10 19.00±3.04 II ECS (Vehicle) 64.33±3.08 47.83±1.92 III EEA 200mg/kg 55.33±2.56 39.50±0.61 IV EEA 300mg/kg 43.00±2.29** 33.30±1.52**

V EEA 400mg/kg 31.66±1.20** 14.83±2.76***

VI Piracetam 250mg/kg 26.16±1.86*** 13.66±1.89***

Values are mean ± SEM (n = 6); **p< 0.01, ***p< 0.001 compared to ECS group

Both the models are normally used for studying short

term memory.

Electro-convulsive shock 10 mA for 0.2 sec was used in

the present study in both the models for producing

amnesia. In passive avoidance response model it was

found out that EEA at all the three doses produced

significant decrease in latency (at 200mg/kg, P<0.01and

at 300 mg/kg and 400 mg/kg, P<0.001), in acute as well

as chronic treatments when compared to the control.

The drug EEA at 300 and 400 mg/kg showed activity

comparable to that of the standard drug piracetam. On

acute treatment at the doses of 300 and 400 mg/kg in

both the models there was a significant decrease

(P<0.001) in the number of mistakes. Where as, on

chronic treatment the drug EEA at all the three doses

showed significant decrease (P<0.001) in the number of

mistakes (Table 1and 2). These results suggest that

Eclipta alba improves both acquisition and retention of

memory.

In the elevated plus maze there was a significant

decrease (P<0.01 and P<0.001) in transfer latency on

both day 1 and 2 respectively at both the doses 300 and

400 mg/kg body weight. On day 2, EEA at 400 mg/kg

showed activity comparable to that of the standard

(Table 3). This indicates that EEA has significant

memory enhancing activity.

The involvement of free radicals in aging and

accelerated aging diseases like Parkinsonism and


372

Alzhiemers diseases is evident from research. The plant

Eclipta alba contains flavanoids which are known to

have antioxidant activity may be aiding in memory

enhancing activity13.

The herb Eclipta alba has significant memory

enhancing activity as indicated by both the models.

Further study has to be conducted regarding the

involvement of neurotransmitters in the enhancement of

memory. Such a study can suggest the possible

mechanism of memory enhancing activity of Eclipta

alba.

ACKNOWLEDGEMENTS

We are thankful to Hill green company, Vasanthnagar ,

Bangalore for providing the drug to carry out the

activity and The Director, K.L.E. Societys college of

pharmacy, Bangalore, for providing the facilities to

carry on the research work.

REFERENCES

1. Kirtikar KR, Basu BD.Indian Medicinal Plants. 2nd

ed. Delhi: Jayyed Press; 1975.

2. Rajagopal V.Standardization of Botanicals.vol I

New Delhi: Eastern Publishers; 2002.

3. Thakur VD, Mengi SA .Neuropharmacological

profile of Eclipta alba (Linn.) Hassk.J of

Ethnopharmacology 2005; 102:23-31.

4. Ashok DB, Vaidya.The status and scope of Indian

Medicinal Plants acting on Central nervous system.

Indian J Pharmacol 1997; 29:340-343.

5. Satyavathi GV, Rana MK, Sharma M. Medicinal

plants of India.Vol I.: Cambridge printing works;

Delhi 1976.

6. Reddy DS.Assessment of nootropic and amnestic

activity of centrally acting agents. Indian J

Pharmacol 1997; 29:208-221.

7. Rang HP,Dale MM, Ritter JM, Moore

PK.Pharmacology.5th ed. London: Churchill

livingstone;2003.

8. www.oecd.org/ehs

9. Kulkarni SK, Anita Verma. Evidence for nootropic

effect of BR-16A (Mentat) a herbal psychotropic

preparation,in mice. Indian J Physiol Pharmacol

1992; 36(1): 29-34.

10. Jiro Itoh, Toshitaka Nabeshima, Tsotomu

Kameyama.Utility of an elevated plus-maze for the

evaluation of memory in mice: effects of

Nootropics, scopolamine and electroconvulsive

shock. Psychopharmacology 1990; 101:27-33.

11. Jaiswal AK, Bhattacharya SK.Effects of Shilajit on

memory, anxiety and brain monoamines in rats.

Indian J Pharmacol 1992; 24:12-17.

12. Anita verma, Kulkarni SK.Effect of herbal

psychotropic preparation, BR-16A (Mentat) on

performance of mice on elevated plus maze. Indian

J of Exp Biol 1991; 29:1120-1123.

13. Nasik S.R. Antioxidants & their role in biological

functions: An overview. Indian Drugs 2003; 40(9):

501-516.

***********


373

Application of Hibiscus Leaves Mucilage as

Suspending Agent Edwin Jarald

∗∗∗∗, Edwin Sheeja, Dosi Shweta, Amal Raj, and Gupta Smita ∗Department of Pharmacognosy, B.R. Nahata College of Pharmacy, Mandsaur-458001, Madhya Pradesh, India.


Abstract

The present study was undertaken to evaluate the mucilage obtained from the leaves of Hibiscus rosasinensis

Linn as a suspending agent. A suspension of CaCO3 was prepared using 2 % w/v of hibiscus mucilage as

suspending agent and it is evaluated for its stability using the parameters like, sedimentation volume, viscosity,

redispersibility and pH. The suspending effect of hibiscus mucilage was compared with CaCO3 suspensions

prepared using 2 % w/v of suspending agents such as acacia and tragacanth. The results obtained indicated

that the hibiscus mucilage could be used as a suspending agent. It has low rate of sedimentation, high viscosity,

slightly basic pH and is easily redispersible. These effects were comparable with that of the standard

suspending agents like acacia and tragacanth. The mucilage isolated from the leaves of Hibiscus rosasinensis

can be used as a pharmaceutical adjuvant.

Key words: Hibiscus rosasinensis, mucilage, suspending agent, CaCO3

INTRODUCTION

Mucilages and gums are well known since ancient

times for their medicinal use. In recent years, plant

gums and mucilages have evoked tremendous interest

due to their diverse application in pharmacy in the

formulation of both solid and liquid dosage forms1.

They are employed as suspending agents in the

formulation containing indiffusible materials2.

Naturally demand for this substance is increasing and

new sources are getting identified. The present study

relates to a mucilage, obtained from the leaves of

Hibiscus rosasinensis Linn that could be used as

suspending agent.

The plant Hibiscus rosasinensis. Linn (Malvaceae)3,

commonly known as Jasavanda is used to treat various

diseases. The main parts used in the plants are roots and

petals, but researchers have explored that even the

leaves possess very good medicinal properties and are

used as antihypertensive4, antifertility agent5, for hair

growth6 and as antidiabetic7

The plant was found to contain mucilage8 and in our

earlier studies9, we have identified the mucilage in the

leaves of the plant. The major constituent of

the mucilage is an acidic polysaccharide composed of

L-rhamnose: D-galactose: D-galacturonic acid: D-

glucuronic acid in the molar ratio of 5:8:3:28. The

present work is an attempt to investigate this mucilage

as a suspending agent in pharmaceutical formulations.


Isolation of mucilage

The leaves were collected and sun dried for at least 7

days. The powdered leaves were defatted using

petroleum ether (60-800C) in a soxhlet apparatus. The

defatted material (50 g) was soaked in distilled water

(1000 ml) at room temperature for 12 h. The resulting

mass was stirred at about 100 rpm for 1 h and strained

through muslin cloth. To the filtrate, acetone was added

until precipitation was complete. The precipitated

mucilage was filtered through muslin cloth and the

mucilaginous residue was spread on glass plates and

dried at 400C. Then it was dispersed in 200 ml water

with stirring for 12 h and ethanol was added in different

proportions. Initially, the concentration of ethanol was

made up to 20% in the solution. Some impurities that

precipitated were removed by centrifugation. The

ethanol concentration was further increased to 60% to

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374

precipitate the mucilage. The precipitated mucilage was

filtered, treated with acetone to remove the traces of

water and dried in an oven at 400C10.

Preparation and evaluation of suspensions

Suspensions of 2 % Caco3 in water was made using 2%

of suspending agents1,10 like acacia, tragacanth, and

hibiscus mucilage. For the preparation of suspension

Caco3 was first levigated with glycerine (1:1) and then

the suspending agents were added in required amounts2.

The test suspension was evaluated using the parameters

like, sedimentation volume, redispersibility, pH and

viscosity and it was compared with acacia and

tragacanth.

Sedimentation Volume: Sedimentation volume is the

most important parameter in the evaluation of

suspension stability. Sedimentation volume F is the

ratio of the ultimate height (Hu) of the sediment as a

suspension settles in a cylinder under standard

conditions to the initial height (Ho) of the total

suspension. It was determined by keeping a measured

volume of the suspension in a graduated cylinder in an

undisturbed position for a definite period of time and

noting the value of Hu and Ho2.

Redispersibility: Redispersibility of a suspension can

be estimated by shaking the suspension with the help of

a mechanical device, which simulates the motion of

human arm during shaking2. Fixed volume (50 ml) of

the each suspension was kept in calibrated tubes, which

were then stored at room temperature for various time

intervals (5, 15, 25 days). At regular intervals (5, 15, 25

days) one tube was removed and shaken vigorously to

redistribute the sediment and the presence of deposit if

any is noted. The time taken to redisperse the

sedimented suspension was recorded10,11.

Determination of pH and viscosity: The pH of the

suspensions was determined at intervals of one week

for 21 days using pH meter and their viscosity was

determined at 250 C using brookfield viscometer at

50rpm by using spindle no. 311. The values expressed

are mean ± SD of three observations.


The average yield of dried mucilage obtained from

hibiscus mucilage was 10.6% w/w. It is quite well

understood that the better is the suspending medium the

lesser the rate of sedimentation. The sedimentation

volume profile of the suspensions with hibiscus

mucilage, acacia and tragacanth are presented in Table

1 and graph 1. The dispersed particles of CaCo3

prepared using hibiscus mucilage was found to

sediment at lower rate than those prepared with

tragacanth and slightly higher than that of acacia.

Graph 1: Determination of suspending property of

Hibiscus mucilage

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25

Time in minutes

Sed

imen

tati

on

vo

lum

e

Blank Hibiscus

Tragacanth Acaccia

Since the suspension produces sediment on storage, it

must be readily dispersible so as to ensure the

uniformity of the dose. Less is the time taken to

redisperse the sediment, the better is the

redispersibility. The suspension prepared by hibiscus

mucilage showed better redispersibility than acacia and

tragacanth on 5th and 15th day and on 25th day it was

similar to the suspension prepared using acacia (Table

2).

Nowadays, the whole world is turning towards natural

drugs and excipients. The natural materials do hold

advantages over the synthetic materials because they

are non toxic, less expensive and freely available.

Further they can be modified to obtain tailor made

materials for drug delivery system and then can

compete with the synthetic products available in the

market. In this aspect, the hibiscus leaves mucilage

tested for suspending effect has shown promising

results and the effects were comparable with that of the

standard suspending agents like acacia and tragacanth.

Toxicity is not at all a concern for this mucilage

because the effective concentration of the suspending

agents in conventional dosage form normally does not

exceed 2% of the formulation10 and earlier studies on

this mucilage states that it is very safe even in higher

doses8.


375

Table 1: Determination of Suspending Property of Hibiscus mucilage Time in Minutes Blank (CaCo ) 3

(F) CaCo + Hibiscus 3

(F) CaCo + Tragacanth 3

(F) CaCo + Acacia 3

(F) 0 1 1 1 1 5 0.8 0.92 0.91 0.917

10 0.058 0.91 0.835 0.91 15 0.058 0.882 0.823 0.91 20 0.058 0.882 0.794 0.9 25 0.058 0.852 0.764 0.882 30 0.058 0.852 0.694 0.882

Sedimentation volume (F) = Hu/Ho

Table 2: Determination of pH, Viscosity and Redispersibility pH after storage for Rate of redispersibility (cycles)

Excipients 2 % w/w

th th th st0 day 7 day 14 day 21 day

Viscosity

(Centipoise) 5 days 15 days 25 days

Tragacanth 4.00 ± 0.10

4.32 ± 0.15

4.52 ± 0.05

4.60 ± 0.10

16.33 ± 1.52

13.00 ± 1.00

17.33 ± 1.52

20.00 ± 1.15

Acacia 4.24 ± 0.10

4.30 ± 0.10

4.70 ± 0.05

5.00 ± 0.10

20.00 ± 2.00

14.33 ± 1.52

16.66 ± 1.00

22.66 ± 1.15

Hibiscus Mucilage

8.00 ± 0.15

8.10 ± 0.10

8.32 ± 0.15

8.52 ± 0.20

24.33 ± 2.55

12.00 ± 1.00

15.33 ± 1.52

23.00 ± 1.00

Values are expressed in mean ± SD, n=3

From the observations it is concluded that the extracted

mucilage from leaves of Hibiscus rosasinensis is non-

toxic, has the potential as a suspending agent even at

low concentration and can be used as a pharmaceutical

adjuvant.

REFERENCES

1. Chandra Sekhara Rao G, Girija Prasad P, Srinivas

K, Bhanoji Rao ME. Application of Moringa

oleifera gum as a suspending agent in the

fformulation of Sulphamethoxazole. The Indian

Pharmacist 2005; 5:75-7.

2. Jain NK, Sharma SN. Text book of Professional th Pharmacy. 4 ed. New Delhi:Vallabh Prakashan;

1998.

3. ndNadkarni AK. Indian Materia Medica. 22 ed. Vol.

1. Mumbai:Popular Book Depot; 1995.

4. Anees AS, Sachin MW, Rajesh R, Alagarsamy V.

Isolation and hypotensive activity of five new

phytoconstituents from chloroform extract of

flowers of Hibiscus rosasinensis Linn. Indian J

Chem 2005; 4:4-7.

5. Nivsarkar M, Patel M, Padh H, Bapu C,

Shrivastava N. Blastocyst implantation failure in

mice due to nonreceptive endometrium:

endometrial alterations by Hibiscus rosa-sinensis

leaf extract. Contraception 2005; 71:227-30.

6. Adhirajan N, Ravikumar T, Shanmughasundaram

N, Babu J. In vivo and in vitro evaluation of hair

growth potential of Hibiscus rosa-sinensis Linn. J

Ethnopharmacol 2003; 88:235-9.

7. Sachdewa A, Khemani LD. Effect of Hibiscus rosa

sinensis Linn ethanol flower extract on blood

glucose and lipid profile in streptozotocin induced

diabetes in rats. J Ethnopharmacol 2003; 89:61-6.

8. Shimizu N, Tomoda M, Suzuki I, Takada K. Plant

mucilages. XLIII. A representative mucilage with

biological activity from the leaves of Hibiscus rosa

sinensis. Bio Pharm Bull 1993; 16:735-9.

9. Edwin E, Sheeja E. Pharmacognostical and

Preliminary Phytochemical Studies on Hibiscus

rosasinensis. Ind J Nat prod 2005; 21:43-5.

10. Anroop B, Bhatnagar SP, Ghosh B, Parcha V.

Studies on Ocimum gratissimum seed mucilage:

evaluation of suspending properties. Ind J Pharm

Sci 2005; 67:206-9.

11. Verma PRP, Razdan B. Evaluation of Leucaenea

leucocephata seed gum as suspending agent in

sulphadimidine suspensions. Ind J Pharm Sci 2003;

67:665-8

*********


376

The New Patent Regime – Implications for Indian Pharma

Industry K.Madhavi

Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Visvavidyalayam, Tirupati


Abstract

The new patent regime implemented in India as a sequel to its commitment to the Trade Related Intellectual

Property Rights Agreement raises several crucial issues, such as public health consideration and its impact on

Indian Pharma Industry. In this paper the status of Pharma Industry, the new Patent Regime and the future

strategies for protection of domestic Pharma Industry are discussed.

Key words: TRIPS, Patent Regime, Pharma Industsry, Public Health Care

INTRODUCTION

The new patent regime, providing protection to

pharmaceutical patents both process and product,

through the instrument of Trade Related Intellectual

Property Rights and the domestic legislation has

assumed considerable significance in the light of

globalization of the market and the national policies

induced by trade liberalization. The new international

patent regime, which has forced drastic changes in the

domestic patent legislation has invoked national debate

on the impact of this new regime on the Pharma

Industry on the one hand and the public health care

system on the other. While developed countries

favored strong protection keeping their commercial

interest in the global market, the developing countries

were never in favour of giving up the advantage of

weak protections of their pharmaceutical industry. The

developing countries have their own priorities like

improving the standards of living, maintaining prices of

drug etc.

The ongoing debate on product patent in

pharmaceutical industry centers around the

accessibility of essential drugs to the poor and price

hikes resulting out of a possible monopolistic

condition. Granting a product patent means conferring

exclusive marketing rights (EMRs) to the patent holder

for a period of 20 years.

Drugs being treated as essential commodities, research-

based Pharma companies, which are mostly Multi-

National Corporations today, will definitely be keen to

sell the patented drugs at maximum possible profits and

at unaffordable prices to the poor and the middle class

sections of the society1.

INDIAN PHARMA INDUSTY – AN OVERVIEW

Pharmaceutical industry is one of India’s most

successful industries. The Industry’s contribution to

the economy of the country is not only mammoth but it

also provides drugs at affordable prices to the different

classes of our society. Almost the entire domestic

demand is met by the industry’s indigenous production.

Today India is amongst the top 15 pharmaceutical

manufacturing countries in the world. It is significant

to note that India accounts for 8% of world’s

pharmaceutical production volume wise and is the fifth

largest country in the world after the US, Japan, Europe

and China in terms of pharmaceutical production.

India is the cheapest producer of drugs since its labour

cost is 50% to 55% less than the west. Infrastructure

cost lower by at least 40%. It has a vast pool of talent,

and the Indian Patent Act, 1970, accepted only process

patent prior to amendment. Thus, overall with these

benefits the industry was able to develop every drug at

a fraction of cost that was being incurred by innovative

company2.The Indian pharmaceutical industry is a

successful, high-technology-based industry that has

witnessed consistent growth over the past three

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Received on 23.9.2005; Accepted on 26.3.2007



377

decades, in spite of the fact that, it in operating under

severe price competition and government price control.

The Indian pharma sector is highly fragmented with

more than 24,000 registered Units. It has expanded

drastically in the last two decades. Formulations

account for 81.5% of the market and bulk drugs

account for the remaining 18.5%3. The leading 250

pharmaceutical companies control 70% of the market,

with market leaders holding nearly 7% of the market

share. Requirement for 85% of bulk drugs and almost

all formulations is met within India itself. There are

around 465 main bulk drugs used in India and out of

these, around 425 bulk drugs are totally manufactured

in India.4,5 The Indian pharmaceutical industry is

expected to register a growth rate of 11 percent to

enhance its size to Rs. 60,000 Crores by 2007-08 as

against Rs. 43,290 Crores in 2004-056. The clinical

research market in India is estimated to be $100 million

and is expected to grow to $300 million by 20107

Further, the Pharma Industry targeted to export

Rs.63,000 in 2010, the non-conventional markets were

expected to earn 60 per cent. These markets now

contribute one fifth of the total export of Rs.16,682

crore.8

There are many factors, which have contributed to the

growth of the Indian pharmaceutical industry. Some of

these factors need a special mention9.

• India has more demand as a result of increased

population.

• Government encouragement to Multi-National

Corporations in the early period resulted in entry

of more Multi-National Corporations.

• The Indian Patent Act (Process Patent) and Drug

Price Control Order (DPCO) supported the

development of domestic companies.

• Wholly developed chemical industries in India

encouraged the development of the bulk drugs

segments.

• Cheap manpower in the Indian pharmaceutical

industry made the export cost-effective.

• Limited pollution control norms and environment

cleanliness made the cost of production in India

lower than that in developed countries.

TRIPS AGREEMENT – PATENT

(AMENDMENT) ACT 2005 - The TRIPS

agreement in Articles 27 to 34 deals with the patent

regime to be followed by the member countries, which

include India. Article 27 of the TRIPS Agreement

clearly provides that patents shall be made available for

any inventions, whether products or processes, in all

fields of technology, provided that they involve

inventive step, are new and capable of industrial

application. This provision is subject to certain

transitional provisions contained in part VI of the

TRIPS Agreement It is also provided that patents shall

be available and patent rights enjoyable without

discrimination as to the place of invention, the field of

technology and whether the products are imported or

locally produced.

The TRIPS agreement required Member countries

under Article 27(1) to make patents available for any

inventions, whether product or processes, in all fields

of technology without discrimination, subject to the

normal tests of novelty, inventiveness and applicability.

There are three permissible exceptions to the basic rule

on patentability. Members may exclude from

patentability10 under Article 27(2) inventions contrary

to public order or mortality, this explicitly inventions

dangerous to human, animal or plant life or health or to

avoid serious prejudices to the environment.

Member countries may also exclude from patentability

under article 27(3)(a) diagnostic, therapeutic and

surgical methods for the treatment of humans or

animals. The article 27(3)(b) stipulates patents cannot

be granted for plants and animals other than micro-

organisms and same article provided that the protection

of plant varieties either by patents or by an effective

sue generic system or by any combination thereof and

shall be reviewed four years after the entry into force

of the Agreement.

Members shall require under Article 29(1) that an

application for a patent shall disclose the invention in a

manner sufficiently clear and complete for the

invention to be carried out by a person skilled in the art

and may require the applicant to indicate the best mode

for carrying out the invention known to the inventor at

the filing date or, where priority is claimed, at the

priority date of the application. Under article 33 of the

TRIPS provided that term of patent is 20 years.

A look at the above provisions shows that at least in

theory, the member states can take steps to protect


378

public order, health of human beings, animals or plants

and also to avoid serious hazards to environment.

Similarly patenting of life may be excluded except in

the case of microorganisms and plant varieties.11

Indian patent legislation, hailed as a model, the world

over, for its far reaching provisions, was amended

through the Patent (Amendment) Acts 1999, 2002 and

2005, to fulfill Indian obligation with the World Trade

Organization’s Trade Related Intellectual Property

Rights Agreement. The Amendment has introduced

wide-ranging changes in the Act, apart from paving the

way for a product patent system. The salient features

of the Patent (Amendment) Act 2005 are:

1. Extension of product patent protection to all

fields of technology i.e. drugs, food and

chemicals.

2. Abolition of exclusive Marketing Rights.

3. Grant of compulsory license for export of

medicines to countries which have insufficient

or no manufacturing capacity to meet emerging

public health situation.

4. Permitting of pre and post-grant applications.

5. The term of patent has been extended to 20 years

In addition to the above, in order to restrict the scope of

patentability inserted the following clauses to the

Sections: Section 2(ja) “Inventive step” means a

feauture of an invention that involves technical advance

as compared to the existing knowledge or having

economic significance or both and that makes the

invention not obvious to a person skilled in the art.

Section 2(l) “New Invention” means any invention or

technology which has not been anticipated by

publication in any document or used in the country or

elsewhere in the world before the date of filing of

patent application with complete specification, i.e. the

subject matter has not fallen in public domain or that it

does not form part of the state of the art.

Section 2(ta) “Pharmaceutical substance” means any

new entity involving one or more inventive steps.

Section 3(d) the mere discovery of a new form of a

known substance which does not result in the

enhancement of the known efficacy of that substance or

the mere discovery of any new property or new use for

a known substance or of the mere use of known

process, machine or apparatus unless such known

process results in a new product or employs at least one

new reactant. The controversial Section 5 of the

principal Act is omitted. On analysis of the new patent

regime especially Section 3(d) is leading to raise more

litigation for grant of patents.

NEW PATENT REGIME – THE PUBLIC

HEALTH CARE - The much-debated Trade Related

Intellectual Property Rights Agreement, a part of the

World Trade Organization Convention, has become a

reality in its application to the Health Care System,

particularly in the developing world. The Trade

Related Intellectual Property Rights Agreement has

raised several apprehensions in the minds of the people

in the third world countries, concerning the adverse

impact of the patent regime on the public health care

system.

It is widely believed that after the implementation of

Trade Related Intellectual Property Rights, the

pharmaceutical market will identify a global middle

class with preference for costly foreign brands leaving

millions without access to essential drugs to treat

common illnesses. Therefore, they perceive

globalization as a feast for the rich and tragedy for the

poor.

However, the proponents of new patent regime argue

that though developing countries have to provide for

product patent, under Article 27 of the Trade Related

Intellectual Property Rights agreement, they have an

option of formulating laws and regulations by adopting

necessary measures to protect public health and

nutrition. Further, the right to health is safeguarded in a

number of international instruments such as the

Universal Declaration of Human Rights 1948 under

Article 25(1), the International Convention on Civil

and Political Rights 1966 under Article 12(1) and under

Article 21 of the Indian Constitution.

On a clear analysis of the TRIPS obligation to be

discharged by the third world countries more

particularly in the developing countries like India, it

becomes clear that a conflict arises between the health

and welfare of the society and the economic rights of

individual patent holders in the case of product

patenting of drugs.

DRUG PRICES – GOVERNMENTAL POLICIES

The drugs patented prior to 1995 would continue to be

available at the same prices for the consumers of India.

Consumers could expect a slight price increase for

medicines that are the subject of mailbox patents. The

prices for medicines that are the subject of patents


379

issued after 2005 would probably be higher. This is to,

because clones of Indian Pharmaceutical Products of

medicines in the mail box that are subsequently granted

product patents are expected to be sold with reasonable

royalty payments. The effective use of the compulsory

licensing provisions could result in continued domestic

access to medicines at heap costs for the drugs in the

mailbox and after. In addition, the National

Pharmaceutical Pricing Authority (NPPA) and India’s

price regulatory policy, the Drug Price Control Order

(DPCO) and Drug Policy could play a key role in

keeping a check on prices12. Further, the Government

should to take appropriate measures for controlling the

prices of essential drugs from time to time.

TRIPS AGREEMENT AND PHARMA

INDUSTRY:

Most of the provisions of the Trade Related Intellectual

Property Rights Agreement pertaining to

Pharmaceuticals and Health care, it was felt, are most

unfavorable to the developing countries, in as much as,

this may result in serious problems in the field of

Pharmaceuticals and Health Care, in the form of

nonavailability of essential drugs etc. Some of the

negative features of the patents of Trade Related

Intellectual Property Rights Agreement, particularly in

the context of Pharmaceutical Industry are that, grant of

product patent will effect the production of many

drugs, which are otherwise available freely in the

market. Further, under the new regime, the protection

of patent is extended to a period of 20 years which too

long a period is given the conditions prevalent in the

developing countries. The apprehensions in the third

world countries about the compliance with the Trade

Related Intellectual Property Rights Agreement

mentioned hereunder under13:

1. The Trade Related Intellectual Property Rights

in its present form, is in favour of developed

nations and the right to trade is being exploited

by developed countries only.

2. It is opined that Trade Related Intellectual

Property Rights in fact, violates the human

rights of people, as the life saving drugs could

not be made available at affordable price.

3. The Trade Related Intellectual Property Rights

would also hinder the preservation and infact

would become a threat to innovation and

practices of indigenous medicines especially,

alternate medicines like Ayurveda and Unani.

4. It is feared that in this big shake up a number of

medium and small pharmaceutical companies

will be forced to close down. This would mean

thousands of employees will loose their jobs.

5. Companies which do not have enough Research

and Development competence and orientation

and who want to be in the race and survival may

be forced to go in for mergers, joint ventures etc.

6. The overall growth rate of industry may slow

down or even stagnate for some time.

7. Above all, drugs could become expensive and

beyond the reach of common man.

The interests of the Indian pharmaceutical industry and

the indigenous communities are likely to be affected by

the Trade Related Intellectual Property Rights

agreement of World Trade Organization. The Trade

Related Intellectual Property Rights agreement is a part

of globalization process aimed at removing all barriers

of economic integration for economic interdependence.

Unlike the other agreement of World Trade

Organization, it is perceived that the Trade Related

Intellectual Property Rights ensures more power to

control the activities of developing countries by

financially powerful developed countries. Under this

agreement, it is mandatory to accept patents, which will

ultimately influence the growth of Indian

pharmaceutical industry and the prices of drugs.

THE NEW PATENT REGIME – IMPLICATIONS

FOR INDIAN PHARMA INDUSTRY

The legal framework of any patent regime reflects the

balance of force in the society in which it exists. In its

essence, it also reflects the social tensions, which

determine how strong or weak the regime is.14 This is

also the case with the stakeholder of the new patent

regime vis-à-vis the pharma industry. This invoked

mixed reactions from different sections of society.

Concerns were expressed about the new patent regime

in various corners. One of the implications of this new

regime, it is said, was that it will result in people being

cut off from the vital source of affordable generic

versions of essential medicines produced in India. That

the amended law would make it easier for multinational

drug companies to get patents granted, while generic

producers would find it more difficult to acquire


380

compulsory licenses for such drugs by issuing

compulsory licensing provisions. The new patent

regime would make all new drugs patentable in India.

The new patent regime adopted in India, as a sequel to

its commitment to the Trade Related Intellectual

Property Rights Agreement raises several crucial

issues, such as public health consideration impact of

Trade Related Intellectual Property Rights on the

Indian Pharma Industry etc. Regarding the first issue,

expressions have been raised in several quarters that

protecting of drugs would raise the cost of the drugs,

thus putting them out of the reach for the poor and

consequential damage to the public health. Though

these apprehensions are not totally unfounded, the size

of Indian Pharma Industry and the growth rate of these

industries will encourage introduction of new drugs,

and will also promote Research and Development in

Pharma Industry15.

Further, it is also argued that Indian Pharma Industry is

highly fragmented with numerous small players

making generic formulations and lack of adequate

capital or technology to invent new drugs; as a result,

they feel that the market will be polarized in favour of

foreign multinationals or Indian Pharma majors. On

the other hand, the larger firms in pharma industry are

in full support of the new patent regime on the ground

that the new regime will not only attract foreign

investment but also promote joint ventures and

research, in the Pharma Industry.

However, it should be noted that, with the introduction

of product patent system Indian companies will have to

compete with Multi-National Companies by focusing

on development of drugs, and thereby produce their

own-patented products. Alternately, under the new

regime Indian companies will have to focus on

producing patented drugs under license from foreign

companies or concentrate on generating revenues from

producing generic drugs.

Once patent protection is available, patent-owning

firms may choose either to export their patented drugs

to India, thereby replacing domestic production, or they

may choose to produce in India through a subsidiary or

under license to Indian firms. The Multi-National

Corporations concern about global differentials make

local, low cost production attractive as a way to justify

Indian prices, which are lower than those charged in

the markets of developed countries.

Further, with the introduction of product patents, the

resulting transfer of profit from domestic to foreign

patent owners, via royalty payments or export profits

on drugs sold to Indian consumers, will have an

adverse effect on India’s balance of payments. Also

Indian Pharma firms will no longer be able to

export/patent drugs to other countries, primarily in the

former Soviet Union and in Africa, which, until now,

also did not offer much protection for pharmaceutical

products. But this effect, it is said, is expected to be

very small.

However, it was argued in some quarters that, it is too

early to predict the impact of product patents on

pharmaceutical industry in India because it depends a

lot on the price of the patented products and their

accessibility. Even competition among generic drug

producers also results in substantial reduction in the

price of drugs, yet an increased competition among

generics does not result in aggressive response in price

behavior by established brand name products It is

feared that the low purchasing power of the common

people in India and other developing countries will

restrict access to the new inventions.

PATENT ACT AMENDMENT – THE NOVARTIS

CASE:

In a recent case, the Swiss Pharmaceutical

multinational Novartis challenged the constitutional

validity of Section 3(d) of the Act as amended by the

Patents (Amendment) Act 2005.The relief sought by

drug major Novartis is two fold: first the quashing of

an order of the Indian Patent Office at Chennai for its

refusal to grant product patent. Secondly it is argued

for a declaration that Section 3(d) of the product patent

regime is unconstitutional as it is not in compliance

with the TRIPS agreement under the aegis of the World

Trade Organization.16

The reasons assigned for refusal of the patent by the

Chennai patent office were firstly, the product patent

regime introduced in India applies only to drugs that

were invented after January 1, 1995 but Gleevec was in

existence prior to the cut-off date as was evident from a

patent taken by the Novertis in 1993 in the United

States; Secondly, the drug by the name of gleevec was

not patentable in India as it was only a new form of a

known substance without any significant improvement

in efficacy and thirdly, due to the disclosures already

made in the patent granted by the United States, the


381

claims made by the Novertis for seeking a fresh patent

in India stood “ anticipated by prior publication”.16

It is argued that an invention with a mere change of

form without any enhanced efficacy could not be

granted patent and if patent was granted, it would be

arbitrary. The amended provision along with the

explanation fully complied with Articles 7 and 8 of

TRIPS. As per Article 7 “The protection and

enforcement of intellectual property rights should

contribute to the promotion of technological innovation

and to the transfer and dissemination of technology, to

the mutual advantage of producers and users of

technological knowledge and in a manner conducive to

social and economic welfare and to a balance of rights

and obligations. Under Article 8 “Members may, in

formulating or amending their laws and regulations,

adopt measures necessary to protect public health and

nutrition, and to promote the public interest in sectors

of vital importance to their socio-economic and

technological development, provided that such

measures are consistent with the provisions of this

Agreement.” Article 27 of TRIPS provided for patents

to be given only to inventions which were new and

involved an inventive step.

The Madras High Court dismissed two Writ Petitions

filed by Novartis AG and Novartis India Limited on the

ground that Sec.3 (d) of the Patent (Amendment) Act

2005 is lawfully valid and the Novartis failed to show

the enhancement of efficacy, for its life saving cancer

drug ‘ Gleevec’ (imatinib mesylate).

The judgment and the impact of Sec.3 (d) must be

understood better by knowing what it permits and what

it prohibits. Patents for pharmaceutical substances

today fall into two broad categories: Original

inventions and incremental innovation. Incremental

innovation is a grey area, what actually amounts to

incremental innovation and the extent to which such

innovations should be protected is debatable. In any

case, the language of Section 3(d) permits incremental

innovation. But it is for the applicant to demonstrate

why a fresh patent should be granted to a known

substance. For this the applicant has to demonstrate an

increase in efficacy of the substance over the known

efficacy. What section 3(d) is trendsetting provision

as it is the first legal provision in the world not to be

found in the patent legislation of any country, which

provides a check on frivolous patenting17. If the ruling

in favour of Novartis would affect the future of India’s

Pharmaceutical Industry and deprive cancer patients

throughout the third world. Further, it would be harder

for Indian companies to produce the cheap generic

versions of existing drugs that are used across the

developing world to treat diseases such as malaria and

HIV/AIDS.

PROTECTION OF DOMESTIC PHARMA

INDUSTRY – THE FUTURE STRATEGIES

Pharmaceutical patent, it is believed, will evoke profit

maximization attitude of the pharma industry against

the public welfare and interest. Now the dilemma

before the Indian government is how to schedule the

changes in the patents so as to ensure that its Trade

Related Intellectual Property Rights compliance and at

the same time ensure that the local industry continue to

develop and prepare themselves to face the global

competition. What is required, however, is to find a

balance of a patent regime where the inventors are

offered incentive and at the same time to check the

abuse of Intellectual Property Rights in patents. The

government should intervene to implement price

control mechanisms wherein a common man should

not be affected due to adoption of product patent

system. It should not be ignored that right to health is a

fundamental right which is guaranteed by the

Constitution, and also the International Human Rights

Law.

Medical research and public policy measures can bring

down the cost of life-saving drugs for the people in

developing countries. At present 95 percent of the

people in developing world cannot afford the prevailing

cost of medicines. Unless, the governments take

measures to reduce the cost of drugs, unnecessary

illness and deaths in the developing world will be on

the increase. A world full of diseases will not be good

for any business17.

CONCLUSION

In accordance with the amendments made to the Patent

Act the Indian Pharmaceutical industry must gear up to

face the challenges and transform into knowledge

based organizations capable of producing research

based medicines at affordable prices to the Indian

people. Further, the emerging patent era should be

looked upon as an opportunity and not as a threat. It


382

should strengthen Science and Technology, Research

and Development on one side and Governmental

policies on the other side to balance the interests of the

patentee and the general public at large.

REFERENCES:

1. Francis, P.A., Patents and Acess to Medicine,

Pharmabiz.com, 2001 August 21, 1.

2. Ashok Ram Kumar, TRIPS-Is it healthy

prescription for Pharma Industry, International

Conference on Innovation and IPR Strategy,

2002 September, 12-13.

3. Sanjeev Chandran, Achna Roy and Lokesh Jain,

Implications of New Patent Regime on Indian

pharmaceutical Industry: Challenges and

Opportunities, Journal of Intellectual Property

Rights, 2003, Vol. 10, 270.

4. Nair, M.D., An industry in transition: The Indian

pharmaceutical industry, Journal of Intellectual

Property Rights 2002, 7(5), 405-415.

5. Jha, S.K., Intellectual Property Rights and

Globalization of the pharmaceutical Industry,

Pharma Times, 2003, 35(5), 12-22

6. The Deccan Chronical, 2006 July 1st.

7. Smita Joshi,India Emerges as Pharma R&D humb,

Deccan Chronicle,2006 August 19, 15.

8. Unnikrishnan,CH,Non-conventional markets to

drive drug exports, Business Standard, 2006 Jan

11, 15.

9. Palanichamy, P., Shanmuga Sundaram, G., Impact

of Globalization on pharmaceutical Industry,

Indian Economic Panorama, 2004, 35.

10. Parikshit, Patent Amendment Act,2005-An

Overview. Available from: www.Legal Service

India.Com.

11. Dr. Reddy,G.B, Intellectual Property Rights and

the Law, 5th Edn, Hyderabad, Gogia Law Agency,

2005, 190.

12. Murti,R.K.P, and Prasanna Rani, K, Patents

(Amendment) Act 2005 and the Indian

Pharmaceutical Industry, paper presented in Two

Day National Seminor on Intellectual Property

Rights, organized by Department of Commerce,

Kakatiya University, Warangal, 2006 November

29-30.

13. Ashok Ramkumar, TRIPS-Is it a Healthy

Prescription for Indian Pharma Industry, Indian

Journal of IPR, 2003, 71-76.

14. Sridhar, V., Siddharth Narain, A Tempered Patent

Regime, Frontline, 2005 April 22nd , 29.

15. http://www.patentmatics.com/pub2002’pub4.htm

16. Vohra,DR.DC, Novartis challenges IP Regime,

2007 Feb 24. Available from: www. Financial

express. Com.

17. Feroz Ali. K, Novartis: do Indian patent laws

stifle research?, The Hindu, 2007 August 9th, 15.

18. Chandramohan, B.P., Impact of TRIPS on

Pharmaceutical Industry in India, Indian

Economic Panorama, 2004, 16.

**********


383

Marketing and Advertising of Prescription and Over the

Counter (OTC) Products – Ethical Issues Manthan D.Janodia and Udupa N.

*

Department of Pharmacy Management, Manipal College of Pharmaceutical Sciences, MAHE, Manipal

576 104, Karnataka, India

*E- mail : [email protected] ; [email protected]

Abstract

Marketing and advertising of pharmaceutical products to healthcare prescribers for prescription only products

and to the consumers for Over the Counter medications has been one of the ethical issues and is a challenge to

pharmaceutical companies. Various measures have been taken by certain regulatory agencies across the world

to effectively control and curb the misleading or false claims related to pharmaceutical products through strict

regulations or with stringent regulatory standards that scrutinizes all the advertisements for pharmaceuticals,

targeted to doctors or consumers directly. These measures are still inadequate to control and restrict

advertisements that claim product to be superior than it actually is. This article tries to explain pharmaceutical

advertising practices in certain countries and draws comparison with various standards that are prevalent-

either due to regulatory restrictions or self imposed restriction by the pharma industry- in certain developed

countries and in India for pharmaceutical advertising.

Key words: PhRMA, IFPMA, DDMAC, Pharmaceutical advertising, Pharmaceutical industry, Drugs and Magic

Remedies (Objectionable Advertising) Act, 1954

INTRODUCTION

Recent advances in drug therapy have brought to the

market newer drugs to alleviate disease at global level.

These drugs, in order to be prescribed, are to be

advertised to the physicians so that they become aware

of various newer therapies to treat or cure certain

ailments. Pharmaceutical companies for a long time

have been advertising these products to their target

customers i.e. physicians through various media. In the

past it was either in medical journal, symposia or

conference that pharma companies were able to

advertise their products to a larger section of

prescribers. With the advent of newer technologies like

Internet companies have got a new platform to reach a

larger target customer group with added advantage of

disseminating information in shortest possible time.

Advertising - the context:

In India according to Drugs and Magic Remedies

(Objectionable Advertisement) Act, 1954,

an advertisement is defined as “any notice, circular,

label, wrapper, or other documents and any

announcement made orally or by means of producing or

transmitting light, sound or smoke 1. As drugs cannot be

sold like commodities, promotion of drugs is regulated

and restricted. The advertisements for pharmaceutical

products could be for drugs that are to be dispensed

against the prescription of a physician or Over the

Counter (OTC) medicines that are used to treat minor

ailments and does not require a physician’s

prescription. Since advertisement of pharmaceutical

products is restricted in most of the countries of the

world except a few like US where drugs can be directly

advertised to the patients, only available means to the

pharmaceutical companies to reach the target customer,

physician, is through medical journals, symposia or

conferences. In the US two types of prescription drug

advertisement is identified – Product claim and

reminder. The reminder advertisement is suppose to not

contain indication and dosage unlike product claim

APTI ijper





384

advertisement. Reminder advertisement intended to

remind the target groups about the brand only 2. The

information provided to physicians by pharmaceutical

companies is supposed to be accurate and educative to

the prescriber for newer therapies that were not

available earlier. Further, these advertisements need to

be educative and informative rather than persuasive for

the physicians and consumers with justifiable benefits

regarding product claims and the risk profile associated

with the drug so that a physician can prescribe the

therapy according to individual patient’s needs. In

addition the aim is to protect public health and avoid

misleading information to prescribers. It is expected

that the claims regarding the product made by a

company needs to be substantiated by sufficient clinical

evidence in the form of clinical trials performed.

USFDA (United States Food and Drugs

Administration) requires advertisements and

promotional items aimed at prescribers to be submitted

to the Division of Drug Marketing, Advertising and

Communication (DDMAC) for review before it is aired

on television or targeted to physician to evaluate its

credibility.

Guidelines on Advertising:

To adhere to high ethical standards of promotion,

comply with the regulatory requirements and provide

useful information to physicians for prescription and

OTC products, PhRMA (Pharmaceutical Research and

Manufacturers of America)- a strong lobbying group of

Multinational Pharmaceutical Companies of the US,

has prescribed guiding principles. These guiding

principles refer to promotion of prescription as well as

OTC products. Thus, promotion of prescription as well

as OTC products forms the basis for DTC (Direct to

Consumer) advertising. PhRMA in its guiding

principles has emphasized to responsibly advertise

products such as pharmaceuticals and this fact is

reiterated in the first guiding principle that focus on

creating awareness about the disease and educating

patients for treatment options available and encourage

dialogue between patient and physician 3. IFPMA

(International Federation of Pharmaceutical

Manufacturers Association) has also published

guidelines for marketing practices termed as “IFPMA

code of Pharmaceutical Marketing Practices”. IFPMA

code is exhaustive in its explanation of advertising that

encompasses nearly all the aspects of pharmaceutical

advertising such as Standards of Promotion, Scientific

evidence, Training and Responsibilities of Medical

Representatives and companies, promotional efforts in

Symposia and conferences, Hospitality extended to

physicians, Printed promotional material and Audio

Visual and Computer based promotional material.

IFPMA code further states that promotional material for

pharmaceutical products should be accurate, fair and

objective 4. In the United States the Federal Food,

Drugs and Cosmetics Act (FD & C Act) requires that

all drug advertisements that are directed to consumers

contain information in brief summary relating to side

effects, contraindications and effectiveness besides

other information that is required. In addition to these

requirements the FD & C Act requires that

advertisements cannot be false or misleading or omit

material facts and present a fair balance between

effectiveness and risk information of drug 5.

Advertising regulations of the United Kingdom (the

UK) sets out the standards for promotion of medicines

in the UK. The UK rules prohibits use of any

advertising that may lead to the use of Prescription

Only Medicine (POM). The regulation further

encourages rational use by stating the proper use of

medicine such as when and how much medicine should

be taken with its route of administration and

precautions to be observed while on medication 6.

According to Medicines and Healthcare Products

Regulatory Agency (MHRA), advertising to general

public should not suggest that one product is better than

another identifiable treatment 7. This is particularly in

view of tendency to compare products by companies to

prove their product superior without any substantial

clinical evidence to support their claim and to mislead

public. Advertising of Prescription drugs to public is

prohibited in all the countries of European Union (EU)

as well. Apart from the US, only New Zealand is the

other developed country that allows for advertisement

of prescription drugs to public 8, 9. In Japan the

advertising of medicinal products is governed by the

“Pharmaceutical Affairs Law” as amended and

“Standards for fair advertising practices concerning

medicinal products”. In addition to this Japan

Pharmaceutical Manufacturer Association (JPMA) has

evolved self discipline code of marketing practices

termed as “Code of Practices for Promotion of ethical

drugs” as amended and “Fair Competition Rules


385

concerning Restriction on Provision of Unjustifiable

Extra or Unexpected Benefit in Manufacture of Ethical

Drugs” as amended 10. In India, OPPI (Organization of

Pharmaceutical Producers of India) has given

guidelines for maintaining ethical standards for

promotion. OPPI guidelines are designed on model

code of conduct of PhRMA’s guiding principles but

OPPI guidelines have elaborated on the terms fair,

accurate and objective that are enshrined in PhRMA’s

guiding principles on DTC advertising 11. In India,

although advertising of OTC products is allowed

directly to public by various means of communication

such as print and electronic media, prescription

medicines cannot be advertised directly to general

public according to Drugs and Magic Remedies

(Objectionable Advertisements) Act. Drug and Magic

Remedies (Objectionable Advertisements) Act prohibits

prescription drug advertisements so as to prevent abuse

and misuse of prescription drugs. Advertisements of

pharmaceutical products aimed at physicians and

healthcare professionals should be advertised during

symposia, conferences, Continuing Medical Education

(CME), and only through medical journals. Schedule J

of Drugs and Cosmetics Act 1940 and rules 1945 of

India gives an exhaustive list of ailments that a drug

may not claim to cure 12.

Compliance to the Guidelines:

Although the guidelines are provided to disseminate

accurate, fair and objective information to physicians

and public, in many instances it is found that not all the

companies follow so called “self imposed” guidelines.

These companies also include member companies of

PhRMA. Division of Drug Marketing Advertising and

Communication (DDMAC) of USFDA that scrutinizes

the promotional messages by pharmaceutical

companies aimed at physicians and the public for the

first time receives almost 53,000 pieces of promotional

material every year for scrutiny 13. The task of DDMAC

becomes more complicated with a meager staff of 35 to

scrutinize these promotional materials. The director of

DDMAC Thomas Abrams says DDMAC does not even

scrutinize all the promotional material before it is

released to the public 14. Several developed countries

like UK, Japan have mechanism either by government

or “self imposed” regulations by association of

pharmaceutical companies in these countries so as to

comply with the ethical standards of promotion of

medicinal products.

Violation and breach of guidelines:

It is found in several instances where companies are

violating the guiding principles of advertising and

promotion of pharmaceutical products in their respected

countries or common guidelines prescribed by IFPMA

or PhRMA. In the US upon finding complaints of

violation of breach of advertising principles, DDMAC

issues two types of letters – Notice of violation or

untitled letters and warning letter - to the companies.

Notice of violation letters are issued for less serious

violations whereas warning letters asks a company to

stop its promotion and disseminate correct message

regarding the product 15. DDMAC has issued several

warning letters to leading pharmaceutical companies in

America that are found to violate Federal Food, Drug,

and Cosmetic Act and regulations for Direct to

Consumer advertising. DDMAC has issued warning

letter dated 5/29/03 to Hoffman La Roche for the

company’s product Xeloda (Capecitabine). DDMAC

had noted that Hoffman La Roche’s sales aid is

misleading as it fails to present risk information about

Xeloda which is associated with serious, potentially life

threatening risks. Further the agency noted that the

company’s video on Xeloda minimizes safety risks

associated with the drug and makes unsubstantiated

claims for the drug 16. Another warning letter issued by

DDMAC dated 8/7/03 is to Bristol Myers Squibb

(BMS) for the company’s product Pravachol

(Pravastatin Sodium). DDMAC noted that BMS

violated several of sections of Federal Food, Drug and

Cosmetic Act. The agency noted that the company’s

promotional material is misleading or false and the

product is misbranded according to the Federal Food,

Drug and Cosmetic Act 17. DDMAC issued warning

letter to Novartis dated 4/21/04 for the company’s

product Diovan (Valsartan) tablets. After reviewing the

sales aid for the product Diovan, the division found that

sales aid for Diovan is misleading or false according to

Federal Food, Drug and Cosmetic Act 18]. According to

Reuters report about 82 percent of FDA’s warning

letters cited companies for not including adequate risk

information 19.

Corrective Actions:

Although IFPMA Code of Pharmaceutical Marketing


386

Practices insists on providing accurate, fair and

objective information, it shows how companies

undermine providing factual information to public

regarding the use and side effects of drugs. If so is the

case it casts serious doubts to what extent “self

imposed” regulation by PhRMA will be followed by

PhRMA member companies. In addition to these, the

warning letters issued by FDA is on rise since 2002. In

2002, only one warning letter was issued by FDA. The

number increased to five in 2003. Out of 23 letters

issued by FDA in 2004 12 were warning letters. As of

October 2005, the number had already increased upto

15 20. In the UK MHRA has a developed procedure to

check for violation or breach of regulation. MHRA has

the power to scrutinize the current advertising material

including health professionals, consumer journals and

other media of advertisement. MHRA also receives

complaints about breach or violation for regulation on

advertising. If breach is found on the part of the

company, MHRA may ask the company to 21:

(a) Amend the advertisement.

(b) Withdraw the advertisement.

(c) Issue a corrective statement.

(d) Submit future advertisement for the product to

the MHRA for review prior to issue.

In Japan the jurisdictional authorities have the power to

stop further publication or issue a correction by

pharmaceutical company to the advertisement, which is

violating the law 22. In India, as of now, there is no

check on misleading or false advertisements by

pharmaceutical companies and no evidence is found to

support that the punitive actions in Drugs and Magic

Remedies (Objectionable Advertisement) Act is

enforced. No action is taken on advertisers that

generally advertise their products occasionally in print

media and purport to “cure” ailments such as baldness,

and some other ailments or conditions, which according

to Drugs and Magic Remedies (Objectionable

Advertisements) Act cannot be cured. With the advent

of stringent rules and regulations it is observed more

and more companies are violating the regulations. As

promotion becomes more extensive and competition

becomes fiercer, money invested in promotion will be

going northwards. Expenditures for the promotion of

drugs in the US increased from $11 billion in 1997 to

$15.7 billion in 2000 23. Currently pharmaceutical

industry in the US spends $ 25 bn a year on promotion

of pharmaceutical products24. In India there is no data

available or compiled as to how much Indian

pharmaceutical industry spends yearly on

advertisements - prescription as well as OTC. It can no

longer be termed as Direct to Consumer Advertising

(DTCA) rather it must mean Direct to Consumer

Communication 25.

CONCLUSION

Although there are several guiding principles and

government enacted laws and regulations that direct

pharmaceutical companies to provide healthcare

professionals and public with correct information

regarding drug therapy for rational use of drugs,

enhance patient – physician interaction, and create

awareness about disease condition and prevention

through DTC advertising, pharmaceutical companies

are sometimes violating these regulations. Now a days

there are more means of disseminating information to

public, government agencies will have be to on its toes

all the time to curb the violation of rules for DTC

advertising. In order to avoid scrutiny by law, certain

times products are advertised through newer means of

communication such as Internet. In India there is a need

to scrutinize advertisements in various media that claim

to cure certain illnesses and need to create more

awareness among general public regarding rational use

of drug or consult physician before starting any

medication.

REFERENCES

1. The Drugs and Magic Remedies (Objectionable

Advertisement) Act 1954, India.

2. Kathryn J.Aikin, John L.Swasy, Amie C.Braman,

Patient and Physician Attitudes and Behaviors

Associated with DTC Promotion of Prescription

Drugs – Summary of FDA survey Research

Results, Final Report Submitted to US Department

of Health and Human Services, Food and Drug

Administration, Center for Drug Evaluation and

Research, November 19,2004 (accessed at

http://www.fda.gov/cder/ddmac/final%20report/FR

final111904.pdf on 16-04-2006.)

3. PhRMA Guiding Principles – Direct to Consumer

Advertisements About Prescription Medicines,

August 2005, (accessed at

www.phrma.org/publications/policy/2005-08-

02.1194 on 25-10-2005)


387

4. IFPMA code of Pharmaceutical Marketing

Practices, 2000 update (accessed at

http://www.ifpma.org/site_docs_News_Code_Engli

sh_2000.pdf at 27.12.2005)

5. http://www.fda.gov/cder/ddmac/FAQs.htm

(accessed on 16-04-2006)

6. The Blue Guide, Advertising and Promotion of

Medicines in the UK, 2005, Chapter 4, General

Rules- 4.3 (2): 15 (accessed at

http://www.mhra.gov.uk/home/groups/pl-

a/documents/publications/con2022589.pdf on 28-12-

2005)


Medicines in the UK, 2005, Chapter 5, Prohibition of

certain Material- 5.3: 19 (accessed at


/documents/publications/con2022589.pdf on 28-12-

2005)

8. Joan Buckley, Pharmaceutical Marketing – Time

for Change, European Journal of Business ethics

and Organization studies, 9(2): 5 (accessed at

http:// ejbo.jyu.fi/pdf/ejbo_vol9_no2_pages_4-

11.pdf on 12-05-06)

9. http://www.web.net/

~desact/anglais/public/dconsumers.htm (accessed

on 04-01-2007)

10. Somuku Iimura and Kotaro Kubo, Japan in “The

international comparative legal guide to:

Pharmaceutical Advertising 2006, A practical

insight to cross-border pharmaceutical advertising

work”, Global Legal Group: 182 (accessed at

www.alo.jp/English/img/pubo9_b_1.pdf on 04-01-

2007)

11. Organization of Pharmaceutical Producers of India

guidelines for promoting pharmaceutical products

in India.

12. Schedule J, Drugs and Cosmetics Act 1940 and

rules 1945, India.

13. Inside DDMAC, Pharmaceutical Executive,

December 2005:48


December 2005: 48


December 2005: 48

16. Warning letter issued by DDMAC, USFDA to

Hoffman La Roche on 5/29/2003 (accessed at

http://

www.fda.gov/foi/warning_letters/g4059d.pdf on

April 16, 2006)


Bristol Myers Squibb on 8/7/03 (accessed at http://

www.fda.gov/foi/warning_letters/g4200d.pdf on

April 16,2006)


Novartis Pharmaceuticals Corporation on 4/21/04

(accessed at

http://www.fda.gov/foi/warning_letters/g4652d.pdf

on April 16, 2006)


December 2005: 48.


December 2005: 50.


Medicines in the UK, 2005, Chapter 7, 7.3 scrutiny

of current advertising material and 7.4 complaints

about medicines advertising: 34,35

(accessed at


/documents/publications/con2022589.pdf on 28-12-

2005)

22. Somuku Iimura and Kotaro Kubo, Japan in “The

international comparative legal guide to:

Pharmaceutical Advertising 2006, A practical

insight to cross-border pharmaceutical advertising

work”, Global Legal Group: 182 (accessed at

www.alo.jp/English/img/pubo9_b_1.pdf on 04-01-

2007)

23. Sidney M.Wolfe, Direct to Consumer Advertising –

Education or Emotion promotion?, New England

Journal of Medicine, 2002,346:524-526


December 2005:50.

25. Its all relative, Pharmaceutical Executive,

November 2005:40

***********


388

Development and Evaluation of Propranolol Hydrochloride

Transdermal Patches by using Hydrophilic and

Hydrophobic Polymer Dey B.K*, Nath L.K, Mohanti B

1, Bhowmik B.B.

Department of Pharmaceutics, Himalayan Pharmacy Institute, Majhitar, Rangpo, East Sikkim- 737136 1Technical Manager, National Health Care Pvt. Ltd, Birgunj, Nepal

* For Correspondence : E-mail: [email protected]

Abstract

Propranolol hydrochloride is a non-selective beta-adrenergic blocking agent and clinically used in angina

pectoris, cardiac arrhythmia and in hypertension. It inhibits response to adrenergic stimuli by competitively

blocking beta-adrenergic receptors in the myocardium, bronchial and vascular smooth muscles. The drug has

been reported for potential administration through transdermal route. Present investigation was carried out to

study the effect of different proportions of ethyl cellulose and polyvinyl pyrrolidone, a hydrophobic and a

hydrophilic polymer respectively, on the permeation profile of the drug across the rat abdomen for the

development of a reproducible transdermal therapeutic system of propranolol hydrochloride. Transdermal films

were prepared using ten different combinations of the two polymers by solvent evaporation technique. Polyvinyl

alcohol was used to prepare the backing membrane and dibutyl phthalate as plasticizer. Several

physicochemical parameters like moisture content, moisture loss, thickness, film folding endurance, tensile

strength and film elongation were studied. For all the formulations, skin permeation of the drug through rat

abdomen was studied using Keshary-Chien diffusion cell. Formulations with highest proportion of polyvinyl

pyrrolidone shows faster release over a day’s span whereas increasing proportion of ethyl cellulose produces a

prolonged regimen of sustained drug delivery through transdermal route for a period of more than a day. The

present study has demonstrated the potential of the fabricated matrix films for prolonged release of propranolol

hydrochloride.

Key words: Propranolol hydrochloride, transdermal therapeutic system (TTS), ethyl cellulose (EC), polyvinyl

pyrrolidone (PVP).

INTRODUCTION

Though the concept of transdermal therapeutic system

(TTS) of drug delivery has been well known since

1924, it is only in the year of 1979, with FDA approval

of scopolamine transdermal systems, the TTS have

received broad impact on the scenario of novel dosage

forms1. Transdermal therapeutic systems are designed

for controlled drug delivery through the skin into

systemic circulation maintaining consistent efficacy and

reducing dose of the drug and it’s related side effects2-3.

It provides an alternate route of drug delivery avoiding

the hepatic first pass effect. It also improves patient

compliance, safety and efficacy of the drug4.

Propranolol hydrochloride is used in the treatment of

angina pectoris, cardiac arrhythmia and hypertension. It

is the drug of choice for sustained release formulation

since it has a low terminal elimination half life of about

3 to 5 hr, which requires frequent dosing necessary to

maintain the therapeutic blood level for a long term

treatment. The drug shows considerable first pass

metabolism in the liver and thereby has poor

bioavailability (15-23%) when administered orally5,6

and the low molecular weight (295.81) of the drug

APTI ijper


Received on 28/12/2006; Modified on : 27/8/2007


Indian J.Pharm. Educ. Res. 41(3), Jul – Sep, 2007

389

again indicates it’s suitability for administration by the

transdermal route. Propranolol hydrochloride

transdermal films were prepared using ten different

combinations of the two polymers namely ethyl

cellulose (EC) and polyvinyl pyrrolidone (PVP) by

solvent evaporation technique. Polyvinyl alcohol (4%

w/v) was used to prepare the backing membrane and

dibutyl phthalate (30% w/w) as plasticizer.

Concentration of drug was maintained at 20% w/w for

all the formulations. Several physicochemical

parameters like moisture content; moisture uptake, film

thickness, film folding endurance, tensile strength and

film elongation were evaluated. For all the

formulations, permeation of the drug through the

excised abdominal skin of rat was studied using

Keshary-Chien diffusion cell7-8.

MATERIALS AND METHOD

Ethyl cellulose (20cps), polyvinyl pyrrolidone (K30),

polyvinyl alcohol (PVA) and dibutyl phthalate were

procured from S.D.Fine Chem. Ltd. Mumbai.

Propranolol hydrochloride was received as generous

gift sample from Sun Pharmaceuticals Ltd., Baroda,

Gujarat. All other chemicals and solvents used were of

analytical grade.

Preparation of the transdermal patches:

Matrix type transdermal patches containing propranolol

hydrochloride were prepared using different ratios

(Table 1) of EC and PVP by solvent evaporation

technique in cylindrical glass moulds opened from both

end. The bottom of the mould was wrapped with

aluminum foil on which the backing membrane was

cast by pouring 4% w/v PVA solution in distilled water

followed by drying at 60°C for 6 h in an oven9. The two

polymers were weighed in requisite ratio and they were

then dissolved in chloroform. The ratios of the

polymers were varied for all the formulations keeping

the total weight fixed at 500 mg. Dibutyl phthalate

30% w/w of polymer composition was added as

plasticizer. Propranolol hydrochloride at a

concentration of 20% w/w of polymer was added and

stirred with a mechanical stirrer to get a homogeneous

dispersion. The dispersion (2ml) was cast on the

prepared PVA backing membrane in each mould. The

rate of evaporation was controlled by inverting a funnel

over the mould and dried at 40°C for 6 h in hot air

oven. After drying they were kept in desiccator for

further study.

Evaluations of transdermal patches:

Moisture content:

The prepared films were marked, weighed and kept in

desiccator containing activated silica at room

temperature for 24 h. The individual films were

weighed on every alternate day until a constant weight

was achieved. The percentage of moisture content was

calculated10 by determining the difference between

initial and final weight with respect to final weight. The

mean value of three replicates of weight was used for

calculation.

Moisture loss:

A weighed film kept in a desiccator at normal room

temperature for 24 h, was taken out and exposed to

84% relative humidity (standard solution of potassium

chloride) in a desiccator until a constant weight for the

film was obtained. The percentage of moisture loss was

calculated10 by determining the difference between final

and initial weight with respect to initial weight. The

mean value of three replicates of weight was used for

calculation.

Folding endurance:

Folding endurance of the film was determined manually

by folding a small strip of the film at the same place till

it breaks. The maximum number of folding operation

done at the same place of the film without breaking,

gives the value of folding endurance, where the

cracking point of the films were considered as the end

point11.

Thickness:

The thickness of each film was measured at five

different sites using a meter gauge (Mercer, USA) and

the mean thickness was calculated12.

Elongation and tensile strength:

The tensile strength measurement was made using an

instrument assembled in the laboratory and following

the method used by Sadhana et al12. The films were

fixed individually to the assembly. The required

weights to break the films were noted. Percentage of

elongation of the films was measured by attaching a

pointer mounted on the assembly. Tensile strength was

calculated by using the following formula. The results

are given in Table 1.

Tensile strength = (break force/a × b) × (1+L/I)


390

Where, a, b, L and I are the width, thickness, length and

elongation of the films.

Skin permeation study:

Skin permeation study of the transdermal therapeutic

systems was carried out using Keshary-Chien

permeation cell. The skin of an albino rat was removed

from the abdominal portion after sacrificing the rat. It

was made free from hair and fat by treating with 0.32 M

ammonia solution for 35 min. The transdermal

therapeutic system of 3.08 cm2 area was mounted

between the donor and receptor compartments of the

diffusion cell keeping intimate contact with the stratum

corneal side of the skin13. The receptor compartment

was filled with 25 ml of phosphate buffer of pH 7.4.

The cell was placed in a water bath maintained at 37 ±

1°C on a magnetic stirrer and stirring was continued

throughout the experiment. At regular intervals over a

period of 24 h, samples were withdrawn and

simultaneously compensation was made with same

volume of buffer. The samples were then analyzed

spectrophotometrically at 290 nm against a blank.

Scanning electron microscopy:

The surface morphologies of the films showed better

permeation were investigated by using Scanning

Electron Microscope, model Jeol JSM-5200, Japan, at

15 kV. Prior to examination, the samples were gold-

coated to render them electrically conductive14.


In this study, various matrix type transdermal patches

containing propranolol hydrochloride with variable

combinations of EC and PVP were prepared and

prolonged release of the drug through the matrix films

was demonstrated.

The physicochemical parameters and the release

characteristics were studied on the fabricated patches.

The moisture content and the moisture loss (Fig 1) of

the various formulations exhibit that with the increase

in the concentration of hydrophilic polymer (PVP), both

the percentage moisture content and the percentage

moisture loss was increased. The low moisture content

in the formulations helps them to remain stable and

prevent from being a completely dried and brittle film.

Again, a low moisture uptake protects the material from

microbial contamination and limits the bulkiness of the

patches. In this respect, formulation TTS6 showed best

result amongst all the formulations.

Folding endurance was found to be varied between 61 ±

3.78 to 260 ± 5.20 (Table 1). It was found that films

with high proportion of PVP showed drastic reduction

in film endurance. Changes in the proportion of EC did

not affect much on the mean folding endurance of the

films but it is evident from the result that higher the EC

proportion more was the film endurance. Formulation

TTS6 showed an optimum endurance (240 ± 5.46).

With the increase in the proportion of the PVP in the

film, the tensile strength and the thickness of the films

was found to be significantly decreased (Table 1), but

the variation in percentage elongation was found to be

insignificant over the different proportions of EC and

PVP used. Formulation TTS6 showed less percentage

elongation and high tensile strength in comparison to

the formulation TTS5, which indicated that the films

containing more proportion of EC are relatively more

strong and tough compared to the films containing more

proportion of PVP.

The graphical representation of the cumulative

percentage of drug permeated as a function of time

through the rat skin is presented in Fig 2. From the

figure it is evident that high proportion of hydrophilic

polymer (PVP) enhances permeation rate. Formulation

TTS6 and TTS7 with higher concentration of EC

showed prolonged permeation of drug in comparison to

other formulated patches. It was observed that from the

formulation TTS6 only 39% of the drug was permeated

in 24 h whereas the permeation was 85% from the

formulation TTS5 within that period.

Fig 3 is the SEM photograph of the film containing

both the polymers and the plasticizer without the drug.

Fig 4 is the SEM photograph of the drug loaded film

before skin permeation study, which exhibits surface

uniformity of the film. The pores on the film as

visualized in Fig 5 are due to the drug released from the

polymer matrix after permeation through skin.

In conclusion, skin permeation of propranolol

hydrochloride from its transdermal patches showed that

the films containing higher proportion of PVP are

suitable for once a day drug delivery and the films

containing higher proportion of EC showed suitability

for a prolonged regimen of sustained drug delivery

through transdermal route for a period of more than 24

h. The results of the study give a rational guideline for

formulating a sustained release transdermal therapeutic


391

Table1: Formulation design and study of various physical parameters of the transdermal therapeutic systems of

propranolol hydrochloride.

Sl. No. Formulation code Ratio of

polymers (EC: PVP)

Mean Thickness

(µm) n = 3

(± = s.d.)

Mean Elongation (%)

n = 3

(± = s.d.)

Mean Tensile strength

(gm/cm2) n = 3

(± = s.d.)

Mean folding endurance

n = 5

(± = s.d.)

1. TTS1 1:2 115 ± 0.9 23.22 251.36 261±5.21 2. TTS2 1:4 116 ± 0.9 24.21 243.67 208±6.74 3. TTS3 1:6 110 ± 0.8 22.28 221.54 183±6.21 4. TTS4 1:8 109 ± 0.4 25.10 215.58 101±5.51 5. TTS5 1:10 104 ± 0.8 23.30 211.45 62±3.78 6. TTS6 10:1 125 ± 0.8 20.40 280.52 240±5.46 7. TTS7 8:1 126 ± 0.4 21.65 280.89 233±6.73 8. TTS8 6:1 119 ± 1.0 20.00 261.48 220±7.84 9. TTS9 4:1 120 ± 1.0 22.10 257.60 211±6.46 10. TTS10 2:1 117 ± 0.9 21.41 253.52 202±5.37

n = number of repeated observation. ; s.d. = standard deviation.

Fig 1: (%) Moisture loss and moisture content profile of the prepared Propranolol hydrochloride transdermal

therapeutic systems.

Fig 2: Comparison of in-vitro permeation rate profile of matrix diffusion controlled Propranolol hydrochloride

TTS through rat abdominal skin. Patches with different concentrations of EC and PVP that include (-●-) 1:2,

(-▪-) 1:4, (-∆-) 1:6, (-\-) 1:8, (-×-) 1:10, (-O-) 10:1, (-■-) 8:1, (-– -) 6:1, (-— -) 4:1, (-♦-) 2:1.


392

Fig 3: SEM photograph of the blank film containing EC

and PVP.

Fig 4: SEM photograph of the drug-loaded film before skin

permeation study.

Fig 5: SEM photograph of the exhausted film after skin permeation study

system of Propranolol hydrochloride for effective

therapy and prophylaxis of angina pectoris, cardiac

arrhythmia and hypertension.

ACKNOWLEDGEMENT

The authors are thankful to Sun Pharmaceuticals Ltd.,

Baroda, for providing the gift sample of propranolol

hydrochloride.

REFERENCES

1. Misra A. Transdermal drug delivery: present and

future. The Pharma Review 2004; 92-102.

2. Barry BW. Dermatological Formulations,

Percutaneous Absorption. New York: Marcel

Dekker; 1987.

3. Robinson JR, Lee VHL. Controlled Drug

Delivery, Fundamentals and Applications. 2nd ed.

New York: Marcel Dekker; 1987.

4. Paudel Kalpana S, Nalluri Buchi N, Hammell

Dana C et al. Transdermal delivery of naltrexone

and its active metabolite 6-β-naltrexol in human

skin in vitro and guinea pigs in vivo. J Pharm Sci

2005; 94(9): 1965-1974.

5. Goodman Gilman A. The Pharmacological Basis

of Therapeutics. 10th ed. New York: McGraw-Hill,

Medical Publishing Division; 2001.

6. Pharmacopoeia of India, 3rd ed. Vol.ll, New Delhi:

Controller of Publications, Ministry of Health,

Govt. of India; 1996.

7. Kumhar SK, Jain SK, Pancholi SS, Agrawal S,

Saraf DK, Agrawal GP. Provesicular transdermal

drug delivery system of ethinylestradiol and

levonorgestrel for contraception and hormone

replacement therapy. Indian J Pharm Sci 2003;

65(6): 620-627.

8. Kulkarni VH, Keshavayya J, Shastry CS, Kulkarni

Preeti V. Transdermal delivery of antiasthmatic


393

drug through modified chitosan membrane. Indian

J Pharm Sci 2005; 67(5): 544-547.

9. Sagar P, Kulkarni Raghavendra V, Doddayya H.

Development and evaluation of membrane

controlled transdermal therapeutic systems. The

Pharma Review 2006; 90-92.

10. Raghuraman S, Velrajan G, Ravi R, Jeyabalan B,

Benito Johnson D, Sankar V. Design and

evaluation of propranolol hydrochloride buccal

films. Indian J Pharm Sci 2002; 64(1): 32-36.

11. Manvi FV, Dandagi PM, Gadad AP,

Mastiholimath VS, Jagadeesh T. Formulation of a

transdermal drug delivery system of ketotifen

fumarate. Indian J Pharm Sci 2003; 65(3): 239-

243.

12. Gupta Sadhana P, Jain SK. Effective and

controlled transdermal delivery of metoprolol

tartarate. Indian J Pharm Sci 2005; 67(3): 346-350.

13. Bharkatiya M, Nema RK, Gupta GD, Gaud RS.

Designing and evaluation of propranolol

hydrochloride transdermal patches. The Pharma

Review 2005; 113-116.

14. Das MK, Bhattacharya A, Ghosal SK. Transdermal

delivery of trazodone hydrochloride from acrylic

films prepared from aqueous latex. Indian J Pharm

Sci 2006; 68(1): 41-46.


394

Free Radical Scavenging Activity of Ficus racemosa roots Surendra Kumar Sharma

* and Vivek Kumar Gupta

Department of Pharm. Sciences, Guru Jambheshwar University of Science & Technology, Hisar 125 001,

Haryana

[email protected]

Abstract

Antioxidant activity of ethyl acetate extract of Ficus racemosa root was investigated for its free radical

scavenging activity by determining nitric oxide radical and superoxide radical scavenging activity. Maximum

scavenging of nitric oxide radical and superoxide radical found were, 27.35% and 48.3% respectively, at 250

µg/ml concentration.

Key words: Antioxidant, free radical, nitric oxide radical, PMS-NADH system

INTRODUCTION

Ficus racemosa Linn. syn. Ficus glomerata Roxb., is a

very common plant, distributed throughout India and is

well-known for its medicinal uses. It is used as anti-

inflammatory, spermatozoic, hypocholesterolemic,

hypoglycaemic, in dysentery, diarrhea and in scabies.

Glycosides, lupeol, α-amyrin, β- sitosterol, are the main

chemical constituents reported in the plant1-5. The

present paper involves the evaluation of antioxidant

potential of the ethyl acetate extract by nitric oxide

radical scavenging and superoxide radical scavenging

assay.


The roots of the plant Ficus racemosa Linn. were

collected from the outskirts of Dist. Sirsa, Haryana and

authenticated by Dr. P. Jayaraman, Scientist, Plant

Anatomy Research Centre, Chennai. One voucher

specimen (Voucher No. PARC/2007/21) has been

procured in department of Pharmacognosy, Guru

Jambheshwar University, Hisar. The roots were dried

under shade, coarsely powdered and 50 g root powder

was extracted with 400 ml of ethyl acetate for 72 h by

hot continuous extraction method. The ethyl acetate

extract was filtered and partitioned by using petroleum

ether to remove impurities. The solvent was evaporated

under reduced pressure and dried in vacuum.

The dried extract (FREA) thus obtained was used for

the assessment of antioxidant activity.

Preliminary chemical tests6,7 were performed to detect

the presence of polyphenolic compounds. The

qualitative chemical tests performed were Shinoda test,

ammonia fuming test, lead acetate test, boric acid test

for flavonoids, and ferric chloride test, nitric acid test,

ammonia hydroxide-potassium ferricyanide test, lead

acetate test for tannins. All the tests confirmed the

presence of flavonoids and tannins.

Nitric oxide radical inhibition assay

Nitric oxide radical inhibition was estimated by the use

of Griess Illosvoy reaction8. In this investigation, Griess

Illosvoy reagent was modified by using naphthyl

ethylene diamine dihydrochloride (0.1% w/v) instead of

1-napthylamine (5%). The reaction mixture (3 ml)

containing sodium nitroprusside (10 mM, 2 ml),

phosphate buffer saline (0.5 ml) and extract (16 – 250

µg/ml) or standard solution (rutin, 0.5 ml) was

incubated at 25°C for 150 minutes. A control without

test compound but equivalent amount of methanol was

taken. After incubation, 0.5 ml of the reaction mixture

mixed with 1 ml of sulfanilic acid reagent (0.33% in

20% glacial acetic acid) and allowed to stand for 5 min

for completing diazotization. Then, 1 ml of naphthyl

ethylene diamine dihydrochloride was added, mixed

and allowed to stand for 30 min at 25ºC. The

concentration of nitrite was assayed at 540 nm and

calculated with reference to the absorbance of the

APTI ijper


Received on 10/04/2007; Modified on : 30/06/2007



395

standard nitrite solutions. Rutin was take as reference.

The percent inhibition was calculated using the

following formula:

% Inhibition = [(Acont-Atest) /Acont] X 100 …… (1)

Where Acont is the absorbance of the control reaction

and Atest is the absorbance in the presence of the sample

of the extracts.

Superoxide anion radical scavenging activity in

PMS-NADH system

Measurement of superoxide anion scavenging activity

of extract was based on the method described by Liu9

with slight modification. Superoxide radicals are

generated in PMS-NADH systems by oxidation of

NADH and assayed by the reduction of nitroblue

tetrazolium (NBT). Tris-HCl buffer (3 ml, 16 mM, pH

8.0) containing 1 ml NBT (50 µM) solution, 1 ml

NADH (78 µM) solution and a sample solution of

extract (16 to 250 µg/ml) in water were mixed. The

superoxide radical-generating reaction was started by

the addition of 1 ml of phenazine methosulfate (PMS)

solution (10 µM) to the mixture. The reaction mixture

was incubated at 25°C for 5 min, and the absorbance

was read at 560 nm against corresponding blank

samples. Quercetin was used as reference. Decreased

absorbance of the reaction mixture indicated increased

superoxide anion scavenging activity. Percentage

inhibition was calculated using the same formula

(1).

Ethyl acetate extract was not fully soluble in the

corresponding solvents (phosphate-buffer or water).

The tests were performed with the filterate (soluble

portion).


Preliminary chemical tests indicated the presence of

tannins and flavonoids. Nitric oxide radical generated

from sodium nitroprusside at physiological pH was

found to be inhibited by FREA. In the PMS/NADH

coupling reaction reduces NBT. The decrease of

absorbance at 560 nm with antioxidants thus indicates

the consumption of superoxide anion in the reaction

mixture.

Table 1 shows that the percentage inhibition of nitric

oxide and superoxide radical generation by FREA. The

percentage inhibition for superoxide radical is moderate

and nitric oxide radical is less as compared to the

reference (Fig.1 and Fig 2).

Table 1. Antioxidant activity of ethyl acetate extract of Ficus racemosa Linn. root

Nitric oxide Radical Scavenging Activity (% inhibition)

Superoxide Radical Scavenging Activity (% inhibition)

Concentration

(µg/ml) Rutin (Std.) FREA Quercetin (Std.) FREA

16 30.0 ± 0.53 7.75 ± 0.32 34.1 ± 0.51 16.9 ± 0.45 32 46.1 ± 1.6 10.05 ± 1.12 52.7 ± 0.85 28.1 ± 1.12 63 53.5 ± 0.79 18.55 ± 1.61 56.9 ± 1.22 35.7 ± 1.07

125 68.9 ± 2.16 21.45 ± 2.25 58.4 ± 1.12 42.6 ± 2.86 250 76.4 ± 0.52 27.35 ± 1.79 60.9 ± 1.6 48.3 ± 2.49

IC50 (µg/ml) 68.7 514.92 63.3 234.1

Data are presented as the mean ± SD (n = 3)

FREA – Ficus racemosa ethyl acetate extract; Std. – Standard

0

20

40

60

80

100

0 16 32 63 125 250

Concenteration (mcg/ml)

Sca

ven

gin

g a

ctiv

ity

(%)

Series4 Series5Rutin FREA

Figure 1. Nitric oxide radical scavenging activity


396

0

20

40

60

80

0 16 32 63 125 250

Concenteration (mcg/ml)S

cav

eng

ing

act

ivit

y

(%)

Series1 Series3FREAQuercetin

Figure 2. Superoxide anion radical scavenging activity in PMS-NADH system

Data are reported as the mean ±SD of three

measurements. Statistical analysis was performed by

the Student t-test and by ANOVA. IC50 values for all

the above experiments were determined by linear

regression analysis. The activity is increasing with

concentration and differences were statistically

significant (p<0.01). Though FREA was not completely

soluble, even then it has shown antioxidant potential,

so, work should be carried out with more antioxidant

models. The results point out some justification of the

therapeutic role of the plant.

REFERENCES

1. Rastogi RP, Mehrotra BN. Compendium of Indian

Medicinal Plants. CDRI Lucknow and NISCAIR,

New Delhi, vol I (1960-1969); 1990.

2. The Wealth of India. First Supplement Series (Raw

Materials) vol 3: D-1, NISCAIR, CSIR, New

Delhi; 2002.

3. Asolkar LV, Kakkar KK, Chakre OJ. Glossary of

Indian Medicinal Plants With Active Principles,

part-1, NISCAIR, CSIR,

New Delhi; 1992.

4. Trivedi CP, Shinde S, Sharma RC. Preliminary

phytochemical and pharmacological studies on

Ficus racemosa (Gular). Ind J Med Res 1969; 57:

1070-1074.

5. Shrotri DS, Aiman R. The relationship of the post-

absorptive state to the hypoglycemic action studies

on Ficus bengalensis and Ficus glomerata. Ind J

Med Res 1960; 48: 162-168.

6. Hyoung Lee S. Antioxidative activity of browning

reaction products isolated from storage-aged

orange juice. J Agric Food Chem 1992; 40(4): 550-

552.

7. Khandelwal KR. Practical Pharmacognosy,

Techniques and Experiments, Nirali Prakashan,

Pune, 2nd Ed.; 2000.

8. Garrat DC. The quantitative analysis of Drugs. Vol.

3, Chapman and Hall Ltd., Japan; 1964.

9. Liu F, Ooi VEC, Chang ST. Free radical

scavenging activity of mushroom polysaccharide

extracts. Life Science 1997; 60: 763 – 771.

*********


397

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for publication in ijper.

All manuscripts submitted from September 2007 onwards should

be submitted in triplicate along with ‘Authorship Responsibility

Undertaking’(see ijper web-site), signed by all the authors of the

paper to, The Editor, Indian Journal of Pharmaceutical Education

and Research (ijper), Association of Pharmaceutical Teachers of

India, H.Q.: Al-Ameen College of Pharmacy, Hosur Road, opp.

Lalbagh Main Gate, Bangalore- 560 027.

Manuscripts will be subjected to peer review process to determine

their suitability for publication provided they fulfill the

requirements of the journal. After the review, manuscript will be

returned for revision along with reviewer’s and /or editor’s

comments. One original copy of the final revised manuscript

should be submitted for publication within one month receiving

the comments. It is also desirable to submit the final revised

manuscript on a CD prepared in MS word version 6.0/95 or a

higher version.

Submission of a manuscript to ijper for publication implies

that the same work has not been either published or under

consideration for publication in another journal. The authors

are required to send Authorship Responsibility Undertaking

signed by all the Authors of the paper along with the

manuscript.

Author publishing results from in-vivo experiments involving

animal or humans should state whether due permission for

conduct of these experiments was obtained from the relevant

authorities /Ethics committee/Institutional Review Board.

Manuscript preparation

Manuscripts should be concisely typewritten in double spaced in

A4 sized sheets, only on one side with a 2 cm margin on all sides.

The manuscript shall be prepared in Times New Roman font

using a font size of 12. Title shall be in a font size 14. All section

titles in the manuscript shall be in font size 12, bold face capitals.

Subtitles in each section shall be in font size 12, bold face lower

case followed by a colon. The pages shall be numbered

consecutively with arabic numbers, beginning with title page,

ending with the (last) page of figure legends. The length of an

Review/ Science Education article should not exceed 25

manuscript pages to include figures, tables and references. No

abbreviations or acronyms shall be used in the Title or Abstract

acronyms, except for measurements.

All the references, figures (Fig.) and tables (Table) in the text

shall be numbered consecutively as they first appear. No sentence

shall start with a numeral. Abbrevaiations like “&” and “etc” shall

be avoided in the paper. There shall not be any decorative borders

anywhere in the text including the title page. The entire MS Word

document with graphs and illustrations pasted in it shall not

exceed 2 MB. Manuscripts must conform to the “Uniform

Requirements for Manuscripts Submitted to Biomedical Journals”

http://www.icmje.org/.

The Content of the manuscript shall be organized in the following

sequence and shall start on separate pages: title page (including

author's name, affiliations and address for correspondence),

abstract (including at least 4 key words), text (consisting of

introduction, materials and methods, results, discussion,

conclusion and acknowledgements), references, figure legends,

tables and figures. Titles should be short, specific, and clear.

Beginning with the first page of text, each page should be

consecutively numbered. All animal experimental procedure

followed must be approved by appropriate ethics committee and

the relevant document furnished to ijper.

For the Review Articles, the author(s) is absolutely free to design

the paper. The Abstract section is needed for review articles too.

The article should not exceed 25 manuscript pages including

figures, tables and references. References, figures, and legends

shall follow the general guidelines described below.

For all other Articles, the following format shall be strictly

followed.

Title Page. The following information should appear: title of

article (A running title or short title of not more than 50

characters), authors’ name, and last name. The author to whom all

correspondence be addressed should be denoted by an asterisk

mark. Full mailing address with pin-code numbers, phone and fax

numbers, and functional e-mail address should be provided of the

author for correspondence. Names of the authors should appear as

initials followed by surnames for men and one given-name

followed by surname for women. Full names may be given in

some instances to avoid confusion. Names should not be prefixed

or suffixed by titles or degrees.

Abstract. The abstract is limited to 250 words, and should

describe the essential aspects of the investigation. In the first

sentence the background for the work should be stated; in the

second sentence the specific purpose or hypothesis shall be

provided; followed sequentially by summary of methods, results

and conclusion. No references should be cited.

Introduction. A brief background information on what has been

done in the past in this area and the importance of the proposed

investigation shall be given. Introduction shall end with a

statement of the purpose or hypothesis of the study.

Material and Methods. This section may be divided into

subsections if it facilitates better reading of the paper. The

research design, subjects, material used, and statistical methods

should be included. Results and discussion shall not be drawn into

this section. In animal experimentation, ethical guidelines shall be

acknowledged.


398

Results. This section may be divided into subsections if it

facilitates better reading of the paper. All results based on

methods must be included. Tables, graphic material and figures

shall be included as they facilitate understanding of the results.

Discussion. Shall start with limited background information and

then proceed with the discussion of the results of the investigation

in light of what has been published in the past, the limitations of

the study, and potential directions for future research. The figures

and graphs shall be cited at appropriate places.

Conclusion. In a separate section, the major findings of the study

and their usefulness shall be summarized. This paragraph should

address the hypothesis or purpose stated earlier in the paper.

Acknowledgments. Acknowledgments should appear on a

separate page.

Tables. Each table should be given on a separate page. Each table

should have a short, descriptive title and numbered in the order

cited in the text. Abbreviations should be defined as footnotes in

italics at the bottom of each table. Tables should not duplicate

data given in the text or figures. Only MS word table format

should be used for preparing tables. Tables should show lines

separating columns with those separating rows. Units of

measurement should be abbreviated and placed below the column

headings. Column headings or captions should not be in bold face.

It is essential that all tables have legends, which explain the

contents of the table. Tables should not be very large that they run

more than one A4 sized page. If the tables are wide which may

not fit in portrait form of A4 size paper, then, it can be prepared in

the landscape form,. Authors will be asked to revise tables not

conforming to this standard before the review process is initiated.

Tables should be numbered as Table No.1 – Title…., Table No.2

– Title…. Etc. Tables inserted in word document should be in

tight wrapping style with alignment as center.

Figures, Photographs and Images. Graphs and bar graphs

should preferably be prepared using Microsoft Excel and

submitted as Excel graph pasted in Word. These graphs and

illustrations should be drawn to approximately twice the printed

size to obtain satisfactory reproduction. [Specification of

Legends/values in Graphs – Font – Arial, size- 10 pt, Italics-

None] Diagrams made with Indian ink on white drawing

paper, cellophane sheet or tracing paper with hand written

captions or titles will not be accepted. Photographs should be

submitted only on photo-glossy paper. Photographs should bear

the names of the authors and the title of the paper on the back,

lightly in pencil. Alternatively photographs and photomicrographs

can also be submitted as ‘jpeg/TIFF with the resolution of 600

dpi or more’ images. Figure and Table titles and legends should

be typed on a separate page with numerals corresponding to the

illustrations. Keys to symbols, abbreviations, arrows, numbers or

letters used in the illustrations should not be written on the

illustration itself but should be clearly explained in the legend. In

case of photomicrographs, magnification should be mentioned

either directly on them or in the legend. Symbols, arrows or letters

used in photomicrographs should contrast with the background.

Method of staining should also be mentioned in the legend.

The complete sets of original figures must be submitted. Legends

should be in the present tense (e.g., ‘Illustration shows ...’).

Subjects’ names must not appear on the figures. Labels should

contrast well with the background. Images should be uniform in

size and magnification. Illustrations should be free of all

identifying information relative to the subject and institution. Line

drawings should be professional in quality. Written permission for

use of all previously published illustrations must be included with

submission, and the source should be referenced in the legends.

Written permission from any person recognizable in a photo is

required. Legends must be double spaced, and figures are

numbered in the order cited in the text. Color prints shall be

submitted only if color is essential in understanding the material

presented. Label all pertinent findings. The quality of the printed

figure directly reflects the quality of the submitted figure. Figures

not conforming to acceptable standards will be returned for

revision.

Figures should be numbered as Fig.1, Fig.2 etc. ; Figures inserted

in word document should be in square wrapping style with

horizontal alignment as center.

Resolution: Drawings made with Adobe Illustrator and

CorelDraw (IBM/DOS) generally give good results. Drawings

made in WordPerfect or Word generally have too low a

resolution; only if made at a much higher resolution (1016 dpi)

can they be used. Files of scanned line drawings are acceptable if

done at a minimum of 1016 dpi. For scanned halftone figures a

resolution of 300 dpi is sufficient. Scanned figures cannot be

enlarged, but only reduced. Figures/Images should be submitted

as photographic quality scanned prints, and if possible attach an

electronic version (TIFF/ JPEG).

Chemical terminology - The chemical nomenclature used must

be in accordance with that used in the Chemical Abstracts.

Symbols and abbreviations - Unless specified otherwise, all

temperatures are understood to be in degrees centigrade and need

not be followed by the letter 'C'. Abbreviations should be those

well known in scientific literature. In vitro, in vivo, in situ, ex

vivo, ad libitum, et al. and so on are two words each and should

be written in italics. None of the above is a hyphenated word. All

foreign language (other than English) names and words shall be in

italics as a general rule. Words such as carrageenan-induced

inflammation, paracetamol-induced hepatotoxicity, isoproterenol-

induced myocardial necrosis, dose-dependent manner are all

hyphenated.

General Guidelines for units and symbols - The use of the

International System of Units (SI) is recommended.

For meter (m), gram (g), kilogram (kg), second (s), minute (m),

hour (h), mole (mol), liter (l), milliliter (ml), microliter (µl). No

pluralization of symbols is followed. There shall be one character

spacing between number and symbol. A zero has to be used

before a decimal. Decimal numbers shall be used instead of

fractions.

Biological nomenclature - Names of plants, animals and bacteria

should be in italics.

Enzyme nomenclature - The trivial names recommended by the

IUPAC-IUB Commission should be used. When the enzyme is the


399

main subject of a paper, its code number and systematic name

should be stated at its first citation in the paper.

Spelling - These should be as in the Concise Oxford Dictionary of

Current English.

PAGE LAYOUT GUIDELINES – ijper

Page size Letter Portrait 8 X 11

Margins All Margins, 1”

Page numbers Numbered as per the assigned page /

Absolutely no break or Missed sections

Indent None, Absolutely, No Tab

Footer / Headers None

Title 14pt Times New Roman, bold, centered

followed by a single blank line.

Text 12pt Times New roman, full justification –

1.5 line spacing between paragraphs. No

indentation

Tables At the end of context with rows and columns

active; Tables should have individual rows

and columns for each values expressed.

All text should be fully justified. Please put all primary section

titles in UPPER CASE letters (Example INTRODUCTION,

MATERIALS AND METHODS, RESULTS, DISCUSSION,

ACKNOWLEDGEMENT, REFERENCES) and subheading in

both Upper and Lower Case letters (Italics). Do not number your

subtitles (for example, 1.0 Introduction; 2.0 Background ; 2.1.1

are not acceptable). Do not use the tab key to indent blocks of text

such as paragraphs of quotes or lists because the page layout

program overrides your left margin with its own, and the tabs end

up in mid-sentence.

References

Literature citations in the text must be indicated by Arabic

numerals in superscript. Each reference separately in the order it

appears in the text. The references should be cited at the end of

the manuscript in the order of their appearance in the text. In case

of formal acceptance of any article for publication, such articles

can be cited in the reference as “in press”, listing all author's

involved. References should strictly adhere to Vancouver style of

citing references

Format:Author(s) of article (surname initials). Title of article.

Journal title abbreviated Year of publication; volume

number(issue number):page numbers.

Standard journal article (If more than six authors, the first three

shall be listed followed by et al.)

You CH, Lee KY, Chey WY, Menguy R. Electrogastrographic

study of patients with unexplained nausea, bloating and vomiting.

Gastroenterology 1980;79:311-4.

Books and other monographs

Format:Author(s) of book (surname initials). Title of book.

Edition. Place of publication: Publisher; Year of publication.

Personal author(s)

Eisen HN. Immunology: an introduction to molecular and cellular

principles of the immune response. 5th ed. New York: Harper and

Row; 1974.

Editor, compiler, as author

Dausser J, Colombani J, editors. Histocompatibility testing 1972.

Copenhagen: Munksgaard; 1973.

Organisation as author and publisher

Institute of Medicine (US). Looking at the future of the Medicaid

program. Washington: The Institute; 1992.

Conference proceedings

Kimura J, Shibasaki H, editors. Recent advances in clinical

neurophysiology. Proceedings of the 10th International Congress

of EMG and Clinical Neurophysiology; 1995 Oct 15-19; Kyoto,

Japan. Amsterdam: Elsevier; 1996.

Dissertation

Kaplan SJ. Post-hospital home health care: the elderly's access

and utilization [dissertation]. St. Louis (MO): Washington Univ.;

1995.

Patent

Larsen CE, Trip R, Johnson CR, inventors; Novoste Corporation,

assignee. Methods for procedures related to the electrophysiology

of the heart. US patent 5529 067. 1995 Jun 25.

Chapter or article in a book

Format: Author(s) of chapter (surname initials). Title of chapter.

In: Editor(s) name, editors. Title of book. Place of publication:

Publisher; Year of publication. page numbers.

Electronic journal article

Morse SS. Factors in the emergence of infectious diseases. Emerg

Infec Dis [serial online] 1995Jan-Mar [cited 1996 Jun 5];1(1):[24

screens]. Available from: URL: http://www.cdc.gov/

ncidod/EID/eid.htm

World Wide Web Format:Author/editor (surname initials). Title

[online]. Year [cited year month day].

Available from: URL:World Wide Web page

McCook A. Pre-diabetic Condition Linked to Memory Loss

[online]. 2003 [cited 2003 Feb 7]. Available from: URL:

http://www.nlm.nih.gov/medlineplus/news/fullstory_11531.ht

ml

Abbreviations for Journals

For More information on medline indexed journals : Download

list of medline journals:

ftp://ftp.ncbi.nih.gov/pubmed/J_Medline.zip

American Journal of Pharmacy- (Amer J Pharm)

Analytical Chemistry- (Anal Chem)

British Journal of Pharmacology and Chemotherapy- (Brit J

Pharmacol)

Canadian Journal of Pharmaceutical Sciences- (Can J Pharm

Sci)

Clinical Pharmacokinetics- (Clin Pharmacokinet)

Drug Development and Industrial Pharmacy- (Drug Develop Ind

Pharm)

Helvitica Chimica Acta- (Helv Chim Acta)

Indian Journal of Medical Sciences- (Indian J Med Sci)

Indian Journal of Pharmaceutical Sciences- (Indian J Pharm Sci)

Journal of the American Chemical Society, The- (J Amer Chem

Soc)

Journal of Biological Chemistry- (J Biol Chem)

Journal of Organic Chemistry, The- (J Org Chem)


400

Journal of Pharmacology and Experimental Therapeutics- (J

Pharmacol Exp Ther)

New England Journal of Medicine- (N Engl J Med)

Pharmaceutical Journal, The (Pharm J)

Pharmacological Research Communications- (Pharmacol Res

Commun)

AUTHOR CHECKLIST FOR SENDING PROOFS TO

EDITORIAL OFFICE

In order to maintain quality and consistency in ijper, we ask you

to perform the following items prior to submitting your final proof

for publication:

• Include the original, hard copy of Author’s Transfer of

Copyright signed by each author

• Thoroughly check the article for typographic errors,

format erros, grammatical errors, in particular: spelling

of names, affiliations, any symbols, equations in the

context, etc.

• Provide graphs and figures in excel format, Pictures are

required as high resolution images (300 dpi).

• Provide laser printed hard copies of all figures and

graphics in black and white or scanned copies can also

be sent to [email protected]

• Submit a proof corrected with RED INK ONLY.

• List out the corrections made in typed format in a

separate page with the proof

Send the Corrected Proof, Copyright Transfer Form, with covering letter in a single envelope to the Following Address

Authors are required to send their contributions or manuscripts through post or courier services.

Dr. Sanjay Pai P.N.

Editor, Indian Journal of Pharmaceutical Education and Research (ijper),

C/o. Association of Pharmaceutical Teachers of India (APTI),

H.Q: Al-Ameen College of Pharmacy,

Opp. Lalbagh Main Gate, Hosur Road, Bangalore 560 027, Karnataka, INDIA.

All enquiries can be made through e-mail : [email protected]

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