“FORMULATION AND DEVELOPMENT OF REPAGLINIDE ...

“FORMULATION AND DEVELOPMENT OF REPAGLINIDE

MICROPARTICLES BY IONOTROPIC GELATION TECHNIQUE”

by

SACHIN E. BHADKE B.PHARM.

Dissertation Submitted to the

Rajiv Gandhi University Of Health Sciences, Karnataka, Bangalore.

In partial fulfillment of the requirements for the award of degree of

MASTER OF PHARMACY

In

PHARMACEUTICS

Under the guidance of Shri. S. P. HIREMATH

SELECTION GRADE LECTURER Department of Pharmaceutics

K.L.E.Society’s College of Pharmacy, Hubli - 580 031. Karnataka

(India)

JUNE-2006

I

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation / thesis entitled “FORMULATION

AND DEVELOPMENT OF REPAGLINIDE MICROPARTICLES BY

IONOTROPIC GELATION TECHNIQUE” is a bonafied and genuine

research work carried out by me under the guidance of Shri. S. P.

HIREMATH Selection grade lecturer, Department of Pharmaceutics,

K.L.E. Society’s College of Pharmacy, Hubli.

SACHIN E. BHADKE Date: Place: Hubli

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

II

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “FORMULATION AND

DEVELOPMENT OF REPAGLINIDE MICROPARTICLES BY

IONOTROPIC GELATION TECHNIQUE” is a bonafied research work

done by SACHIN E. BHADKE in partial fulfillment of the requirement for the

award of degree of MASTER OF PHARMACY IN PHARMACEUTICS.

Shri. S.P. HIREMATH Selection Grade Lecturer

Date: Place: Hubli

Department of PharmaceuK.L.E. Society’s College o Hubli - 580 031, Karnatak(India)

III

tics, f Pharmacy,a,

ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE

INSTITUTION

This is to certify that the dissertation entitled “FORMULATION AND

DEVELOPMENT OF REPAGLINIDE MICROPARTICLES BY

IONOTROPIC GELATION TECHNIQUE” is a bonafied research work

done by SACHIN E. BHADKE under the guidance of Shri. S.P. HIREMATH

Selection Grade Lecturer, Department of Pharmaceutics, K.L.E. Society’s

College of Pharmacy, Hubli.

Dr. B.M.PATIL M. Pharm, Ph.D.

Principal K.L.E. Society’s College of Pharmacy, Hubli - 580 031, Karnataka, (India)

Shri. V.G. JAMAKANDI M. Pharm, Head of the Department Department of Pharmaceutics, K.L.E. Society’s College of Pharmacy, Hubli - 580 031, Karnataka (India)

Date: Place: Hubli

IV

COPYRIGHT

Declaration by the Candidate

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka

shall have the rights to preserve, use and disseminate this dissertation / thesis in

print or electronic format for academic / research purpose.

© Rajiv Gandhi University of Health Sciences, Karnataka

SACHIN E.BHADKE

Date: Place: Hubli

V

DEDICATED TO MY BELOVED

PARENTS AND

SPECIAL THANKS

TO MY

BROTHER

VI

VII VII

VIII

Acknowledgement

I consider myself most lucky to work under the able guidance of Shri. S. P.

Hiremath, Selection Grade Lecturer, Department of Pharmaceutics, K.L.E.S’s College of

Pharmacy, Hubli. I take this opportunity to express my heartfelt gratitude to my reverend

guide. His discipline, principles, simplicity, caring attitude and provision of fearless work

environment will be cherished in all walks of my life. I am very much greatful to him for

his invaluable guidance and ever-lasting encouragement throughout my course.

I am immensely thankful to Dr. V. I. Hukkeri, Ex-Principal and Dr.B.M.Patil,

Principal, K.L.E.S’s College of Pharmacy, Hubli.

I owe my warmest and humble thanks to Mr. Umesh Zope, Research and

Development (EMCURE PHARMACEUTICALS) Pune for providing complete details

regarding drug.

I owe my warmest and humble thanks to Mr. V. G. Jamakandi, Associate

Professor, HOD of Pharmaceutics, and Mr. S. A. Sreenivas, Mrs. Fatima, Mr. S .S.

Biradar, Mr. S .T. Bhagawati, Mr. Jameel S. Mulla, Mrs. K. R. Praveena, K.L.E.S’s

College of Pharmacy, Hubli, for their timely help, encouragement, boosting my

confidence in the progress of my academics.

I also take this opportunity to express my sincere thanks to teaching and non-

teaching staff of K.L.E.S’s College of Pharmacy, Hubli, for their kind co-operation and

help throughout my course.

I express my deepest and very special thanks to my batch mates Nilesh, Anupam,

Siddu, Ashwin, Manoj, Rohan, Prashant, Shrikant, Muzzaffar, Saurabh, Prashant,

Sanjay and afrin, siddhart and my colleagues Nilesh, Sanjay, Rahul, Meena, Ramdas,

Tushar, for their kind co-operation, help and encouragement throughout my post-

graduation.

My heartfelt thanks to my seniors especially Rajiv, Alok, Manish, Pranshu,

Gauda, Kiran, Johnson, Sandeep, Mangesh, Yadvindar, Adesh, Faheem, Pasha, and

Zakir for their help and encouragement.

I am thankful to all my juniors, especially, Mahesh, Nitesh, Ashish, Ratan,

Vinay, Avinash, Nagraj, Ashwini, Sandip, Ajit, Ram, Abdulla, Umesh, Anant, and

Rashmi, and others who have contributed directly or indirectly during my dissertation.

I am really thankful to my family members Nilesh, Mom, Anna, Narendra and

others for constant encouragement, moral support throughout my life.

It is with deep gratitude and humbleness; I express my thanks to Mr. Akshay

Baheti, Mr.V.P Chowdhary, and Mr. Dayanand Kunnur for their constant

encouragement and moral support throughout my dissertation work, with out him

experiments would not have come to reality.

The completion of this dissertation is not only fulfillment of my dreams but also the

dreams of my Parents who have taken lots of pain for me in completion of my higher

studies.

Lastly I thank ‘God’ the Almighty, to show the path to the ladder of success.

Thankful I ever remain....

Sachin E. Bhadke

CONTENTS

No. Particulars Page No.

1. INTRODUCTION …………………………………………. 1-14

2. NEED FOR STUDY……………………………………….. 15-17

3. REVIEW OF LITERATURE………………….…………... 18-37

4. MATERIALS AND METHODS…………………………… 38-53

5. RESULTS AND DISCUSSION …………………………. 54-70

6. SUMMARY ………………………………………………… 71-72

7. CONCLUSION …………………………………………….. 73

8. BIBLIOGRAPHY……………….………………………….. 74-80

LIST OF TABLES Sl.No. PARTICULARS OF THE TABLES Page No.

1 Microencapsulation processes & their Applicabilities 11

2 Pharmacokinetic data of Repaglinide 25

3 Materials were used as supplied by manufacturers 38

4 Equipments used for experimental work 38

5 Formulation Design of Microparticles

45

6 Common techniques for measuring fine particles of various sizes 46

7 Designations and Dimensions of IP Specification Sieves 48

8 Relation Between Angle of Repose and Flow property of the

microparticles

50

9 Standard Calibration Curves of Repaglinide 59

10 Comparison of I.R. Spectra of Repaglinide and in

Combination with Polymers

60

11 Percentage weight remained on various sieve size

60

12 Angle of Repose of Microparticles 61

13 Drug entrapment Efficiency of Microparticles

61

14 In-vitro Dissolution Profile for Formulation F1 62







21 Values of Correlation-coefficient (r) of Repaglinide 69

22 Curve Fitting Data of the Release Profile for Repaglinide 69

23 Results of assay of Formulations F1 & F4 after Accelerated

Stability Studies

70

Abstract

DEPT. OF Pharmaceutics KLESCOP, HUBLI

ABSTRACT

Repaglinide is an anti-diabetic, oral blood-glucose lowering drug of the

meglitinide class used in the management of type-II diabetes mellitus.

The present investigation involves formulation and evaluation of microparticles

with repaglinide as model drug for prolongation of drug release time. An attempt was

made to prepare microparticles of repaglinide by ionotropic gelation technique, with a

view to deliver the drug at sustained or controlled manner in gastrointestinal tract and

consequently into systemic circulation.

The microparticles were formulated by calcium chloride cross-linking method

using various concentration of Hydroxy Propyl Methyl Cellulose and Chitosan by

dropping the Drug-Polymer solution along with sodium alginate in calcium chloride

solution.

The prepared microparticles were evaluated for Flow behavior, Compatibility

study, Drug Entrapment Efficiency, In-vitro Dissolution, Scanning Electron Microscopy

and Sieving method. Among the seven formulations prepared and evaluated F1 and F4 are

found to show satisfactory results.

The Prepared Microparticles shows entrapment efficiency of 78.62% to 91.25%

Fourier transform Infrared spectroscopy confirmed the absence of any Drug-Polymer

interaction. In-vitro release studies carried out in 1.2 pH and 7.2 pH phosphate buffer

solution shows 93.78% and 91.78% of drug was released from F1 and F4 respectively.

The results of Flow behavior and Particle size analysis were found to be in the

official limits.

Abstract

DEPT. OF Pharmaceutics KLESCOP, HUBLI

The In-vitro release studies shows that formulation F1, F2 and F3 releases 91.99 %,

81.66 % and 71.66 % respectively after 12 hours.

Formulation F4, F5 and F6 shows release of 92.11%, 81.93% and 81.76%

respectively.

Formulation F7 which is a combination of hydroxy propyl methyl cellulose and

Chitosan shows drug release of 71.88%.

Chapter-I

Introduction

“ That is good study, which is opened with expectation and closed with delight ”

Chapter-II

Need for study

“ The real act of discovering is not a finding in new lands, but in seeing with new eyes ”

Chapter-III

Review of Literature

Chapter-IV

Materials and Methods

“ Art and science have their meeting point in method ”

Chapter-V

Results and

Discussion “ He, who is not open to conviction , is not qualified for discussion ”

Chapter-VI

Summary

Chapter-VII

Conclusion

Chapter-VIII

Bibliography

Chapter I INTRODUCTION

Dept. of Pharmaceutics KLECOP, HUBLI

1

INTRODUCTION

DIABETES MELLITUS1 :

It is a metabolic disorder characterized by hyperglycaemia, glycosuria, negative

nitrogen balance and sometimes ketonemia. A wide-spread pathological change is

thickening of capillary basement membrane, increase in vessel wall matrix and cellular

proliferation resulting in vascular complications like lumen narrowing, early

atherosclerosis, sclerosis of glomerular capillaries, retinopathy, neuropathy and

peripheral vascular insufficiency.

Two major types of diabetes mellitus are:

Type I Insulin dependent diabetes mellitus (IDDM), juvenile onset diabetes mellitus:

There is a β-cell destruction in pancreatic islets; majority of cases autoimmune (type

I A) antibodies that destroy β-cells are detectable in blood, but some are idiopathic (type I

B)-no β-cell antibody is found. In all type I cases circulating insulin levels are low, and

patients are more prone to ketosis. This type is less common and has low degree of

genetic predisposition.

Type II Non insulin dependent diabetes mellitus (NIDDM), maturity onset diabetes

mellitus:

There is no loss or moderate reduction in β-cell mass; insulin in circulation is low,

normal or even high, no anti- β-cells antibody is demonstrable; has a high degree of

genetic predisposition; generally has a late onset (past middle age). Over 90% cases are

type II DM.



2

Causes may be:

• Abnormality in gluco-receptor of β-cells so that they respond at higher glucose

concentration.

• Reduced sensitivity of peripheral tissues to insulin: reduction in number of insulin

receptors, ‘down regulation’of insulin receptors. Many hypertensives are

hyperinsulinemic but normoglycaemic; exhibit insulin resistance.

Hyperinsulinemia perse has been implicated in causing angiopathy.

• Excess of hyperglycemic hormones (glucagons etc.)/obesity: cause relative

insulin deficiency—the β-cells lag behind.

ORAL HYPOGLYCAEMIC DRUGS

These drugs are blood-glucose lowering agents are effective orally. The chief draw

back of insulin is—it must be given by injection. Orally active drugs have always been

searched. The early sulfonamides tested in 1940s produced hypoglycaemia as side effect.

Taking this lead, the first clinically acceptable sulfonylurea tolbutamide was introduced

in 1957. Others followed soon after. In the 1970s many so called ‘second generation’

sulfonylureas have been developed which are 20-100 times more potent. A diguanidine

synthalin was found to be hypoglycaemic in the 1920s, but was toxic. Clinically useful

biguanide phenformin was developed parallel to sulfonylureas in 1957s. Recently 3

newer classes of drugs viz. α-glucosidase inhibitors, meglitinide analogues and

thiazolidinediones have been inducted.


CLASSIFICATION

SULFONYLUREA

First generation Second generation

Tolbutamide Glibenclamide (glyburide)

Chlorpropamide Glipizide

Gliclazide

Glimepiride

BIGUANIDES


3

Phenformin Metformin

MEGLITINIDE ANALOGUES

Repaglinide Nateglinide

THIAZOLIDINEDIONES

Rosiglitazone Pioglitazone

α-GLUCOSIDASE INHIBITORS

Acarbose Miglitol



4

MEGLITINIDE ANALOGUES

These are recently developed quick and short acting insulin releases.

Repaglinide:

It is the first member of new class of oral hypoglycaemics designed to normalize the

meal time glucose excursions. Though not a sulfonylurea, it acts in an analogous manner

by binding to sulfonylurea as well as to other distinct receptors –closer of ATP dependent

K+ channels – depolarization --- insulin release.

Repaglinide induces rapid onset short lasting insulin release. It is administered

before each major meal to control postprandial hyperglycaemia; the dose may be omited

if a meal is missed. Because of short lasting action it may have a lower risk of serious

hypoglycemia. Side effects are mild headache, dyspepsia, arthralgia, and weight gain.

Repaglinide is indicated only in type II DM as an alternative to sulfonylureas, or to

supplement metformin/long acting insulin. It should be avoided in the liver disease.

Nateglinide:

Another nonsulfonylurea drug which principally stimulates the 1st phase insulin

secretion resulting in rapid onset and shorter duration of hypoglycaemic action than

repaglinide. Ingested 10-30 min. before meal, it limits postprandial hyperglycaemia in

type II DM without producing late phase hypoglycaemia. There is effect on fasting blood

glucose level. Episodes of hypoglycaemia are less frequent than with sulfonylureas. Side

effects are dizziness, nausea, flu like symptoms and joint pain. It is used in the type II

DM along with other antidiabetics, to control postprandial rise in blood glucose.



5

CONTROLLED DRUG DELIVERY SYSTEM

For many decades, medication of an acute disease or a chronic illness has been

accomplished by delivering drugs to the patients via various pharmaceutical dosage

forms like tablets, capsules, pills, creams, ointments, liquids, aerosols, injectables

and suppositories as carriers. To achieve and then to maintain the concentration of

drug administered within the therapeutically effective range needed for medication, it is

often necessary to take this type of drug delivery systems several times a day. This results

in a fluctuated drug level and consequently undesirable toxicity and poor efficiency. This

factor as well as other factors such as repetitive dosing and unpredictable absorption lead

to the concept of controlled drug delivery systems.2, 3

The objectives of controlled release drug delivery includes two important aspects

namely spatial placement and temporal delivery of drug.

Spatial placement relates to targeting a drug to a specific organ or tissue, while

temporal delivery refers to controlling the rate of drug delivery to the target tissue.

An approximately designed controlled release drug delivery system can be a major

advance towards solving these two problems. It is this reason that the science and

technology responsible for development of controlled release pharmaceuticals have been

and continue to be the focus of a great deal of attention in both industrial and academic

laboratories.

Controlled release systems includes any drug delivery system that “achieves slow

release of the drug over an extended period of time.” If the system can provide some

control weather this is of a temporal or spatial nature, in other words, if the system is



6

successful in maintaining predictable and reproducible kinetics in the target tissue or cell,

it is considered as a controlled release system.

If the system only extends the duration of release without reproducible kinetics it is

considered as a prolong release system.

The objectives in designing a controlled release system is to deliver the drug at a

rate necessary to achieve and maintain a constant drug blood level. This rate should be

analogous to that achieved by continuous intravenous infusion where a drug is provided

to the patient at a rate just equal to its rate of elimination. This implies that the rate of

delivery must be independent of the amount of drug remaining in the dosage form and

constant over time. That is release from the dosage form should follow zero-order

kinetics.4

The several advantages of a controlled drug delivery system over a conventional

dosage forms:

• Improved patient convenience and compliance due to less frequent drug

administration.

• Reduction in fluctuation in steady-state levels and therefore better control of

disease condition and reduced intensity of a local or systemic side effects.

• Increased safety margin of high potency drugs due to better control of plasma

levels.

• Maximum utilization of drug enabling reduction in total amount of dose

administered.

• Reduction in health care costs through improved therapy, shorter treatment



7

period, less frequency of dosing and reduction in personnel time to dispense,

administer and monitor patients5.

Microencapsulation :

Microencapsulation is a rapidly expanding technology. As a process, it is a means of

applying relatively thin coatings to small particles of solids or droplets of liquids and

dispersions. Microencapsulation is arbitrarily differentiated from macrocoating

techniques in that the former involves the coating of particles ranging dimensionally from

several tenths of a micron to 5000 microns in size.6

Microencapsulation provides the means of converting liquids to solids, of altering

colloidal and surface properties, of providing environmental protection, and of

controlling the release characteristics or availability of coated materials.

Microenacapsulation is a process where by small discrete solid particles or small

liquid droplets are surrounded or enclosed, by an intact shell. Two major classes of

microencapsulation methods have evolved i.e. chemical and physical.

The first class of encapsulation method involves polymerization during the process

of preparing the microcapsules. The second type involves the controlled precipitation of a

polymeric solution where in physical changes usually occur.7, 8



8

Microencapsulation process:

Basic microencapsulation processes can be divided into chemical and mechanical.

Chemical processes involved :-

Complex coacervation

Polymer-polymer compatibility

Interfacial polymerization in liquid media

In-situ polymerization

In-liquid drying

Thermal and ionic gelation in liquid media

Mechanical processes involved:-

Spray drying

Spray coating

Fluidized bed coating

Electrostatic deposition

Centrifugal extrusion

Spinning disk or rotational suspension separation

Polymerization at liquid-gas or solid-gas interface

Pressure extraction or spraying into solvent extraction bath.9, 10



9

Ideal characteristics of drug for microencapsulation5 :

• Particle size requirement.

The lower the molecular weight, faster and complete is the absorption of the drug.

The drugs having size 150-600 daltons they can easily diffuse through the membrane but

diffusivity (the ability of drug to diffuse through the membrane) is inversely related to

molecular size.

• The drug or the protein should not be adversely affected by the process.

• Reproducibility of the release profile and the method.

• No stability problem.

Drugs unstable in GI environment cannot be administered as oral controlled release

formulation because of bioavailability problems e.g. Nitroglycerine.

• There should be no toxic product associated with the final product.

• Therapeutic range:

A candidate drug for controlled delivery system should have a therapeutic range

wide enough such that variations in the release rate do not result in a concentration

beyond this level.

• Therapeutic index:

The ratio of maximum safe concentration to the minimum effective concentration of

drug is called as therapeutic index. The release rate of a drug with narrow therapeutic

index should be such that the plasma concentration is attained between the therapeutically

safe and effective range. It is necessary because such drugs have toxic concentration

nearer to their therapeutic range.



10

• Elimination half life:

Smaller the half life larger the amount of drug to be incorporated in the controlled

release dosage form. Drugs with t1/2 in the range of 2 to 4 hours make a good candidates

for such a system e.g. propranolol.

• Plasma Concentration-Response Relationship:

Drugs whose pharmacologic activity is independent of its concentration are poor

candidates for controlled release systems.

Microencapsulation of pharmaceuticals is undertaken for the following

applications:

Microencapsulation has been employed to provide protection to the core material

against atmospheric effects. The separation of incompatible substances, for example

pharmaceutical eutectics, has been achieved by encapsulation. Toxic chemicals such as

insecticides may be microencapsulated to reduce hazards. Also the hygroscopic

properties of many core materials such as sodium chloride may be reduced by

microencapsulation.

Many drugs have been microencapsulated to reduce the gastric and other

gastrointestinal tract irritation. The local irritation and release properties of a number of

topically applied products can be altered by microencapsulation. This process is also used

to mask the taste of bitter drugs.

Microencapsulation has been widely employed in the design of controlled release

and sustained release dosage forms. It is the most recent addition to oral prolonged



11

release mechanisms. The use of microencapsulation for the production of sustained

release dosage forms has been widely employed in the last 30 years since the successful

introduction by Smith, Nine and French in the early 1950’s.8,11

The physical nature of the core materials and the particle size ranges applicable to

each process are given in following table.

The process generally is considered to be applicable only to the encapsulation of

solid core materials as indicated in following table:6

Table No. 1

Microencapsulation Processes & their Applicabilities

Microencapsulation

process

Applicable core material Approximate

particle size

(µm)

Air suspension Solids 35-5000

Coacervation-phase separation Solids & liquids 2-5000

Multiorifice centrifugal Solids & liquids 1-5000

Pan coating Solids 600-5000

Solvent evaporation Solids & liquids 5-5000

Spray drying and congealing Solids & liquids 600

Two general structures are exists – Microcapsules and Microparticles

A microcapsules is a system that contains a well defined core and a well defined

envelop. The core can be solid, liquid or gas; the envelope is a made of a continuous,

porous or non-porous, polymeric phase. The drug can be dispersed inside the



12

microcapsule as solid particulates with regular or irregular shapes, pure or dissolved

solution, suspension, emulsion or a combination of suspension and emulsion.

A microparticle is a structure made of a continuous phase of one or more miscible

polymers in which particulate drug is dispersed at either the macroscopic or molecular

levels. However, difference between the two system is the nature of the microparticle

matrix in which no well defined wall or envelop exists.7, 8

On the basis of classification these microcapsules and microspheres are prepared by

following techniques:12

Single emulsion technique

Double emulsion technique

Polymerization technique

Normal polymerization technique

Interfacial polymerization technique

Phase separation coacervation technique

Spray drying & spray congealing technique

Solvent extraction technique

Solvent evaporation technique

Solvent diffusion technique

Ionotropic gelation technique



13

Release characteristics of microparticles :

Release of core material from a non-erodible microparticle can occur in several

ways. Non-erodible spherical microparticles releases the encapsulated material by

steady-state diffusion through a coating of uniform thickness. The rate of release remains

constant as long as the internal and external concentration of core material and

concentration gradient through the membrane are constant.

If some of the encapsulated material migrates through the microcapsule membrane

during storage a burst effect occurs. If the microcapsule acts as inert matrix particle in

which core material is dispersed (microparticles) the Higuchi model is valid up to 60%

release.7

Microencapsulation by Ionotropic Gelation Technique:

In this method strong spherical beads with a narrow particle size distribution and

low friability could be prepared with high yield and a drug content approaching 91.25 %.

The flow properties of micronized or needle like drug crystals were significantly

improved by this agglomeration technique when compared with non-agglomerated drug

crystals. The ionic character of the polymers results from pH dependent disintegration of

the beads.

One of the most important and useful properties of alginates is the ability to form

gels by reaction with calcium salts. Alginic acid is composed of D-mannuronic acid and

L-gluronic acid residues at varying proportions of GG-, MM- and MG-blocks.

Crosslinking takes place only between the carboxylate residue of GG-blocks and Ca+2



14

ions via egg-box model to give a tight gel network structure. These gels resembles a solid

in retaining their shape and resisting stress and consist of almost 100% water.

A gel in classical colloidal terminology is defined as a system which owes its

characteristic properties to a cross-linked network of polymer chains which form at the

gel point. A considerable amount of research has been carried out in recent years to

elucidate the nature of the cross-links and determine the structure of alginate gels.

It has been suggested that the cross-links were caused either by simple ionic

bridging of two carboxyl groups on adjacent polymer chains via calcium ions or by

chelating of a single calcium ions by hydroxyl and carboxyl groups on each of a pair of

polymer chains.13

Chapter III Research Investigated


15

Need for study:

Microparticles have been widely accepted as a means to achieve oral and parenteral

controlled release. The microsphere requires a polymeric substance as a coat material or

carrier. A number of different substances both biodegradable as well as non-

biodegradable have been investigated for the preparation of microparticles17.

It not only reduces the dose of the drug, reaching to the effective biological sites

rapidly but also results in reduced toxicity of the targeting. But in the past few years,

pharmacists have been focused their research in colloidal drug delivery system/colloidal

carriers, like liposomes, microspheres and nanoparticles as a targeting carriers, which has

given selective targeting41.

Diabetes mellitus is a group of syndrome characterized by hyperglycaemia, altered

metabolism of lipids, carbohydrates, proteins and an increased risk of complication from

the vascular disease42.

Repaglinide is a nonsulfonylurea oral hypoglycaemic agent of the meglitinide

class, is mainly used in the management of type II diabetes mellitus. Chemically it is (S)-

2-ethoxy-4-{2-[3-methyl-1-[2-(1-piperidinyl) phenyl] butyl] amino]-2-oxoethyl} benzoic

acid. It has short biological half-life of less than one hour and rapidly eliminated from

body43.

The main aim of the present work is to develop the repaglinide microparticles by

using chitosan and hydroxyl propyl methyl cellulose as a polymer for prolonged,

relatively constant effective level of repaglinide and improve patient compliance.



16

OBJECTIVES

Repaglinide is antidiabetic agent used in the treatment of Diabetes Mellitus. It is

used orally in the dose of 0.5-8 mg 3-4 times a day.

The present study “Repaglinide microparticles by ionotropic gelation technique”

was meticulously designed to improve the therapeutic efficacy and to prolong its release.

The following experimental protocol was therefore designed to allow a systematic

approach to the study:

1) Compatibility study:

Compatibility of drug with various polymers by using IR.

2) Preparation of standard curve for Repaglinide in acid buffer (pH 1.2),

and alkaline buffer (pH 7.2).

3) To prepare microparticles of Repaglinide using polymers Chitosan and

Hydroxy Propyl Methyl Cellulose by using Ionotropic Gelation Technique.

4) The following evaluation parameters were carried out based on

laboratory experiments:

i) Drug Entrapment Efficiency.

ii) Flow property of prepared microparticles.

iii) In-vitro dissolution studies by using dissolution tester USP (XXIII)

basket method release.

iv) Analysis by Scanning Electron Microscopy (SEM).

v) Particle size analysis by Sieving Method.



17

The objectives of the proposed study are as follows:

• To overcome the rapid elimination of drug and to develop the oral controlled drug

delivery system.

• To increase the biological half-life of the drug or to maintain the constant drug

concentration in the body.

Chapter IV ----------------------------------------------------------------------------------- Review of Literature

17 Dept. of Pharmaceutics KLESCOP, HUBLI



Review of Literature :

A great deal of work has been done by the scientists about microspheres or

microparticles.

Bhalla H. L. et. al., (1988) worked on the development of sustained release tablets,

beads and coated pellets of Indomethacin using guar gum, sodium alginate and acrylic

resins. In comparison with the marketed slow release product of Indomethacin, the

prepared formulations showed considerable in-vitro drug release patterns.18

Chowdary K.P.R. et al., (1989) carried out microencapsulation of aspirin, diazepam

and nitrofurantoin by calcium alginate. The microparticles were found to be discrete,

spherical and free flowing. The release mechanism was found to be of diffusion type and

release depended on solubility of the core material in the dissolution fluid.19

Udupa N. et. al., (1994), developed implantable formulations of Flubiprofen using

biodegradable aliphatic polyesters, hydrophilic polymers like alginates and HPMC by

ionotropic gelation technique, in the form of films, microspheres and pellets for treating

chronic inflamed conditions associated with arthritis.20

Kakkar A.P. (1995) developed and characterized Ibuprofen loaded microcapsules

with sodium alginate and calcium chloride by ionotropic gelation technique. Spherical,

smooth surfaced alginate microcapsules of Ibuprofen were obtained by this method. The

preparation was based on dispersion of sodium alginate-Ibuprofen matrix in liquid

paraffin followed by coating process by calcium chloride.21

M. Guzman, J. Molepeceres et. al., (1996) have Prepared, characterized poly-e-

caprolactone and hydroxyl propyl methyl cellulose phathalate ketoprofen loaded



microspheres by encapsulating the ketoprofen within poly-e-caprolactone and hydroxyl

propyl methyl cellulose phathalate22.

M. Cuna, M. L. Lorenzo-Lamosa et. al., (1997) have prepared pH dependent

cellulosic microspheres containing Cefuroxime Axetil: Stability and In-vitro release

behavior by using CAT (cellulose acetate trimellitate) and two types of hydroxyl propyl

methyl cellulose phathalate, HPMCP-55 and HPMCP-50 were obtained by solvent

extraction procedure.23

Propranolol hydrochloride was incorporated into formed calcium alginate beads or

incorporated simultaneously with the gelation of alginate beads by calcium ions, and the

interaction of the drug with alginate molecular chains in the beads was studied by Lim-Ly

and Wan-LS, (1997)24.

Lim-Ly et. al., (1997), carried out the preparation of chitosan microspheres by an

emulsion phase separation technique and ionotropic gelation technique. They reported

that the microspheres, so prepared were spherical and free flowing and had smooth

surfaces25.

Pillay V., Dangor C.M. et. al., (1998) worked on encapsulation of indomethacin in

calcium alginate gel disks by ionotropic gelation technique.26

A. Manna, I. Ghosh, et. al., (1999) prepared and evaluated of an oral controlled

release microparticulate drug delivery system of Nimesulide by ionotropic gelation

technique and statistical optimization by factorial analysis by using HPLC and sodium

alginate as a polymer and calcium chloride as a crosslinking agent. Finally they have

done statistical optimization with two-way analysis of varience (ANOVA) and linear

regression analysis27.



Alf Lamprecht, Ulrich Schafer, et. al., (2000) demonstrated the potential of

confocal laser scanning microscopy as a characterization tool for different types of

microparticles which are prepared by various methods including complex coacervation,

spray drying, double emulsion solvent evaporation technique and ionotropic gelation

technique.28

Patil V. B. and Pokharkar Varsha B., (2001) prepared and evaluated sustained

release nimesulide microspheres from sodium alginate. In this investigation a 23 full

factorial design was used to study the joint influence of three variables. The polymer

concentration, Drug concentration, and cross-linker concentration and also various

dependent variables like percent efficiency, spericity, particle size, and drug release.29

Choi B.Y., Park H. J., et. al., (2002) prepared alginate beads for floating drug

delivery system in which they prepared floating beads by adding the sodium alginate

drop by drop in calcium chloride solution and the floating properties were investigated.

Bead porosity and volume average pore size, as well as surface and cross sectional

morphology of beads were examined with mercury porosimetry and scanning electron

microscopy30.

Chowdary K.P.R. et al., (2003) developed indomethacin microcapsules with a coat

consisting of alginate and Mucoadhesive polymers such as HPMC, MC etc. by an

emulsification-ionic gelation process and were investigated to develop Mucoadhesive

microcapsules. The resulting microcapsules were discrete, large, spherical and free

flowing.31

Jelena Filipovie-Grcie, et. al., (2003) have evaluated Spray-dried Carbamazepine-

loaded Chitosan and HPMC microspheres and characterized by X-ray powder



diffractometry (XRD) and differential scanning calorimetry (DSC), were also studied

with particle size distribution, drug content analysis and drug release.32

Nikhil O. Dhoot et. al., (2003) studied the release of fluorescein isothiocynate

labled bovine serum albumin from alginate-microencapsulated liposomes and evaluated

the properties of the system for controlled drug delivery.33

Mukherjee A. et. al., (2003) have conducted a nebulization technique for

nanocapsulation of methotrexate using calcium alginate as a coating material.

Nanocapsules formed were compared with those produced by in-situ gelling technique,

which showed that by controlling various process parameters superior drug nanocapsules

were produced by pneumatic nebulization technique34.

Rajesh K.S., Khanrah A. and Biswanath Sa, (2003) studied preparation of

ketoprofen-loaded microparticles by dropping alginate-drug suspension, with or without

aqueous colloidal polymer dispersions, into calcium chloride solution. The effect of

various formulation variables on the physical characteristics of the microparticles were

studied. In-vitro release showed that drug release from the microparticles depended only

on the concentration of alginate and the nature of aqueous colloidal polymer dispersion13.

Arul B., Kothai R., Sangameswaran B., et. al., (2003) have conducted formulation

and evaluation of chitosan microspheres containing isoniazid in this they have prepared

microspheres by glutaraldehyde cross-linking method using various concentration of

chitosan15.

Pralhad T. Tayade, et. al., (2004) they have done Encapsulation of water-insoluble

drug by a cross-linking technique: Effect of process and formulation variables on



encapsulation efficiency, particle size, and in vitro dissolution rate they have prepared

Ibuprofen-gelation micropellets by cross-linking technique formaldehyde.35

Mingshi Yanga, Fudea et. al., (2004) have conducted a novel pH dependent

gradient-release delivery system for nitrendipin microspheres. By using Eudragit E-100,

Hydroxy propyl methyl cellulose phthalate and Hydroxy propyl methyl cellulose acetate

succinate which dissolve at an acid condition, the pH of ≥ 5.5 and ≤ 6.5, respectively.36

Chowdary K.P.R., Koteswara Rao N., et. al., (2004) prepared ethyl cellulose

microspheres of glipizide. In this study glipizide was incorporated into Ethyl cellulose

and were investigated by in-vitro and in-vivo methods.14

Govindrajan G., Muttusamy K., et. al., (2004) have formulated and developed

lansoprazole floating microspheres in which they have studied improvement of half-life

and bioavailability of lansoprazole by using chitosan as polymer in which it acts as a

carrier and characterized by scanning electron microscope (SEM)37.

Jamagondi L. N., Patil V. B. et. al., (2004) studied the microencapsulation of

metformin hydrochloride using ethyl cellulose as a polymer. By this they have tried to

improve glucose tolerance in patients with type-II diabetes38.

Dandagi P. M., Manvi F.V., et. al., (2004) worked on “Microencapslulation of

verapamil hydrochloride by ionotropic gelation technique”, By using hydroxyl propyl

methyl cellulose and hydroxyl propyl cellulose as a polymer and characterized by

scanning electron microscopy.39



DRUG PROFILE 40, 41 :

REPAGLINIDE

Molecular formula: C28 H36 O4 N

Molecular weight: 452.6

Description :

Repaglinide (Prandin) is an oral insulin secretagogue of the meglitinide class. This

agent is a derivative of benzoic acid & chemically it is: (S)-2-ethoxy-4-{2-[3-methyl-1-

[2-(1-piperidinyl) phenyl] butyl] amino]-2-oxoethyl} benzoic acid.

Structure:

CH3

COOH

H3C O

N O C2H5

N H



Clinical Pharmacology:

Mechanism of action:

It is the first member of new class of oral hypoglycaemics designed to normalize the

meal time glucose excursions. Though not a sulfonylurea, it acts in an analogous manner

by binding to sulfonylurea as well as to other distinct receptors –closer of ATP dependent

K+ channels ---- depolarization ------ insulin release.

Repaglinide induces rapid onset short lasting insulin release. It is administered

before each major meal to control postprandial hyperglycaemia; the dose may be omited

if a meal is missed. Because of short lasting action it may have a lower risk of serious

hypoglycemia. Side effects are mild headache, dyspepsia, arthralgia, and weight gain.

Repaglinide is indicated only in type II DM as an alternative to sulfonylureas, or to

supplement metformin/long acting insulin. It should be avoided in the liver disease.

Pharmacokinetic data of Repaglinide:

After oral administration, Repaglinide is rapidly and completely absorbed from the

gastrointestinal tract. After single and multiple oral doses inn healthy subjects or in

patients, peak plasma drug levels (Cmax) occurs within 1 hour (Tmax). Repaglinide is

rapidly eliminated from the blood stream with a half life of approximately 1-hour. The

mean absolute bioavailability is 56%. When repaglinide was given with food.

Repaglinide is 98% bound to plasma proteins, primarily to albumin. The drug is

completely metabolized by oxidative biotransformation and direct conjugation with

glucuronic acid after either an intravenous or oral dose. Metabolites do not contribute to

the glucose-lowering effect of repaglinide.



Table No. 2

Pharmacokinetic data of Repaglinide

No. Drug Repaglinide

1. Elimination half life (t1/2) 1 hrs.

2. Duration of action 2-3

3. Clearance route Liver

4. Daily dose 1.5-8 mg

5. Number of doses per day 3-4 per day

6. Elimination rate constant (Ke) 0.693 hrs.

7. Peak plasma level (Cmax) 37.5 ng/ml

8. Apparent volume of distribution (Vd) 31.0 L

9. Therapeutic concentration range 0.5-4 mg

10. Bioavailability 56 %

Indications :

Rapilin is indicated as an adjunct to diet and exercise to lower the blood glucose

levels in patients with type II diabetes mellitus (NIDDM) whose hyperglycaemia cannot

be controlled satisfactorily by diet and exercise alone.

Rapilin is also indicated for use in combination with metformin to lower blood

glucose levels in patients whose hyperglycaemia cannot be controlled satisfactorily by

diet, exercise and either repaglinide or metformin alone.



Drug interactions:

Invitro data indicate that antifungal agents like ketoconazole and miconazole may

inhibit repaglinide metabolism, and antibacterial agents like erythromycin. Drugs that

induce the cytochrome P-450 enzyme system 3A4 may increase repaglinide metabolism;

such drugs include troglitazone, rifampin, barbiturates, and carbamazepine.

Repaglinide had no clinically relevant effect on the pharmacokinetics properties of

digoxin, theophylline, or warfarin,. Thus no dosage adjustment is required for digoxin,

theophylline, or warfarin on co-administration of cimetidine with repaglinide did not

significantly alter the absorption and disposition of repaglinide.

Side effects:

In various clinical trials, the most common side effects leading to withdrawal were

hyperglycaemia, hypoglycaemia and related symptoms. Other commonly reportedly side

effects were upper respiratory tract infections, nausea, vomiting, arthralgia, backpain and

headache. The incidence of serious cardiovascular side effects added together, including

ischaemia was slightly higher for repaglinide (4 %) than for sulfonylurea drug (3 %) in

controlled comparator clinical trials.

Overdosage:

There were few adverse effects other those associated with the intended effect of

lowering blood glucose in the patients who received increasing doses of repaglinide upto

80 mg a day for 14 days. Hypoglycaemia did not occur when meals were given with

these high doses. Hypoglycaemia symptoms without loss of consciousness or neurologic

findings should be treated aggressively with oral glucose and adjustment in drug dosage

and / or meal pattern.



If hypoglycaemic reactions with coma is diagnosed or suspected, the patients

should be given a rapid intravenous injection of concentrated (50%) glucose solution.

This should be followed by a continuous infusion of more dilute (10%) glucose solution

at a rate that will maintain the blood glucose at a level above 100 mg/dl.

Dosage and Administration :

There is no fixed-dosage regimen for the management of type II diabetes with

repaglinide. Short-term duration of repaglinide may be sufficient during periods of

transient loss of control in patient’s usually well-controlled on diet. Repaglinide doses are

usually taken within 15 minutes of the meal but time may vary from immediately

preceding the meal to as long as 30 minutes before the meal.

Starting dose :

For patients not previously treated or whose glycosylated haemoglobin is < 8 % the

starting dose should be 0.5 mg with each meal. For patients previously treated with blood

glucose-lowering drug and whose glycosylated haemoglobin is < 8% the initial dose is 1

or 2 mg with each meal preprandially.

Dose adjustment:

Dosage adjustment should be determined by blood glucose response, usually

fasting blood glucose. The preprandial dose should be doubled upto 4 mg with each meal

until satisfactory blood glucose response is achieved. At least one week should elapse to

asses response after each dose adjustment.



The recommended dose range is 0.5 mg to 4 mg taken with meals. Repaglinide

may be preprandial 2, 3, or 4 times a day in response to changes in the patient’s meal

pattern. The maximum dose recommended daily dose is 16 mg.

No dosage adjustments are required for the elderly.

Combination Therapy :

The starting dose adjustments for repaglinide combination therapy is the same as

for repaglinide monotherapy. The dose of each drug should be carefully adjusted to

determine the minimal dose required to achieve the desired pharmacological effects.

Appropriate monitoring of fasting plasma glucose and glycosylated haemoglobin

measurements should be used to ensure that the patient is not subjected to excessive drug

exposure or increased probability of secondary drug failure.



POLYMER REVIEW

HYDROXY PROPYL METHYL CELLULOSE42, 43 :

Hydroxy propyl methyl cellulose (HPMC) is one of the cellulose ether, commonly

used in the formulation of controlled release dosage forms. These polymers hydrates in

water, forming a gel layer at the matrix periphery. Drug is released by a combination of

diffusion through and erosion of gel.

HPMC offers the advantage of being non-toxic and relatively inexpensive, it can be

directly compressed into matrices and the many grades available allow a wide latitude in

the ability to tailor desired drug release profiles.

It is believed that provided the drug and the resin are oppositely charged they will

bind together in-situ within the HPMC matrix, leading to reduced drug release rates.

Once the drug is released sufficient ions are available to displace it from the binding site.

It was not surprising, to find that the ionic strength of the dissolution fluid affected the

action of the resin. Nevertheless, the HPMC matrix has shown to have an inherent

buffering capacity, which allowed a virtually pH independent release profile to be

obtained.

Structural Formula of HPMC :

CH2OCH3 H OCH3 H OH HO H H O O H OCH3 H H OCH3 H H OH H H H OH H O O O CH2OCH3 H OCH2CHCH3

OH



Empirical formula :

C8H15O6 - (C10H18O6)N - C8H15O5

Synonyms :

Methyl hydroxypropyl cellulose, propylene glycol ether of methyl cellulose.

Solubility :

Soluble in cold water forming a viscous colloidal solution, insoluble in alcohol,

ether and chloroform but soluble in mixtures of methyl alcohol and methylene chloride.

Functional categories :

USP: Suspending and or viscosity increasing agent, tablet binder, coating agent.

BP: Viscosity increasing agent, adhesive anhydrous ointment ingredient.

Others: Film former, emulsion stabilizer.

Method of manufacture :

Cellulose fibers, obtained from cotton linters or wood pulp, are treated with caustic

solution. The alkali cellulose thus obtained is in turn treated with methyl chloride and

propylene oxide to produce methyl hydroxyl propyl; ethers of cellulose. The fibrous

reaction products is then purified and ground to a fine, uniform power.

Stability and storage condition :

Very stable in dry condition, solutions are stable at pH 3.0-11.0. Aqueous solutions

are liable to be affected by microorganisms.

Safety :

Human and animal feeding studies have shown hydroxypropyl methyl cellulose to

be safe.



Application in Pharmaceutical formulation :

Film former in tablet film coating (perhaps the most commonly used film forming

agent). Lower viscosity grades are used in aqueous film coating and higher viscosity

grades are used in solvent film coating. The concentration varies from 2 to 10 %

depending on the viscosity grade of polymer. High viscosity grade are used to retard the

release of water soluble drugs.

Review of past work on HPMC :

Microparticulates drug delivery system of nimesulide was prepared with HPMC as

a polymer. HPMC microparticles are sufficiently hard, spherical in shape, capable of

loading drug upto 90%. Drug leaching from the surface of microparticles is negligible

and capable of releasing drug up to 12 hrs.28

Microencapsulation of drug with aqueous colloidal polymer was studied with

HPMC, sodium alginate, and chitosan as a polymers. About 80% to 90% encapsulation

efficiency was achieved with Ibuprofen, Theophylline, Guaifenesin. Water soluble drugs

with HPMC shows highest drug loading capacity.



SODUM ALGINATE :44, 45

Sodium alginate consists of mainly of the sodium salt of alginic acid which is a

mixture of polyuronic acids [(C6H8O6) n] composed of β-D-mannuronic acid and residue

linked so that the carboxyl group of each unit is free while the aldehyde group is shielded

by a glycosidic linkage.

Empirical formula :

(C6H7O6Na) n

Structural formula : H COOH H H H O O OH OH HO O O H H O

H COONa n

Description :

Sodium alginate occurs as a white or buff powder which is odourless and tasteless.

Sodium alginate produces aqueous solutions that forms gel on the addition of small

amount of soluble calcium salt.

Grades:

Various grades of sodium alginate are available yielding aqueous solutions of

varying viscosities within a range of 20 to 400 centipoises in 1% solution at 200C.



Solubility :

Sodium alginate is very slowly soluble in water forming a viscous colloidal

solution practically it is insoluble in alcohol, chloroform and ether and in hydroalcoholic

solutions in which alcohol content is greater than 30% by weight.

Stability and Storage Conditions :

Sodium alginate is hygroscopic, the moisture content at equilibrium is a function of

relative humidity. Dry storage stability is excellent when the powder is stored in a well

closed container at temperature of 250C or less.

Incompatibility :

Depending on the concentration, sodium alginate is incompatible with phenols and

parabens.

Uses :

Alginic acid and alginates such as propylene glycol alginate and sodium alginate

are used as suspending and thickening agents, aid in the preparation of water miscible

pastes, creams and gels. They may be used as stabilizers for oil in water emulsions and as

binding and disintegrating agents in tablets. Alginic acid and alginates (ammonium

alginate, calcium alginate, potassium alginate, propylene glycol alginate and sodium

alginate) are also employed as emulsifiers and stabilizers in food industry.



CHITOSAN 46:

Chitosan is the term applied to deacetylated chitins in various stages of

deacetylation and depolarization and it is therefore not easily defined in terms of its exact

chemical composition. Partial deacetylation of chitin results in the production of chitosan,

which is a polysaccharide comprising copolymers of glucosamine and N-

acetylglucosamine.

Molecular Weight :

Chitosan is commercially available in several types and grades that vary in

molecular weight between 10,000 and 1,000000, and vary in degree of deacetylation and

viscosity.

Synonyms :

2-amino-2-deoxy-(1,4)- β-D-glucopyranan; deacetylated chitin; deacetylchitin;

β-1,4-poly-D-glucosamine; poly-D-glucosamine; poly-(1,4- β-D-glucopyranosamine ).

Chemical name :

Poly- β-(1,4) -2-amino-2-deoxy-D-glucose.

Description :

Chitosan occurs as odorless, white or creamy-white powder or flakes. Fibre

formation is quite common during precipitation and the chitosan may look ‘cotton like.’

Structural formula : CH2OH CH3 O OH O CH3 NHR n R = H or COCH3



Functional category :

Coating agent; disintegrant; film-forming agent; mucoahesive; tablet binder;

viscosity-increasing agent.

Typical properties :

Acidity :

pH = 4.0-6.0 (1% W/V aqueous solution)

Density :

1.35-1.40 g/cm3

Glass transition temperature : 2030C

Moisture content :

Chitosan absorbs moisture from the atmosphere, the amount of water absorbed

depending upon the initial moisture content and the temperature and relative humidity of

the surrounding air.

Particle size distribution : < 30 µm

Solubility :

Sparingly soluble in water; practically insoluble in ethanol (95%), other organic

solvents, and neutral or alkali solutions at pH above 6.5.

Incompatibility :

Chitosan is incompatible with strong oxidizing agents.

Method of manufacturing :

Chitosan is manufactured commercially by chemically treating the shells of

crustaceans such as shrimps and crabs. The basic manufacturing process involves the

removal of proteins by treatment with alkali and of minerals such as calcium carbonate



and calcium phosphate by treatment with acid. Before these treatment the shells are

ground to make as accessible and then deproteinized by treatment with aqueous sodium

hydroxide 3-5 % solution. The resulting product is neutralized and calcium is removed by

treatment of hydrochloric acid at room temperature to precipitate chitin. The chitin is

dried and by deacetylation of this chitin forms chitosan.

N-acetylation of chitin is achieved by treatment with an aqueous sodium hydroxide

at elevated temperature (1100C), the precipitate is washed with water. The crude sample

is dissolved in acetic acid 2% and insoluble part is removed and the resulting clear

supernatant solution is neutralized with sodium hydroxide solution to give purified white

precipitate of chitosan.

Safety :

Chitosan is investigated widely for use as an excipient in oral and other

pharmaceutical formulations. It is biocompatible with both healthy and infected skin.

Chitosan has been shown to be biodegradable.

Stability and storage conditions :

Chitosan powder is a stable material at room temperature, although it is

hygroscopic after drying. Chitosan should be stored in tightly closed container in a cool,

dry place and it should be stored at a temperature of 2-80C.

Applications in Pharmaceutical Formulation or Technology :

The suitability and performance of chitosan as a component of pharmaceutical

formulations for drug delivery applications has been investigated in numerous studies.

These include controlled drug delivery applications, used as a component of

mucoadhesive dosage forms, rapid release dosage forms, improved peptide delivery,



colonic drug delivery systems, and use for gene delivery. Chitosan has been processed in

to several pharmaceutical dosage forms, including gels, films, beads, microspheres,

tablets and coating for liposomes. Furthermore, chitosan may be processed into drug

delivery systems using several techniques including spray-drying, coacervation, direct

compression, and conventional granulation processes.

Chapter V -------------------------------------------------------------------------------------------Methodology

37

Dept. of Pharmaceutics, KLECOP, Hubli


38

MATERIALS AND METHODS

The following materials were used as supplied by the manufacturers without

further purification:

Table No.3

Materials were used as supplied by manufacturers

No. Chemicals Grade Supplied by

1. Repaglinide ------ Sun Pharmaceuticals Ltd., Mumbai.

2. Hydroxy Propyl Methyl Cellulose

K4 Grade

Colorcon, Goa.

3. Chitosan

Central Institute of Fisheries Cochi, Kerala.

4. Sodium Alginate AR S. D. Fine Chem. Ltd. Mumbai.

5. Calcium Chloride AR S. D. Fine Chem. Ltd. Mumbai.

Following equipments were used for experiment:

Table No.4

Equipments were used by for experimental work

No. Equipment Supplied by

1. Dissolution

test apparatus

USP rotating basket dissolution apparatus USP

(XXIII).

2.. Remi Stirrer No. 1A323 Instruments and Appliances Mfg. Corporation,

Ajmeer (India).



39

3. UV-visible Spectrophotometer Model No.1201

JASCO V-530. UV/Vis spectrophotometer.

4. Dryer: : Hot Air Oven

PSM Industries, Bangalore

5. Electronic Balance

K. Roy & Company, Varanasi.

6. Scanning Electron microscope

JSM 35CF,JEOL, Japan

7. Sieve Shaker

Toshniwal, India.

Reagents and solutions :

• Preparation of standard curve

Preparation of standard curve for Repaglinide in acid buffer (pH 1.2),

and alkaline buffer (pH 7.2) and in 0.5 N HCl solution.

• 0.5 N Hydrochloric acid :

Dilute 42.5 ml of Hydrochloric acid in 1000ml of distilled water.

• Acid buffer ( pH 1.2 ) :

Place 50 ml of 0.2 M Potassium chloride in a 200 ml of volumetric flask, add 85 ml

of 0.2 M Hydrochloric acid and then add water to volume.

• Phosphate buffer ( pH 7.2 ) :

Place 50 ml of 0.2 M Potassium Dihydrogen Phosphate in a 200 ml of 0.2 M

sodium hydroxide and then add water to volume.

• 0.2 M Potassium chloride solution :

Dissolve 14.911 gm of Potassium Chloride in water and dilute with water to

1000ml.



40

• 0.2 M Hydrochloric acid :

Dilute 17 ml of hydrochloric acid in 1000ml of distilled water.

• 0.2 M Potassium Dihydrogen Phosphate :

Dissolve 27.218gm of Potassium Dihydrogen Phosphate in water and dilute with

water to 1000ml.

• 0.2 M Sodium Hydroxide solution 31:

Dissolve sodium hydroxide in water to produce 40% to 60% w/v solution and allow

to stand. Taking precautions to avoid absorption of carbon dioxide, siphone off the

clear supernatant liquid and dilute with carbon dioxide free water a suitable volume

liquid to contain 8gm of sodium hydroxide in 1000 ml.

Formulation and preparation of microparticles

Formulation and preparing microparticles of Repaglinide using polymers Hydroxy

Propyl Methyl Cellulose by using Ionotropic Gelation Technique.

• Sodium alginate solution ( 2% ) :

Accurately weighed amount of sodium alginate (2 gm) mixed in the 100 ml of

distilled water.

• Calcium chloride solution ( 4% ) :

Accurately weighed amount of calcium chloride (2 gm) mixed in the 100 ml of

distilled water.



41

• Hydroxy Propyl Methyl Cellulose ( 1% ) :

Accurately weighed amount of Hydroxy Propyl Methyl Cellulose (1 gm) mixed in

the 100 ml of cold water.

• Hydroxy Propyl Methyl Cellulose (1.5%) :

Accurately weighed amount of Hydroxy Propyl Methyl Cellulose (1.5 gm) mixed

in the 100 ml of cold water.

• Hydroxy Propyl Methyl Cellulose ( 2%) :

Accurately weighed amount of Hydroxy Propyl Methyl Cellulose (2 gm) mixed in

the 100 ml of cold water.

• Chitosan ( 1%) :

Accurately weighed amount of Chitosan (1 gm) mixed in the 100 ml of 1% Acetic

acid solution.

• Chitosan (1.5%) :

Accurately weighed amount of Chitosan (1.5 gm) mixed in the 100 ml of 1%

Acetic acid solution.

• Chitosan (2%) :

Accurately weighed amount of Chitosan (2 gm) mixed in the 100 ml of 1% Acetic

acid solution.



42

METHODS :

1) Preformulation study13 :

Prior to the development of the dosage forms the preformulation study was carried

out. Hence Infrared spectra of the physical mixture of the drug and the polymers chosen

were taken. The infra-red spectra of the drug57 and polymers were also taken.

The application of infra-red spectroscopy lies more in the qualitative identification

of substances either in pure form or in the mixtures and as a tool in establishment of the

structure. Since I.R. is related to covalent bonds, the spectra can provide detailed

information about the structure of molecular compounds. In order to establish this point,

comparisons can be made between the spectrum of the substance and the drug.

The above discussions imply that infra-red data is helpful to confirm the identity of

the drug and to detect the interaction of the drug with the carriers.

2) Standard Plot for Repaglinide48 :

a) Standard Graph by using 0.5N HCl :

Accurately weighed 10 mg of Repaglinide was dissolved in 100 ml of 0.5 N HCl

buffer solution to form 100 µg/ml stock solution.

From this stock solution aliquots of 2.5 ml, 5 ml, 7.5 ml, 10 ml, 12.5 ml, 15 ml,

17.5 ml, 20 ml, 22.5 ml, 25 ml, were pipetted out into a series of 50 ml volumetric

flask and volume was made up to 50 ml in order to get a concentration ranging from

5-50µg/ml.



43

The absorbance of the resulting solution was then measured at 247nm using UV

spectrophotometer against respective parent solvent as a blank. The standard curve was

obtained by plotting absorbance V/s. concentration in µg/ml.

b) Phosphate Buffer (pH 7.2):

Accurately weighed 10 mg of Repaglinide was dissolved in 100 ml of 7.2 pH

buffer solution to form 100 µg/ml stock solution.

From this stock solution aliquots of 2.5 ml, 5 ml, 7.5 ml, 10 ml, 12.5 ml, 15 ml,

17.5 ml, 20 ml, 22.5 ml, 25 ml, were pipetted out into a series of 50 ml volumetric

flask and volume was made up to 50 ml in order to get a concentration ranging from

5-50µg/ml.

The absorbance of the resulting solution was then measured at 247nm using UV

spectrophotometer against respective parent solvent as a blank. The standard curve was

obtained by plotting absorbance V/s. concentration in µg/ml.

c) Acid Buffer (pH 1.2):

The above procedure was followed but instead of (pH7.2), phosphate buffer (pH

1.2) was used.

Preparation of Microparticles28:

In the present study, microparticles of Repaglinide were prepared by ionotropic

gelation technique. In this method weighed quantity of Repaglinide was added to 50 ml

sodium alginate solution and thoroughly mixed with a stirrer at 500 rpm. For the



44

formation of microparticles, 50 ml of this solution was extruded dropwise from a needle

into 100 ml aqueous calcium chloride solution and stirred at 100 rpm. After stirring for

10 minutes the obtained microparticles were washed with water and dried at 700 C for 2

hrs in an oven.

Three sets of microparticles were prepared. In the first set microparticles of

repaglinide were prepared using only hydroxyl propyl methyl cellulose in different

concentrations.

In the second set, microparticles of the drug were prepared by using only chitosan

in a different concentrations.

In the third set, microparticles of the drug were prepared in a combination of

polymers like hydroxyl propyl methyl cellulose and chitosan.

Sodium alginate + Drug + Polymer

Calcium chloride

Microparticles



45

Table No. 5

Formulation Design of Mcroparticles

Formulation

No.

Sodium

Alginate

(W/V)

Calcium

Chloride

(W/V)

HPMC

(W/V)

Chitosan

(W/V)

Drug

(Repaglinide)

in mg

F1

2 %

4 %

1 %

---

8 mg

F2

2 %

4 %

1.5 %

---

8 mg

F3

2 %

4 %

2 %

---

8 mg

F4

2 %

4 %

---

1 %

8 mg

F5

2 %

4 %

---

1.5 %

8 mg

F6

2 %

4 %

---

2 %

8 mg

F7

2 %

4 %

1 %

1 %

8 mg



46

EVALUATION PARAMETERS :

Particle size determination49, 50 :

The particle size of a pharmaceutical substance is strictly maintained in order to get

optimal biological activity.

Methods to estimate particle size are :

a. Optical Microscopy

b. Sieving Method

c. Sedimentation Method

d. Elutriation Method

e. Centrifugal defractometry

f. Permeability Method

g. Light scattering Method

Table No.6

Common techniques for measuring fine particles of various sizes

No. Technique Particles sizes in (µm)

1. Optical Microscopy 1-100 µm

2. Sieving >50 µm

3. Sedimentation >1 µm

4. Elutriation 1-50 µm

5. Centrifugal <50 µm

6. Permeability >1 µm

7. Light scattering 0.5-50 µm



47

Sieving Method :

Particles having size range between 50 and 1500 µm are estimated by sieving

method. In this method the size is expressed as dsieve , which describes the diameter of a

sphere that passes through the sieve aperture as the assymmetric particle. This method

directly gives weight distribution. The sieving method finds application in dosage form

development of tablets and capsules.

Sieves for pharmaceutical testing are constructed from wire cloth with square

meshes, woven from wire of brass, bronze, stainless steel or any other suitable material.

Standard sieves and their dimensions as per IP are given as follows :



48

Table No.7

Designations and Dimensions of IP Specification Sieves

Sieve No. Aperture size micrometer

10

12

16

22

25

30

36

44

60

85

100

120

150

170

1700

1400

1000

710

600

500

425

325

250

35

36

34

36

35

Method:

Standard sieves of different mesh numbers are available commercially as per the

specifications of IP and USP. Sieves are arranged in a nest with the coarsest at the top. A

sample (50gms) of the powder is placed on the top sieves. This sieve set is fixed to the

mechanical shaker apparatus and shaken for a certain period of time (20 minutes).

The powder retained on each sieve is weighed. Frequently, the powder is assigned the

mesh number of the screen through which it passes or on which it is retained. It is



49

expressed in terms of arithmetic or geometric mean of the two sieves.

Flow Properties49, 50:

Irregular flow of powder from the hopper produces tablets and capsules with

nonuniform weights. Flow property depends on particle size, shape, porosity and density

of the powder.

Angle of Repose :

The flow characteristics are measured by angle of repose. Improper flow is due to

frictional forces between the particles. These forces are quantified by angle of repose.

Angle of repose is defined as the maximum angle possible between the surface of the pile

of the powder and the horizontal plane. The flow of powder and the angle of repose is

depicted in following fig. By definition :

tan θ = h / r

θ = tan-1 (h / r)

Where, h = height of pile

r = radius of the base of the pile

θ = angle of repose

The lower the angle of repose, the better the flow property. Rough and irregular

surface of particles gives higher angle of repose. Decreased in the particle size leads to a

higher angle of repose.



50

Method :

A glass funnel is held in place with a clamp on a ring support over a glass plate.

The glass plate is placed on a stand. Approximately 100 g of particles is transfered into

funnel keeping the orifice of the funnel blocked by the lower thumb. As the thumb is

removed, the particles are emptied from funnel, and the angle of repose is determined by

above mentioned formula.

Table No.8

Relation Between Angle of Repose and Flow of the Particles

Angle of repose (θ)

(degrees)

Flow

<25

25-30

30-40

>40

Excellent

Good

Passable

Very poor

Drug Entrapment Efficiency28, 56 :

Drug entrapment efficiency of repaglinide was performed by accurately weighing

100 mg of microparticles and suspended in 100 ml of simulated intestinal fluid of pH

7.2±0.1 and it was kept for 24 hrs. Next day it was stirred for 15 mins, and subjected for

filtration. After suitable dilution, Repaglinide content in the filtrate was analyzed

spectrophotometrically at 247 nm using Shimadzu 1201 UV-visible spectrophotometer.

The absorbance found from the UV-spectrophotometer was plotted on the standard



51

curve to get the concentration of the entrapped drug. Calculating this concentration with

the dilution factor we get the percentage drug encapsulated in microparticles.

In-vitro Dissolution Studies :

A drug is expected to release from the solid dosage forms (granules, tablets,

capsules etc) and immediately go into molecular solution. This process is called as

Dissolution50.

Drug Release Studies :

The method specified in USP for the drug release study was followed.

Apparatus :

USP XXIII dissolution test apparatus employing the round bottom dissolution

vessel and rotating basket assembly.

Acid Stage :

900 ml of simulated gastric fluid TS (acid buffer, pH 1.2 without enzymes).

Buffer stage :

900 ml of pH 7.2 and intestinal fluid Ts (phosphate buffer).

Time :

Acid stage : pH : 1.2 -2 hrs.

Buffer stage : pH :7.2: 6 hrs.



52

Procedure :

In-vitro release profile of the microparticles was evaluated using rotating basket

dissolution apparatus. 900 ml of acid buffer (pH1.2), and phosphate buffer (pH7.2)

maintained at 37±0.5 0C were used as dissolution medias respectively, and the basket was

rotated at a constant speed of 50 rpm. Accurately weighed amount of microparticles

equivalent to 200 mg of drug were placed in the baskets.

Aliquotes of samples were withdrawn at the interval of 1 hour for pH 1.2 and 2 hrs

for 7.2 pH. The samples withdrawn were filtered, diluted suitably and analyzed at 247 nm

spectrophotometrically for drug release51.

Kinetic treatment :

The data obtained from the in-vitro dissolution studies was subjected for kinetic

treatment to obtain the order of release and best fit model for the formulations by using

PCP-Disso-V2 software.

Scanning electron microscopy:

Procedure :

Morphology details of the specimens were determined by using a scanning electron

microscope (SEM), Model JSM 35CF, JEOL, Japan.

The samples were dried thoroughly in vacuum desicator before mounting on brass

specimen studies. The samples were mounted on specimen studies using double sided

adhesive tape, and gold-palladium alloy of 120Ao kness was coated on the sample using



53

sputter coating unit (Model E5 100 Polaron U.K.) in an Argon ambient of 8-10 pascal

with plasma voltage about 20 MA. The sputtering was done for nearly 3 minutes to

obtain uniform coating on the sample to enable good quality SEM images. The SEM was

operated at low accelerating voltage of about 15 KV with load current of about 80 MA.

The condenser lens position was maintained between 4.4--5.1. The objectives lens

aperture has a diameter of 240 microns and the working distance WD = 39 mm52, 53.

Accelerated Stability Studies:

The formulations were stored in a oven at 37 ± 10C and 60 ± 10C for a period of six

weeks. The samples were analyzed for drug content every week by spectrophotometer at

247 nm54, 55.


Chapter V RESULTS AND DISCUSSION

Dept. of Pharmaceutics KLESCOP, HUBLI

53



54

Results and Discussion:

Microparticles of repaglinide were prepared by ionotropic gelation technique and

various evaluation parameters were assessed, with a view to obtain oral controlled release

of Repaglinide.

In the present work, total seven formulations were prepared and the detailed

composition is shown in Table No. 5. The prepared microparticles were then subjected to

granulometric study, angle of repose, scanning electron microscopy, drug entrapment

efficiency, in-vitro dissolution and stability studies.

1. Standard curve of Repaglinide:

A standard calibration curve for the drug was obtained by measuring absorbance at

247 nm, and by plotting the graph of absorbance V/s concentration. Table No.9 shows the

absorbance readings of repaglinide in triplicate 0.5 N HCl, pH 1.2, pH 7.2 between 5-50

µg/ml concentrations. The standard plots of repaglinide are shown in graph No.1, 2, and 3.

Estimation of drug content and in-vitro drug release studies are based on this

standard curve.

2. Preformulation Study:

To check the compatibility of the drug with various polymers, IR spectra of drugs,

polymers and combination of the drug and polymers were taken. The IR spectra of the

drug, polymers and their combinations are shown in Spectra No.1 to 5.

The characteristics absorption peaks of repaglinide were obtained at1687.3cm-1,

2935.03 cm-1, 1217.12 cm-1, and 3308.38cm-1. The IR spectras of the drug and polymer



55

combinations were compared with the spectra of pure drug and individual polymers. The

principle peaks obtained for the combinations were almost similar to that of the drug.

The details of IR spectra are mentioned in Table No.10.

The IR spectra of the Drug-HPMC, Drug –chitosan, and Drug-Sodium alginate, did

not show any changes. The possibility of interaction was ruled out as there was no major

shift in the absorption bands of drug and the formulations as shown in Spectra No.1 to 5.

Evaluation parameters:

Granulation study:

In the granulometric study it is observed from the Table No.11 that about 64 % to

90 % and 40 % to 55 % percent of microparticles were of 16 and 20 mesh size, which

proves the flexibility of the method. It is observed that, with the increase in the

concentration of HPMC maximum amount of microparticles of desired size were

obtained and with the increase in the concentration of chitosan the distribution of particle

size shifts to the higher sieve size due to increase in viscosity of the medium.

Flow Property:

The flow property of the prepared formulations was checked by the method, angle

of repose. Acceptable range of angle of repose is 22061' to 31060'. All the formulations

showed an angle of repose within the range as shown in Table No.12.

Formulations F1 to F7 showed an angle of repose in the acceptable range, which

indicates a good flow property.



56

Drug Entrapment Efficiency:

The drug entrapment efficiency of all the formulations were in the range between

78.62 % to 91.25 %. The results of drug entrapment efficiency are shown in Table No.13.

Drug entrapment efficiency of microparticles increases with increase in

concentration of HPMC and chitosan.

In-vitro Dissolution Studies:

Dissolution studies of all the formulations were carried out using dissolution tester

USP XXIII.

The dissolution studies were conducted by using two different dissolution medias,

PH 1.2 and PH 7.2.

The data obtained in the in-vitro dissolution studies were grouped according to

modes of data treatment as follows :-

Cumulative percent drug release V/s. Time (Zero-order).

Cumulative percent drug retained V/s. Square root of Time (Higuchi Matrix Model).

Log Cumulative percent drug retained V/s. Time (First-order).

Cumulative percent drug release in (mg) V/s. Time (Krosmeyer-Peppas Model).

The results of the in-vitro dissolution studies of formulations F1 to F7 are shown in

table 14 to 20. The plots of Cumulative percentage drug release V/s. Time, Cumulative

percent drug retained V/s. Time, Log Cumulative percent drug retained V/s. Time and

Cumulative percent drug release in (mg) V/s. Time were drawn and represented

graphically as shown in Graph No. 4 to 15, respectively.

The formulation F1, F2 and F3 Containing 1%, 1.5%, and 2% Hydroxy Propyl

Methyl Cellulose respectively showed a release of 91.99%, 81.66% and 71.66% after 12



57

hours. This shows that more sustained release was observed with the increase in

percentage of Hydroxy Propyl Methyl Cellulose.

The formulation F4, F5 and F6 Containing 1%, 1.5%, and 2% Chitosan respectively

showed a release of 92.11%, 81.93% and 81.79% after 12 hours. This shows that more

sustained release was observed with the increase in percentage of Chitosan.

The formulation F7 containing both 1% HPMC and 1% chitosan showed a release

of 71.88%. This shows that the particles formulated with HPMC and Chitosan prolongs

the release but without satisfactory surface characteristics.

The formulations F1, F2 and F3 containing 1%, 1.5%, and 2% Hydroxy Propyl

Methyl Cellulose respectively showed a release that more sustained release was observed

with the increase in percentage of HPMC after 12 hours. This indicates that the release

rate is further retarded due to addition and in percentage of Hydroxy Propyl Methyl

Cellulose because of the strong bonds between the HPMC and sodium alginate. As the

percentage of HPMC increased the release was further sustained.

The formulations F4, F5 and F6 containing 1%, 1.5%, and 2% chitosan respectively

showed a release that more sustained release was observed with the increase in

percentage of Chitosan after 12 hours. This indicates that the release rate is further

retarded due to addition and in percentage of chitosan because of the strong bonds

between the chitosan and sodium alginate. As the percentage of chitosan increased the

release was further sustained.

Further these drug releases were subjected for mathematical treatment to check

weather the release is following first order or zero-order kinetics.

The co-efficient of correlation values are shown in table No.13



58

The values of co-efficient of correlation were found to be best fitted to Krosmeyer-

Peppas model and Higuchi model.

The calculated values of various kinetic models are shown in table No. 22.

The values of diffusion co-efficient (n) for formulations F1 to F7 are shown to be

0.453, 0.499, 0.547, 0.418, 0.515, 0.469, 0.596 respectively which indicates that the

release of drug occurs by diffusion following Fickian transport and Anomalous

mechanism.

Scanning Electron Microscopy:

Morphology of the microparticles were investigated by Scanning electron

microscopy. The photographs of formulations taken by scanning electron microscope

are shown in the figure No.1 and 2.

Microparticles of formulation F1 were approximately spherical and their surface

was rough giving them a sandy appearance.

The surface characteristics observed for Formulation F4 shows oval or slightly

spherical structure with smooth appearance.

From the photographic observation it can be stated that bridging and dense nature

of formulation indicated to retard the release of Repaglinide.

Stability study :

Among the seven formulations prepared F1 and F4 were used, which showed the

best release from in-vitro dissolution data selected for stability studies.

Stability study was carried out for the formulations F1 and F4 at 400C ± 10C RH

for a period of 45 days. The samples were analyzed for drug content at different time

intervals, and it is evident that there were slight changes in the content of drug as shown



59

in Table No.23. This indicates that the formulations F1 and F4 were stable for a period of

45 days at the above mentioned temperature.

Table No. 9

Standard Calibration Curves of Repaglinide

Sl. No. Concentration (mcg/ml) Absorbance 0.5 N HCl pH 1.2 pH 7.2

1

5 0.1549 0.0969 0.0763

2

10 0.2092 0.1859 0.1396

3

15 0.3607 0.2913 0.2156

4

20 0.4030 0.3843 0.2663

5

25 0.5333 0.4700 0.3303

6

30 0.6394 0.5732 0.4049

7

35 0.7032 0.6664 0.4725

8

40 0.8100 0.7612 0.5309

9

45 0.8934 0.8964 0.6022

10

50 0.9978 0.9469 0.6618



60

Table No.10

Comparison of I.R. Spectra of Repaglinide and in Combination with Polymers

Sl. No.

System C=O (cm-1) C – H (cm-1) --CH3 (cm-1) N-H (cm-1)

1.

Repaglinide (RPG)

1687.36

2935.03

1217.12

3308.38

2.

RPG-HPMC

1687.39

2934.99

1217.75

3308.74

3.

RPG-Chitosan

1687.44

2934.86

1217.78

3308.49

4.

RPG-Sodium alginate

1688.39

2935.91

1217.07

3308.82

5.

RPG- HPMC- Chitosan- SA

1698.39

2935.91

1217.07

3308.82

Table No. 11

Percentage weight remained on various sieve size

Batch

No.

# 16

(1.19 mm) 840-1190 µm

# 20

(0.84 mm) 590-840 µm

# 25

(0.600 mm) 600 µm

# 30

(0.59 mm) 297-590 µm

F1

F2

F3

F4

F5

F6

F7

0.64

0.68

0.81

0.90

0.10

0.03

0.25

0.21

0.28

0.17

0.06

0.40

0.04

0.55

0.08

0.03

0.01

0.04

0.26

0.86

0.12

0.07

0.01

0.01

0.0

0.24

0.07

0.08



61

Table No. 12

Angle of Repose of Microparticles

Sl. No.

Formulations

Angle of Repose

1.

F1

25070'

2.

F2

28029'

3.

F3

29074'

4.

F4

31060'

5.

F5

29024'

6.

F6

30096'

7.

F7

22061'

Table No. 13

Drug entrapment Efficiency of Microparticles

Formulations

Absorbance

at 247nm

Theoretical

content (mg)

Actual content

(mg)

% Drug

Entrapment Efficiency

F1

0.0521

8

6.29

78.62

F2

0.0569

8

6.87

85.87

F3

0.0601

8

7.26

90.75

F4

0.0549

8

6.63

80.15

F5

0.0591

8

7.14

86.35

F6

0.0604

8

7.30

91.25

F7

0.0537

8

6.49

81.12

Table No. 14.

In-vitro Dissolution Profile for Formulation F1

me

Absorbance

Conc. in mcg/ml

Conc. In

10 ml

Conc. in 900 ml

CLA (mg)

Cum. Drug

Release (mg)

Cum.

%Drug released

Cum. %

drug Retained

Log Cum. % Drug Retained

1

0.0132

0.5

0.005

0.45

-------

0.45

10.00

90.00

1.9542

2

0.0241

1.0

0.01

0.9

0.005

0.905

20.11

79.89

1.9024

4

0.0150

1.0

0.01

0.9

0.015

1.820

40.44

59.56

1.7749

6

0.0212

1.5

0.015

1.35

0.025

2.280

50.66

49.34

1.6931

8

0.0310

2.0

0.02

1.8

0.040

2.745

60.99

39.01

1.5911

0

0.0391

2.5

0.025

2.25

0.060

3.215

71.44

28.56

1.4557

2

0.0490

3.5

0.035

3.15

0.085

4.140

91.99

8.01

0.9036

Table No. 15.


me

Absorbance

Conc. in mcg/ml

Conc. In

10 ml

Conc. In 900 ml

CLA (mg)

Cum. Drug

Release (mg)

Cum.

%Drug released

Cum. %

drug Retained


1

0.0175

0.5

0.005

0.45

-------

0.45

10.00

90.00

1.9542

2

0.0301

1.5

0.015

1.35

0.005

1.355

30.11

69.89

1.8441

4

0.0137

0.5

0.005

0.45

0.020

1.825

40.55

59.45

1.7741

6

0.0163

1.0

0.010

0.9

0.025

2.280

50.66

49.34

1.6931

8

0.0221

1.5

0.015

1.35

0.035

2.740

60.88

39.12

1.5923

0

0.0320

2.0

0.02

1.8

0.050

3.205

71.22

28.78

1.4590

2

0.0359

2.5

0.025

2.25

0.070

3.675

81.66

18.34

1.2633

Table No. 16.


me

Absorbance

Conc. in mcg/ml

Conc. In

10 ml

Conc. in 900 ml

CLA (mg)

Cum. Drug

Release (mg)

Cum.

%Drug released

Cum. %

drug Retained


1

0.0197

0.5

0.005

0.45

--------

0.45

10.00

90.00

1.9542

2

0.0241

1.0

0.01

0.9

0.005

0.91

20.22

79.78

1.9018

4

0.0116

0.5

0.005

0.45

0.015

1.375

30.55

69.45

1.8416

6

0.0173

1.0

0.010

0.9

0.020

1.830

46.66

53.34

1.7270

8

0.0232

1.5

0.015

1.35

0.030

2.290

50.88

49.12

1.6912

0

0.0301

2.0

0.02

1.8

0.045

2.755

61.22

38.78

1.5886

2

0.0366

2.5

0.025

2.25

0.065

3.255

71.66

28.34

1.4523

Table No. 17.


me

Absorbance

Conc. in mcg/ml

Conc. In

10 ml

Conc. in 900 ml

CLA (mg)

Cum. Drug

Release (mg)

Cum.

%Drug released

Cum. %

drug Retained


1

0.0174

0.5

0.005

0.45

--------

0.45

10.00

90.00

1.9542

2

0.0296

1.0

0.01

0.9

0.005

0.905

20.11

79.89

1.9024

4

0.0121

0.5

0.005

0.45

0.015

1.370

30.44

69.56

1.8423

6

0.0213

1.5

0.015

1.35

0.020

2.275

50.55

49.45

1.6941

8

0.0380

2.4

0.025

2.25

0.035

3.185

70.77

29.23

1.4658

0

0.0408

3.0

0.03

2.7

0.060

3.665

84.44

15.66

1.1947

2

0.0484

3.5

0.035

3.15

0.090

4.145

92.11

7.89

0.8970

Table No. 18.


me

Absorbance

Conc. in mcg/ml

Conc. In

10 ml

Conc. In 900 ml

CLA (mg)

Cum. Drug

Release (mg)

Cum.

%Drug released

Cum. %

drug Retained


1

0.0253

1.0

0.001

0.9

--------

0.9

20.00

80.00

1.9030

2

0.0306

1.5

0.015

1.35

0.001

1.351

30.02

69.98

1.8449

4

0.0201

1.5

0.015

1.35

0.016

2.717

60.37

39.63

1.5980

6

0.0249

1.5

0.015

1.35

0.031

2.732

60.70

39.30

1.5943

8

0.0289

2.0

0.02

1.8

0.046

3.197

71.04

28.96

1.4617

0

0.0309

2.0

0.02

1.8

0.066

3.217

71.48

28.52

1.4551

2

0.0371

2.5

0.025

2.25

0.086

3.687

81.93

18.07

1.2569

Table No. 19.


me

Absorbance

Conc. in mcg/ml

Conc. In

10 ml

Conc. in 900 ml

CLA (mg)

Cum. Drug

Release (mg)

Cum.

%Drug released

Cum. %

drug Retained


1

0.0163

0.5

0.005

0.45

--------

0.45

10.00

90.00

1.9542

2

0.0279

1.0

0.010

0.9

0.005

0.901

20.02

79.98

1.9029

4

0.0108

0.5

0.005

0.45

0.015

1.501

33.35

66.65

1.8238

6

0.0215

1.5

0.015

1.35

0.020

2.271

50.46

49.54

1.6949

8

0.0305

2.0

0.02

1.8

0.035

3.096

68.79

31.21

1.4942

0

0.0352

2.5

0.025

2.25

0.055

3.206

71.24

28.76

1.4587

2

0.0409

3.0

0.030

2.7

0.080

3.681

81.79

18.21

1.2603

Table No. 20.


me

Absorbance

Conc. in mcg/ml

Conc. In

10 ml

Conc. in 900 ml

CLA (mg)

Cum. Drug

Release (mg)

Cum.

%Drug released

Cum. %

drug Retained


1

0.0212

1.0

0.010

0.9

--------

0.9

20.00

80.00

1.9030

2

0.0356

1.5

0.015

1.35

0.010

1.360

30.22

69.78

1.8437

4

0.0119

0.5

0.05

0.45

0.025

1.835

40.77

59.23

1.7725

6

0.0169

1.0

0.01

0.9

0.030

2.26

50.88

49.12

1.6912

8

0.0219

1.5

0.015

1.35

0.040

2.75

61.10

38.9

1.5899

0

0.0257

2.0

0.02

1.8

0.055

3.215

71.44

28.56

1.4557

2

0.0279

2.0

0.02

1.8

0.075

3.235

71.88

28.12

1.4490



69

Table No.21

Values of Correlation-coefficient (r) of Repaglinide

Formulations

Zero Order

First Order

F1

0.9757

0.8576

F2

0.9202

0.9835

F3

0.9566

0.9835

F4

0.9884

0.9473

F5

0.7267

0.9345

F6

0.9648

0.981

F7

0.8167

0.9811

Table No. 22

Curve Fitting Data of the Release Profile for Repaglinide Formulations

Matrix

Krosmeyer-

Peppas

n-values

Mechanism

F1

0.9521

0.9841

0.453

Fickian

F2

0.9768

0.9575

0.499

Fickian

F3

0.9702

0.9878

0.547

Anomalous

F4

0.9326

0.9912

0.418

Fickian

F5

0.9636

0.8575

0.515

Anomalous

F6

0.9563

0.9750

0.469

Fickian

F7

0.9943

0.9267

0.596

Anomalous



70

Table No. 23

Results of assay of Formulations F1 & F4 after Accelerated Stability Studies

Days

F1 370C 600C

F4 370C 600C

1

83.12 82.06

84.89 82.21

7

81.09 79.05

83.67 81.85

14

78.98 77.77

79.54 80.77

21

77.86 76.84

78.45 79.99

38

76.04 75.29

77.43 78.35

45

75.24 72.40

76.01 77.04

Spectra No. 1

Repaglinide

761.

75

861.

7591

8.96

1040

.15

1091

.02

1149

.52

1174

.82

1217

.12

1299

.73

1340

.4513

83.6

0

1448

.42

1490

.93

1567

.55

1608

.46

1637

.3516

87.3

6

2805

.34

2852

.71

2866

.84

2935

.03

3308

.38 76

1.75

861.

7591

8.96

1040

.15

1091

.02

1149

.52

1174

.82

1217

.12

1299

.73

1340

.4513

83.6

0

1448

.42

1490

.93

1567

.55

1608

.46

1637

.3516

87.3

6

2805

.34

2852

.71

2866

.84

2935

.03

3308

.38

-10

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

% T

rans

mitt

ance

Wavenumber cm-1

Spectra No. 2

Repaglinide + HPMC

761.

92

1040

.23

1090

.94

1149

.63

1216

.75

1300

.00

1383

.2714

48.7

114

90.7

9

1567

.63

1608

.85

1637

.58

1687

.39

2806

.86

2867

.49

2934

.99

3308

.74

761.

92

1040

.23

1090

.94

1149

.63

1216

.75

1300

.00

1383

.2714

48.7

114

90.7

9

1567

.63

1608

.85

1637

.58

1687

.39

2806

.86

2867

.49

2934

.99

3308

.74

-10

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

% T

rans

mitt

ance

Wavenumber cm-1

Spectra No. 3

Repaglinide + Chitosan

762.

79

1039

.42

1090

.93

1112

.15

1150

.14

1217

.7812

99.5

213

83.4

91436

.65

1490

.92

1567

.74

1609

.81

1638

.23

1687

.44

1723

.1328

05.3

728

64.0

2

2934

.86

3068

.37

3308

.49

-15

-10

-5

0

5

10

15

20

25

30

35

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

% T

rans

mitt

ance

Wavenumber cm-1

Spectra No. 4

Repaglinide + Sodium Alginate

541.

62

647.

5969

3.77

762.

65

863.

3494

0.49

1037

.61

1090

.22

1149

.18

1217

.07

1297

.85

1342

.30

1434

.15

1492

.58

1566

.44

1637

.56

1688

.39

2363

.83

2804

.70

2935

.91

3308

.82

10

15

20

25

30

35

40

45

50

55

60

65

%T

500 1000 1500 2000 2500 3000 3500 4000 Wa

% T

rans

mitt

ance

venumbers (cm-1)Wavenumber cm-1

Spectra No. 5

Repaglinide + HPMC + Chitosan + Sodium Alginate

541.

62

647.

5969

3.77

762.

65

863.

3494

0.49

1037

.61

1090

.22

1149

.18

1217

.07

1297

.85

1342

.30

1434

.15

1492

.58

1566

.44

1637

.56

1688

.39

2363

.83

2804

.70

2935

.91

3308

.82

10

15

20

25

30

35

40

45

50

55

60

65

%T

500 1000 1500 2000 2500 3000 3500 4000 Wavenumbers (cm-1)

% T

rans

mitt

ance

Wavenumber cm-1

Figure No. 1

SEM of Formulation F1 Under Low Magnification

Figure No. 2

SEM of Formulation F4 Under Low Magnification

Graph No. 1

Standard Calibration Curve By 0.5 N HCl

Graph No. 2

Standard Cal y 1.2 pH

Chart Title

y = 0.0204xR2 = 0.9919

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20

Concentrat

Abs

orba

nce

ibration Curve B

30 40 50 60

ion in mcg/ml

Calibration Curve by0.5N HCL

Linear (Calibration Curveby 0.5N HCL)

Calibration curve by 0.5 N

HCl

Linear Calibration curve by

0.5 N HCl

Graph No. 2

Standard Calibration Curve By 1.2 PH

Chart Title

y = 0.0192xR2 = 0.9986

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 30 40 50 60

Concentration in mcg/ml

Abs

orba

nce

Calibration Curve bypH 1.2

Linear (CalibrationCurve by pH 1.2)

Graph No. 3

Standard Calibration Curve By 7.2 pH

Chart Title

y = 0.0134xR2 = 0.999

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30 40 50 60

Concentration in mcg/ml

Abs

orba

nce

Calibration Curve by 7.2pH

Linear (Calibration Curveby 7.2 pH)

Graph No. 4

% Cumulative Drug Release Vs Time

% CDR Vs Time

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

Time in Hrs.

% C

DR

F1F2F3

Graph No. 5


% CDR Vs Time

0

10

20

30

40

50

60

70

80

90

0 2 4 6 8 10 12 14

Time in Hrs.

% C

DR

F4F5

Graph No. 6


% CDR Vs Time in Hrs

0

10

20

30

40

50

60

70

80

90

0 2 4 6 8 10 12 14

Time in Hrs.

% C

DR

F6F7

Graph No. 7

% Cumulative Drug Retained Vs Time

% CDRetained Vs Time

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

Time in Hrs.

% C

DRet

aine

d

F1F2F3

Graph No. 8



0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

Time in Hrs

% C

DRet

aine

d

F4F5

Graph No. 9



0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

Time in Hrs

%C

DRet

aine

d

F6F7

Graph No. 10

Cumulative Drug Release Vs Square root of Time of Formulation F1

F1

0

10

20

30

40

50

60

70

80

90

100

0 0.5 1 1.5 2 2.5 3 3.5 4

Square root of Time

% C

DR

F1

Graph No. 11

Cumulative Drug Release Vs Square root of Time of Formulation F4

F4

0

10

20

30

40

50

60

70

80

90

100

0 0.5 1 1.5 2 2.5 3 3.5 4

Square of Time

% C

DR

F4

Graph No. 12

Cumulative Drug Release Vs Time for F1 formulation

F1

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 2 4 6 8 10 12 14

Time in Hrs

CDR

in (m

g)

F1

Graph No. 13

Cumulative Drug Release Vs Time for F4 formulation

F4

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 2 4 6 8 10 12 14

Time in Hrs

CDR

in (m

g)

F4

Graph No.14

Krosmeyer-Peppas Model

Krosmeyer-Peppas model

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 2 4 6 8 10 12 14

Time in Hrs.

CDR

in m

g

F1F2F3F4F5F6F7

Graph No. 15

Higuchi-Matrix Model

Higuchi-Matrix Model

0

10

20

30

40

50

60

70

80

90

100

0 0.5 1 1.5 2 2.5 3 3.5 4

Time in Hrs.

% C

DR

F1F2F3F4F5F6F7

Chapter VI SUMMARY

Dept. of Pharmaceutics KLESCOP HUBLI

71

SUMMARY

• The goal of any drug delivery system is to provide a therapeutic amount of drug

to the proper site in the body and also to achieve and maintain the desired drug

concentration.

• Microencapsulation is a process whereby small discrete solid particles or small

liquid droplets are surrounded or enclosed, by an intact shell.

• Repaglinide is an antidiabetic agent used in the treatment of type II diabetes

mellitus. The plasma half life of Repaglinide is about one hour.

• An attempt is made to microencapsulate repaglinide by Ionotropic Gelation

Technique with a view to prevent the gastric side effects and to achieve an oral

controlled release of the drug.

• The literature survey discussed reveals the research work done to develop

microparticulates drug delivery system containing anti-diabetic, anti-hypertensive

and NSAID’s drugs by ionotropic gelation technique.

• Polymers used in this technique are Hydroxy Propyl Methyl Cellulose and

Chitosan, calcium chloride as a crosslinking agent, and sodium alginate as a

viscosity builder.

• In the present study seven formulations were formulated by using Hydroxy Propyl

Methyl Cellulose and Chitosan in various proportions.

• All the formulations were subjected for evaluation. Results of preformulation

studies, granulometric study, angle of repose, entrapment efficiency, in-vitro

dissolution study have shown satisfactory results.

Chapter VI SUMMARY


72

• The in-vitro release study of formulations F1 to F7 shows a retarded release with

increase percentage of Hydroxy Propyl Methyl Cellulose and Chitosan.

• On the basis of release data and graphical analysis formulation F1 and F4 showed

a good controlled release profile with maximum entrapment efficiency.

• The release kinetics of formulations F1 and F4 shows a good correlation of

Krosmeyer-Peppas with a good ‘n’ values. According to ‘n’ values obtained F1

and F4 follows Fickian diffusion as release mechanism.

• The surface study of F1 and F4 viewed through SEM shows an uniform matrix

formulation with dense nature and low porosity.

• The formulation were subjected to accelerated stability study. Both the

formulation were found to be stable as there was no change in the drug content.

Chapter VII CONCLUSION


73

CONCLUSION

From the above experimental results it can be concluded that :-

• Oral controlled release of Repaglinide can be achieved by ionotropic gelation

technique using HPMC and Chitosan as a polymer.

• The IR spectras revealed that, there was no interaction between polymers and

drug. All the polymers used were compatible with the drug.

• Prepared microparticles exhibited Krosmeyer-Peppas kinetics/Higuchi model and

the release profile was by Fickian and Anomalous.

• From the study it is evident that a promising controlled release microparticulate

drug delivery of repaglinide can be developed. Further in-vivo investigation is

required to establish efficacy of these formulations.

• The study also indicated that the amount of drug release decreases with an

increase in the polymer concentration.

• Microparticles formulated with a combination of HPMC and Chitosan prolonged

the release but gave unsatisfactory kinetic results with low correlation coefficient

values.

Chapter VIII References


73



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