FORMULATION AND EVALUATION OF A CONTROLLED RELEASE ORAL HYPOGLYCAEMIC TABLET

Thesis submitted in

the partial fulfilment for the award of

Degree of Doctor of Philosophy

in Pharmaceutical Sciences

By

Deepu S

(Reg. No. J863600003)

VINAYAKA MISSION UNIVERSITY

SALEM, TAMILANDU, INDIA.

JANUARY 2015

I

VINAYAKA MISSION UNIVERSITY, SALEM

CERTIFICATE BY THE GUIDE

I, Dr. (Sr.) Molly Mathew, certify that the thesis entitled “Formulation

and evaluation of a controlled release oral hypoglycemic tablet” submitted

for the degree of Doctor of Philosophy by Mr. Deepu S, is the record of

research work carried out by him during the period from July 2008 to July

2014 under my guidance and supervision and that this work has not

formed the basis for the award of any degree, diploma, associate-ship,

fellowship or other titles in this University or any other University or

Institutions of higher learning.

Place: Kasaragod

Date:

Dr. (Sr). Molly Mathew, B.Sc, M.Pharm, Ph.D Principal, Malik Deenar College of Pharmacy, Seenthangoli, Bela Post, Kasaragod-671 321, Kerala, India.

II

VINAYAKA MISSION UNIVERSITY, SALEM

CERTIFICATE BY THE CO-GUIDE

I, Dr. K L Senthilkumar, certify that the thesis entitled “Formulation and

evaluation of a controlled release oral hypoglycemic tablet” submitted for the

degree of Doctor of Philosophy by Mr. Deepu S, is the record of research

work carried out by him during the period from July 2008 to July 2014 under

my guidance and supervision and that this work has not formed the basis

for the award of any degree, diploma, associate-ship, fellowship or other

titles in this University or any other University or Institutions of higher

learning.

Place: Dharmapuri

Date:

Dr. K L Senthilkumar Principal, Padmavathy college of Pharmacy, Krishnagiri main road, Periyanahalli, Dharmapuri - 635205 Tamil Nadu.

III

VINAYAKA MISSION UNIVERSITY, SALEM

DECLARATION

I, Mr. Deepu S, declare that the thesis entitled “Formulation and

evaluation of a controlled release oral hypoglycaemic tablet” submitted by me

for the degree of Doctor of Philosophy is the record of research work carried out

by me during the period from July 2008 to July 2014 under the guidance of Dr.

(Sr). Molly Mathew and co-guidance of Dr. K L Senthilkumar and, has not formed

the basis for the award of any degree, diploma, associate-ship, fellowship or

other titles in this University or any other University or Institutions of higher

learning.

Place: Trivandrum Date:

Mr. Deepu S Mar Dioscorus College of pharmacy Mount Hermon, Alathara, Sreekariyam, Trivandrum-695017 Kerala, India.

IV

ACKNOWLEDGEMENT

I humble myself before my God the Almighty for the countless blessings

showered upon me all through my life and for giving me the ultimate guidance

for the successful completion of the dissertation.

This thesis would not have been materialized without the immeasurable

help from many people who gave their support in different ways. To them I would

like to convey the heartfelt gratitude and sincere appreciation.

It is with great pleasure that I acknowledge my esteemed guide Dr. (Sr).

Molly Mathew, Principal, Malik Deenar College of Pharmacy, Kasaragod,

Kerala for her invaluable guidance, effective criticism and for creating an

environment so conducive for learning. She has been instrumental for the

smooth flow of this research work. This period has been an enriching experience

of working under her guidance. I thank her whole heartedly.

I pay my profound gratefulness and indebtedness to Dr. K. Rajendran,

Dean–research of Vinayaka Missions University, Salem for his timely support. I

express my sincere thanks to my Co-guide Dr. K L Senthilkumar, Principal,

Padmavathy College of pharmacy, Dharmapuri, for his valuable suggestion and

support to accomplish this research work.

I gratefully acknowledge my dearest Friend, Mrs. Shamna.M.S, who has

shared her time, patience and care, with all the full support and encouragement,

all of which have contributed to the successful completion of the thesis work.

Without her this thesis would not have been a reality. I would like to extend my

deep sense of gratitude to Mr. Elayaraja R, Scientist, Dr Reddy’s Laboratories,

V

who had kindly provided me the gift sample required for my work and for his

advice, discussion, and help throughout the work.

I extend my gratitude to management of St. Joseph College of

Pharmacy for helping me to carry out the pharmaceutical and evaluation

studies.

I am grateful to Prof. Dr. M. A. Kuriachan, Principal, Mar Dioscorus

College of Pharmacy, Trivandrum, for his encouragement and advice.

I take this opportunity to thank the Management of Mar Dioscorus

College of Pharmacy for the support to accomplish this research work.

I owe my deepest gratitude to my colleagues Mr. Ganesh Sanker S, Mrs.

Sivazeena T. S, Mr. Praveenraj. R, and Sujith S Nair for helping me to get

through the difficult times and for all the emotional support, entertainment and

caring they provided.

My deepest love and gratitude go to my dear parents, family and my child,

Neha for their unflagging love, prayers and support throughout my life, all of

which helped me to complete my dissertation successfully.

DEEPU S

VI

CONTENTS

LIST OF FIGURES .................................................................................................... XI

LIST OF TABLES ................................................................................................... XIII LIST OF ABBREVATIONS AND SYMBOLS ........................................................... XV

1. INTRODUCTION ................................................................................................ 1

1.1. Oral controlled drug delivery system .............................................................. 2 1.2. Challenges in controlled release formulations ................................................ 4 1.3. Rationale for designing controlled drug delivery ............................................. 5 1.4. Matrix tablets .................................................................................................. 6 1.5. Polymers used in matrix tablets ..................................................................... 7 1.6. Classification of matrix tablets ........................................................................ 9 1.6.1. Hydrophilic matrix tablet ............................................................................ 10

1.6.2. Hydrophobic matrix tablets ........................................................................ 11

1.7. Mechanism of drug release from matrix tablet ............................................. 12 1.8. Bimodal Release .......................................................................................... 15 1.9. Boundaries of gel layer and relevant fronts .................................................. 17 1.10. Swellable matrix tablets as drug delivery systems .................................... 18 1.11. Factors affecting drug release from a matrix system ................................ 19 1.12. Biological factors influencing release from matrix tablets .......................... 23 1.13. Physicochemical factors influencing release from matrix tablets .............. 25 1.14. Evaluation of controlled release matrix tablets .......................................... 28 1.15. In vitro Evaluation ..................................................................................... 28 1.16. In vivo performance evaluation ................................................................. 29 1.17. Data treatments ........................................................................................ 30 1.17.1 Zero-order treatment ................................................................................ 30

1.17.2 First-order treatment ................................................................................. 30

1.17.3 Higuchi’s model ......................................................................................... 30

1.17.4 Korsmeyer – Peppas model ...................................................................... 30

2. REVIEW OF LITERATURE .................................................................................. 33

2.1. Drug profile ................................................................................................... 42 2.1.1 Metformin HCl ........................................................................................... 42

2.1.2 Mechanism of action ................................................................................. 43

2.1.3 Dosage and administration ....................................................................... 44

VII

2.1.4 Adverse effects ......................................................................................... 44

2.1.5 Contraindications ...................................................................................... 45

2.1.6 Precautions ............................................................................................... 45

2.1.7 Identification test for Metformin HCl (I.P) .................................................. 45

2.1.8 Assay ........................................................................................................ 46

2.1.9 Packaging and storage ............................................................................. 46

2.2 Methocel ......................................................................................................... 47

2.2.1 Synonym: Hypromellose. .......................................................................... 47

2.2.2 Nomenclature ............................................................................................ 47

2.2.3 Preparation ............................................................................................... 48

2.2.4 Shelf life .................................................................................................... 48

2.2.5 Characteristics .......................................................................................... 48

2.2.6 Solubility .................................................................................................... 49

2.2.7 Properties .................................................................................................. 49

2.2.8 Substitution ............................................................................................... 49

2.3 Carboxy methyl cellulose ............................................................................... 50

2.3.1 Chemical family: Carbohydrate, Cellulose Derivative ............................... 50

2.3.2 Molecular structure ................................................................................... 50

2.3.3 Synonyms ................................................................................................. 50

2.3.4 Functional category ................................................................................... 50

2.3.5 Stability and storage ................................................................................. 51

2.3.6 Application ................................................................................................ 51

2.4 Cellulose acetate ............................................................................................ 52

2.4.1 Synonym ................................................................................................... 52

2.4.2 Introduction ............................................................................................... 52

2.4.3 Molecular structure ................................................................................... 52

2.4.4 Synthesis .................................................................................................. 52

2.4.5 Applications ............................................................................................... 53

2.4.6 Storage ..................................................................................................... 53

2.4.7 Stability ..................................................................................................... 53

2.5 Magnesium stearate ....................................................................................... 54

2.5.1 Synonym ................................................................................................... 54

2.5.2 Chemical name ......................................................................................... 54

VIII

2.5.3 Functional category ................................................................................... 54

2.5.4 Description ................................................................................................ 54

2.5.5 Application in formulation .......................................................................... 54

2.5.6 Stability and storage ................................................................................. 54

2.5.7 Incompatibilities ........................................................................................ 55

2.6 Aerosil ............................................................................................................. 56

2.6.1 Synonym ................................................................................................... 56

2.6.2 Chemical name ......................................................................................... 56

2.6.3 Storage ..................................................................................................... 56

2.6.4 Application ................................................................................................ 56

3. NEED FOR THE STUDY ....................................................................................... 58

4. OBJECTIVES AND HYPOTHESIS .................................................................. 61

6. METHODOLOGY ............................................................................................. 64

5.1 Preformulation studies..................................................................................... 64

5.1.1 Drug excipient compatibility studies .......................................................... 64

5.1.1.1 FTIR characterization ............................................................................ 65

5.1.1.2 Differential scanning calorimetry (DSC) ................................................. 65

5.1.1.3 Powder X-ray diffraction ........................................................................ 65

5.2 Pre-optimisation studies. ................................................................................. 66

5.2.1 Optimisation of polymer concentration. ........................................................ 66

5.3 Formulation development: .............................................................................. 66

5.3.1 Preparation of matrix tablets of Metformin HCl ......................................... 66

5.3.2 Preparation of 5% Cellulose acetate (CA) film: ......................................... 67

5.3.3 Preparation of swelling restricted matrix tablets: .......................................... 67

5.4 Evaluation of prepared tablets: ....................................................................... 69

5.4.1 Pre formulation studies................................................................................. 69

5.4.1.1 Bulk density ............................................................................................... 69

5.4.1.2 Hausner’s ratio .......................................................................................... 69

5.4.1.3 Carr’s compressibility index ....................................................................... 69

5.4.1.4 Angle of repose ......................................................................................... 70

5.4.2 Evaluation of physical properties of matrix tablets: ................................... 71

5.4.2.1 Thickness and diameter ............................................................................ 71

5.4.2.2 Weight variation test .................................................................................. 71

IX

5.4.2.3 Hardness test ............................................................................................ 71

5.4.2.4 Friability test .............................................................................................. 71

5.5 Swelling index of matrix tablets ....................................................................... 72

5.6 Drug content estimation .................................................................................. 73

5.7 In vitro release studies .................................................................................... 73

5.7.1 Preparation of standard curve for Metformin ............................................. 73

5.7.2 In vitro drug release studies ...................................................................... 74

5.8 RELEASE KINETIC MODELS ........................................................................ 75

5.8.1 Zero-order treatment .................................................................................... 75

5.8.2 First-order treatment .................................................................................... 76

5.8.3 Higuchi’s model ............................................................................................ 76

5.8.4 Korsmeyer – Peppas model ......................................................................... 77

5.9 SIMILARITY FACTOR..................................................................................... 78

5.10 STATISTICAL ANALYSIS 102 ........................................................................ 79

5.11 STABILITY STUDIES .................................................................................... 79

5.12 In vivo studies ............................................................................................... 80

5.12.1 Pharmacokinetics studies ......................................................................... 80

5.12.1.1 Blood Sample Collection ......................................................................... 80

5.12.1.2 Plasma samples extraction ..................................................................... 81

5.12.1.3 Method Validation of Metformin HCl in HPLC system ............................. 81

5.12.2 Pharmacodynamic studies ........................................................................ 83

5.12.2.1 Induction of Diabetes ............................................................................... 83

5.12.2.2 Experimental design ................................................................................ 83

5.12.2.3 Administration of drugs ............................................................................ 84

5.12.2.4 Blood Sample Collection and determination of blood glucose ................. 84

5.12.2.5 Body weight ............................................................................................. 84

6. RESULTS AND DISCUSSIONS ....................................................................... 86

6.1 Preformulation studies .................................................................................... 86

6.2 Compatibility studies ....................................................................................... 86

6.3 Pre-optimisation studies .................................................................................. 93

6.3.1 Optimisation of polymer concentration ......................................................... 93

6.4 Design and preparation of swelling restricted matrix tablet of ......................... 93

6.5 Evaluation of flow properties of powder .......................................................... 94

X

6.6 Evaluation of tablets ........................................................................................ 96

6.6.1 General appearance .................................................................................... 96

6.6.2 Hardness ...................................................................................................... 96

6.6.3 Thickness ..................................................................................................... 96

6.6.4 Weight variation test ..................................................................................... 97

6.6.5 Friability test ................................................................................................. 97

6.6.6 Drug content ................................................................................................. 97

6.7 Swelling index study ........................................................................................ 98

6.8 In vitro dissolution studies ............................................................................. 102

6.8.1 Preparation of calibration curve .................................................................. 102

6.9 In vitro drug release studies: ......................................................................... 103

6.12 Similarity factor: ........................................................................................... 112

6.13 In vivo release studies ................................................................................ 113

6.13.1 Pharmacokinetic study of Metformin HCl ................................................ 113

6.13.2 Pharmacodynamic study of Metformin HCl ............................................. 114

6.14 Statistical analysis ....................................................................................... 118

7. CONCLUSION ................................................................................................ 120

8. REFERENCES ............................................................................................... 124

9. ANNEXURE ........................................................................................................ 139

9.1 Annexure 1: List of Materials ......................................................................... 139

9.2 Annexure 2: List of Equipment ...................................................................... 140

9.3 Annexure 3: Published Journal Copy ............................................................ 141

9.4 Annexure 4: Animal ethical committee certificate ......................................... 144

XI

LIST OF FIGURES

Fig 1.1: Plasma drug concentration-time profile of conventional, zero order and

sustained release dosage forms. .................................................................. 2

Fig 1.2: Drug diffusion through matrix tablet ............................................................... 7

Fig 1.3: Diagram shows the three fronts of a swelling matrix tablets ........................ 18

Fig 1.4: Freely swellable matrix tablet ....................................................................... 19

Fig 1.5: Swelling restricted matrix tablet ................................................................... 19

Fig 6. 1 : FT-IR peak of different functional groups of Metformin HCl ....................... 87

Fig 6. 2 : FT-IR peak of different functional groups of Metformin HCl and HPMC K4M

.................................................................................................................................. 88

Fig 6. 3 : FT-IR peak of different functional groups of Metformin HCl and HPMC 15M

.................................................................................................................................. 89

Fig 6. 4 : FT-IR peak of different functional groups of Metformin HCl and HPMC K100M

.................................................................................................................................. 90

Fig 6. 5: DSC of pure Metformin HCl (a), Physical mixture of Metformin HCl with HPMC

K100M, Physical mixture of Metformin HCl with HPMC K100M and CMC. ............... 91

Fig 6. 6: X-ray diffraction studies of pure Metformin HCl and formulation blend

containing Metformin HCl, HPMC K100M and CMC. ................................................ 92

Fig 6. 7: Shows the swelling index of the formulations from MFH1 – MFH14 ........... 99

Fig 6. 8: Swelled tablet in SGF while conducting swelling index study. .................... 99

Fig 6. 9: Tablets formed gel like mass when conducting swelling index study ........ 100

Fig 6. 10: Calibration curve of Metformin HCl in distilled water ............................... 102

Fig 6. 11: Tablet removed at Q2 interval form dissolution study ............................. 103

Fig 6. 12: Performing dissolution test in type I apparatus ....................................... 104

Fig 6. 13 : In vitro dissolution profile of Metformin HCl matrix tablet. ...................... 107

XII

Fig 6. 14 : Dissolution profile of MFH 14 Vs SF14 (formulation after stability study)

................................................................................................................................ 111

Fig 6. 15:Dissolution profile of MFH 14 after long term stability study ..................... 112

Fig 6. 16: Comparative Plasma level of Metformin HCl in Rabbits .......................... 113

Fig 6. 17 : Effect of MFH 14 formulation on alloxan induced rabbits ....................... 115

Fig 6. 18: Effect of new formulation on body weight. .............................................. 116

Fig 6. 19: Chromatogram showing Metformin HCl and internal standard Glipizide . 117

XIII

LIST OF TABLES

Table 2.1: Parameters of Metformin HCl .................................................................. 43

Table 2.2: Physical and chemical properties of Avicel .............................................. 50

Table 2.3: Physical and chemical properties of Cellulose acetate ............................ 53

Table 2.4: Physical and chemical properties of Aerosil ............................................. 56

Table 5.1: Composition of matrix tablets containing Metformin HCl. ......................... 68

Table 5.2: Scale of flowability of powders ................................................................. 70

Table 5.3: Angle of repose and corresponding flow property .................................... 70

Table 5.4: Maximum allowable deviation for tablets ................................................. 71

Table 5.5: Value of ‘n’ with corresponding drug release mechanism ........................ 77

Table 5.6: Conditions as per ICH Guidelines. ........................................................... 80

Table 5.7: Treatment of different formulations to various groups of rabbit ................ 80

Table 5.8: Experimental Design for Pharmacodynamic studies in rabbit ................. 83

Table 6. 1: Result of visual inspection of Metformin HCl ........................................... 86

Table 6. 2: Solubility of Metformin HCl in different media ......................................... 86

Table 6. 3: Values of pre – compression parameters of developed formulations ...... 95

Table 6. 4: Observational report of various parameters of tablets ............................ 96

Table 6. 5: Results of post-compression parameters ................................................ 98

Table 6. 6: Showing results of % swelling index value of all formulations. .............. 101

Table 6. 7: Absorbance values of Metformin HCl in distilled water. ........................ 102

Table 6. 8: In Vitro %CDR of drug from Metformin HCl matrix tablets MFH1 to MFH 7

................................................................................................................................ 105

Table 6. 9: In vitro %CDR of drug from Metformin HCl matrix tablets MFH8 to MFH14

................................................................................................................................ 106

XIV

Table 6. 10: Correlation coefficient values and release kinetics of Metformin HCl

matrix tablets ........................................................................................................... 109

Table 6. 11: Results of short term stability study of MFH14 .................................... 110

Table 6. 12: Results of Long term stability study of MFH14 .................................... 112

Table 6. 13: Pharmacokinetic parameters obtained from three different formulations

of Metformin HCl in rabbits (using Residual method PK analysis) ........................... 117

XV

LIST OF ABBREVATIONS AND SYMBOLS

AVG Average

AUC Area under curve

MEC Minimum effective concentration

GI Gastro intestine

Cl Clearance

GIT Gastro intestinal tract

h Hour

CR Controlled release

ER Extended release

CRDDS Controlled release drug delivery system

CA Cellulose acetate

SR Sustained Release

SF Standard formulation

HPMC Hydroxypropyl methyl cellulose

CMC Carboxy methyl cellulose

USP United States Pharmacopoeia

IP Indian Pharmacopoeia

ICH International Conference on Harmonisation

PEG Poly ethylene Glycol

FTIR Fourier transform infrared spectroscopy

SLS Sodium lauryl sulphate

SGF Simulated Gastric Fluid

SIF Simulated Intestinal Fluid

DSC Differential scanning calorimetry

LDL Low density lipoprotein

IR Infra-Red

HPLC High performance liquid chromatography

XVI

CDR Cumulative drug release

PK Pharmacokinetic

cc Cubic centimetre

i.p Intra peritoneal

p.o Per-oral

rpm Rotations per minute

Mg Magnesium

mPa.s Millipascal - seconds

i.e. That is

e.g. Example

Tg Glass transition temperature

mg Milligram

ng Nano gram

ºC Degree Celsius

ºF Degree Fahrenheit

ml Millilitre

cm centimetre

mm millimetre

nm nanometre

kg Kilogram

% Percentage

s Seconds

min Minutes

W Weight

w/v weight by volume

SD Standard Deviation

RH Relative Humidity

Fig Figure

q.s Quantity sufficient

XVII

Q2 Drug release at 2 h

Q8 Drug release at 8 h

Q12 Drug release at 12 h

g/mol Gram per mol

mmol/L millimol per litre

mg/dl milligram per decilitre

g Gram

cPs Centipoise

µm Micro meter

Å Angstrom

λ Lambda

I INTRODUCTIONCHAPTER 1

1

1. INTRODUCTION

Oral route has been one of the most popular commonly employed routes

of drug delivery due to its ease of administration, patient compliance, least

sterility constraints, flexible design of dosage forms and cost effectiveness to

manufacturing process1. Tablets are most popular oral formulations available in

market and preferred by patients and physicians alike. This type of drug delivery

system is called conventional drug delivery system and is known to provide an

immediate release of drug. Such immediate release products results in relatively

rapid drug absorption and onset of accompanying pharmacodynamic effects.

However, after absorption of drug from the dosage form is complete, plasma

drug concentrations decline according to the drug’s pharmacokinetics profile.

Eventually, plasma drug concentrations fall below the minimum effective plasma

concentration (MEC), resulting in loss of therapeutic activity2. Before this point

is reached another dose is usually given if a sustained therapeutic effect is

desired. These dosage forms have been found to have the following serious

limitations.

Inconvenient due to periodic administration

Difficult to monitor

Non-specific administration

Careful calculation necessary to prevent overdosing

Drug goes to non-target cells and can cause damage

Low concentrations can be ineffective

2

1.1. Oral controlled drug delivery system

An alternative to administration of another dose is to use a dosage form

that will provide sustained drug release, and therefore, maintain plasma drug

concentrations. Oral extended release drug delivery system becomes a very

promising approach for those drugs that are given orally but having the shorter

half-life and high dosing frequency. Controlled release formulations are much

desirable and preferred for such therapy because they offer better patient

compliance, maintain uniform drug levels, reduced dose and side effects and

increased margin of safety for high potency drugs3.

Plasma drug concentration profiles for conventional tablet formulation, a

sustained release formulation, and a zero order controlled release formulation

is shown in Fig 1. 1.

Fig 1. 1 : Plasma drug concentration-time profile of conventional, zero order and sustained release dosage forms4.

3

An ideal dosage form for the treatment of any disease is the one which

immediately attain a therapeutic plasma level and maintain it constant for the

entire period of treatment. This is possible through administration of

conventional dosage form at a particular frequency. But with conventional

dosage form there is unavoidable fluctuation in the drug plasma level which can

be overcome by use of sustain release dosage form5. Sustain release is a term

use to characterize a delivery system which is designed in such a manner to

achieve a prolonged therapeutic effect by continuously releasing the drug over

an extended period of time after administration of a single dose. The term

“controlled release” has been associated with those systems which release their

active principle at a predetermined rate6. Physician can achieve certain

desirable therapeutic benefit by prescribing controlled release dosage forms;

since the frequency of drug administered is reduced the patient compliance gets

improved. The blood level oscillation characteristic of multiple dosing of

conventional dosage form is also reduced, as a more even blood level is

maintained.

Advantages7:

1. Maintains therapeutic concentrations over prolonged periods.

2. Avoids the high blood concentration.

3. Reduction in toxicity by slowing drug absorption.

4. Minimize the local and systemic side effects.

5. Improvement in treatment efficacy.

6. Better drug utilization

4

7. Minimize drug accumulation with chronic dosing.

8. Can be made to release high molecular weight compounds.

9. Improved patient compliance.

10. Economical (Although the initial cost of treatment is high the overall

treatment cost will be less due to less dosing frequency).

Disadvantages7:

1. The remaining matrix must be removed after the drug has been released.

2. Greater dependence on GI residence time of dosage form.

3. Increased potential for first-pass metabolism.

4. Delay in onset of drug action.

5. Release rates are affected by food and the rate transit through the gut.

6. Release rate continuously diminishes due to increased diffusional

resistance and decrease in effective area at the diffusion front.

1.2. Challenges in controlled release formulations:

1. Cost of formulation i.e. preparation and processing.

2. Fate of controlled release system if not biodegradable.

3. Biocompatibility.

4. Fate of polymer additives, e.g., plasticizers, stabilizers, antioxidants.

5. Dose dumping (Chewing or grinding of oral formulation by the patients).

7. Retrieval of drug is difficult in case of toxicity, poisoning or hypersensitivity

reaction.

5

1.3. Rationale for designing controlled drug delivery7:

Reducing the frequency and quantity of dose.

To increase effectiveness of the drug by localization at the site of action.

To avoid an undesirable local action within the GIT.

To provide programmed and uniform drug delivery pattern.

To increase extend of absorption/bioavailability.

To extend the time of action of drug after administration.

The basic rationale of a controlled drug delivery system is to optimize the

biopharmaceutic, pharmacokinetic and pharmacodynamic properties of a drug

in such a way that its utility is maximized through reduction in side effects and

cure or control of condition in the shortest possible time by using smallest

quantity of drug administered by the most suitable route.

Ideal drug candidates for controlled drug delivery systems must meet the

following criteria’s:

1. It should be orally effective and stable in GIT medium.

2. Drugs with short half-lives, ideally a drug with half-life in the range of 2 –

4 H makes a good candidate for formulation into CR dosage forms.

3. The dose of the drug should be less than 0.5 g as the oral route is suitable

for drugs given in dose as high as 1.0 g.eg. Metronidazole.

4. A drug for CRDDS should have therapeutic range wide enough such that

variations in the release do not result in concentration beyond the

minimum toxic levels.

6

Potential areas to be considered are:

The various pH that the dosage form would encounter during its transit

The gastrointestinal motility

The enzyme system and its influence on the drug and the dosage form

1.4. Matrix tablets:

Historically, the most popular drug delivery system till date is the matrix

because of its low cost and ease of fabrication. Introduction of matrix tablet as

sustained release has given a new break through from the novel drug delivery

system in the field of pharmaceutical technology. The drug release from the

dosage form is controlled mainly by the type and proportion of polymer used in

the preparation.

Matrix tablet may be defined as “oral solid dosage form in which the drug

or active ingredient is homogeneously dispersed throughout the hydrophobic or

hydrophilic matrices which serves as release rate retardants”7.

These systems release drug in continuous manner by dissolution

controlled or diffusion controlled mechanisms (as shown in Fig 1. 2). Usually the

drug release from these matrices includes penetration of fluid, followed by

dissolution of drug particles and diffusion through fluid filled pores. The diffusion

of drug through a matrix is a rate limiting step.

Matrix tablets serves as an important tool for oral extended- release dosage

forms. They can be formulated by wet granulation or direct compression

methods by dispersing solid particles within a porous matrix formed of

7

hydrophilic and hydrophobic polymers. The use of different classes of polymers

in controlling the release of drugs has become the most important aspect in the

formulation of matrix tablets.

Fig 1. 2: Drug diffusion through matrix tablet 8

1.5. Polymers used in matrix tablets9:

There are number of polymers which may be used to formulate matrix

tablets depending on the physicochemical properties of the drug substance to

be incorporated into matrix system and type of drug release required. Polymers

used for matrix tablets may be classified as:

1. Hydrogels

a. Polyhydroxy ethyl methyl acrylate (PHEMA)

b. Cross linked polyvinyl alcohol (PVA)

c. Cross linked polyvinyl pyrrolidone (PVP)

d. Polyethylene oxide (PEO)

e. Polyacrylamide (PA)

8

2. Soluble polymers

a. Polyethylene glycol (PEG)

b. Polyvinyl alcohol (PVA)

c. Polyvinyl pyrrolidone (PVP)

d. Hydroxypropyl methyl cellulose (HPMC)

3. Biodegradable polymers

a. Polylactic acid (PLA)

b. Polyglycolic acid (PGA)

c. Polycaprolactone (PCL)

d. Polyanhydrides

e. Polyorthoesters

4. Non-biodegradable polymers

a. Polydimethyl siloxane (PDS)

b. Polyethylene vinyl acetate (PVA)

c. Polyether urethane (PEU)

d. Polyvinyl chloride (PVC)

e. Cellulose acetate (CA)

5. Mucoadhesive polymers

a. Polycarbophil

b. Sodiumcarboxy methyl cellulose

c. Polyacrylic acid

d. Tragacanth

e. Methyl cellulose

9

6. Natural gums

a. Xanthan gum

b. Guar gum

c. Karaya gum

d. Gum Arabic

e. Locust bean gum

Various synthetic and natural polymers have been examined in drug

delivery applications. The three key advantages that polymeric drug delivery

products can offer are:

Localized delivery of drug

Sustained delivery of drug

Stabilization of drug (protects the drug from GIT environment)

1.6. Classification of matrix tablets9, 10:

1. On the basis of type of polymer/release rate retardant used matrix tablets

may be divided into two types.

a) Hydrophilic matrix tablet

b) Hydrophobic matrix tablet

2. On the basis of porosity of the matrix system used in the formulation.

a) Macro porous system.

b) Micro porous system.

c) Non porous system.

10

1.6.1. Hydrophilic matrix tablet11

Hydrophilic matrix tablets may be defined as “Homogeneous dispersion

of drug molecules within a skeleton of hydrophilic polymers, such as cellulose

derivatives, sodium alginate, xanthan gum, polyethylene oxide, or carbopol

among others, that swells upon contact with water”.

These systems are called swellable-controlled release systems. Apart

from swelling and diffusion mechanisms polymer dissolution is another

important mechanism that can modulate the drug delivery rate. Swelling or

dissolution can be the predominant factors for a specific type of polymers, in

most cases drug release kinetics is a result of a combination of these two

mechanisms13. The release rate observed is possibly the zero-order release.

The polymers used in the preparation of hydrophilic matrices are divided in to

two broad groups 14.

A. Cellulose derivatives: Methylcellulose 400 and 4000 cPs, hydroxyl ethyl

cellulose, hydroxypropyl methylcellulose (HPMC) 25, 100, 4000 and

15000cPs and sodium carboxy methylcellulose.

B. Non cellulose natural or semi synthetic polymers: Agar-Agar, carbo gum,

alginates, molasses, polysaccharides of mannose and galactose,

chitosan and modified starches.

11

1.6.2. Hydrophobic matrix tablets

This system was first introduced in 1959. In this method, drug is mixed

with an inert or hydrophobic polymer and then compressed into a tablet.

Sustained release is produced due to the fact that the dissolving drug has

diffused through a network of channels that exist between compacted polymer

particles. This is the only system where the use of polymer is not essential to

provide controlled drug release, although insoluble polymers have been used.

The primary rate-controlling components of hydrophobic matrix are water

insoluble in nature. Examples of materials that have been used as inert or

hydrophobic matrices include waxes, glycerides, polyethylene, polyvinyl

chloride, ethyl cellulose and acrylate polymers and their copolymers 15, 16.

The rate-controlling step in these formulations is liquid penetration into

the matrix. The possible mechanism of release of drug in such type of tablets is

diffusion. Such types of matrix tablets become inert in the presence of water and

gastrointestinal fluid. The presence of insoluble ingredient in the formulations

helps to maintain the physical dimension of hydrophobic matrix during drug

release. To modulate drug release, it may be necessary to incorporate soluble

ingredients such as lactose into formulation13.

Macro porous Systems: In such systems the diffusion of drug occurs through

pores of matrix, which are of size range 0.1 to 1 μm. This pore size is larger than

diffusant molecule size.

12

Micro porous System: Diffusion in this type of system occurs essentially

through pores. For micro porous systems, pore size ranges between 50 – 200

Å, which is slightly larger than diffusant molecules size.

Non-porous System: Non-porous systems have no pores and the molecules

diffuse through the network meshes. In this case, only the polymeric phase

exists and no pore phase is present.

1.7. Mechanism of drug release from matrix tablet12:

Drug in the outside layer exposed to the bathing solution is dissolved first

and then diffuses out of the matrix. This process continues with the interface

between the bathing solution and the solid drug moving toward the interior. It

follows that for this system to be diffusion controlled, the rate of dissolution of

drug particles within the matrix must be much faster than the diffusion rate of

dissolved drug leaving the matrix.

Derivation of the mathematical model to describe this system involves the

following assumptions:

1. A pseudo-steady state is maintained during drug release.

2. The diameter of the drug particles is less than the average distance of

drug diffusion through the matrix.

3. The bathing solution provides sink conditions at all times.

13

The release behaviour for the system can be mathematically described by the

following equation:

dM/dh = C0. dh – Cs/2 ……………… (I)

Where,

dM = Change in the amount of drug released per unit area.

dh = Change in the thickness of the zone of matrix that has been depleted

of drug.

C0 = Total amount of drug in a unit volume of matrix.

Cs = Saturated concentration of the drug within the matrix.

Additionally, according to diffusion theory:

dM = ( Dm. Cs / h) dt........................... (II)

Where,

Dm = Diffusion coefficient in the matrix.

h = Thickness of the drug-depleted matrix.

dt = Change in time.

By combining equation (i) and equation (ii) and integrating:

M = [Cs. Dm (2C0 –Cs) t] 1/2 ………… (III)

When the amount of drug is in excess of the saturation concentration then:

M = [2Cs.Dm.C0.t] 1/2 ……………….… (IV)

Equation (III) and eq. (IV) relate the amount of drug release to the square-

root of time. Therefore, if a system is predominantly diffusion controlled, then it

is expected that a plot of the drug release Vs square root of time will result in a

straight line. Drug release from a porous monolithic matrix involves the

simultaneous penetration of surrounding liquid, dissolution of drug and leaching

14

out of the drug through tortuous interstitial channels and pores. The volume and

length of the openings must be accounted for in the drug release from a porous

or granular matrix:

M = [Ds.Ca.p/T. (2Co – p.Ca) t] 1/2 …………… (V)

Where,

p = Porosity of the matrix

t = Tortuosity

Ca = solubility of the drug in the release medium

Ds = Diffusion coefficient in the release medium.

T = Diffusional path length

For pseudo steady state, the equation can be written as:

M = [2D.Ca .C0 (p/T) t] 1/2 …………………... (VI)

The total porosity of the matrix can be calculated with the following equation:

p = pa + Ca/ ρ +Cex/ρex…………………….… (VII)

Where,

p = Porosity

ρ = Drug density

pa = Porosity due to air pockets in the matrix

ρex = Density of the water soluble excipients

Cex = Concentration of water soluble excipients

For the purpose of data treatment, equation (VII) can be reduced to:

M = k. t 1/2 …………………………… (VII)

Where, k = constant.

15

So the amount of drug released versus the square root of time will be

linear, if the release of drug from matrix is diffusion-controlled. If this is the case,

the release of drug from a homogeneous matrix system can be controlled by

varying the following parameters:

1. Initial concentration of drug in the matrix

2. Porosity

3. Tortuosity

4. Polymer system forming the matrix

5. Solubility of the drug

1.8. Bimodal Release17, 18:

In some systems there is anomalous release of the active ingredient. In

these systems release is primarily by diffusion. Sometimes the ER polymer may

become hydrated and begin to dissolve leading to release upon erosion. These

systems are complex and difficult to mathematically model since the diffusional

path length undergoes change due to the polymer dissolution. A series of

transport phenomena are involved in the release of a drug from a swellable,

diffusion/erodible matrix:

1. Initially, there are steep water concentration gradients at the

polymer/water interface, resulting in absorption of water into the matrix.

2. Due to the absorption of water, the polymer swells, resulting in dramatic

changes of drug and polymer concentration, increasing the dimensions of

the system and increasing macromolecular mobility.

16

3. Upon contact with water the drug dissolves and diffuses out of the device.

4. With increasing water content, the diffusion coefficient of the drug

increase substantially.

5. In the case of a poorly water-soluble drug, dissolved and undissolved drug

coexist within the polymer matrix.

6. Finally, the polymer itself dissolves.

Swellable matrix tablets are activated by water, and drug release control

depends on the interaction between water, polymer and drug. Water penetration

is the first step leading to polymer swelling and polymer and drug dissolution.

The presence of water decreases the glassy rubbery temperature (e.g., for

HPMC from 184°C to lower than 37°C), giving rise to the transformation of

glassy polymer in a rubbery phase (gel layer). The enhanced mobility of the

polymeric chain favours the transport of dissolved drug. Polymer relaxation

phenomena determine the swelling or volume increase of the matrix. The latter

may add a convective contribution to the drug transport mechanism in drug

delivery.

The gel layer thickness depends on the relative contributions of water

penetration, chain disentanglement, and mass (polymer and drug) transfer in

water. At the beginning the water penetration is more rapid than chain

disentanglement and a quick build-up of gel layer thickness takes place. But

when the water penetrates slowly, due to the increase of the diffusional distance,

little chance in the gel thickness is obtained because water penetration and

17

polymer disentanglement rates are similar. Thus the gel layer thickness

dynamics in swellable matrix tablet shows three distinct phases:

1. It increases when the penetration of water is the fastest phenomenon.

2. Stays constant when the disentanglement rate is similar to the

penetration.

3. Decreases when the entire polymer is in the rubbery phase.

1.9. Boundaries of gel layer and relevant fronts19:

It is common knowledge that the gel layer thickness is defined by the front

separating the matrix from the dissolution medium. The penetration of the

medium into the matrix is accompanied by the formation of a series of fronts (Fig

1. 3) which later disappear along the process of matrix dissolution. The following

fronts have been defined with regard to anomalous release systems:

1. The swelling front: The boundary between the still glassy polymer and its

rubbery phase. With the entry of water into the matrix, the polymer passes

from the crystalline state to a hydrated or jellified state.

The rubbery zone is characterized by being the one into which more

solvent has entered and hence the vitreous transition temperature

(Tg) at 37°C of the polymer is lower than the experimental

temperature.

The glassy region is the one into which the least solvent has

entered and hence its Tg is higher than the experimental

temperature.

18

2. Diffusion front (solid drug–drug solution boundary): The boundary

between the solid as yet undissolved drug and the dissolved drug in gel

layer.

3. The erosion front or dissolution front: The boundary between the matrix

and dissolution medium.

By using sufficient soluble polymers, the gel layer thickness remains

constant, since the fronts in the matrix move in a synchronised way. Keeping

constant the releasing area, this situation leads to zero-order release.

Erosion Front Diffusion Front Swelling Front

Fig 1. 3 : Diagram shows the three fronts of a swelling matrix tablets

1.10. Swellable matrix tablets as drug delivery systems19, 94:

Swelling controlled release systems for drug delivery are very often

prepared as monoliths, i.e., matrices formed by compression of hydrophilic

micro particulate powders. The amount of swellable polymers usually range

from 10-30% of the total weight of the matrix. Different types of swellable matrix

tablets can be prepared by the use of hydrophilic polymers, such as:

19

1. Free swellable matrix tablets: Polymers and solid drug mixed and

compressed, in which swelling is unhindered (as shown in Fig 1. 4).

Fig 1. 4 : Freely swellable matrix tablet

2. Swelling restricted matrix tablets: Their function is to alter the swelling

behaviour and then the drug release. The partial coating of swellable

matrix tablets containing soluble polymers with impermeable films

(Cellulose acetate) created conditions for attainment of zero-order

release (as shown in Fig 1. 5).

A B C

Fig 1. 5 : Swelling restricted matrix tablet (Blue colour illustrates coating with Polymer)

3. Swelling controlled reservoir system: Swellable polymers are used as

coating for delaying or controlling the diffusion of drug from the core.

1.11. Factors affecting drug release from a matrix system20:

1. Drug solubility: Molecular size and water solubility of drug are important

determinants in the release of drug from swelling and erosion controlled

A B C

20

polymeric matrices. For drugs with reasonable aqueous solubility, release

of drugs occurs by dissolution in infiltrating medium and for drugs with

poor solubility release occurs by both dissolution of drug and dissolution

of drug particles through erosion of the matrix tablet.

2. Polymer diffusivity: The diffusion of small molecules in polymer structure

is energy activated process in which the diffusant molecules moves to a

successive series of equilibrium position when a sufficient amount of

energy of activation for diffusion, Ed has been acquired by the diffusant is

dependent on length of polymer chain segment, cross linking and

crystallinity of polymer. The release of drug may be attributed to the three

factors:

a. Polymer particle size: e.g. when the content of hydroxyl propyl

methylcellulose (HPMC) is higher, the effect of particle size is less

important on the release rate of propranolol hydrochloride, the

effect of this variable is more important when the content of polymer

is low. Results may be justified by considering that in certain areas

of matrix containing low levels of HPMC led to the burst release.

b. Polymer viscosity: With cellulose ether polymers, viscosity is used

as an indication of matrix weight. Increasing the molecular weight

or viscosity of the polymer in the matrix formulation increases the

gel layer viscosity and thus slows drug dissolution. Also, the greater

viscosity of the gel, the more resistant the gel is to dilution and

erosion, thus controlling the drug dissolution.

21

c. Polymer concentration: An increase in polymer concentration

causes an increase in the viscosity of gel as well as formulation of

gel layer with a longer diffusional path. This could cause a decrease

in the effective diffusion coefficient of the drug and therefore

reduction in drug release. The mechanism of drug release from

matrix also changes from erosion to diffusion as the polymer

concentration increases.

3. Thickness of polymer diffusional path: The controlled release of a drug

from both capsule and matrix type polymeric drug delivery system is

essentially governed by Fick’s law of diffusion:

JD = D dc/dx

Where,

JD = Flux of diffusion across a plane surface of unit area.

D = diffusibility of drug molecule.

dc/dx = is conc. gradient of drug molecule across a diffusion path

with thickness dx.

4. Thickness of hydrodynamic diffusion layer: It was observed that the drug

release profile is a function of the variation in thickness of hydrodynamic

diffusion layer on the surface of matrix type delivery devices. The

magnitude of drug release value decreases on increasing the thickness

of hydrodynamic diffusion layer.

5. Drug loading dose: The loading dose of drug has a significant effect on

resulting release kinetics along with drug solubility. The effect of initial

drug loading of the tablets on the resulting release kinetics is more

22

complex in case of poorly water soluble drugs, with increasing initial drug

loading the relative release rate first decreases and then increases,

whereas, absolute release rate increases. In case of freely water soluble

drugs, the porosity of matrix upon drug depletion increases with

increasing initial drug loading. This effect leads to increased absolute drug

transfer rate. But in case of poorly water soluble drugs, another

phenomenon also has to be taken in to account. When the amount of drug

present at certain position within the matrix, exceeds the amount of drug

soluble under given conditions, the excess of drug has to be considered

as non-dissolved and thus not available for diffusion. The solid drug

remains within tablet, on increasing the initial drug loading of poorly water

soluble drugs, the excess of drug remaining with in matrix increases.

6. Surface area and volume: The dependence of the rate of drug release on

the surface area of drug delivery device is well known theoretically and

experimentally. Both the in-vitro and in-vivo rate of the drug release, are

observed to be dependent upon surface area of dosage form. Siepman

et al. found that release from small tablet is faster than large cylindrical

tablets.

7. Diluent’s effect: The effect of diluent or filler depends upon the nature of

diluent. Water soluble diluents like lactose, mannose cause marked

increase in drug release rate and release mechanism is also shifted

towards Fickian diffusion; while insoluble diluents like dicalcium

phosphate reduce the Fickian diffusion and increase the relaxation

23

(erosion) rate of matrix. The reason behind this is that water soluble filler

in matrices stimulate the water penetration in to inner part of matrix, due

to increase in hydrophilicity of the system, causing rapid diffusion of drug,

leads to increased drug release rate.

1.12. Biological factors influencing release from matrix tablets95:

1. Biological half-life: SR product aims to maintain therapeutic blood levels

over an extended period of time. In order to achieve this, drug must enter

the circulation at approximately the same rate at which it is eliminated.

The elimination rate is quantitatively described by the half-life (t1/2). Each

drug has its own characteristic elimination rate, which is the sum of all

elimination processes, including metabolism, urinary excretion and all

over processes that permanently remove drug from the blood stream.

Therapeutic compounds with short half-life are generally are excellent

candidate for SR formulation, as this can reduce dosing frequency. In

general, drugs with half-life shorter than 2 h such as furosemide or

levodopa are poor candidates for SR preparation. Compounds with long

half-lives, more than 8 h are also generally not used in sustaining form,

since their effect is already sustained. E.g. Digoxin and phenytoin.

2. Absorption: Since the purpose of forming a SR product is to place control

on the delivery system, it is necessary that the rate of release is much

slower than the rate of absorption. If we assume that the transit time of

most drugs in the absorptive areas of the GI tract is about 8-12 h, the

24

maximum half-life for absorption should be approximately 3-4 h;

otherwise, the device will pass out of the potential absorptive regions

before drug release is complete. Thus corresponds to a minimum

apparent absorption rate constant of 0.17-0.23 to give 80-95% over this

time period. Hence, it assumes that the absorption of the drug should

occur at a relatively uniform rate over the entire length of small intestine.

If a drug is absorbed by active transport or transport is limited to a specific

region of intestine, SR preparation may be disadvantageous to

absorption. One method to provide sustaining mechanisms of delivery for

compounds tries to maintain them within the stomach. This allows slow

release of the drug, which then travels to the absorptive site. These

methods have been developed as a consequence of the observation that

co-administration results in sustaining effect.

3. Metabolism: Drugs those are significantly metabolized before absorption,

either in the lumen or the tissue of the intestine, can show decreased

bioavailability from slower-releasing dosage form. Hence, criteria for the

drug to be used for formulating SR dosage form is:

Drug should have short half-life (2-4 h.)

Drug should be soluble in water

Drug should have large therapeutic window

Drug should be absorbed throughout the GIT

25

Even a drug that is poorly water soluble can be formulated in SR dosage

form. For the same, the solubility of the drug should be increased by the

suitable system and later on that is formulated in the SR dosage form.

4. Distribution: Drugs with high apparent volume of distribution, which

influence the rate of elimination of the drug, are poor candidate for oral

SR drug delivery system e.g. Chloroquine.

5. Protein Binding: The Pharmacological response of drug depends on

unbound drug concentration drug rather than total concentration and all

drug bound to some extent to plasma and or tissue proteins. Proteins

binding of drug play a significant role in its therapeutic effect regardless

the type of dosage form as extensive binding to plasma increase

biological half-life and thus sometimes SR drug delivery system is not

required for this type of drug.

6. Margin of safety: As we know larger the value of therapeutic index safer

is the drug. Drugs with low therapeutic index are usually poor candidate

for formulation of oral SR drug delivery system due to technological

limitation of control over release rates.

1.13. Physicochemical factors influencing release from matrix

tablets21, 94:

1. Dose size: For orally administered systems, there is an upper limit to the

bulk size of the dose to be administered. In general, a single dose of 0.5-

26

1.0 g is considered maximal for a conventional dosage form. This also

holds true for sustained release dosage form. Compounds that require

large dosing size can sometimes be given in multiple amounts or

formulated into liquid systems. Another consideration is the margin of

safety involved in administration of large amount of a drug with a narrow

therapeutic range.

2. Ionization, pka and aqueous solubility: Most drugs are weak acids or

bases. Since the unchanged form of a drug preferentially permeates

across lipid membranes, it is important to note the relationship between

the pka of the compound and the absorptive environment. Presenting the

drug in an unchanged form is advantageous for drug permeation. Delivery

systems that are dependent on diffusion or dissolution will likewise be

dependent on the solubility of the drug in aqueous media. These dosage

forms must function in an environment of changing pH, the stomach being

acidic and the small intestine more neutral, the effect of pH and release

process must be defined. Compounds with very low solubility

(<0.01mg/ml) are inherently sustained, since their release over the time

course of a dosage form in the GI tract will be limited by dissolution of the

drug. So it is obvious that the solubility of the compound will be poor

choices for slightly soluble drugs, since the driving force for diffusion,

which is the drug’s concentration in solution, will be low.

3. Partition Coefficient: When a drug is administered to the GI tract, it must

cross a variety of biological membranes to produce a therapeutic effect in

27

another area of the body. It is common to consider that these membranes

are having lipophilic nature; therefore the partition coefficient of oil-soluble

drugs becomes important in determining the effectiveness of membrane

barrier penetration. Compounds which are lipophilic in nature having high

partition coefficient are poorly aqueous soluble and it retain in the

lipophilic tissue for the longer time. In case of compounds with very low

partition coefficient, it is very difficult for them to penetrate the membrane,

resulting in poor bioavailability. Furthermore, partitioning effects apply

equally to diffusion through polymer membranes. The choice of diffusion-

limiting membranes must largely depend on the partitioning

characteristics of the drug.

4. Stability: Orally administered drugs can be subject to both acid-base

hydrolysis and enzymatic degradation. Degradation will proceed at a

reduced rate for drugs in solid state; therefore, this is the preferred

composition of delivery for problem cases. For the dosage form that are

unstable in stomach, systems that prolong delivery over entire course of

transit in the GI tract are beneficial; this is also true for systems that delay

release until the dosage form reaches the small intestine. Compounds

that are unstable in small intestine may demonstrate decreased

bioavailability when administered from a sustaining dosage form. This is

because more drugs is delivered in the small intestine and, hence, is

subject to degradation. Propentheline and probanthine are representative

example of such drug.

28

1.14. Evaluation of controlled release matrix tablets:

Before marketing a controlled release product it is necessary to assure

the strength, safety, stability and reliability of the product by performing in vitro

and in vivo analysis and correlation between the two.

1.15. In vitro Evaluation:

For solid oral controlled release dosage forms, drug release

characterisation is the most important among various in vitro tests because the

in vivo absorption is determined by the release kinetics of the dosage forms. A

validated in vitro dissolution test can serve the purposes of

1. Providing necessary quality and process control

2. Determining stability of the relevant release characteristics of the product

3. Facilitating certain regulatory determinations and judgments concerning

minor formulation changes, change in site of manufacture

However the dissolution rate of a specific dosage is essentially arbitrary

parameter that may vary with the dissolution methodology, such as type of

apparatus, medium, agitation, etc. The key elements during the dissolution

evaluation include:

a) Reproducibility of the method

b) Maintenance of sink condition

c) Dissolution profile with a narrow limit on 1-h specification to assure lack

of dose dumping

29

d) At least 75% of drug released at the last sampling interval to assure

complete release

Commonly used USP dissolution methods are recommended for determination

of drug release from oral controlled release dosage forms are96;

I. USP apparatus I (basket method): Preferred for capsules and dosage

forms that tend to float or disintegrate slowly.

II. USP apparatus II (Paddle method): Preferred for tablets.

III. USP apparatus III (Bio-Dis dissolution method, or modified

disintegration): Useful for bead type dosage form.

IV. USP apparatus IV (Flow-through cell method): For insoluble drugs.

1.16. In vivo performance evaluation:

Once the satisfactory In vitro profile is achieved, it becomes necessary to

conduct in vivo evaluation and establish an in vitro - in vivo correlation. The

various in vivo evaluation methods are:-

a) Clinical response

b) Blood level data

c) Urinary excretion studies

d) Nutritional studies

e) Toxicity studies

f) Radioactive tracer techniques

30

1.17. Data treatments

1.17.1 Zero-order treatment 22

Qt = Q0+K0t

Where, Qt = Amount of drug released in time (t).

Q0 = Initial amount of drug in solution,

K0 = Zero order release constant.

1.17.2 First-order treatment 23, 24

Log c = Log c0 – kt / 2.303

Where, c = amount of drug remaining unreleased at time t.

C0 = initial amount of drug in solution.

K = first order rate constant.

1.17.3 Higuchi’s model 24, 25

Qt = kt1/2

Where, Qt = amount of drug released in time t

K = Higuchi’s constant.

A linear relationship between amount of drug released (Q) versus square root

of time (t1/2) is observed if the drug release from the matrix is diffusion controlled.

1.17.4 Korsmeyer – Peppas model 27

It relates that the drug release is exponentially to time. It is described by the

following equation;

Mt / Minf = atn

31

Where, Mt / Minf = fraction release of drug.

a = constant depending on the structural and geometric

characteristics of the drug dosage form.

n = release exponent.

The value of n indicates the drug release mechanism.

For slab:

n = 0.5 (indicates Fickian diffusion)

n = 0.5 – 1.0 or n = 1 (indicates non – Fickian mechanism)

For cylinder:

n = 0.45 instead of 0.5 and 0.89 instead of 1.0.

This model is used to analyse the release of drug from polymeric dosage form,

when the release mechanism is not understood or when there is a possibility of

more than one type of release mechanisms are involved.

32

REVIEW OF LITERATURECHAPTER 2

33

2. REVIEW OF LITERATURE

Sandhya28 et. al in 2014 prepared bilayered tablet containing Glimepiride

(immediate release) and Metformin HCl (Sustained release). Tablets were

prepared using different polymers (HPMC, Povidone, Ethyl cellulose) which

were evaluated and in vitro release profiles were recorded. It had sufficient

floating properties and developed formulations gave near to zero order release

pattern which followed higuchi model.

Damodar29 et. al in 2014 developed immediate and sustained release

Metformin HCl tablet. Immediate release was prepared by direct compression

and sustained release beads were prepared by inotropic gelation method and

its evaluation were done. All tablets contained micro beads up to 35% and were

within specified limits.

Babu30 et. al in 2014 prepared Metformin HCl sustained release tablets using

wet granulation technique with polymers such as Xanthan gum, Guar gum,

HPMC and Eudragit. Different batches were prepared using varying

concentration of polymers and its evaluation were done. From the dissolution

studies it was found that guar gum used tablets gave a drug release of 12 h and

showed slower release rate when compared to others. Most of the formulations

followed zero order release pattern.

Hasan31 et. al in 2014 developed extended release Metformin HCl tablet by

direct compression using varying drug-polymer ratio. Drug release were

retarded with Methocel K100MCR premium and Xanthan Gum. Precompression

and post compression parameters were evaluated and were within acceptable

34

limits. F3, F4 and F5 gave better release than others. Data were fitted into

various kinetic models and found that that diffusion along with erosion could be

the mechanism of drug release.

Solanki32 et. al in 2014 formulated Metformin HCl sustained release tablets by

wet granulation technique. Prepared tablets exhibited sustained release profile

for an extended period of time. It showed non-fickian diffusion release

mechanism and compatibility studies showed that there was no interaction

between the excipients.

Reddy33 et. al in 2013 formulated sustained release tablets of Metformin HCl by

wet granulation using different ratio of polymers (Xanthan Gum, Guar Gum).

Prepared tablets using natural polymers gave better sustained release profile

than synthetic polymers. Prepared tablets had 87.02% release at 8h whereas

marketed once had 105.6% drug release at 8h.

Saluja34 in 2013 designed once daily sustained release matrix tablet containing

Metformin HCl using Chitosan and HPMC phthalate by wet granulation method.

From 8 formulations A-H granulating agent for A was PVP in isopropyl alcohol

and formulation B-H by decreasing concentration of Chitosan and HPMCP.

Formulation G sustained the drug release for 10h which was the best and

showed non-fickian diffusion mechanism.

Kumar35 et. al in 2013 developed bilayered tablet containing Pioglitazone HCl

for immediate release using cross Povidone (super disintegrant) and Metformin

HCl for sustained release using poly ethylene oxide as matrix forming agent.

35

Formulation F5 exhibited first order release and diffusion was the dominant

mechanism for drug release.

Sahoo36 et. al in 2013 prepared Metformin HCl matrix tablets by wet granulation

technique using HPMC and Xanthan gum. HPMC alone couldn’t retard the drug

release for 12h but with Xanthan gum it gave satisfactory release profile. Short

term stability studies were performed for best formulation.

Charulatha et.al37 in 2012 prepared sustained release matrix tablets of

Acebrophylline (200mg) by wet granulation technique using HPMC K 100M with

Sodium CMC of various concentrations. Dissolution profile showed that as

polymer ratio increased, the release was retarded in the prepared matrix tablets

with different polymers.

Hadi et.al38 in 2012 had described about sustained release tablets of

Montelukast sodium prepared by direct compression method using various

polymers. The drug release was extended for a period of 12 h. The kinetic

treatment showed that the release of drug followed first order models. There

was no significant change in drug content after stability studies for optimised

formulation.

Potnuri et.al39 in 2012 prepared Diltiazem HCl bi-layered matrix tablets using

natural polymer (Gum olibanum) and hydrophilic polymer HPMC and

investigated the effect of binders (Starch, Gelatin PEG 6000), diluents and fillers

influencing drug release. The release rate of drug from matrices were affected

36

by an increase in the binder concentration except gelatin. Most dissolution

profiles followed zero order than first order and higuchi model.

Kamlesh et al40 in 2011 prepared Metformin HCl matrix tablet using different

pH dependent polymers like eudragit L100 and S100 and pH independent

polymers like eudragit RLPO and RSPO. Tablets were prepared by direct

compression. Exepients used didn’t react with the ingredients in the tablet which

was confirmed by FTIR studies. In vitro dissolution data’s were fitted into various

kinetic models. Korsmeyers peppas data’s revels that it followed diffusion along

with erosion.

Nanjwade et.al41 in 2011 developed oral extended release matrix tablet using

a combination of hydrophobic and hydrophilic polymers. They prepared the

tablets by two techniques such as direct compression and melt granulation

technique and evaluated their release characteristics and found that the melt

granulation technique was more effective in retarding the release than direct

compression and followed closely to korsmeyers peppas mechanism with a

correlation coefficient of 0.991.

Bangale et.al42 in 2011 made an attempt to formulate sustained release matrix

tablets of Nimodipine using various natural matrix former gums like xanthan

gum, olibanum gum and locust bean gum separately. Majority of designed

formulations displayed nearly zero order release. Korsmeyer and Peppas

equations gave release patterns of R =0.9925 and n=0.6054 respectively

indicating non-fickian or Anomalous types of diffusion through matrix of Locust

37

bean gum. The results demonstrated the feasibility of natural gum in the

development of matrix tablets for controlled delivery of nimodipine.

RajaSekharan et.al43 in 2011 formulated HPMC based controlled release

matrix tablets of theophylline with varying drug- polymer ratios (1:1 and 1:2).

The results indicated that high drug- polymer ratio (1:2) and hardness value (7

kg/cm2), prolonged the drug release rather than the low drug- polymer ratio.

(1:1). Release kinetics followed korsmeyers-peppas model and the mechanism

of drug release was by non-fickian.

Gupta et.al44 in 2011 prepared sustained release tablets (F1 to F4) using

different drug and polymer ratios by direct compression method .Polymers like

Sodium carboxymethyl cellulose (Sod.CMC), hydroxyl propyl methyl cellulose

(HPMC K100), Xanthan gum and HPMC K4 were used. In vitro dissolution study

was carried out for 16 h using paddle method in phosphate buffer (pH 6.8) as

dissolution media. Among all the formulations, F6 showed 100.42% of drug

release at the end of 16 h. This finding revealed that above a particular

concentration of Sod. CMC, HPMC K100 and xanthan gum were capable of

providing sustained drug release.

Samal et.al45 in 2011 prepared matrix tablet by wet granulation method using

hydrophilic Sodium CMC, HPMC, Eudragit‐L155, and Xanthan gum alone or in

combination with hydrophobic polymer ethyl cellulose. The release kinetics was

analysed using Zero order, First order, Higuchi and Hixson Crowell and that

presence of sodium CMC gave zero‐order release kinetics and the linearity

38

ranged from 0.990 to 0.996, with good drug entrapment efficiency ranging from

96 to 106% of drug.

Uner et.al46 in 2011 developed hydralazine HCl matrix tablet to overcome its

side effects and to enhance the bioavailability. They formulated matrix tablets

using different polymers (HPMC, carbomer, glyceryl dibehenate and Cetyl

alcohol) at various concentrations. Among these the slowest release was

obtained by carbomer followed by HPMC. Prepared tablets followed higuchi

kinetic model and non fickian drug release mechanism.

Rahman et.al47 in 2011 formulated sustained release matrix tablets of

Ronolazine by using Eudragit L 100-55 and different grades of HPMC (Methocel

E50 and Methocel K15M). Study showed that an increase in the polymer

concentrations resulted in a decrease of drug release.

Rojas et.al48 in 2011 designed a study using a simplex centroid experiment with

over 69 runs from which best combination of some hydrophilic polymers were

taken which extended the release of drug up to 24 h. The data were fitted to

korsmeyers- peppas model as it gave the best fit. A cubic model predicted best

release of Metformin HCl i.e., up to 24 h by combination of polymers such as

PVP, EC, HPMC, carrageenan, calcium alginate and gum arabic. Confirmation

of cubic model was done by validation.

Amish et.al49 in 2011 studied the effect of various polymers and additives.

Various ratio of polymers were taken and the water uptake study data indicated

that HPMC K100M containing tablets swelled higher than the additives present

39

in the tablet. Soluble and insoluble additives present in the tablets affected

release of the drug from HPMC K100M polymer. From the dissolution data it

was clear that starch showed better drug extending release among other

insoluble additives whereas SLS had a dramatic drug delaying property among

soluble additives.

Chandira et.al50 in 2010 formulated Metformin HCl extended release tablets

using different combination of polymers such as HPMC K100M and Carbopol

71 G with wet granulation (PVP K30) technique. The tablets were subjected to

physical and chemical evaluations and there were no significant changes

observed. In vitro dissolution studies were carried out and F10, considered as

optimised batch, gave a satisfactory release as that of innovator.

Ganesh et.al51 in 2010 prepared sustained release Diclofenac sodium matrix

tablets using cashew nut tree gum HPMC and Carbopol. In vitro release studies

were conducted for twelve h which stated that an increase in the polymer

concentration retarded the release of the drug to a great extent.

Shankar et.al52 in 2010 prepared modified release ciprofloxacin HCl matrix

tablets using different polymers. In vitro dissolution data showed that formulation

containing chitosan and guar gum in the ration of 2:1 (F6) showed 93.6%

release after 12 h whereas those containing lactose in the concentration of 50

and 75% (F7 & F8) showed 96.15% and 99.90% release after 12 h respectively.

Dixit et.al53 in 2009 prepared once daily matrix tablets of Metformin HCl by wet

granulation technique using non aqueous solvent as granulating agent

40

(isopropyl alcohol containing PVP K30) and polymers such as HPMC and locust

bean gum. Tablets were subjected to in vitro dissolution studies and results

indicated that formulation M5 containing HPMC and locust bean gum in the

ration of 200:30 could extend the release of tablets up to 8 h.

Nair et.al54 in 2009 prepared controlled release uncoated tablet by direct

compression method using various grades of HPMC (K100M and K4M) with a

hydrophilic drug enalapril maleate. Results showed that both the polymers alone

were enough to retard the release of drug from the tablet and gave higher r2

values for Higuchi and zero order release. Hence they concluded that HPMC

alone could retard the release of drug for 14 h.

Rao et.al55 in 2009 developed water soluble Tramadol HCl matrix tablet using

HPMC and natural polymers like Karaya gum and carrageenan. Tablets were

subjected to in vitro dissolution studies and data were fitted into different kinetic

models; it was observed that matrix tablet containing HPMC and carrageenan

successfully retarded the release of drug up to 12 h. DSC and FTIR studies

revealed that there was no interaction between the drug and exepients.

Thapa et.al56 in 2008 made Indomethacin containing matrix tablets using

different grades of HPMC (HPMC K4M, HPMC K15M, and HPMC K100M) and

were compared with marketed products. The dissolution profiles of formulations

containing different viscosity grades of HPMC in same concentrations were

different. The dissolution profile of developed formulations were compared with

41

marketed products and showed similarity with a similarity factor of 74.59 and

68.04.

Abdelkader et.al57 in 2007 developed Baclofen matrix tablets containing 25 mg

with different types and levels of polymers such as methylcellulose, sodium

alginate and carboxymethylcellulose. Among these, methylcellulose and sodium

alginate containing formulations showed high drug release retarding efficiency

and good reproducibility. These were stable when stored for 6 months in

ambient room condition which suggested that methylcellulose and sodium

alginate are good candidates for preparing modified release baclofen tablet

formulations.

42

2.1. Drug profile

2.1.1 Metformin HCl58

Fully synthesised and found to reduce blood sugar in 1920.

First described in scientific literature in 1922 by Emil Werner and James

bell

In 1957 French physician Jean Sterne published the first clinical trial

report of Metformin HCl as a treatment for diabetes.

Metformin HCl in synthesised using reaction of dimethyl amine

hydrochloride (dicyandiamide) and 2 – cyanoguanidine which are dissolved in

toluene with cooling to make a concentrated solution and an equimolar amount

of hydrogen chloride is slowly added59. When the mixture begins to boil at its

own, it is cooled and Metformin HCl precipitates with a yield of 96%.

Metformin HCl is an antidiabetic drug belonging to biguanide class which

is a first-line drug of choice for the treatment of type 2 diabetes. Particularly

given to obese patients with overweight (reduces LDL Cholesterol, triglycerides

and doesn’t cause weight gain) and normal kidney function 60. It is further

indicated in the treatment of polycystic ovary syndrome and has been

investigated for other diseases where insulin resistance is an important factor.

It is the only drug supressing the cardiovascular complications of diabetes. From

2010 onwards it is only one among two oral antidiabetics in the World Health

Organisation Model List of Essential Medicines (other being Glibenclamide).

43

Table 2. 1 : Parameters of Metformin HCl

2.1.2 Mechanism of action61

Metformin decreases hyperglycaemia primarily by suppressing glucose

production by the liver (hepatic gluconeogenesis). Metformin improves glucose

tolerance in type 2 diabetic patients. Its pharmacological mechanism of action

is different from other class of drugs. Metformin decreases

Hepatic glucose production

Intestinal glucose absorption and also

Improves insulin sensitivity by increasing peripheral glucose uptake and

utilisation.

Unlike sulphonylureas Metformin doesn’t produce hypoglycaemia and

hyperinsulinemia with normal and diabetic subjects. With Metformin therapy

Properties

Molecular Formula C4H11N5 Chemically Molar weight 129.16 g/mol Log P 1.254

Pharmacokinetics Bioavailability 50-60%

Cmax 1-3 H of Immediate release

4-8 H of Extended release

Tmax 1-3.1 H Route of administration Oral Elimination half life 6.2 H Excretion Renal (unchanged) Plasma protein binding Negligible Steady state reaches 1-2 days

44

insulin secretion remains unchanged hence decreased fasting insulin levels and

day-long plasma insulin response.

2.1.3 Dosage and administration

Given as 1000-2000 mg daily in one or divided doses. Starting dose one

tab/day, after 10-15 days with slow increase of dose. The maximum

recommended daily dose of Metformin HCl, USP is 2550 mg in adults and 2000

mg in paediatric patients (10 to 16 years of age). Metformin HCl, should be given

in divided doses with meals. Metformin HCl, should be started at a low dose,

with gradual dose escalation, both to reduce gastrointestinal side effects and to

permit identification of the minimum dose required for adequate glycaemic

control of the patient.

2.1.4 Adverse effects

GI- Nausea, vomiting, diarrhoea, anorexia, abdominal pain or cramps,

flatulence, colitis inducing pseudomembranous colitis.

Genitourinary: Pyuria, renal function impairment, dysuria, reversible

intestinal nephritis, haematuria, toxic nephropathy.

Haematological: Eosinophilia, neutropenia, lymphocytosis, decreased

platelet function, aplastic anaemia, haemorrhage.

Pregnancy: Category B

Lactation: Excreted in milk.

Lactic acidosis: A rare but serious complication that can occur due to

Metformin accumulation during treatment with Metformin. When it occurs

45

it is fatal in 50% of cases. It is characterised by increased blood lactate

levels (>5 mmol/L).

2.1.5 Contraindications

Renal disease or renal dysfunction.

Congestive heart failure requiring pharmacological treatment.

Known hypersensitivity to Metformin HCl.

Chronic or acute metabolic acidosis including diabetic ketoacidosis which

should be treated with insulin.

2.1.6 Precautions

Metformin should be temporarily discontinued in patients undergoing

radiologic studies involving intravascular administration of iodinated contrast

material, as this may result in acute alteration of renal function hence lactic

acidosis.

2.1.7 Identification test for Metformin HCl (I.P)

A. Determined by IR absorption spectrophotometry (2.4.6). Compare the

spectrum with that obtained with Metformin HCl RS or with the reference

spectrum of Metformin HCl.

B. Dissolve 25 mg in 5 ml of water , add 1.5 ml of 5 M sodium hydroxide, 1

ml of 1- naphthol solution and drop wise with shaking, 0.5 ml of sodium

hypochlorite solution (3% Cl); an orange red colour is produced which

darkens on keeping.

46

C. Dissolve 10 mg in 10 ml of water and add 10 ml of a solution prepared by

mixing equal volumes of a 10% w/v solution of potassium ferricyanide and

a 10% w/v solution of NaOH and allowing to stand for 20 minutes; a wine

red colour develops within 3 minutes.

D. Gives reaction A of chlorides (2.3.1).

E. Melts between 222°C and 226°C, Appendix 8.8

2.1.8 Assay

Weigh accurately about 60 mg, dissolve in 4 ml of anhydrous formic acid

and add 50 ml of acetic anhydride. Titrate with 0.1M perchloric acid, determine

the end point potentiometrically (2.4.25). Carry out a blank titration.

2.1.9 Packaging and storage

It is preserved in tight container at a temperature not exceeding 25°C

and protect from light and moisture.

47

POLYMERS PROFIE

2.2 Methocel62 2.2.1 Synonym: Hypromellose. 2.2.2 Nomenclature

METHOCEL is a trademark of The Dow Chemical Company for a line of

cellulose ether products. An initial letter identifies the type of cellulose ether, its

“chemistry.” “A” identifies methylcellulose (MC) products. “E,” “F,” and “K”

identify different hydroxypropyl methylcellulose (HPMC) products. METHOCEL

E and METHOCEL K are the most widely used for controlled-release drug

formulations. The number that follows the chemistry designation identifies the

viscosity of that product in millipascal-seconds (mPa·s), measured at 2%

concentration in water at 20°C. In designating viscosity, the letter “C” is

frequently used to represent a multiplier of 100, and the letter “M” is used to

represent a multiplier of 1000. Several different suffixes are also used to identify

special products. “P” is sometimes used to identify METHOCEL.

These cellulose ethers are water-soluble methyl cellulose and hydroxyl propyl

methyl cellulose polymers. They are derived from the pine pulp, the most

abundant polymer in nature, and used as thickeners, binders, film formers, and

for water retention. They are also used as suspension aids, protective colloids

and emulsifiers.

Does the job of two or more ingredients in many applications.

Delivers optimum performance at a lower concentration than required by

other water soluble polymers.

Peerless range of product types and performance.

48

Viscosity grades range from 15 to over 200,000 mPas for optimum

thickening, binding, moisture retention and other properties.

HPMC polymers are very versatile release agents. They are non-

ionic, so they minimize interaction problems when used in acidic, basic, or other

electrolytic systems. HPMC polymers work well with soluble and insoluble drugs

and at high and low dosage levels. And they are tolerant of many variables in

other ingredients and production methods.

2.2.3 Preparation

The cellulose obtained from cotton linters and wood pulp are treated with

an alkali like NaOH to produce swollen alkali cellulose. The alkali cellulose is

then treated with chloromethane and propylene oxide due to which it gets

converted to methyl hydroxyl propyl ether of cellulose. The final product is then

purified and ground to powder or granules.

2.2.4 Shelf life: 3 years

2.2.5 Characteristics

It is a white, yellowish white or greyish-white powder, inert, odourless,

tasteless, non-ionic, hydrophilic polymer.

49

2.2.6 Solubility

It is practically insoluble in hot water, in absolute ethanol, acetone, ether,

and in toluene. It dissolves in cold water forming a colloidal solution.

2.2.7 Properties

Its physicochemical properties like solubility, glass transition temperature

and viscosity depend upon the ration of methoxy and hydroxyl propoxyl groups

and the molecular weights. Various grades of Methocel are available which differ

in viscosity and extend of substitution. The different grades can be identified by

a number indicative of apparent viscosity, in mPas of a 2% aqueous solution at

20°C.

2.2.8 Substitution

The major chemical differences are in degree of methoxyl substitution

(DS), moles of hydroxyl propoxyl substitution (MS), and degree of

polymerization (measured as 2% solution viscosity). There are four established

product “chemistries” or substitution types for METHOCEL products, defined

according to the combination of their percent methoxyl/DS and percent hydroxyl

propoxyl/MS.

50

2.3 Carboxy methyl cellulose63 - AVICEL®

2.3.1 Chemical family: Carbohydrate, Cellulose Derivative

Chemically CMC is a cellulose derivative with carboxymethyl groups (-

CH2-COOH) bound to some of the hydroxyl groups of the glucopyranose

monomers that make up the cellulose backbone.

2.3.2 Molecular structure

2.3.3 Synonyms

Microcrystalline cellulose (MCC), cellulose gel, Sodium Carboxy

methylcellulose: Carboxy methylcellulose, Carboxy methyl ether, Sodium CMC,

Sodium salt, Cellulose gum

2.3.4 Functional category: Polymer for Tablets and Capsule.

Table 2. 2 : Physical and chemical properties of Avicel

Odour Odourless

Appearance Off-white, free flowing powder

Percentage volatile Approx. 4% by weight

pH 6.0 - 8.0 (in solution)

Solubility in water Dispersible

Specific gravity (H2O = 1) Bulk Density, 0.6 g/cc

51

2.3.5 Stability and storage

It is a stable chemical substance. It should be stored in a well closed, air

tight container in a cool and dry place.

2.3.6 Application

Used as viscosity modifier or thickening agent to stabilise emulsions. It is

used as lubricant in non-volatile eye drops. It is used in controlled release matrix

formulation.

52

2.4 Cellulose acetate64

2.4.1 Synonym

Cellacephate, Cellulose acetate, Cellulosi acetas phthalas, Cellulose

acetate monopthalate, Acetylphthalylcellulose.

2.4.2 Introduction

Cellulose is a natural polymeric polysaccharide composed of β-D-glucose

subunits. Hydroxyl groups of cellulose can be modified chemically to form esters

or ethers that differ in physicochemical properties allowing for a wide range of

applications. This is an enteric coating polymer which withstands prolonged

contact with acidic gastric fluids, but dissolves readily in the mildly acidic to

neutral environment of the small intestine. It can be applied to tablets or granules

from solutions of organic solvent.

2.4.3 Molecular structure

2.4.4 Synthesis

The most common way to prepare cellulose acetate phthalate consists of

the reaction of a partially substituted cellulose acetate (CA) with phthalic

anhydride in the presence of an organic solvent and a basic catalyst. The

53

organic solvents widely used as reaction media for the phthaloylation of

cellulose acetate are acetic acid, acetone or pyridine. The basic catalysts

employed are anhydrous sodium acetate when using acetic acid, amines when

using acetone, and the organic solvent itself when using pyridine as reaction

medium.

Table 2. 3: Physical and chemical properties of Cellulose acetate

2.4.5 Applications

1. Used in enteric coating of tablets and capsules.

2. Matrix binder for tablets and capsules.

2.4.6 Storage

Stable for several years if stored in a cool, dry place. Keep container

closed when not in use. Store in a tightly closed container. Store in a cool, dry,

well ventilated area away from incompatible substances.

2.4.7 Stability

Stable under normal temperature and pressure.

Appearance White to off white powder

Odour Faint, characteristic

Molecular weight 2534.12 g/mol

Molecular formula C116H116O64

pH solubility ≥6.2

Viscosity cP at 25ºC 68

Specific Gravity (H2O = 1)1.08

54

2.5 Magnesium stearate

2.5.1 Synonym

Magnesium octadeconate, Octadeconate acid magnesium salt, stearic

acid magnesium salt.

2.5.2 Chemical name Octadecanoic acid magnesium salt.

2.5.3 Functional category

Tablets and Capsules lubricant.

2.5.4 Description

It is fine, white, precipitated or milled, impalpable powder with low bulk

density. Insoluble in water, powder shows a faint odour of Stearic acid, tasteless.

The powder is greasy to touch and readily adhere to skin.

2.5.5 Application in formulation

1. Used extensively in cosmetic formulations (barrier creams), food and

pharmaceutical industry.

2. Primarily used as lubricant in tablets and capsules in a concentration of

0.25 – 0.5%.

2.5.6 Stability and storage

It is a stable chemical substance. It should be stored in a well closed, air

tight container in a cool and dry place.

55

2.5.7 Incompatibilities

It is incompatible with strong acids, iron salts and should not be mixed

with strong oxidising agents. It should not be included in the formulations

containing aspirin, some vitamins and most of the alkaloidal salts.

Safety: It is one of the mostly used pharmaceutical excipient as it is nontoxic

when ingested through oral route. When consumed in large amounts produces

laxative effect and can irritate mucosal layer of GIT.

56

2.6 Aerosil65

2.6.1 Synonym Aerosil 200, amorphous fumed silica.

2.6.2 Chemical name Silicon dioxide.

Table 2. 4: Physical and chemical properties of Aerosil

Odour Odourless

Taste Tasteless

Molecular weight 60.08 g/mol

Colour White

pH 4

Loss on Drying ≤ 1.5

Melting point 1610ºC (2930ºF)

Specific gravity 2.2 (Water = 1)

2.6.3 Storage

Keep container tightly closed. Keep container in a cool, well-ventilated

area. Do not store above 23ºC (73.4ºF).

2.6.4 Application

1. Used as glidant to improve the flow property of granules.

2. Used to stabilise emulsions.

3. Thickening and suspending agent in gels and semisolids.

57

NEED FOR THE STUDYCHAPTER 3

58

3. NEED FOR THE STUDY

Metformin is a first line drug of choice for the treatment of type II diabetes

which act by decreasing hepatic glucose output and peripheral insulin

resistance. It can be given to obese patients with overweight having normal

kidney function. The conventional therapy has the following drawbacks:

1. Relatively short half-life of 1.5 to 4.5 h.

2. High dose of 1.5 to 2.0 g/day with conventional therapy.

3. Single 500mg gives a bioavailability of 50 – 60%.

4. High incidence of GI side effects such as abdominal discomfort, nausea

and diarrhoea (up to 30%) which particularly occurs in the early 3 weeks

of treatment.

5. Food delays the Tmax of drug up to 35 min.

6. Reduced patient compliance due to increased dosing frequency.

By formulating the drug in controlled release system, GI side effects had

reduced considerably as reported by Harry Howlett66 and also a decrease in

dosing frequency increases patient compliance. So, it was decided to formulate

it in a controlled release dosage form. Although there are marketed formulation

available Glycomet SR 500mg was taken for comparison, which works under

the diffusion controlled release mechanism. This system has certain limitations

such as

High cost of production,

High molecular weight drugs are difficult to deliver and

59

Dose dumping chances are high.

Incomplete drug release

In order to overcome the following limitations of the marketed formulation a new

formulation should be developed. By developing such a formulation the

advantages are:

1. Reduction in dose can be achieved which can reduce the toxic side

effects of conventional release systems

2. High incidence of GI side effects can be reduced

3. Patient compliance can be improved

Disadvantage:

1. Despite of the following advantage of the new formulation the drug itself

is having a rare side effect of lactic acidosis, which cannot be eliminate

by any type of formulation.

60

OBJECTIVES AND HYPOTHESISCHAPTER 4

61

4. OBJECTIVES AND HYPOTHESIS

Primary objective of the study was:

1. To study the effect of different ratios of polymers and its drug release

properties in a swelling restricted matrix tablet of Metformin HCl.

2. To develop a swelling restricted matrix tablets of Metformin HCl using

different ratios of polymers.

3. To optimize the formula.

4. To compare the in vitro studies like dissolution, disintegration, swelling

index etc. of developed formulation with marketed product

5. To perform bio equivalence study of newly developed formulation and

compare it with marketed formulation (Glycomet SR).

This could be achieved by:

1. Formulating and evaluating controlled release matrix tablets of Metformin

HCl, as this type of formulation can achieve therapeutically effective

concentration of drug in the systemic circulation over an extended period

of time. This type of formulation will eliminate the above said issues and

will exhibit additional advantages such as low cost, simple processing,

improved efficacy, reduces adverse effects, flexibility in terms of range of

release profiles, increased convenience and patient compliance.

2. Formulating Metformin into a controlled release formulation using various

hydrophilic polymers like HPMC (K4M, K15M and K100M) in different

ratios, with hydrophobic polymer CMC to act as a release modulator.

62

Finally the best formulation selected to give partial coating with cellulose

acetate (swelling restricted matrix tablets) to further control the release.

63

METHODOLOGYCHAPTER 5

64

6. METHODOLOGY

5.1 Preformulation studies67, 99:

Preformulation studies are conducted before the development of any

formulations as it is necessary to find out drug characteristics and its stability in

the formulation.

Description: Drug was visually inspected for any change of colour, odour,

etc. as specified in the quality certificate.

Melting point: Capillary method was selected for determining the melting

point. The test was performed in triplicate.

Solubility in dissolution media: Screw caped glass vials of 20 ml capacity

were taken and filled with 10 ml of media (i.e. simulated gastric fluid pH-

1.2, simulated gastric fluid, pH-6.8, and simulated intestinal fluid, pH-7.4).

Drug was added to this till saturation occurred and shaken at room

temperature for 48 h. After that, samples were filtered, appropriately

diluted and analysed at 233nm using UV visible spectrophotometer.

5.1.1 Drug excipient compatibility studies98:

These studies are conducted prior to formulation development, to

determine the physicochemical properties, drug-excipient compatibility, etc. To

establish drug-excipient compatibility, drug alone was first taken and then the

mixture of powder and excipients were taken in 1:1 ratio. The sample was

ground in a mortar and filled in vials, sealed with rubber stoppers and stored for

a period of 2 weeks at 60ºC (except for Mg stearate for which 40ºC is used) and

65

the same samples were retained for 2 months at 40ºC. After storage the drug

was observed physically for liquefaction, caking, odour and gas formation and

discolouration. Further FTIR characterization was done for drug excipient

compatibility.

5.1.1.1 FTIR characterization 68: The desired drug concentration was checked

by assaying the sample; pellets were prepared by mixing with KBr and

scanned. The IR spectrum of drug was recorded using Shimadzu FTIR

Spectrophotometer with scanning range at 250-4500 cm-1.

5.1.1.2 Differential scanning calorimetry (DSC) 69: DSC analysis were

conducted in order to evaluate possible solid-state interaction between

the components and consequently to assess the actual drug excipient

compatibility. Physical mixtures were prepared (Pure Metformin HCl and

F14) dried and examined using DSC (Mettler Toledo DSC 822) to find

its thermal behaviour. Samples are dried thoroughly and placed in an

aluminium sealed pan and preheated to 200ºC. Samples were cooled to

room temperature and reheated from 40ºC to 450ºC at a scanning rate

of 10ºC/min.

5.1.1.3 Powder X-ray diffraction 69: Pure drug Metformin HCl and its physical

mixture with exepients (Metformin HCl, HPMC K100M and CMC) were

subjected to PXRD studies. The crystalline behaviour of pure Metformin

HCl and formulation blend containing Metformin HCl and exepients

66

(HPMC K100M and CMC) were studied using instrument Bruker AXS

D8 Advanced, scanning was done up to 2θ of 70º.

5.2 Pre-optimisation studies. 5.2.1 Optimisation of polymer concentration.

For fixing the desired range of variables (polymer concentration) required

for the final formulation, a pre-optimisation study was conducted with different

concentration of polymers. As the concentration of polymers changed there was

a direct effect on swelling index and drug release.

Tablets were prepared using concentration of polymers form low to high

in each batch. HPMC different grades and CMC were used as polymers for

retarding the drug release. Micro crystalline cellulose was used as filler as the

tablets were prepared by direct compression. Talc and Magnesium stearate

were used as lubricant and glidant.

5.3 Formulation development: 5.3.1 Preparation of matrix tablets of Metformin HCl

Metformin HCl (500mg) matrix tablets were prepared by mixing the drug

with various concentrations of polymers (shown in Table 5. 1) such as HPMC

(various grades), CMC and other excipient’s like micro crystalline cellulose,

magnesium stearate, talc etc. in a polythene bag. Tablets were prepared by

direct compression technique for which prepared powder blend was sieved

through sieve no 40 and dried in a hot air oven below 60ºC. Powder blend was

then mixed with magnesium stearate and aerosil to get it lubricated, which was

then compressed into tablets in a rotary tablet compression machine using 12

mm caplet punches. Before compression the surfaces of the dies and punches

67

were lubricated with magnesium stearate. Prepared tablets were stored in an

airtight container at room temperature for further studies.

5.3.2 Preparation of 5% Cellulose acetate (CA) film:

To 100ml of acetone, 5g of cellulose acetate was gradually added. After

all the cellulose acetate was added the solution was stirred for 2 h to completely

dissolve the CA. The CA solution was degassed for 3 h and it is ready for

coating.

5.3.3 Preparation of swelling restricted matrix tablets:

Swelling restricted matrix tablets were prepared by giving the tablet a

partial coating with 5% cellulose acetate solution. Different levels of coating

were given as shown in Figure 1.5, by dipping the tablet into the solution for

three times. MFH 13 was coated on both of the crown portions of tablets (Figure

1.5 C) whereas MFH 14 was coated only on one of the crown of tablets (Figure

1.5 A).

68

Table 5. 1: Composition of matrix tablets containing Metformin HCl.

Sl. No Ingredients MFH 1 MFH 2 MFH 3 MFH 4 MFH 5 MFH 6 MFH 7 MFH 8 MFH 9 MFH10 MFH11 MFH12 Batch 1 Batch 2 Batch 3 Batch 4

1 Metformin HCl 500 500 500 500 500 500 500 500 500 500 500 500

2 HPMC K4M 150 200 250 X X X X X X X X X

3 HPMC K15M X X X 150 200 250 X X X X X X

4 HPMC K100M X X X X X X 150 200 250 200 150 125

5 CMC X X X X X X X X X 50 100 125

6 MCC 200 150 100 200 150 100 200 150 100 100 100 100

8 Mg Stearate q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s

9 Aerosil q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s

Total Weight (mg) 850 850 850 850 850 850 850 850 850 850 850 850

69

5.4 Evaluation of prepared tablets:

5.4.1 Pre formulation studies 5.4.1.1 Bulk density 70:

Loose bulk density

An accurately weighed quantity of powder was transferred to a 10 ml

measuring cylinder and the volume occupied by the powder in terms of ml

was recorded.

Weight of powder in gm Loose bulk density (LBD) =

Volume packed in ml

Tapped bulk density

Loosely packed powder in the cylinder was tapped 100 times on a

plane hard surface and volume occupied in ml was noted.

Weight of powder in gm Tapped bulk density (TDB) =

Tapped volume in ml 5.4.1.2 Hausner’s ratio 71:

It is the number that is related to the flowability of powder or granules.

A Hausner’s ratio of <1.25 indicates a powder that is free flowing whereas >1.25

indicates poor flow ability.

Hausner’s ratio = TDB / LDB

5.4.1.3 Carr’s compressibility index 72:

It is an indication of the compressibility of a granule or powder. In

pharmaceutics it is an indication of flowability of powder. A Carr’s index with

value more than 25 is considered to be an indication of low flowability and less

70

than 25 is having good flow property. The smaller the Carr’s Index the better the

flow properties. For example 5-15 indicates excellent, 12-16 good, 18-21 fair

and > 23 poor flow.

Carr’s index (%) = [(TBD – LBD) x 100] ÷ TDB

Table 5. 2: Scale of flowability of powders

Compressibility index (%) Flow description 5 -15 Excellent 12-16 Good 18-21 Fair 23-28 Poor (Very fluid powder) 28-35 Poor (Fluid cohesive powder) 35-38 Very poor ˃40 Extremely poor

5.4.1.4 Angle of repose70:

A funnel is fixed and is secured with its tip at a height (h) of 2cm above

graph paper which is placed on a horizontal surface. The powder is dropped

and the radius (r) is measured. Angle of repose can be measured by the

following equation

ϴ = tan-1 (h/r) or Tan ϴ = h / r

Values ≤ 30 indicates free flowing powder and 30 ≥ 40 are poor flowing powders.

Table 5. 3: Angle of repose and corresponding flow property

Angle of repose (θ) Flow property ˂25 Excellent

25-30 Good 31-40 Passable (may hang up) 41-50 Poor (must agitate, vibrate) ˃50 Very poor

71

5.4.2 Evaluation of physical properties of matrix tablets:

5.4.2.1 Thickness and diameter 71, 100

10 tablets were randomly picked from each batch and their thickness

and diameter were measured using a calibrated dial Vernier calliper (± 5% is

allowed).

5.4.2.2 Weight variation test 72, 100

20 tablets were randomly selected from each batch and weighed on

an electronic balance. Weight of 10 tablets and individual tablets were taken;

their mean and standard deviation of weight were calculated from each batch.

Table 5. 4: Maximum allowable deviation for tablets

5.4.2.3 Hardness test 73, 99

10 tablets were randomly selected from each batch and hardness of

each tablet was determined by using a Pfizer type hardness tester. Mean of

standard deviation was calculated for each batch.

5.4.2.4 Friability test 74, 100

It is the ability of tablets to withstand mechanical shocks during

handling and transportation. 10 tablets were selected randomly from each batch

and weighed and placed in a friability test apparatus and operated at a speed of

25 rpm for 4 minutes. Tablets were collected and weighed again. The loss of

Average weight of tablet (mg)

Maximum percentage deviation allowed (%)

130 mg or less ± 10.0 130 – 324 ± 7.5

˃ 324 ± 5

72

tablet weight was calculated and measured in terms of % friability. Acceptable

value of friability is less than 1.

F = [(WINITIAL – WFINAL) x 100] ÷ WINITIAL

5.5 Swelling index of matrix tablets 75

The swelling property of matrix tablets was measured in terms of

percentage weight gain by the tablet. The swelling behaviour of all formulations

were studied. One tablet from each formulation was kept in a petridish

containing pH 7.4 phosphate buffer. At the end of 0.5 h and 1h, the tablets were

withdrawn and soaked with tissue paper, then weighed. Then after each hour,

weight of tablets were weighed and continued till 8h percentage weight gain of

tablets were calculated by the formula

S.I = {(Mt – Mo) / Mo} x 100

Where, S.I = swelling index, Mt = weight of tablet at time t (h), Mo = weight of

tablet at zero time.

Fig 5. 1 : Tablet immersed in dissolution media for swelling index study

73

5.6 Drug content estimation 76:

a) Standard solution: 100mg of pure Metformin HCl (drug) was dissolved in

water in a volumetric flask and the volume was made up to the mark and

sonicated for 5 minutes.

b) Sample solution: 20 tablets from each batch were randomly selected and

weighed accurately and finely powdered. To a powder equivalent to

100mg of Metformin HCl about 70ml of water was added and dissolved

with the aid of shaker for 15 minutes; then sufficient quantity of water was

added to produce 100ml in a volumetric flask, mixed well and filtered. To

1ml of the filtrate methanol was added to produce 100ml and mixed well.

The absorbance of the resulting solution was measured at 233nm using

standard solution as blank. This test was conducted in triplicate.

5.7 In vitro release studies:

5.7.1 Preparation of standard curve for Metformin HCl77:

Preparation of standard stock solution:

Accurately weighed 100mg of Metformin HCl and transferred into a 100ml

volumetric flask, dissolved in 50ml of distilled water and made up to to obtain a

standard stock solution of 1000µg/ml drug concentration. From this Standard

stock solution of Metformin HCl, 100µg/ml (Stock Solution) was prepared by

pipetting 10 ml of stock solution to a 100ml volumetric flask and making up to

100ml with distilled water.

74

Determination of wavelength of maximum amplitude (D2 value) of

Metformin HCL.

10ml of the above solution was diluted to 100ml with the same solvent to

get 10µg/ml of concentration. The UV spectrum of final solution was scanned in

the range of 200 – 400 nm against distilled water as blank. The λmax was found

at 233.8 nm.

STD Curve of Metformin HCl

One mille litre (1ml) of the standard stock solution was taken and diluted

to 10ml with distilled water (100µg/ml), from the above solution 0.2, 0.4, 0.6, 0.8

and 1 ml were pipetted out and diluted to 10 ml with distilled water to get the

final concentration of 2, 4, 6, 8 and 10µg/ml respectively.

5.7.2 In vitro drug release studies78, 79.

Fig 5. 2 : Type I Dissolution apparatus used for study

75

In vitro release studies of prepared matrix tablets and marketed

(Glycomet SR) were conducted for a period of 12 h using an eight station USP

XXII type I apparatus (Fig 5. 2) at 37±0.5ºC; speed of basket was set at 100±1

rpm. In each flask 900ml buffered media (pH 6.8) was used as dissolution

media.

An aliquot (5ml) was withdrawn at every 1 h interval and replaced with fresh

medium to maintain sink condition. Samples were filtered through whatman filter

paper no.1 and diluted appropriately and analysed at 233nm by double beam

UV / visible spectrophotometer using dissolution medium as blank. Experiments

were performed as: n = 3. The amount of drug present in the samples was

calculated by using calibration curve constructed from reference standard.

5.8 RELEASE KINETIC MODELS.

RELEASE MECHANISM OF DRUG

While developing novel drug release system it is essential to understand

the drug release mechanism. There are various types of drug release

mechanisms such as zero order, first order, higuchi model and peppas model.

The most appropriate model is selected by fitting the data into the following

mathematical models and finding which model gives the best fit to them.

5.8.1 Zero-order treatment 80

Zero order takes place at a constant rate independent of existing

concentration or initial concentration. This system is used to describe drug

dissolution of several modified release pharmaceutical dosage forms such as

76

transdermal systems and sustained release tablets (osmotic and matrix) with

low soluble drugs. This model can be expressed as

Qt = Q0+K0t

Where, Qt = Amount of drug released in time (t). Q0= Initial amount of drug in solution, K0= Zero order release constant.

5.8.2 First-order treatment 81

The application of this model was first introduced by Gibaldi and Feldman

(1967) and later by Wagner in (1969). First order treatment takes place at a

constant proportion of drug concentration available at that time so that the

process is depending on the initial concentration. The following equation is used

to express this model.

Log c = Log c0 – kt / 2.303

Where, c = amount of drug remaining unreleased at time t. C0= initial amount of drug in solution. K = first order rate constant.

5.8.3 Higuchi’s model 82

This model was used to study the release of water soluble and low soluble drug

incorporated in semisolid and solid matrices. The following equation is used to

express the model.

Qt = kt1/2

Where, Qt = amount of drug released in time t K = Higuchi’s constant.

A linear relationship between amount of drug released (Q) versus square root

of time (t1/2) is observed if the drug release from the matrix is diffusion controlled.

77

5.8.4 Korsmeyer – Peppas model 83, 84

Kosermeyer in 1983 developed a simple and semi empirical model

relating exponentially the drug release to elapsed time. If diffusion is the main

drug release mechanism, a graph representing the drug amount released

versus square root of time gives a straight line. Under certain experimental

conditions the release mechanism deviated from ficks equation follows an

anomalous behaviour (non fickian). In such condition the following equation is

generally used.

Mt / Minf = atn

Where, Mt / Minf = fraction release of drug. a = constant depending on the structural and geometric characteristics of the drug dosage form. n = release exponent.

Peppas used this ‘n’ value to characterise different release mechanisms.

The value of n indicates the drug release mechanism.

Interpretation of diffusion release mechanisms

Table 5. 5: Value of ‘n’ with corresponding drug release mechanism

Release Exponent ‘n’ Mechanism of drug transport

< 0.5 Fickian transport

0.5< n < 1.0 Non – Fickian Transport

1.0 Case II transport

> 1.0 Super case II transport

78

For cylinder:

n = 0.45 instead of 0.5 and 0.89 instead of 1.0.

This model is used to analyse the release of drug from polymeric dosage forms,

when the release mechanism is not understood or when there is a possibility of

using more than one type of release mechanisms.

5.9 SIMILARITY FACTOR 85, 101

Similarity factor (f2) is a logarithmic reciprocal square root transformation

of the sum of squared error and is a measurement of the similarity in percentage

(%) dissolution between two curves. To evaluate and compare dissolution data,

the dissolution data are statistically analysed using dissolution similarity factor.

Similarity factor can be found out by using the following equation

Where,

n = number of dissolution time points.

Wt = Optional weight factor.

Rt = Reference dissolution point at time t.

Tt = Test dissolution point at time t.

The f2 factor between 50 and 100 suggests that the dissolution is similar

and the f2 values ranging from 100 suggest that the two dissolution profiles are

similar, whereas the smaller values suggest that they are not similar.

79

5.10 STATISTICAL ANALYSIS 102:

Statistical analysis was performed using Minitab software on the

experimental data and the data obtained from regression analysis are shown

below.

5.11 STABILITY STUDIES 86, 87:

It is defined as “the capacity of a drug product to remain within the

specifications established to ensure its identity, strength, quality, and purity”. It

can be simply explained as the ability of a drug to resist deterioration”.

Short-term stability study: Was performed at temperature 40 ± 2ºC over

a period of three months on the matrix tablet (MFH 14). Sufficient number of

tablets (10) were packed in amber colored screw capped bottles and kept in

stability chamber maintained at 40 ± 2ºC. Samples were taken at one month

interval for drug content estimation. At the end of three months, tablets were

evaluated for drug content, percentage friability, swelling index and dissolution

test to determine the drug release profiles.

Long-term stability study: Was performed at a temperature of 25 ± 2ºC

60% ± 5% RH over a period of twelve months on the matrix tablet (MFH 14).

Sufficient number of tablets (10) were packed in amber colored screw capped

bottles and kept in stability chamber maintained at 25 ± 2ºC. Samples were

taken at 0, 3, 6, 9 and 12 month interval for drug content estimation. On each

specified interval tablets were evaluated for drug content, percentage friability,

swelling index and dissolution test to determine the drug release profiles.

80

Table 5. 6: Conditions as per ICH Guidelines

5.12 In vivo studies 5.12.1 Pharmacokinetics studies 5.12.1.1 Blood Sample Collection

Three groups of Rabbit, each comprising of three, were used for the

pharmacokinetic analysis. Each rabbit of all the groups were given 400 mg/kg

of drug.

Table 5. 7: Treatment of different formulations to various groups of rabbit

Group Treatment

A STD Drug solution of Metformin HCl (p.o)

B Marketed tablet of Metformin HCl (p.o)

C Sustained Release F14 tablet formulation (p.o)

The rabbits were acclimated with laboratory conditions for one week.

Before pharmacokinetic study, all the rabbits were fasted overnight. At zero

hour 1 ml of blood sample was collected from marginal ear vein of each animal

and this was considered as blank. No food or liquid other than water was

permitted until 4 h following administration of product (normal t1/2 of drug 2 h).

Blood samples were collected at 0.5,1, 2, 3, 4, 6, 8, 16, and 24 h intervals from

marginal ear vein into heparinized centrifuge tubes.

Study Storage condition

Minimum time Temperature Relative humidity %

Long term 25ºC ± 2ºC 60% ± 5% RH 12 Months

Intermediate 30ºC ± 2ºC 65% ± 5% RH 6 Months

Accelerated 40ºC ± 2ºC 75% ± 5% RH 3 Months

81

5.12.1.2 Plasma samples extraction

For spiked samples as well as animal blood plasma sample, the following

earlier procedure was used for HPLC analysis to extract Metformin HCl. 100 µl

of Metformin HCl solution of appropriate concentration and 100 µl of Glipizide

solution (5 µg mol-1) were added to 900 µl of drug free plasma contained in a

clean 5 ml Ria vial properly mixed (for spiked samples). After centrifugation at

3000 rpm for 15 minutes, 700 µl of the supernatant was evaporated to dryness

at 45o C under nitrogen. The residue was reconstituted in 100 µl of mobile phase

and 20 µl of this was injected to HPLC system (Shimadzu LC-10AT with SPD-

10A detector).

5.12.1.3 Method Validation of Metformin HCl in HPLC system88

Instead of developing a new method, the already reported method was

used for Metformin HCl in plasma level. According to this method, HPLC using

C18 ODS (5 μ) 250 × 4.60 mm column, mobile phase selected for this method

contained acetonitrile: phosphate buffer (65:35) pH adjusted to 5.75 with o-

phosphoric acid which was filtered through 0.2 μ membrane filter. Flow rate

employed was 1.0 ml/ min. Detection of eluent was carried out at 233.0 nm.

Glipizide was used as the internal standard. Column was saturated with mobile

phase for about an hour at the above specified conditions. HPLC method was

used for validation of specificity, linearity and range, precision, accuracy,

robustness and solution stability according to USP and ICH guidelines.

After setting the chromatographic conditions the instrument was stabilized

to obtain a steady base line and a mixed standard dilution of pure drug

82

containing 10 μg/ml of Metformin HCl and 5 μg/ml of glipizide (internal standard)

were prepared in mobile phase, filtered through 0.2 μ membrane filter and

loaded in the injector of instrument fitted with 20μl fixed volume loop. The

solution was injected three times and the chromatogram recorded. The mean

retention time for Metformin HCl and glipizide were found to be 2.30 and 3.95

min, respectively.

Standard stock solutions of Metformin HCl and glipizide with a

concentration of 100 μg/ml was prepared separately in the mobile phase. For

the preparation of drug solutions for the calibration curve, aliquots of standard

stock solution of Metformin HCl (0.25, 0.5, 1.0, 1.5, 2.0 and 2.5 ml) were

transferred into a series of 10 ml volumetric flask and to each flask 0.5 ml of

glipizide standard stock solution was added and the volume made up to the

mark with mobile phase. Each solution was injected after filtration through 0.2 μ

membrane filter and a chromatogram was recorded. The calibration curve was

plotted between concentration of drug and ratio of peak area of Metformin HCl

and Glipizide (internal standard). Linearity was found to be in a concentration

range of 0 to 25 μg/ml of Metformin HCl with linear regression equation as y=

0.0204x+0.0012 and the correlation coefficient value of 0.9990.

After setting the chromatographic conditions and stabilizing the

instrument, the formulated sample solution was injected and a chromatogram

was recorded. The injection was repeated three times and the peak area of

Metformin HCl and Glipizide were recorded. The peak area ratio of drug to

internal standard was calculated and the amount of drug present was estimated

83

from the respective calibration curve. The analysis of formulated tablet was

compared with the commercially available tablet formulation of Metformin HCl.

5.12.2 Pharmacodynamic studies 5.12.2.1 Induction of Diabetes 89

Diabetes mellitus (DM) was induced by a single intravenous (IV) injection

of Alloxan monohydrate (150 mg/kg, body wt.), dissolved in 0.1 M sodium citrate

buffer (pH 4.5). Only vehicle (citrate buffer, 1 ml/kg) was given to fasted-alloxan

treated rabbits80. In order to reduce the death rate due to hypoglycaemic shock,

alloxan-treated rabbits received 5% of glucose instead of water for 24 h after

diabetes induction (Barbosa et al., 2008).Hyperglycaemia was confirmed by

elevated glucose levels in plasma, which was determined at 72 h, following 7th

day after injection (stabilization period). The threshold value of fasting plasma

glucose to diagnose diabetes was taken as >200mg/dl. Only rabbits having this

blood glucose levels were used for the study.

5.12.2.2 Experimental design

Rabbits used for experiments were divided into groups of 6 animals each.

Table 5. 8: Experimental Design for Pharmacodynamic studies in rabbit

Group Treatment

I Diabetic rabbits - given only Alloxan (150 mg/Kg, i.p.)

II Diabetic rabbits treated with Standard Tablet(p.o)

III Diabetic rabbits treated with Formulation Tablet (p.o)

84

5.12.2.3 Administration of drugs

On day one (01), fasted-treated rabbits were administered with standard

and test tablets through intragastric tube and this was repeated for three

consecutive days (1-3) ( orally) and fasted alloxan treated rabbits were given

vehicle only (citrate buffer, 1 ml/kg).

5.12.2.4 Blood Sample Collection and determination of blood glucose

At the end of the 3rd day of drug treatment, blood was collected from

marginal ear veins with the help of sterilized needle and syringe, at 0h, 2nd h, 3rd

h, 4thh, 6thh, 8thh, and 10th h and transferred into the appropriate sterilized micro

centrifuge tube. The blood was used to determine Fasting blood glucose (FBG)

level.

5.12.2.5 Body weight

Body weight of animals in each group was recorded on 0,7th and 14th day

and difference in weight were noted.

85

RESULT AND DISCUSSIONCHAPTER 6

86

6. RESULTS AND DISCUSSIONS

6.1 Preformulation studies Description: Visual inspection of the drug was done.

Table 6. 1: Result of visual inspection of Metformin HCl

Property Observation Organoleptic

properties A white crystalline powder, without any characteristic

odour

Melting point: was found to be in the range of 222ºC to 226ºC

Solubility of drug in different media:

Table 6. 2: Solubility of Metformin HCl in different media

Solvent Solubility (mg/ml)

Distilled water 145

SGF (pH-1.2) 256

SIF (pH-6.8) 282

SIF (pH-7.4) 156

6.2 Compatibility studies FT-IR: The compatibility study of Metformin HCl was carried out using FT-

IR spectrum. The IR spectra of pure drug and drug polymer mixtures (1:1) are

given in figures

Fig 6. 1, Fig 6. 2, Fig 6. 3 and Fig 6. 4. Figures shows qualitative

identification for Metformin HCl. FT-IR characterisation indicated that no

interactions of drug were observed neither with exepients nor with additives.

This is comparable to a study done by Kamlesh J et . al in 2011 confirmed in

their study that excipients didn’t react with the ingredients.

87

Fig 6. 1 : FT-IR peak of different functional groups of Metformin HCl

88

Fig 6. 2 : FT-IR peak of different functional groups of Metformin HCl and HPMC K4M

89

Fig 6. 3 : FT-IR peak of different functional groups of Metformin HCl and HPMC K15M

90

Fig 6. 4 : FT-IR peak of different functional groups of Metformin HCl and HPMC K100M

91

DSC: Has been used to measure the amount of heat energy absorbed

(endothermic) or released (exothermic) by the drug when it is heated or cooled.

The thermal curve of Metformin HCl showed an initial flat profile followed by a

sharp endothermic peak representing the melting of the substance in the range

of 223 - 237ºC. The thermal curves of both mixtures obtained by simple blending

gave a superimposition as that of single component indicating the absence of

solid-state interaction as shown in Fig 6.5. A similar study was conducted by

Raghavendra Rao N et al in 2009 prepared Tramadol HCl matrix tablet revealed

Fig 6. 5: DSC of pure Metformin HCl (a), Physical mixture of Metformin HCl with HPMC K100M, Physical mixture of Metformin HCl with HPMC K100M and CMC.

92

PXRD: Solid substances are usually characterised as either crystalline or

amorphous state. Spacing of atoms or molecules in crystals are of repetitious

spacing whereas in amorphous form they are randomly placed which is similar

to liquids. Amorphous forms are of higher thermodynamic energy than the

crystalline form due to random arrangement of atoms and molecules hence the

energy required for separation is low, so their solubility and dissolution rates are

higher.

The physical mixture of formulation drug peak was observed with varying

intensity. This suggests that in the formulation drug remains in crystalline form

and it doesn’t transform into amorphous state (Fig 6.6).

Fig 6. 6: X-ray diffraction studies of pure Metformin HCl and formulation blend containing Metformin HCl, HPMC K100M and CMC.

93

6.3 Pre-optimisation studies

6.3.1 Optimisation of polymer concentration

Matrix tablets of Metformin HCl were prepared in each batch using

different concentration of polymers i.e. from lower to higher concentration. The

formulated tablets were evaluated for its swelling index and drug release at Q2,

Q8 and Q12.

The concentration of polymers and its effect on response variables helped

to fix the higher and lower concentration of polymers. The higher concentration

of various grades of HPMC were selected as it gave more controlled release of

drug with sufficient swelling index and drug release at Q2, Q8 and Q12 h. among

batch 1, batch 2 and batch 3, MFH9 was selected as it gave the desired release

and swelling index pattern when compared to others. Batch 4 was prepared by

using a combination of HPMC K100M and CMC in order to reduce the burst

release at Q2 interval. This results can be compared with R. Charulatha et al in

2012, which states that HPMC K100M with Na CMC was effective in retarding

the release as the polymer ratio increased.

6.4 Design and preparation of swelling restricted matrix tablet of

Metformin HCl

Tablets from Batch 4 (MFH 12) were selected as they met the fixed

parameters of release and swelling index. This formulation was converted to

swelling restricted matrix tablet by partially coating them with 5% cellulose

acetate solution. By doing so, there swelling was restricted and hence release

was further retarded and also no studies were found in literature to compare it.

94

6.5 Evaluation of flow properties of powder

Powders prepared for direct compression method were evaluated by

measuring the following parameters such as bulk density, angle of repose,

Hausner’s factor, compressibility index, and drug content. The results are shown

in Table 6. 3.

Angle of repose: The result of angle of repose (<30) indicate good flow

properties and the values for prepared formulations ranges from 21.59 –

26.37.

Hausner’s factor: The values of Hausner’s factor were under satisfactory

range.

Compressibility index: the values up to 15% results were in good to

excellent flow properties and values of all formulations ranges from 17.29

– 20.60%.

Drug content: the values of all the formulations were in the range from

97.48% - 99.83%.

All these results obtained indicated that the granules possessed satisfactory

flow properties, compressibility, and uniform drug content.

95

Table 6. 3: Values of pre – compression parameters of developed formulations, n = 3

Sl.No LBD (gm/cc)

TBD (gm/cc)

Hausner’s Factor

Carr's compressibility Index (%)

Angle of repose (º)

MFH 1 0.4244 0.5345 1.26 20.60 21.59±0.012

MFH 2 0.4156 0.5126 1.23 18.92 23.52±0.013

MFH 3 0.4136 0.5164 1.25 19.91 26.37±0.021

MFH 4 0.4247 0.5135 1.21 17.29 22.74±0.026

MFH 5 0.4275 0.5274 1.23 18.94 23.93±0.069

MFH 6 0.4172 0.5164 1.24 19.21 25.49±0.010

MFH 7 0.4225 0.5203 1.23 18.80 21.73±0.040

MFH 8 0.4394 0.5428 1.24 19.05 25.30±0.062

MFH 9 0.4199 0.5194 1.24 19.16 22.38±0.042

MFH 10 0.4291 0.5273 1.23 18.62 24.28±0.064

MFH 11 0.4315 0.5349 1.24 19.33 22.66±0.055

MFH 12 0.4174 0.5158 1.24 19.08 25.44±0.028

96

6.6 Evaluation of tablets

6.6.1 General appearance

The formulated tablets were evaluated for its organoleptic characteristics

as shown in Table 6. 4.

Table 6. 4: Observational report of various parameters of tablets

Parameters Observation

Shape All tablets remained in caplet shape with no visible cracks.

Colour Off white

Odour No characteristic odour

Appearance All tablets were elegant in appearance

6.6.2 Hardness Normally for oral tablets the hardness range is in between 5 – 10 kg/cm2.

Hardness of the tablets were tested using Pfizer type hardness tester and the

results are shown in Table 6. 5. Hardness of all the tablets were within the

acceptable limit i.e. from 5.23 kg/cm2 – 5.87 kg/cm2. Hence all the formulated

tablets pass the hardness test.

6.6.3 Thickness

All formulated tablets were evaluated for uniformity of thickness using a

Vernier calliper and the results are shown in Table 6. 5. The thickness of all the

formulated tablets were in the range of 3.20 ± 2.00 mm which indicated that all

the tablets were of uniform thickness.

97

6.6.4 Weight variation test

The test was performed as per the official method. Twenty tablets from

each batch were selected randomly and individually weighed. Each tablet was

evaluated for percentage deviation and their results are shown in Table 6. 5.

Allowable percentage deviation for prepared tablets are ±5 % for weight more

than 324 mg. and the results showed that all were within the acceptable limits.

6.6.5 Friability test

The apparatus used for conducting friability test was Roche friabilator.

Pre-weighed 10 tablets were taken and loaded into the apparatus and the

revolution speed was adjusted to 100. After 100 revolutions the tablets were

dusted and reweighed. The results are shown in Table 6. 5. The acceptable

weight loss for tablets should not be more than 1% of the weight of tablets. The

friability of all tablets were within the prescribed limits, which showed that there

was a good adhesion property between the excipients used in the formulation.

6.6.6 Drug content

The percentage content of Metformin HCl in the formulation was

estimated at 234 nm using spectrophotometer. The limit for content uniformity

should be in the range of 90 – 110% and the results showed that content

uniformity of Metformin HCl in the formulation was between 97.48 ± 0.54 – 99.83

± 0.72% (Table 6. 5), which is within the acceptable limits.

All the formulation in batches from MFH1 – MFH 12 showed values within

the specified limits for all test and hence complying with pharmacopoeial

specifications indicating that all prepared tablets were of standard quality.

98

Table 6. 5: Results of post-compression parameters

Batch Hardness Thickness %

Friability Weight

variation % Drug content

MFH 1 5.23 3.40 0.8 860.54 99.83

MFH 2 5.44 3.60 0.75 870.67 98.45

MFH 3 5.57 3.80 0.64 854.98 99.38

MFH 4 5.27 3.50 0.56 848.63 99.68

MFH 5 5.47 3.70 0.58 859.3 97.48

MFH 6 5.50 3.50 0.64 855.37 98.37

MFH 7 5.30 3.60 0.65 851.84 99.25

MFH 8 5.50 3.40 0.59 862.75 98.57

MFH 9 5.50 3.50 0.54 847.85 99.73

MFH 10 5.87 3.60 0.63 857.83 98.49

MFH 11 5.60 3.50 0.68 860.37 99.28

MFH 12 5.63 3.70 0.53 854.48 99.63

6.7 Swelling index study

Swelling index study was performed on all batches for a period of 2 h to

12 h. From the result of this evaluation test it was observed that there was a

linear relationship between swelling index and concentration of polymer till 8 h

which was due to the formation of a viscous gel type mass. Afterwards the gel

mass got eroded or dissolved in the media from the outermost layer of tablet.

The results of swelling index study are shown in Table 6. 6.

99

Fig 6. 7: Shows the swelling index of the formulations from MFH1 – MFH14

Fig 6. 8: Swelled tablet in SGF while conducting swelling index study.

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

% S

WEL

LIN

G IN

DEX

TIME

Swelling Index

MFH1 MFH2 MFH3 MFH4 MFH5 MFH6 MFH7

MFH8 MFH9 MFH10 MFH11 MFH12 MFH13 MFH14

100

Fig 6. 9: Tablets formed gel like mass when conducting swelling index study

101

Table 6. 6: Showing results of % swelling index value of all formulations.

Time In Hours % Swelling Index

MFH1 MFH2 MFH3 MFH4 MFH5 MFH6 MFH7 MFH8 MFH9 MFH10 MFH11 MFH12 MFH13 MFH14

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 40 42 43 41 44 45 42 44 41 44 41 47 26 20

4 49 52 55 52 56 58 51 55 51 53 53 59 34 29

6 57 60 64 58 62 66 59 60 63 64 71 76 52 41

8 82 85 87 81 84 88 83 84 84 81 82 88 65 59

10 74 75 75 73 71 77 74 76 82 73 76 91 75 66

12 64 66 72 66 69 70 69 71 74 68 72 85 68 61

102

6.8 In vitro dissolution studies

6.8.1 Preparation of calibration curve

Calibration curve of Metformin HCl was plotted with concentration in x-axis and

absorbance in y-axis (Fig 6.10).

Table 6. 7: Absorbance values of Metformin HCl in distilled water.

Concentration (µG/ML) Absorbance at 234nm 2 0.1915 4 0.4186 6 0.5465 8 0.7371

10 0.9155

Fig 6. 10: Calibration curve of Metformin HCl in distilled water

y = 0.0906x + 0.0152R² = 0.9963

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2 4 6 8 10 12

Ab

sorb

ance

Concentration

Metformin HCl STD Curve

Absor

Linear (Absor)

103

6.9 In vitro drug release studies:

The results of cumulative percentage of drug release are given in the

Table 6. 8 and Table 6. 9 which clearly indicated that the release rate and

percentage drug release showed a wide variation. From the obtained result it

was understood that the drug release was strongly affected by the concentration

of polymer in the formulation (Fig 6. 13). The matrix tablet during swelling is an

aggregate mass of water swollen polymer, drug and excipients which

experiences various degree of hydration. The solid content in the tablet of

various regions varied from 0 – 100%. The area of 100% solid was just a wetted

mass of powder. When the water content of wetted mass increased the polymer

became hydrated and formed a gel. In the outermost layer the polymer had no

structural integrity and hence got eroded.

Fig 6. 11: Tablet removed at Q2 interval form dissolution study

104

Formulations fabricated with different grades of HPMC alone i.e. MFH1 –

MFH 9 showed an initial burst release but release was retarded by increasing

the concentration of polymer, which may be due to the formation of a thick

viscous gel layer around the tablet. Among these formulations (MFH 1 – MFH

9), the best formulation i.e. MFH 9 was selected and treated with combination

of HPMC K100M and CMC in different ratios (MFH 10 – MFH 12), which resulted

in a decrease in the initial burst release and hence a more retarding effect was

observed. From MFH 10 – MFH 12 formulations MFH 12 was selected as there

was a decrease in the drug release, particularly at Q2 H interval and was

converted to swelling restricted matrix tablet (MFH 13 & MFH 14), where the

release was more effectively retarded to the desired frequency.

Comparative results of optimised formulation and marketed formulation

are shown in Fig 6.14.

Fig 6. 12: Performing dissolution test in type I apparatus

105

Table 6. 8: In Vitro %CDR of drug from Metformin HCl matrix tablets MFH 1 to MFH 7 (n = 3)

Time in h MFH1 MFH2 MFH3 MFH4 MFH5 MFH6 MFH7

1 45.00±0.577

34.07±0.581

27.00±0.635

42.74±0.491

25.64±0.670

25.07±0.581

26.63±0.591

2 59.20±0.325

41.25±0.557

31.44±0.694

48.47±0.664

42.02±0.578

32.28±0.582

40.05±0.580

3 62.09±0.550

46.55±0.652

40.58±0.462

56.38±0.665

48.45±0.557

41.21±0.532

52.08±0.583

4 67.49±0.550

53.05±0.580

46.78±0.665

62.46±0.559

57.09±0.585

46.65±0.614

59.07±0.581

5 71.09±0.550

61.15±0.522

58.48±0.580

70.36±0.560

62.81±0.724

57.52±0.553

63.31±0.701

6 76.56±0.606

54.82±0.318

62.06±0.580

76.21±0.723

70.77±0.722

63.02±0.578

69.11±0.587

7 83.70±0.665

71.64±0.664

66.56±0.580

78.06±0.581

75.12±0.590

71.06±0.580

71.40±0.401

8 87.26±0.696

76.09±0.585

71.06±0.580

81.40±0.666

79.51±0.609

77.33±0.668

74.17±0.639

9 90.93±0.521

80.78±0.434

80.06±0.617

84.40±0.723

83.35±0.486

81.03±0.578

82.43±0.705

10 97.90±0.458

87.05±0.580

86.26±0.617

93.31±0.724

89.03±0.578

85.46±0.638

90.48±0.640

11 98.03±0.469

91.63±0.611

90.73±0.536

97.51±0.701

95.21±0.645

91.60±0.680

94.31±0.667

12 98.25±0.531

97.10±0.586

96.53±0.811

98.45±0.476

98.32±0.562

94.43±0.669

97.66±0.599

106

Table 6. 9: In vitro %CDR of drug from Metformin HCl matrix tablets MFH8 to MFH14, (n = 3)

Time in h MFH8 MFH9 MFH10 MFH11 MFH12 MFH13 MFH14

1 23.30±0.723

22.20±0.917

25.07±0.581

21.40±0.700

22.30±0.700

20.73±0.433

18.32±0.610

2 39.28±0.676

33.03±0.578

37.44±0.747

36.77±0.754

36.01±0.577

34.07±0.582

31.31±0.701

3 51.08±0.583

36.09±0.584

49.21±0.740

48.22±0.646

40.38±0.583

38.85±0.522

39.48±0.525

4 55.59±0.621

47.98±0.591

54.82±0.725

53.05±0.580

46.95±0.580

47.61±0.552

45.81±0.725

5 61.56±0.617

54.89±0.948

61.09±0.584

59.19±0.641

58.52±0.405

60.28±0.550

58.03±0.578

6 68.11±0.588

61.86±0.940

65.66±0.810

65.03±0.611

64.32±0.439

66.07±0.581

66.07±0.582

7 74.17±0.639

69.32±0.659

70.12±0.611

71.78±0.755

68.32±0.496

71.06±0.580

74.62±0.554

8 77.78±0.722

72.41±0.463

72.03±0.578

75.12±0.589

73.80±0.724

75.53±0.611

80.06±0.581

9 82.42±0.741

76.43±0.869

76.33±0.704

80.66±0.882

78.08±0.583

79.09±0.585

83.11±0.588

10 87.16±0.617

79.09±0.585

81.03±0.578

89.03±0.578

84.11±0.588

85.56±0.549

91.20±0.643

11 93.26±0.655

82.42±0.712

85.46±0.696

93.73±0.722

91.00±0.577

89.07±0.581

92.77±0.722

12 97.05±0.580

89.70±1.193

90.10±0.635

95.17±0.601

93.50±0.529

91.80±0.723

94.70±0.723

107

Fig 6. 13 : In vitro dissolution profile of Metformin HCl matrix tablet.

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0 2 4 6 8 10 12 14

Mea

n %

CD

R

Time

MEAN % CDR Vs TIME

MFH1 MFH2 MFH3 MFH4 MFH5 MFH6 MFH7

MFH8 MFH9 MFH10 MFH11 MFH12 MFH13 MFH14

108

Fig 6.14: In vitro dissolution profile of Glycomet SR and Formulation F14

6.10 Release kinetic model:

To find out the drug release from the obtained data, all the data were fitted

into various kinetic models such as zero order, first order and higuchi model.

Linear regression analysis was done for all the batches and their results are

shown in the following Table 6. 10. The higuchi square root of time model had

the higher r2 values which when compared to zero order and first order kinetic

models showed that they followed higuchi model.

Further, to understand the drug release mechanism, the data were fitted

in to peppas model and the obtained ‘n’ values indicated the drug transport

mechanism. From the obtained data for peppas model, ‘n’ values were within

the range of 0.5 < n < 1.0 as shown in Table 6. 10, which clearly indicated that

the drug release followed non – fickian anomalous transport diffusion

mechanism which is comparable to a study done by T. Raja Sekharan et al in

2011 explained released kinetic followed korsmeyers peppas model and

mechanism of drug release was non-fickian for the preparation of HPMC based

controlled release matrix tablet.

0.00

20.00

40.00

60.00

80.00

100.00

120.00

1 2 3 4 5 6 7 8 9 10 11 12

Con

cent

ratio

n

Time

Glycomet SR Vs F14

Glycomet SR F14

109

Table 6. 10: Correlation coefficient values and release kinetics of Metformin HCl matrix tablets

Formulation Zero order First order Higuchi model Peppas model

AVG k AVG r2 AVG k AVG r2 AVG k AVG r2 AVG n AVG r2 MFH 1 4.7751 0.9637 -0.3372 0.8951 0.2218 0.9851 0.3192 0.9818 MFH 2 5.6638 0.9961 -0.2394 0.8642 0.2605 0.9884 0.4367 0.9785 MFH 3 6.3877 0.9919 -0.2400 0.8709 0.2937 0.9836 0.5463 0.9771 MFH 4 5.0957 0.9788 -0.3068 0.8537 0.2365 0.9891 0.3568 0.9798 MFH 5 6.1117 0.9685 -0.2900 0.8588 0.2863 0.9967 0.5196 0.9936

MFH 6 6.4548 0.9845 -0.2262 0.9484 0.2992 0.9924 0.5679 0.9899 MFH 7 5.9096 0.9635 -0.2679 0.8669 0.2762 0.9873 0.5003 0.9888 MFH 8 6.0788 0.9619 -0.2519 0.8985 0.2854 0.9947 0.5421 0.9876 MFH 9 5.9328 0.9638 -0.1673 0.9762 0.2777 0.9908 0.5804 0.9902

MFH 10 5.2974 0.9507 -0.1634 0.9699 0.2498 0.9914 0.4920 0.9903 MFH 11 6.3020 0.9698 -0.2406 0.9281 0.2946 0.9942 0.5762 0.9894 MFH 12 6.2578 0.9810 -0.2106 0.9403 0.2908 0.9937 0.5722 0.9935 MFH 13 6.3349 0.9632 -0.2014 0.9800 0.2969 0.9926 0.6043 0.9920 MFH 14 7.0509 0.9647 -0.2537 0.9683 0.3300 0.9913 0.6765 0.9936

110

The Precompression and post compression in this study were evaluated

and were within acceptable limits. Data were fitted into various kinetic models

which was comparable to a study done by Mohammed Raquibul Hasan et al in

2014 for the preparation of ER Metformin HCl.

6.11 Stability studies:

Short term stability study: Accelerated stability studies were conducted

to prove how the manufactured tablets may change with respect to time under

the influence of environmental factors like temperature and humidity. As per the

guidelines, short term stability study was conducted for a period of 6 months (

Fig 6. 14) with temperature at 40 ± 2ºC and 75 ± 5% relative humidity. It

showed negligible changes in respect to appearance, drug content, dissolution

and assay. The obtained results are shown in table 6.11.

Optimised formulations are considered to be stable as they were considerably

stable even after the storage of 6 months and there was no change from the

initial assay of 5% or more. This confirmed that the formulations were stable

during the stability study period.

Table 6. 11: Results of short term stability study of MFH14

Evaluation MFH 14 SF14 Colour Off white No change

Drug content % 99.63 ± 0.53 99.56 ± 0.48

Swelling index Q2 (h) Q8 (h) Q2 (h) Q8 (h)

20.0% 58.82% 21.2% 58.67%

Hardness 5.63 5.60

Friability 0.53% 0.60%

111

Fig 6. 14 : Dissolution profile of MFH 14 Vs SF14 (formulation after stability study)

Long Term Stability Study: As per the guidelines, long term stability

study was conducted for a period of 12 months (Fig 6.15) with temperature at

25 ± 2ºC and 60 ± 5% relative humidity. It showed negligible changes in respect

to appearance, drug content, dissolution and assay. The obtained results are

shown in.table 6.12. Optimised formulations are considered to be stable as they

were considerably stable even after the storage of 12 months and there was no

change from the initial assay of 5% or more. This confirmed that the formulations

were stable during the stability study period. A similar studies was found in the

literature by Abdelkader H et al in 2007 and Mohammed Abdul Hadi et al in 2012

found that there were no significant changes in drug content after stability

studies for optimised formulation.

0.0

20.0

40.0

60.0

80.0

100.0

120.0

1 2 3 4 5 6 7 8 9 10 11 12

Co

nce

ntr

atio

n

Time

SHORT TERM STABILITY STUDY

MFH 14

SF14

112

Table 6. 12: Results of Long term stability study of MFH14

Fig 6. 15: Dissolution profile of MFH 14 after long term stability study

6.12 Similarity factor:

Similarity factor of formulation MFH14 when compared with reference

formulation was 70.228.

Evaluation 0 Month 12 Month Colour Off white No change

Drug content % 99.63 ± 0.53 95.56 ± 0.47

Swelling index Q2 (h) Q8 (h) Q2 (h) Q8 (h)

20.0% 58.82% 22.6% 60.15%

Hardness 5.63 5.85

Friability 0.53% 0.84%

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14

Co

nce

ntr

atio

n

Time

Long Term Stability Study

0 Month 3 Month 6 Month 9 Month 12 Month

113

6.13 In vivo release studies

6.13.1 Pharmacokinetic study of Metformin HCl

Overnight fasted rabbits were treated with standard drug (Group A),

marketed formulation (Group B) and MFH 14 formulation (Group C). Blood

samples were collected, plasma extraction was done, method was validated and

the obtained data were plotted with time along y – axis and concentration of

drug in plasma along x – axis. Fig 6. 16 is showing the graph of standard drug,

marketed formulation and MFH14 formulation which was administered to the

animal. From the graph it is understood that the MFH 14 formulation can

effectively maintain the blood plasma level up to 24 h which is more than the

marketed formulation.

Fig 6. 16: Comparative Plasma level of Metformin HCl in Rabbits

-1

-0.5

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30

Co

ncn

etr

atio

n

Time

Plasma Concnetration Vs Time

Reference STD Marketed MFH 14

114

6.13.2 Pharmacodynamic study of Metformin HCl

After the injection of alloxan for inducing diabetes in healthy rabbits the

following stages were observed.

1. Within few minutes of alloxan injection a sudden hypoglycaemic phase

was observed, which lasted for 20 minutes of onset of hypoglycaemic

stage.

2. After one hour of administration of alloxan an increase in the

concentration of blood glucose level was observed. This hyperglycaemic

phase lasted for 2 – 4 h.

3. After 4 – 8 h of alloxan injection again hypoglycaemic stage was

developed which persisted throughout the study.

After the 3rd stage it was confirmed that alloxan had induced pancreatic

beta cell toxicity which formed diabetogenicity in the alloxan treated rabbits.

Before injecting alloxan, rabbits were orally administered with 2g of glucose /kg

body weight in 10 ml of distilled water. After injection of alloxan, rabbits were

stabilised for a period of 7 days, with free access to food and water. On the

seventh day all surviving diabetic rabbits were randomly tested for blood sugar

levels and the results were ranging from 200mg/dl to 650mg/dl, which confirmed

that all rabbits had developed type 2 diabetes, adequately required for the study.

115

Rabbits were divided into 3 groups and each group were treated

differently i.e. group A (standard drug), group B (MFH 14 Formulation) and

group C –control- (only alloxan). From the obtained data shown and plotted in

Fig 6. 17, MFH 14 formulation was effective in controlling the blood glucose level

in a more consistent and steady form, which clearly indicated that MFH14

formulation was effective in controlling diabetes rather than marketed

formulation.

Fig 6. 17 : Effect of MFH 14 formulation on alloxan induced rabbits

6.13.3 Effect of body weight on alloxan treated rabbits

Alloxan treated rabbits were divided in to 3 groups and treated with

standard formulation, MFH 14 formulation, and the last group was kept as

control. Weight of all the rabbits were taken from initial day up to 10 days of

treatment. From the obtained data it was observed that groups treated with

standard formulation and MFH 14 formulation had a marginal increase in the

25

6.6

7

28

2

27

2

25

6.3

3

21

6.6

7

15

8.3

3

12

8.3

3

26

7.5

25

4.2

5

21

7.7

5

20

1.7

5

19

2

18

3.2

5

14

4.7

5

32

0.6

7

36

7.5

45

6.6

7

34

2.6

7

33

4.3

3

31

9

29

0.3

31 2 3 4 5 6 7

PLA

SMA

GLU

CO

SE M

G/D

L

TIME (H)

PLASMA GLUCOSE VS TIME

Alloxan + STD Alloxan + MFH 14 Only Alloxan

116

body weight while the group that was kept as control had a marginal decrease

in the body weight (Fig 6. 188).

Fig 6. 18: Effect of new formulation on body weight.

Pharmacokinetic parameters were found by collecting blood samples

from marginal ear vein of rabbits, extraction of plasma, and method validation in

HPLC system (Fig 6. 199) and was compared with a marketed formulation as

shown in Fig 6. 16. From the obtained data it could be understood that MFH 14

formulation maintained plasma concentration for a period of 24 h. Hence it could

effectively control the release of Metformin from the formulation.

1.2

1.1 1

.151

.26

1.1

5

1.1

1.3

1.2

2

1.0

8

A L L O X A N + S T D A L L O X A N + M F H 1 4 O N L Y A L L O X A N

BO

DY

WEI

GH

T

GROUPS

GROUP VS BODY WEIGHTDay 0 Day 7 Day 10

117

Fig 6. 19: Chromatogram showing Metformin HCl and internal standard Glipizide

Table 6. 13: Pharmacokinetic parameters obtained from three different formulations of Metformin HCl in rabbits (using Residual method PK analysis)

Sl.No PK parameters Type of formulation

Reference STD solution

Marketed product

Formulated SR tablet

1 Cmax 2.2 ng/ml 1.4 ng/ml 1.2 ng/ml 2 Tmax 2 h 2 h 2 h

3 Elimination rate constant (Ke)

0.1388/h 0.0506/h 0.0455 /h

4 Absorption rate constant (Ka)

0.1524/h 0.6222/h 0.6284 /h

5 AUC0t 61.64 ng h/ml 7905.23 ng h/ml

9040.22 ng h/ml

6 Elimination half-life (t1/2)

0.5 h 13.69 h 15.23 h

7 Vd 6.45 litres 23.66 litres 25.85 litres 8 ClT 14.92 ml/min 19.95 ml/min 20.50 ml/min

118

6.14 Statistical analysis

Mathematical relationships for the measured dependent variable

(response) and the independent variables were developed using statistical

software, Graph pad. The two output variables (responses), such as Release

Time (R1) and percentage drug release (R2) were evaluated. The predicated

and actual values of the responses were calculated and found to be in good

agreement with experimental values.

From the t-test comparison of MFH 14 and marketed product,

calculated ‘t’ value was 0.0009. There was no difference between the MFH 14

and marketed formulations. The MFH 14 formulations were subjected to short

term stability studies, namely, for a period of 3 months as per ICH guidelines at

room temperature (25ºC ± 2ºC and 60% ± 5% RH) and accelerated conditions

of temperature and humidity, (40ºC ± 2ºC and 75% ± 5% RH) respectively for

finding the effect of ageing on release pattern. Both the release data were

subjected to t- test and the ‘p’ value was ˂ 0.0001 which was statistically very

significant. Hence there was no significant difference between the formulations.

Therefore MFH 14 can be taken as the best formulation. From the in vivo studies

of marketed and MFH 14 formulations ‘p’ value was 0.0016 which suggested

that they were statistically significant. There were no similar in vivo studies were

found in the literature to compare.

119

CONCLUSIONCHAPTER 7

120

7. CONCLUSION

In the present study an attempt was made to formulate and develop a

controlled release swelling restricted matrix tablet containing Metformin HCl

using various ratios of synthetic polymers. Initially chemical interactions were

found out using Fourier transform infrared spectrophotometer, Differential

scanning calorimetry and powdered X-ray diffraction technique. From the study

it was concluded that there was no chemical interaction between the drug and

the exepients used for the formulation of matrix tablets and at the same time the

drug existed in crystalline nature and didn’t changed to its amorphous form.

From the pre optimisation studies it could be concluded that lower and

higher concentration of polymers gave undesired release patterns. It helped in

fixing the ratio of polymers in final batches of formulations, which gave sufficient

swelling index and release patterns. The effect of different ratio of polymers

were studied and it clearly showed that the drug release rate for the entire batch

had a wide variation. The results clearly indicated that the drug release was

strongly affected by the polymers selected for the study.

The obtained pre compression and post compression study data revealed

that the prepared tablets comply with the requirements necessary to pass official

quality control test. The findings from the dissolution study revealed that

hydrophilic matrix tablet of HPMC K100M alone could not retard the release of

Metformin HCl as it gave an initial burst release at Q2 interval. This burst release

121

was controlled by combining with CMC which retarded the release rate of

Metformin HCl from the matrices and reduced the burst release at Q2 interval.

A slow and constant release was achieved by coating the best formulation (MFH

12) with cellulose acetate 5% solution and a swelling restricted matrix tablet was

developed. By converting to swelling restricted matrix tablet the release was

further retarded and the initial burst release at Q2 interval was controlled

effectively and the fixed parameters were achieved. Release kinetic models

revealed that drug release profiles of all formulations confirmed higuchi model

and followed non – fickian diffusion transport.

MFH 14 was taken as the optimised formulation where diffusion coupled

with erosion could be the mechanism for drug release which help to reduce the

frequency of administration and decrease the dose dependent side effects

associated with the repeated administration of Metformin HCl. Thus it can be

concluded that the prepared swelling restricted matrix tablets had the ability to

control the release of Metformin HCl from the formulation at a pre-determined

rate for a period of 12 h.

From the data of stability studies it could be concluded that the optimised

formulations were stable when they were stored for a period of 6 months (Short

term stability study) at a temperature of 40 ± 2ºC and 75 ± 5% humidity and 12

Months (Long term stability study) at a temperature of 25 ± 2ºC and 60 ± 5%

humidity.

122

The in vivo study of MFH 14 tablets were compared with marketed

formulation. The marketed formulation had certain disadvantages such as;

Glycomet had a diffusion controlled reservoir release mechanism which

works under the principle of diffusion and erosion, whose drawbacks are

high cost of production, more chances of dose dumping, incomplete

release and larger molecular weight drugs cannot be incorporated.

The newly developed formulation is a swelling restricted matrix tablet which

overcomes the above said drawbacks. The in vivo study conducted using rabbits

showed that these tablets were able to ensure controlled drug release for a

longer period and also it has the potential to lower the plasma glucose level.

Advantages of newly developed formulation:

Newly developed formulation is a matrix dissolution controlled system.

Hence it is easy to manufacture, its cost of production is low, low chance

of dose dumping and gives a complete release profile.

123

REFERENCESCHAPTER 8

124

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ANNEXURECHAPTER 9

139

9. ANNEXURE 9.1 Annexure 1: List of Materials

Sl.No List of Materials Source

1 Metformin HCl Caplin point laboratories Ltd

2 HPMC K4M Dr Reddys Laboratories

3 HPMC K15M Dr Reddys Laboratories

4 HPMC K100M Dr Reddys Laboratories

5 Carboxy methyl Cellulose Nice Chemicals, Kochi

6 Cellulose acetate Loba Chemicals, Mumbai

7 Aerosil 200 Loba Chemicals, Mumbai

8 Magnesium stearate Nice Chemicals, Kochi

9 Isopropyl alcohol Nice Chemicals, Kochi

10 Ethanol Nice Chemicals, Kochi

11 Alloxan monohydrate K M C , Coimbatore

12 Glipizide K M C , Coimbatore

13 Acetonitrile Loba Chemicals, Mumbai

14 Ortho phosphoric acid CDH Chemicals, New Delhi

15 Sodium citrate CDH Chemicals, New Delhi

16 Hydrochloric acid Nice Chemicals, Kochi

17 Disodium phosphate CDH Chemicals, New Delhi

18 Sodium hydroxide SD Fine Chemicals.

19 Disodium hydrogen phosphate SD Fine Chemicals.

20 Potassium chloride SD Fine Chemicals.

140

9.2 Annexure 2: List of Equipment

Sl. No List of equipment Manufacturer

1 Electronic precision balance Shimadzu, Analytical India Pvt Ltd

2 Tablet Punching machine Karnavati engineering, Gujarat.

3 Hot air oven Rotex India Pvt Ltd.

4 Pfizer type hardness tester Lyzer India

5 Roche Friabilator Lyzer India

6 UV Visible spectrophotometer Shimadzu, Analytical India Pvt Ltd

7 FT-IR spectrophotometer Shimadzu, Analytical India Pvt Ltd

8 HPLC Shimadzu, Analytical India Pvt Ltd

9 pH meter Lyzer India

10 Dissolution apparatus Electrolab India Pvt Ltd

11 Stability chamber Labline equipments Pvt Ltd

12 Centrifuge Lyzer India

141

9.3 Annexure 3: Published Journal Copy

142

143

144

9.4 Annexure 4: Animal ethical committee certificate

145