i

IN VITRO BIOEQUIVALENCE STUDIES OF DIFFERENT

BRANDS OF AZITHROMYCIN AND CLARITHROMYCIN

TABLET DOSAGE FORMS MARKETED IN NIGERIA

BY

OKORIE, JAMES EKEMEZIE

(PG/M.Pharm/06/41043)

DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY

AND INDUSTRIAL PHARMACY FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

JULY, 2010.

i

IN VITRO BIOEQUIVALENCE STUDIES OF

DIFFERENT BRANDS OF AZITHROMYCIN AND

CLARITHROMYCIN TABLET DOSAGE FORMS

MARKETED IN NIGERIA

BY

OKORIE, JAMES EKEMEZIE

(PG/M.Pharm/06/41043)

A DISSERTATION SUBMITED

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR

THE AWARD OF DEGREE OF MASTER OF PHARMACY

(M.PHARM) IN THE FACULTY OF PHARMACEUTICAL

SCIENCES UNIVERSITY OF NIGERIA, NSUKKA

SUPERVISOR: PROF. S. I. OFOEFULE

JULY, 2010.

ii

CERTIFICATION

Okorie, James Ekemezie, a postgraduate student in the department of Pharmaceutical

Technology and Industrial pharmacy, Faculty of Pharmaceutical Sciences, University of

Nigeria, Nsukka, Reg.No: PG/M.Pharm/06/41043, has satisfactorily completed the

requirements for the research work for the degree of Master of Pharmacy in

Pharmaceutical Technology and Industrial Pharmacy.

The work embodied in this dissertation is original, and has not been submitted in

part or in full for any other diploma or degree of this or any other university.

________________________

Prof.S.I.Ofoefule

Supervisor

--------------------------------

Dr G.C. Onunkwo

Head of Department

iii

DEDICATION

To my mother, Evang (Mrs) Uzoamaka Okorie for her constant care and prayers

that have sustained me in all I do, and to my late father, Mr. Johnson M.Okorie whose

vision has propelled me to the height I have attained so far in the journey of life. Also to

my dear wife, Dr (Mrs) Pamela Okorie and to God, almighty.

iv

ACKNOWLEDGEMENT

I want to use this medium to thank my lovely wife, Dr (Mrs) Pamela O.Okorie, for

all her support and encouragement towards the completion of this programme. May I

thank immensely Prof. S. I. Ofoefule, my resourceful and caring supervisor. His input,

patience, understanding and support strengthened my commitment and resolve to forge

ahead even in the face of most glaring difficulties.

I am particularly grateful to Pharm Emeka Okpani, Mrs. Chinwe Chukwura and

Paucco Pharmaceutical Industries Ltd, Awka, for their assistance in the course of this

work.

Dr. C.S Nworu, Pharm Osita Eze and Pharm Abali Sunday Okorie are friends

whose encouragement have helped this work to come out successfully. The effort of Mr.

Chijioke Muogbo will always be appreciated and to all staff of deparment of

Pharmaceutical Technology and Industrial Pharmacy, University of Nigeria Nsukka, i say

thank you.

To my brothers Rev. Joel, John and Elijah, I am grateful.

Finally, my thanks go to the most high God, my creator, who makes all things beautiful

in His own time. To Him be all the glory.

v

ABSTRACT

Quality control assessment and in vitro bioequivalent studies of four different

brands of azithromycin (Zithromax, Azith, Azifast and Nobaxin) and five different

brands of clarithromycin (Klabax, Klatril, Thromyc, Acem and Clariwin) tablets

marketed in Nigeria were carried out. In vitro quality control parameters were used to

assess the quality and the bioavailability of the different brands. The tests carried out

included: weight uniformity, hardness, disintegration, friability, dissolution studies and

assay (spectrophotometrical and microbiological assays). Disintegration and dissolution

were carried out using the Erweka multiple disintegration unit and dissolution apparatus

respectively. The dissolution media consisted of sodium phosphate buffer for

azithromycin and sodium acetate buffer for clarithromycin.

Dissolution data were fitted into the Food and Drug Administration (FDA) F1 similarity

factor equation by Moore and Flanner and the dissolution efficiency parameter equations.

The coefficient of weight variation for azithromycin tablets ranged from 0.0052 to 0.0082

% while that for clarithromycin ranged from 0.0092 to 0.0104 %. Hardness values of

11.58 ± 0.58, 7.80 ± 0.22, 5.83 ± 0.52 and 6.00 ± 0.38 kgf were obtained for Zithromax,

Azith, Azifast and Nobaxin brands of azithromycin tablets respectively. The five brands

of clarithromycin tablets had hardness values of 9.90 ± 0.58 kgf (Klabax),7.75 ± 0.28 kgf

(Klatril), 9.41 ± 0.38 kgf (Thromyc), 7.67 ± 0.28 kgf (Acem) and 12.10 ± 0.67 kgf

(Klariwin). All the tablet brands of azithromycin and clarithromycin exhibited friability

values less than 1%. All the brands disintegrated within the time limit of 30 minutes, time

limit for film coated tablets. Results of the dissolution profiles of the azithromycin and

clarithromycin tablet brands showed over 75% drug release within 60 minutes. The

spectrophotometric and microbiological assay results indicated compliance to compendia

(USP) requirements. There were no significant (P 0.05) differences between the assay

results obtained from this two assay methods. Statistical analysis further showed

significant differences (P<0.05) between the FDA F1 factor and the dissolution efficiency

(DE) results of all the tablet brands. Results of the FDA F1 factor or the DE parameter

indicated that all the brands of azithromycin tablets evaluated in this study are

bioequivalent. Similar results were obtained with all the brands of clarithromycin.

Interchanging of brands within the azithromycin dosage form or within the

clarithromycin tablet dosage form may not result in any significant variation in

bioavailability.

vi

TABLE OF CONTENTS

Title page - - - - - - - - - i

Certification - - - - - - - - ii

Dedication - - - - - - - - - iii

Acknowledgement - - - - - - - - iv

Abstract - - - - - - - - - v

Table of Contents - - - - - - - - vi

List of Tables - - - - - - - - xi

List of Figures - - - - - - - - xv

List of Appendices - - - - - - - - xvii

CHAPTER ONE

1.1 General Introduction - - - - - - 1

1.2 Importance of Bioavailability - - - - - 3

1.2.1 Comparing Commercial Drug Formulations - - - 3

1.2.2 Quality Control and Quality Assurance - - - - 4

1.2.3 In vitro /in vivo Correlation - - - - - - 4

1.3 Factors Affecting Bioavailability - - - - - 4

1.3.1 Physicochemical Properties of the Active Drug Substance - - 5

1.3.2 Manufacturing Process - - - - - - 5

1.3.3 Formulation Factors - - - - - - - 5

1.4 Determination of Bioequivalence/Bioavailability - - - 6

1.4.1 Some in vitro Tablet Characteristics. - - - - 7

vii

1.4.1.1 Weight Uniformity - - - - - - - 8

1.4.1.2 Hardness - - - - - - - - 8

1.4.1.3 Friability - - - - - - - - 9

1.4.1.4 Disintegration Test - - - - - - - 9

1.4.1.5 Dissolution Profile - - - - - - - 10

1.5 Biological Method - - - - - - - 11

1.5.1 Microbiological Assay - - - - - - 12

1.5.2 Factors Affecting Microbiological Assay - - - - 12

1.5.2.1 Selection of Test Micro-organism - - - - - 12

1.5.2.2 Size of Innoculum - - - - - - - 13

1.5.2.3 Medium - - - - - - - - 13

1.5.2.4 Sample Preparation - - - - - - - 13

1.5.2.5 Temperature and Time of Incubation - - - - 13

1.6 Methods of Microbiogical Assay - - - - - 14

1.6.1 Serial Dilution Method - - - - - - 14

1.6.2 Agar Diffusion Method - - - - - - 14

1.7 Factors which Affect Antimicrobial Action - - - 15

1.7.1 Factors Associated with the Microorganism - - - 15

1.7.2 Factors Associated with the Environment - - - 16

1.7.2.1 Temperature - - - - - - - - 16

1.7.2.2 The Hydrogen ion Concentration (pH) - - - - 16

1.7.3 Factors Relating to the Antimicrobial Agent - - - 17

1.8 Dissolution Data Analysis - - - - - - 18

viii

1.8.1 Effect of Formulation Related Factors on Dissolution Rate - - - - - 19

1.8.1.1 Effect of Disintegrants - - - - - - - 19

1.8.1.2 Effect of Lubricants - - - - - - - 19

1.8.1.3 Effect of Binder - - - - - - - - 20

1.9 Methods used to Compare Dissolution Profile Data - - - 20

1.9.1 Mathematical Method - - - - - - - 21

1.9.2 Exploratory Data Analysis Method- - - - - - 21

1.10 Test organism - - - - - - - - 23

1.10.1 Staphylococcus aureus - - - - - - 23

1.10.2 Escherichia coli- - - - - - - - 24

1.11 Azithromycin - - - - - - - - 25

1.11.1 Mechanism of Action - - - - - - - 26

1.11.2 Antibacterial Spectrum - - - - - - 27

1.11.3 Therapeutic Uses- - - - - - - - 27

1.12 Clarithromycin - - - - - - - 28

1.12.1 Mechanism of Action- - - - - - - 28

1.12.2 Antibacterial Spectrum - - - - - - 28

1.13 Objectives of this Study - - - - - - 29

CHAPTER TWO

2.0 Materials and Method - - - - - - - 30

2.1 Materials - - - - - - - 30

2.1.1 Culture Media - - - - - - - - 30

ix

2.1.2 Equipment - - - - - - - - 30

2.1.3 Test Organism - - - - - - - - 30

2.1.4 Brands of Azithromycin and Clarithromycin - - - - 30

2.1.5 Pure Drug Sample - - - - - - - 32

2.1.6 Dissolution Medium - - - - - - - 32

2.2 Methods - - - - - - - - 32

2.2.1 Evaluation of in vitro Tablet Properties - - - - 32

2.2.1.1 Uniformity of Weight - - - - - - - 32

2.2 .1.2 Hardness Test (Crushing Strength) and Friability - - - 32

2.2.1.3 Disintegration Test - - - - - - - 33

2.2.1.4 Drug Content - - - - - - - 33

2.2.1.5 Standard Beer-Lambert plot- - - - - - 34

2.2.1.6 Dissolution Test - - - - - - - 34

2.3.1 Preparation of Media - - - - - - - 35

2.3.1.1 Preparation of Nutrient Agar - - - - - - 35

2.3 .1.2 Preparation of Nutrient Broth- - - - - - 35

2.3.2 Preparation of Drug Samples - - - - - - 36

2.3.2.1 Standard Concentrations of Pure Drug Brands - - - 36

2.3.2.2 Test Concentrations of Drug Samples - - - - 36

2.3.3 Standardization of the Micro-organsm- - - - - 37

2.3.4 One Point Microbiological Assay - - - - - 37

x

CHAPTER THREE

3.0 Results and Discussion - - - - - - 39

3.1 In vitro Tablet Properties - - - - - - 39

3.1.1 Uniformity of Weight - - - - - - - 39

3.1.2 Crushing Strength (Hardness) - - - - - - 39

3.1.3 Friability - - - - - - - - 40

3.1.4 Disintegration Time Test - - - - - - 40

3.2. Beer’s Plot- - - - - - - - - 41

3.3 Drug Content - - - - - - - - 46

3.4 Dissolution Profile - - - - 48

3.5 Microbiological Assay - - - - - - 51

3.6.1 Bioequivalence Prediction (F1 Values) - - - - 53

3.6.2 Dissolution Efficiency - - - - - - 53

3.6.3 Comparison of the Results of the Spectrophotometric and the Microbiological

Assay Methods - - - - - - - - - - - - - - - 54

3.6.4 Correlation between F1 value and Dissolution Efficiency - - 54

CHAPTER FOUR

Conclusion - - - - - - - - 57

References - - - - - - - - 59

Appendices - - - - - - - - 66

Figures - - - - - - - - - 119

xi

LIST OF TABLES

Table 1: Some Physical Properties of brands of Azithromycin and Clarithromycin

Tablets - - - - - - - - - - - - - - - - - 31

Table 2: Weight Uniformity Analysis Results for

Azithromycin brands - - - - - - - 42

Table 3: Weight Uniformity Analysis Results for

Clarithromycin brands - - - - - - 42

Table 4: Summary of Hardness, Friability and Disintegration time for

Azithromycin Brands. - - - - - - 43

Table 5: Summary of Hardness, Friability and Disintegration time for

Clarithromycin Brands. - - - - - - 43

Table 6: Absolute Drug content of Azithromycin Brands - - - 47

Table 7: Absolute Drug content of Clarithromycin Brands - - - 47

Table 8: Microbiological Assay Result for Azithromycin brands - - 52

Table 9: Microbiological Assay Result for Clarithromycin brands - - 52

Table 10: F1 Values for Azithromycin Brands - - - - 55

Table 11: F1 Values for Clarithromycin Brands - - - - 55

Table 12: Dissolution Efficiency for Azithromycin Tablet brands - - 56

Table 13: Dissolution Efficiency for Clarithromycin Tablet brands - 56

Table 14: Azithromycin Calibration table - - - - - 76

Table 15: Clarithromycin Calibration table - - - - - - 76

Table 16: Uniformity of Weight for Azithromycin Brands - - - 77

Table 17: Uniformity of Weight for Clarithromycin Brands- - - 78

xii

Table 18: Weight Uniformity Summary Azithromycin Tablet Brands - 79

Table 19: Weight Uniformity Summary of Clarithromycin Tablet Brands - 79

Table 20: Azithromycin brands Disintegration Time results- - - 80

Table 21: Clarithromycin brands Disintegration Time results- - - 80

Table 22: Disintegration Time analysis results for Azithromycin brands - 81

Table 23: Disintegration Time analysis results

for Clarithromycin brands - - - - - 81

Table 24: Friability results for Azithromycin brands - - - - 82

Table 25: Friability results for Clarithromycin brands - - - 82

Table 26: Zithromax Dissolution result - - - - - - 83

Table 27: Nobaxin Dissolution result - - - - - 84

Table 28: Azith-250 Dissolution result - - - - - 85

Table 29: Azifast Dissolution result - - - - - - 86

Table 30: Summary of Azithromycin Brands release profile - - - 87

Table 31: Clariwin Dissolution result - - - - - - 88

Table 32: Klabax Dissolution result - - - - - - 89

Table 33: Klatril Dissolution result - - - - - - 90

Table 34: Acem Dissolution result - - - - - - 91

Table 35: Thromyc Dissolution result - - - - - 92

Table 36: Summary of Clarithromycin Brands release profile - - 93

Table 37: IZD of Azifast against Staphyloccocus aureus - - - 94

Table 38: IZD of Nobaxin against Staphyloccocus aureus - - - 94

Table 39: IZD of Zithromax against Staphyloccocus aureus - - - 95

xiii

Table 40: IZD of Azith against Staphyloccocus aureus - - - 95

Table 41: IZD of Azifast against Eschericia coli - - - - 96

Table 42: IZD of Nobaxin against Eschericia coli - - - - 96

Table 43: IZD of Zithromax against Eschericia coli - - - - 97

Table 44: IZD of Azith against Eschercia coli - - - - 98

Table 45: IZD of Klatril against Staphyloccocus aureus - - - 99

Table 46: IZD of Klabax against Staphyloccocus aureus - - - 99

Table 47: IZD of Acem against Staphyloccocus aureus - - - 100

Table 48: IZD of Thromyc against Staphyloccocus aureus - - - 100

Table 49: IZD of Clariwin against Staphyloccocus aureus- - - - 101

Table 50: IZD of Klatril against Eschericia coli - - - - 101

Table 51: IZD of Klabax against Eschericia coli - - - - 102

Table 52: IZD of Acem against Eschericia coli - - - - 102

Table 53: IZD of Thromyc against Eschericia coli - - - - 103

Table 54: IZD of Clariwin against Eschericia coli - - - - 103

Table 55: IZD Summary table for Azifast against Staph aureus - - 104

Table 56: IZD Summary table for Nobaxin against Staph aureus- - - 104

Table 57: IZD Summary table for Zithromax against Staph aureus - - 105

Table 58: IZD Summary table for Azith against Staph aureus - - 105

Table 59: IZD Summary table for Azifast against Eschericia coli - - 105

Table 60: IZD Summary table for Nobaxin against Eschericia coli - - 106

Table 61: IZD Summary table for Zithromax against Eschericia coli - 106

Table 62: IZD Summary table for Azith against Eschericia coli - - 107

xiv

Table 63: IZD Summary table for Klatril against Staph aureus - - 107

Table 64: IZD Summary table for Klabax against Staph aureus - - 107

Table 65: IZD Summary table for Acem against Staph aureus - - 108

Table 66: IZD Summary table for Thromyc against Staph aureus- - - 108

Table 67: IZD Summary table for Clariwin against Staph aureus- - - 108

Table 68: IZD Summary table for Klatril against Eschericia coli - - 109

Table 69: IZD Summary table for Klabax against Eschericia coli - - 109

Table 70: IZD Summary table for Acem against Eschericia coli - - 109

Table 71: IZD Summary table for Thromyc against Eschericia coli - - 110

Table 72: IZD Summary table for Clariwin against Eschericia coli - - 110

Table 73: F1 Pre-analysis table for Nobaxin - - - - - 111

Table 74: F1 Pre-analysis table for Azith - - - - - 112

Table 75: F1 Pre-analysis table for Azifast - - - - - 113

Table 76: F1 Pre-analysis table for Klabax - - - - - 114

Table 77: F1 Pre-analysis table for Klatril - - - - - 115

Table 78: FI Pre-analysis table for Acem - - - - - 116

Table 79: FI Pre-analysis table for Thromyc - - - - - 117

xv

LIST OF FIGURES

Fig 1: A typical Dissolution profile curve - - - - - - - - - 22

Fig.2: Structure of Azithromycin - - - - - - 26

Fig.3: A Cup plate illustration for Assay of Azithromycin Brands- - - 38

Fig.4: A Cup plate illustration for Assay of Clarithromycin Brands - - 38

Fig.5: Azithromycin Beer Lambert Plot - - - - - 44

Fig.6: Clarithromycin Beer Lambert Plot - - - - - 45

Fig.7: Dissolution Profile of Azithromycin Brands - - - - 49

Fig.8: Dissolution Profile of Clarithromycin Brands - - - - 50

Fig.9: Dissolution Profile of Zithromax - - - - - 119

Fig.10: Dissolution Profile of Nobaxin - - - - - 120

Fig.11: Dissolution Profile of Azith - - - - - - 121

Fig.12: Dissolution Profile of Azifast - - - - - - 122

Fig.13: Dissolution Profile of Thromyc - - - - - 123

Fig.14: Dissolution Profile of Klabax - - - - - - 124

Fig.15: Dissolution Profile of Acem - - - - - - 125

Fig.16: Dissolution Profile of Klatril - - - - - - 126

Fig.17: Dissolution Profile of Clariwin - - - - - 127

Fig.18: IZD Vs Log Conc. plot of Azifast for Staph aureus - - - 128

Fig.19: IZD Vs Log Conc. plot of Nobaxin for Staph aureus - - 129

Fig.20: IZD Vs Log Conc. plot of Zithromax for Staph aureus - - 130

Fig.21: IZD Vs Log Conc. plot of Azith for Staph aureus - - - 131

Fig.22: IZD Vs Log Conc. plot of Azifast for Eschericia coli - - 132

xvi

Fig.23: IZD Vs Log Conc. plot of Nobaxin for Eschericia coli - - 133

Fig.24: IZD Vs Log Conc. plot of Zithromax for Eschericia coli - - 134

Fig.25: IZD Vs Log Conc. plot of Azith for Eschericia coli - - - 135

Fig.26: IZD Vs Log Conc. plot of Klatril for Staph aureus - - - 136

Fig.27: IZD Vs Log Conc. plot of Klabax for Staph aureus - - - 137

Fig.28: IZD Vs Log Conc. plot of Acem for Staph aureus - - - 138

Fig.29: IZD Vs Log Conc. plot of Thromyc for Staph aureus- - - 139

Fig.30: IZD Vs Log Conc. plot of Clariwin for Staph aureus- - - 140

Fig.31: IZD Vs Log Conc. Plot of Klatril for Eschericia coli - - - 141

Fig.32: IZD Vs Log Conc. plot of Klabax for Eschericia coli- - - 142

Fig.33: IZD Vs Log Conc. plot of Acem for Eschericia coli - - - 143

Fig.34: IZD Vs Log Conc. plot of Thromyc for Eschericia coli- - - 144

Fig.35: IZD Vs Log Conc. plot of Clariwin for Eschericia coli- - - 145

xvii

LIST OF APPENDICES

Appendix 1: Certificate of analysis of pure Azithromycin and Clarithromycin

pure drug samples. - - - - - - 66

Appendix 2: Statistical analysis results - - - - - 71

Appendix 3: Beers’ plot table for Azithromycin and Clarithromycin pure drug

sample - - - - - - - - 76

Appendix 4: Uniformity of weight for Azithromycin and Clarithromycin brand 77

Appendix 5: Weight uniformity Summary result for all brands - - 79

Appendix 6: Disintegration time for Azithromycin and Clarithromycin brand 80

Appendix 7: Disintegration time analysis results for all brands - - 81

Appendix 8: Friability results - - - - - - - 82

Appendix 9: Dissolution results for all brands - - - - 83

Appendix 10: IZD of all brands against the two test organisms - - 94

Appendix 11:IZD summary table for all brands against the two test organisms. 104

Appendix 12: FI Pre-analysis table - - - - - - 111

Appendix 13: Formula for Some Calculations - - - - - 118

1

CHAPTER ONE

1.1 General Introduction

Increasing economic activities in many parts of the world especially in

developing countries like Nigeria, has resulted in the proliferation of pharmaceutical

manufacturing industries and importation of different brands of the same drugs into

Nigeria at cheaper prices. With increasing incidence of drug counterfeiting and the use of

different grade and quality of excipients in solid dosage formulation, bioavalability and

efficacy become major concern. In a case where affordability of certain brands of the

same drugs is a major consideration, interchangeability of the brands is usually an

affordable alternative. Interchangeability of brands of the same drug can only be

undertaken when a reliable in vitro or in vivo studies establishes bioequivalence of the

brands of the same drug.

Biopharmaceutical studies have shown that the bioavailability and hence the

therapeutic efficacy of many drugs are significantly affected by formulation factors (1).

Those factors have been studied extensively with respect to tablet dosage forms (2).

The sales of drug product with bogus claims and the”get rich quick” syndrome

associated with the Nigerian society of today, is a challenge. This has given rise to the

tendency to fake or adulterate expensive antibiotics like azithromycin and clarithromycin

which ultimately leads to development of bacterial resistance or therapeutic failures.

It is no longer tenable to consider the safety and efficacy of a drug administered

orally simply on the basis of its compliance with standards laid down in the official

compendia. Bioavailability requirements are now an essential parameter in quality control

2

as a number of medicinal products, especially those that are cheap or possess high

therapeutic indices, or poor water-solubility are in good use today in therapy.

The expectation of the public is that a prescribed drug when administered to a

patient would yield maximum obtainable efficacy. With bioavalability being one of the

most determinants of the therapeutic activity of the drug (3) and patient’s response to a

drug (4). One wonders whether two or more formulation containing the same active

ingredient are bioequivalent. The consideration of two types of bioavailability namely in

vitro and in vivo, as relative terms means that the drug preparation is always being

compared to a reference standard. An innovator product is that which was first authorized

for marketing (as a patented drug) on the basis of documentation ie safety, quality and

efficacy (5).

The marked variation in clinical response with the administration of drugs from

different sources, different manufacturers or different batches of drugs from the same

manufacturers has been reported in literature (5). The equivalence of drug products may

be of three kinds (5), namely: (a) Chemical or pharmaceutical equivalence, (b) Biological

equivalence, (c) Clinical or therapeutic equivalence.

Chemical or pharmaceutical equivalence applies to multiple source drug products

which contain equal amount of the same therapeutically active ingredient in identical

dosage forms and which meet existing physio-chemical standard in the official

compendia.

Biological equivalence describes chemically identical substances, which give rise

to similar bioavailability when used therapeutically. When they are used in the same

3

dosage regimen to the same individuals, they would produce essentially the same

biological or physiological concentrations as measured by blood or urine levels.

Therapeutic equivalence describes the pharmaceutical forms which when used at

the same dose to treat the same condition, in the same individual, give rise to therapeutic

effects of similar intensity as measured by the control of symptom of disease.

1.2 Importance of Bioavailability

The importance of the concept of bioavailability can not be readily over-

emphasized. The prediction of in vivo bioavailability of most oral drugs depends mostly

on the in vitro dissolution studies as in vitro disintegration tests do not always give in

vivo correlation (6).

Bioavailability studies are used in establishing therapeutic

equivalence of two or more drug products or formulations.

1.2.1 Comparing Commercial Drug Formulations

Bioavailability is an important parameter in the comparism of commercial drug

formulations. A lot of emphasis have been placed on generating bioavailability data on a

product marketed, so that community and hospital based pharmacists can adequately

judge the equivalence or otherwise of products made by different manufacturers.

Bioavailability is a concept that the pharmacist can exploit to provide information on

optimum drug absorption and availability of the medication dispensed to patients.

4

1.2.2 Quality Control and Quality Assurance

Owing to the problems of therapeutic inequivalence arising from prescriptions

using either brand or generic names, bioavailability studies are helpful in ensuring that a

patient with a prescribed dose gets the same quantity of drug in the same biological fluids

at comparatively the same rate, no matter the manufacturer of the prescribed drug. It also

helps to ensure that any variation in clinical responses that is observed in a patient, who is

switched from one drug product to another is due to inter-subject variability to the tablets.

Generally the quality assurance of tablets would involve these tests: hardness /tensile

strength, uniformity of diameter /thickness, weight uniformity/variation, disintegration

tests, content uniformity test, friability and dissolution rate tests.

1.2.3 In Vitro/In Vivo Correlation

Bioavailability studies are important in the study of in vivo/in vitro correlation

between bioavailability and pharmacological activity. As a result of the fact that the

response of a drug is influenced by many factors, bioavailability measurements are

usually based on an assumption that the drugs are absorbed systemically. Bioavailability

measurements are for drugs that clearly show therapeutic in-equivalence because of

differences in drug bioavailability (7).

1.3 Factors Affecting Bioavailability

Biopharmaceutical studies have shown that the bioavailability and hence the

therapeutic efficacy of many drugs are significantly affected by formulation factors (8).

But in science generally, evidence has shown that bioavailability may vary for a variety

5

of reasons and such variations have been identified as factors responsible for certain

failures in drug therapy.The factors which affect bioavailability include;

(a)Physicochemical properties of active drug substance

(b)Manufacturing process

(c)Formulation factors

1.3.1 Physicochemical Properties of Active Drug Substance

The dissolution rate of a drug depends on the physicochemical properties of the

drug after the release of the drug particles. These properties include particle size, salt

form, hydration, polymorphic and stereo-isometric forms, pKa-pH profiles, partition

coefficient (log P)

1.3.2 Manufacturing Process

The difference in manufacturing techniques employed by different pharmaceutical

companies, may manifest themselves in in-equivalence of product from various firms

making same drugs in the same dosage forms (9).

These may be due to method of

granulation and compression of the granules.

1.3.3 Formulation Factors

The difference in product formulation may be due to:

(i) Dosage forms

(ii) The nature and amount of pharmaceutical adjuvants (excipients).

6

Poor and inappropriate formulations can result in product, which releases the drug at too

slow, or too fast a rate, thus leading to unacceptable variation in the performance of

individual dose units. Fast absorption is desirable to provide rapid onset of action or to

achieve effective drug concentration. In the manufacture of dosage forms, inactive

ingredients (excipients or formulation ingredients) are usually added to the active

ingredient in order to improve the quality of the formulation. Examples of such adjuvants

include; diluents, binders, disintegrant, surfactants, lubricants. The nature and quality of

these excipients other than the drug can drastically influence the efficacy or toxicity of

the final product. For example Wells (10) measured the dissolution rate of chlopropamide

tablets containing starch, hydrolyzed gelatin, methylhydroxyethylcellulose (MHEC) and

polyvinylpyrrolidonine(PVP)as binders. It was discovered that tablets containing soluble

binders (PVP and hydrolyzed gelatin) had rapid dissolution rate whereas slow and

incomplete disintegration of tablets formulated with starch paste, led to prolonged release

of drug and therefore poor bioavailability (10).

1.4 Determination of Bioequivalence/Bioavailability

Relative bioavailability studies compare drug absorption, distribution and excretion

from the same dosage form, when they are administered by the same route (11, 12, 13).

In order to demonstrate that certain oral pharmaceutical products are therapeutically

equivalent and therefore interchangeable, their bioequivalence is established based on

bioavailability data. Interchangeability is the process of dispensing a different brand or

unbranded drug product in place of the prescribed drug product (14).

7

Two pharmaceutically equivalent drug products are considered to be bioequivalent

when the rates and extents of the bioavailability of the active ingredient in the two

products are significantly similar under suitable test condition (13,15). There are in vitro

and in vivo tests that can be used to estimate the bioequivalence of drug product. The

dissolution rate tests is the major in vitro tests performed for drugs as an indication of

their bioavailability and in certain cases, in vitro dissolution studies are sufficient to

characterize formulation properties (16,17,18).

In vitro tests and in vivo studies in animals may be the only option for some drugs,

where ethical considerations preclude the use of normal healthy volunteers for in vivo

studies in man.

1.4.1 Some In Vitro Tablet Characteristics

There are several tests that are employed for analysis of finished dosage forms like

tablets or capsules. They include:

(a) Weight uniformity

(b) Hardness

(c) Friability

(d) Disintegration test

(e) Dissolution test

(f) Content of active ingredient

8

1.4.1.1 Weight Uniformity

This is a function of granulation quality, flow of granulation and machine

performance. Weight uniformity is checked routinely in tablet production to ensure that

proper weights of tablets are being made. Official tolerance level of variation of tablet

containing up to 50% or more active ingredient is that out of 20 tablets weighed

individually and the mean calculated not more than 2 tablets should deviate from the

mean by a specific percentage of deviation and that none should deviate by twice that

percentage. This is summarised below:

Average weight %Deviation

80mg or less 10

> 80mg, < than 250mg 7.5

> 250mg 5

The weight variation test would be a satisfactory method of assessing the content

uniformity where the active ingredient comprises a major portion of the tablet and where

control of weight may be presumed to be an adequate control of drug content uniformity

(19).

1.4.1.2 Hardness

This is a term routinely applied to several tablet parameters, including resistance,

crushing strength, axial or radial, impact strength and resistance to attrition or abrasion.

The degree of hardness of the tablets depends on its physical size or shape together with

the characteristics of the chemical that go into the formulation and the pressure applied

during compression. Robert et al (20), stated that the resistance of the tablet to chipping

9

and abrasion under condition of storage and handling before usage depends on its

hardness. Hardness testers like Pfizer, Monsanto and Erweka are used in its

determination.

1.4.1.3 Friability

When compressed, tablets rub against one another or when dropped suddenly

when in a container, they may chip or break. In order to ascertain that such losses are

within limit, friability-testing equipment were developed. They include the Roche

friabilator.

1.4.1.4 Disintegration Test

Disintegration is a process of fragmentation of a solid into a soft mass with no

palpable firm core. For most tablets, breaking up of the tablet mass into smaller particles

or granules is the initial step to dissolution. This is so because the break up or

disintegration tends to increase the surface area and thus facilitates dissolution, although

research has established that there is no automatic correlation between disintegration and

dissolution (21). The test determines whether tablets or capsules disintegrate within a

prescribed time when placed in a liquid medium under the prescribed experimental

condition. The test is essentially for disintegration of one tablet in an enclosed tube

capable of moving up and down and its breakup timed with or without guided disc in the

disintegration apparatus. This is replicated six times. Disintegration is also used as an in-

process control test to ensure lot –to –lot uniformity. Limits are set, for example, 5

minutes for effervescent tablets, 3 minutes for soluble (dispersible tablets), 15 minutes for

10

uncoated tablets, 30 minutes for film- coated tablets and 60 minutes for enteric-coated

tablets.

1.4.1.5 Dissolution Profile

Dissolution test is a parameter used to evaluate the time required for a given

percentage of the drug in a tablet to go into solution under a specified set of conditions. It

is a more predictive indicator of absorption in vitro than disintegration test. It is intended

to provide a step towards the evaluation of physiological availability of the drug . Several

methods are available for study of dissolution rates, the most commonly adopted ones

being the “rotating” basket and paddle method (USP, BP). A popular official Conpendial

alternative (USP, EUr Pharm) to the paddle or basket method is the “flow through cell”

method. It is a valid dissolution method for poorly water-soluble drug and is based on the

mass transfer to fixed bed of drug material transversed by a continuous flow of solvent

liquid in a vertical exchange column.

Dissolution of tablets is known to be influenced by various formulation and

manufacturing processes.

These include:

a) nature of active ingredient,

b) type and concentration of the binder used,

c) presence and amount of disintegrant employed,

d) compression force,

e) nature of coating material used for coated tablets.

11

Underwood et al (22) showed that increase in binder concentration and force of

compression resulted in decrease of dissolution rate. In general, for dissolution to take

place, whether in vitro or in vivo, the dissolution rate of the drug is governed by one law

when the process is diffusion controlled and involves no chemical reaction. This can be

illustrated by Noyes-Whitney equation.

KAdt

dm (Cs-C)....................................................equation 1

Where: m is mass of solute that has passed into solution in time, t, A is surface area of

the undissolved solid in contact with the solvent at experimental temperature, C is solute

concentration at time t, K is instrinsic dissolution rate constant, d is diffusion coefficient

of the solute in the dissolution medium, and Cs is concentration of solvate at saturation.

The relevance of polymorphic and solid state properties to this equation lies in the

fact that A is determined by particle size. If it leads to a change in the polymorph, a

change in Cs occurs and if dissolution is the rate limiting step in absorption, then

bioavailability is affected.

1.5 Biological Method

It involves the quantitative assay of pharmaceutical preparations by biological

methods as well as application of relevant qualitative biological tests (23). It remains

generally the standard for resolving doubts with respect to possible loss of bioactivity in a

drug product or even in assaying.

12

1.5.1 Microbiological Assay

Microbiological assays are based on the inhibition of growth of microorganism in

cultures as a function of the concentration of a certain drug or its derivatives (23). They

are inherently prone to variability, so strict use of controls and reference standards of

known potencies is advocated. They also require replication of observations and

measurements, which will increase the precision of average values. Microbiological

assays when conducted under proper conditions and when the proper test organisms are

used may give some predictions on antibiotic bioequivalence. Microbiological response

(usually measured by inhibition of growth), often permit assays of antibiotic in low

concentration, such as obtained in urine/blood. They also provide a standard method for

resolving doubt with respect to possible loss of bioactivity in some antibiotic

preparations.

1.5.2 Factors Affecting Microbiological Assay

1.5.2.1 Selection of Test Microorganisms

The importance of the selection of appropriate microorganisms must be stressed.

This is so because wide variations do occur in susceptibility between various microbes of

different species or strains, and this may introduce difficulties and errors in respect of

how they affect the zone of inhibition. In general terms, the susceptibility of the

microorganism to chosen antibiotics must be ensured, and must not change with

successive cultivation. The organism must be able to grow rapidly in the assay media.

13

1.5.2.2 Size Of Innoculum

An appropriate size of innoculum is essential as the number of microorganism in

innoculum determines the length of the lag phase of growth. The minimum inhibitory

concentration (MIC) of an antibacterial substance may increase greatly with an increase

in the concentration of bacteria used in the test because of the constant increase in the

number of bacteria resistant to it. A 0.1ml volume of uniform innoculum is therefore used

in microbiological assay to give uniform circles and well defined inhibition zones.

1.5.2.3 Medium

The type of medium used affects the results obtained from microbiological assay.

The volume (depth), pH and composition of the medium influence the growth rate of the

test organism, the size of the zones and the development of specific metabolic pathways.

The pH of the medium, in addition, affects stability and hence the bioactivity of

antibiotics.

1.5.2.4 Sample Preparation

The preparation of solution of both the standard and test drug is very important. For

the purpose of accuracy, it is better to duplicate the assay including the preparation of the

solutions.

1.5.2.5 Temperature and Time of Incubation

The temperature of incubation should be optimum for satisfactory growth of the

organism. The temperature of incubation is usually 32-37oC for 24 hours (23). The time

14

of incubation is also critical for most antibiotic assays. It is recommended that the time of

incubation should neither be too short nor too long as these will produce unclear edges of

zones of inhibition (24).

1.6 Methods of Microbiological Assay

There are two methods of microbiological assay, namely: Serial dilution and Agar

diffusion methods.

1.6.1 Serial Dilution Method

This method involves serial dilution of the test antibacterial agent in test tubes and

inoculating the dilutions with the microorganism. Usually dilution in nutrient broth is

suitable but in some cases, dilution in a nutrient agar is more satisfactory. The lowest

concentration of the agent, which causes observable complete inhibition of growth is

taken as the minimum inhibitory concentration (MIC). This is usually determined after a

period of 18 – 24 hours of incubation from the different dilutions or concentrations used.

1.6.2 Agar Diffusion Method

Prepared nutrient agar in Petri dish shows evidence of growth after seeding with a

susceptible microorganism for an appropriate period of incubation. If a solution that

contains an antimicrobial agent is spotted on the surface of the agar before incubation, a

zone of inhibition, which can be related to concentration subsequently appears. These

events constitute the basis for diffusion method, which is a form of microbiological

15

assay. The solidified agar plates are generally treated with the agent responsible for

growth inhibition in one of these three ways before incubation:,

i) a solution of the agent (antibiotic) may be put in sterile, porous stainless steel

or porcelain cylinder and placed on the surface of the agar (cylinder-plate

method)

ii) the agent may be put in a cup scooped out of the agar with a sterile borer (The

cup-plate method)

iii) a cellulose filter paper impregnated with antibiotic solution may be placed on

the surface of the medium (Disc method).

On incubation at 30-37oC for a period of 24 hours, the antibiotic diffuses into

the agar, causing zone of inhibition around the cup, stainless steel or disc. The zone of

inhibition is measured with the highest possible accuracy after incubation and it is

proportional to the logarithrim of the concentration of the antibiotic. A graph of log

concentration of standard against, zone diameter is plotted. The same is done for the

test on the same graph. The two graphs should be parallel over the range of doses

used in the calculation and from the graph the concentration of the test solution can

be determined.

1.7 Factors which Affect Antimicrobial Action.

1.7.1 Factors Associated with the Micro-Organism

The number of organisms exposed to the agent and the physiological state of

organism may affect the action of the antimicrobial agent (25). The greater the number

of cells of the bacterium, the higher the number of resistant cells which may act as in

16

activators for some chemical agents. Also, actively metabolizing cells are more destroyed

than the old dormant cells.

There is usually higher resistance in bacteria with slime layers or capsules

because these may prevent penetration of the antimicrobial agent. Sensitivity of some

organisms to the agent vary and some may even be resistant to it.

1.7.2 Factors Associated with Environment

The environment affects the rate as well as degree of microbial destruction by

antimicrobial agent. Some of these environmental factors are:

1.7.2.1 Temperature

This is one of the major factors that influence the rate of action of physical and

chemical germicide (24). The rate of action of germicide normally increases with

increase in temperature although the effect is more marked with some agents.

1.7.2.2 The Hydrogen Ion Concentration (pH)

This may affect both the rate of microbial growth and the activity of the

antimicrobial agent. Changes in pH can markedly affect the nature of the bactericide and

its receptor sites in the bacterial cell. It is crucial to note that the state of the microbial

surface, the stability of the agent, the water solubility of the agent and the degree of

ionization of the agent all depend on the operational pH.

17

1.7.3 Factors Relating to the Antimicrobial Agent

The concentration of the agent affects antimicrobial action (24). In general, the

rate of antimicrobial action varies directly with the concentration of the agent. But in

many cases, there is a minimal concentration below which no major action takes place

because insufficient agent reaches the vital reactor sites of the organism. There is also a

maximal concentration above which, the rate does not increase. Above this concentration,

all reaction sites which have been saturated with molecules of the agent produce no

significant difference. Under these two extremes, the relationship between the

concentration of the agent and the kill time is expressed by the following equation (26).

Cnt = K ------------------------------------------------------ (2)

or nlogC + log t = K ………………………….………… (3)

Where C is concentration of the agent, K is microbial Killing rate constant, n is

concentration exponent of the agent, and t is time required to kill a bacterial population.

The value of n determines the extent to which dilution would affect the

antimicrobial efficacy. Water solubility of the chemical agent is equally important

because, for an agent to reach its site of action, either as a molecule entity or in the

ionized form, it must first dissolve in aqueous medium. The ionization constant of the

agent is also an important factor because some agents are more active as ions. Therefore,

the activity of the agent will depend on its ionization constant in a particular medium.

The level of germicidal action of the agent is of great value in consideration of their

spectrum of activity against different group of micro-organisms.

18

1.8 Dissolution Data Analysis

In recent years, dissolution testing has gained prominence among the

pharmaceutical industries and regulating authorities. Comparism of dissolution profiles

can be used to establish similarities of pharmaceutical dosage forms for which

composition, manufacturing site, scale of manufacturing, manufacturing process and/or

equipment many have changed within defined limit. Also it can be used to develop in-

vitro-in-vivo correlations and establish final dissolution specification for the

pharmaceutical dosage forms. Dissolution profile can be defined as the measured fraction

or percentage of the labeled amount of drug that is released from a dosage form or unit

(tablet and capsule) at a number of predetermined time points when tested in a dissolution

apparatus as USP I or II dissolution systems (27). It is intended to provide a step towards

the evaluation of the physiological availability of the drug substance. USP requirements

for immediate release dosage forms are that 75% of the active ingredients from the

dosage unit should be dissolved in water or dilute acid at 37oC within 45 minutes. This

applies when the dissolution experiment is carried out with USP I and II apparatus. The

apparatus is operated at appropriate speed (Typically 100rpm for USP I and 50 rpm for

USP II) (27).

As a result of emphasis placed on the comparism of dissolution profiles data in

FDA guidelines, the interest is now focused on methodology used to compare dissolution

profile data.

19

1.8.1 Effect of Formulation Related Factors on Dissolution Rate

Poor or inappropriate formulation can lead to a product which releases the drug at

too slow or too fast a rate. This leads to unacceptable variations in the performance of

individual dose units. It has been shown that the dissolution of a pure drug can be altered

significantly when mixed with various excipients during the manufacturing process of its

solid dosage form. Yamatoto, et al (28) demonstrated that the dissolution rate and

bioavailability of griseofulvin was significantly improved when it was mixed with

microcrystalline cellulose than as a micronised griseofulvin powder. This is as a result of

deaggregation of griseofulvin by microcrystalline cellulose resulting in difference in

crystalinity. Also they may affect dissolution kinetics of the drug either by altering the

medium in which the drug is dissolving or by reacting with the drug itself.

1.8.1.1 Effect of Disintegrant

It has been observed that ion exchange resin, amberlite, causes rapid disintegration of

chlopropamide tablet. Its effect on the dissolution rate of the drug was found to be

inferior to the effect observed with sodium starch glycollate (10). Measurement of the

swelling and hydration capacity of the disintegrant indicated that amberlite and sodium

starch glycolate possess high swelling capacities. An exceptional high hydration capacity

was observed in the case of sodium starch glycollate (10).

1.8.1.2 Effect of Lubricants

Most tablets contain lubricants to prevent the dosage form from sticking to the

processing machinery. Levy, et al (29) showed that the hydrophylic lubricant, sodium

20

lauryl sufate, allowed the drug to dissolve more rapidly than the control tablet containing

no lubricant. However, incorporating a hydrophobic lubricant, magnesium state, resulted

in a decrease in the dissolution rate. Most effective lubricants are hydrophobic and

therefore must be properly incoporaed at the appropriate concentration to avoid reducing

dissolution rate and oral bioavailability (30).

1.8.1.3 Effect of Binder

These are usually polymeric materials which possess both cohesive and adhesive

properties.They function by holding filler and drug particles together in agglomerates

thereby converting them into granules which are free flowing. Wells (10) observed that

tablets containing soluble binders like PVP and hydrolysed starch undergo faster

dissolution than those containing starch paste as binder.

1.9 Methods used to Compare Dissolution Profile Data

Two methods are frequently used to compare dissolution data. The methods are:

(a) mathematical Method

(b) exploratory data analysis

1.9.1 Mathematical Method

In this method, the Food and Drug Administration (FDA) equation proposed by

Moore and Flanner is employed (30). The equation is expressed as:

F1 = {[Σ t=1n |Rt-Tt|] / [Σ t=1

n Rt]} ×100..........................................(4)

21

Where: n is number of dissolution time point, Rt is reference dissolution values at time

(t),Tt is test dissolution values at time (t). The difference factor (F1) calculates the (%)

difference between the reference (R) and test (T) dissolution curves at each time point

and this is a measurement of the relative error between the two curves. The FI equation is

zero where the mean profiles are identical and increases proportionally as the difference

between profiles increase. FDA has adopted it in various guidance documents, which

recommend their use when data are available for at least three dissolution time points.

Valves of F1, between zero and fifteen (0-15) ensure “sameness” or equivalence of the

two dissolution profiles.

1.9.2 Exploratory Data Analysis

This method is useful as first step to compare dissolution profile data in both a

graphical and numerical manner. The data may be illustrated graphically by plotting the

mean dissolution profile data for each formulation with error bars extending to two

standard errors at each dissolution time point. If for example, the dissolution profile for

two formulations test (T) and reference (R) are being compared, the dissolution profile

may be considered to differ significantly from each other if the error bars at each

dissolution time point do not overlap.

Also the data may be summarized numerically presenting the mean and standard

deviation of the dissolution data at each dissolution time point for the test and reference

formulations.

In addition to the mathematical method and exploratory data analysis, dissolution

efficiency (DE) parameter could be employed to rank the relative in vivo bioavailability /

22

bioequivalence of tablet dosage forms (31). The concept of DE is based on the ratio of

the area under under the graph (AUG) of the in vitro drug release profile and the area of

the extrapolated rectangle at 100 % drug release as shown in figure 1.

Figure 1: A Typical Dissolution Profile Cuve

The AUG is similar to th area under the plasma level – time curve (AUC) where the AUC

represents the amount of of drug absorbed that reaches the systemic circulation. The

AUG just like the AUC can be computed using the trapezoid rule and integral calculus

(32). The DE parameter saves time and it avoids the problem of analysis of drugs in

biological fluids.

30

Time (min)

60 0

100

AUG

% R

elea

sed

23

1.10 Test Organism

1.10.1 Staphylococcus aureus

Staphylococcus aureus is a gram-positive, coagulase positive, non-motile coccus

bacterium that causes a variety of human infection in all age groups (33). It is the major

causative agent in surgical wound infections and epidermal skin disease in new-born

infants. Infection may also be superimposed on superficial dermatological disease such as

eczema, pediculosis mycosis (34). They live as commensals in anterior nares of over half

the population of human (35). They are spread from these sites to the environment by the

hands, handkerchief, clothing and dust.

Staphylococcus aureus is an opportunistic pathogen in the sense that it causes

infection most commonly in tissues and sites with lower host resistance such as in

individuals with diabetes, old malnourished person and other chronic cases (36).

Staphylococcus aureus causes folliculitis, boil, furunculosis, scalded skin syndrome,

conjunctivitis, paromychia and mastitis. Staphylococcal pneumonia can occur if

Staphylococcal infection spread to the lungs (37). Hospital acquired infections are

common in new born babies, surgical patients and hospital staff. Some patients develop

sepsis in operation wounds, which take place in the theater during operation, while others

develop it during post operations in the ward (38). Food poisoning can also occur when a

toxin produced by the bacteria is ingested with food. Food with high salt or sugar content

favours the growth of Staphylococcus aureus (38). Many out-breaks of Staphylococcal

food poisoning result from hand contacts (39). Staphylococcus aureus strains carry a

wide variety of multi drug resistant genes on plasmids, which can be exchanged and

spread among different species of Staphylococci (40). Hospital strains of Staphylococcus

24

aureus are usually resistant to a variety of different antibiotics. Few strains are resistant

to all clinically useful antibiotics except vancomycin. Some workers have reported,

however, the presence of vancomycin resistant strains (41,42). Exposure to new antibiotic

often results in further selection of homologous resistant strains (43). Infection with such

resistant strains is likely to be more severe and requires longer hospitalization with

increased costs, than infection with susceptible strains (44).

1.10.2 Escherichia coli

Escherichia coli is a common bacterium that normally inhabits the intestinal

tracts of humans and animals. It can also cause infection in other parts of the body,

especially the urinary tract. It is the most common member of the genus Escherichia,

named after Theodor Escherich, a German physician. E. coli is a Gram-negative, rod-

shaped bacterium propelled by long, rapidly rotating flagella. It is part of the normal flora

of the mouth and gut and helps protect the intestinal tract from bacterial infection, aids in

digestion, and produces small amounts of vitamins B12 and K. The bacterium, which is

also found in soil and water, is widely used in laboratory research and is said to be the

most thoroughly studied life form. In genetic engeneering it is the microorganism

preferred for use as a host for the gene-splicing techniques used to clone genes. E. coli is

one of several types of bacteria that normally inhabit the intestine of humans and animals

(commensal organism). Some strains of E. coli are capable of causing disease under

certain conditions when the immune system is compromised or disease may result from

an environmental exposure. E. coli bacteria may give rise to infections in wounds, the

urinary tract, biliary tract, and abdominal cavity (peritonitis). This organism may cause

25

septicemia, neonatal Meningitis, infantile gasroenteritis, tourist diarrhoea, and

hemorrhagic diarrhoea. An E. coli infection may also arise due to environmental

exposure. Infections with this type of bacteria pose a serious threat to public health with

outbreaks arising from food and water that have been contaminated with human or

animal faeces or sewage. This type of bacteria has been used as a biological indicator for

safety of drinking water since the 1890s. Exposure may also occur during hospitalization,

resulting in pneumonia in immunocompromised patients or those on a ventilator.

1.11 Azithromycin

Azithromycin tablets contain the active ingredient, azithromycin, an azalide, a

subclass of macrolide antibiotics, for oral administration. Azithromycin has the chemical

name (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-dideoxy-3-C-methyl-3-O-

methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-

heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-1-

oxa-6-azacyclopentadecan-15-one. Azithromycin is derived from erythromycin; however,

it differs chemically from erythromycin in that a methyl-substituted nitrogen atom is

incorporated into the lactone ring. Its molecular formula is C38H72N2O12, and its

molecular weight is 749. Azithromycin has the following structural formula (Fig 2):

1.11.1 Mechanism of Action

Azithromycin acts by binding to the 50s ribosomal subunit of susceptible

microorganisms and, thus, interfering with microbial protein synthesis. Nucleic acid

synthesis is not affected. Azithromycin concentrates in phagocytes and fibroblasts as

26

demonstrated by in vitro incubation techniques. Using such methodology, the ratio of

intracellular to extracellular concentration was greater than 30 after one hour incubation.

In vivo studies suggest that concentration in phagocytes may contribute to drug

distribution to inflamed tissues.

Fig 2: Structure of Azithromycin.

Azithromycin, as the monohydrate, is a white crystalline powder with a molecular

formula of C38H72N2O12•H2O and a molecular weight of 767.

27

1.11.2 Antibacterial Spectrum

Azithromycin has a broad spectrum of activity. It has activity against the following

organisms:

a) aerobic and facultative gram-positive microorganisms such as: Staphylococcus

aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus

pyogenes. Azithromycin demonstrates cross-resistance with erythromycin-

resistant gram-positive strains. Most strains of Enterococcus faecalis and

methicillin-resistant staphylococci are resistant to azithromycin.

b) aerobic and facultative gram-negative microorganisms such as:

Haemophilus ducreyi, Haemophilus influenzae, Moraxella catarrhalis, Neisseria

gonorrhoeae. In addition, it has activity against Echericia Coli and Chlamydia

pneumoniae.

1.11.3 Therapeutic Uses

Azithromycin tablets are indicated for the treatment of patients with mild to

moderate infections (pneumonia) caused by susceptible strains of the designated

microorganisms. Hence it can be used for both upper and lower respiratory tract

infections, skin and soft tissue infections and sexually transmitted diseases.

1.12 Clarithromycin

Clarithromycin is a semi-synthetic derivative of erythromycin. It is chemically

designated as 6-O-Methyl erythromycin, with empirical formula C38H69NO13 and

molecular formula 747.95.

28

1.12.1. Mechanism of Action

Clarithromycin prevents bacteria from growing by interfering with their protein

synthesis. Clarithromycin binds to the subunit 50s of the bacterial ribosome and thus

inhibits the translation of peptides.

1.12.2 Antibacterial Spectrum and Therapeutic Uses

Clarithromycin has similar antimicrobial spectrum as erythromycin but is more

effective against certain gram-negative bacteria, particularly Legionella pneumophila.

Besides this bacteriostatic effect, clarithromycin also has bactericidal effect on certain

strains such as Haemophilus influenza, Streptococcus pneumoniae and Neisseria

gonorrhoeae.

It is used for the treatment of some bacterial infections (pharyngitis/tonsillitis,

sinusitis, bronchitis, pneumonia, uncomplicated skin and skin structure infections) caused

by H. influenzae, M. catarrhalis, M. pneumoniae, S. pneumoniae, C. pneumoniae

(TWAR), S. aureus, S. pyogenes, Mycobacterium avium and Mycobacterium

intracellulare.

1.13 Objective of this Study

Majority of people in Nigeria are low-income earners and so ‘Branded’ drugs are

unaffordable to them. This has encouraged the trade in generics to thrive. However, the

efficacy of these drugs has always been in doubt considering the prevalence of fake,

substandard and adulterated antibiotics known to be largely responsible for treatment

29

failures, toxicity and development of bacterial resistance. As a result of these, there is

loss in confidence in the Healthcare delivery by the stakeholders.

This work is therefore aimed at:

1. Evaluating the authenticity of label claims by manufacturers of brands of

azithromycin and clarithromycin tablets marketed in Nigeria.

2. Assessing the physical properties of the azithromycin and clarithromycin tablet

brands to verify their compliance to compendial and non-compendial standards.

3. Establishing the bioequivalence or other-wise of the brands of the azithromycin and

clarithromycin tablets using Food and Drug Administration (FDA) F1 similarity factor

and dissolution efficiency (DE) parameter.

4. Ascertaining whether the studied brands in each of the two drugs are interchangeable.

30

CHAPTER TWO

2.0 Materials and Methods

2.1 Materials

2.1.1 Culture Media

The following media were prepared as specified by the manufacturers: Muller

Hinton agar and Nutrient broth (Oxoid) using nutrient agar, nutrient broth , sterile molten

nutrient agar 20 ml (maintained at 45oC), distilled water.

2.1.2 Equipment

The equipment used includes the following: autoclave, hot air oven, incubator

refrigerator, sterile Petri dishes, sterile test tubes, innoculating loop, sterile pipettes (5ml

and 1ml capacity) conical flasks.

2.1.3 Test Organism

The test organism include the laboratory strains of Staphylococcus aureus and

Escherichia coli.

2.1.4 Brands of Azithromycin and Clarithromycin Tablets

Four brands of azithromycin and five brands of clarithromycin tablets marketed in

Nigeria were evaluated. The descriptions of each brand are presented in Table 1.

31

Table 1: Some Physical Properties of brands of Azithromycin and Clarithromycin

Tablets

Brand

Name

Generic Name Manufacturer

(Company)

Manufacturing

Date/Batch

Number

Expiry

Date

Dia-

Meter

(mm)

Thick-

Ness

(mm)

Description

Feature

Zithromax Azithromycin

250mg

Pfizer Pharm

USA (Pfizer)

1st Sept 2008

(0011k07A)

1st Oct

2010

13.5 5.5 Deep Pink

Coated

Caplet

Azith- 250 Azithromycin

250mg

Baroque Pharm

India (Zoomota)

Dec 2008

(BFK001128)

Nov.

2010

13.0 5.0 Yellow

biconcave,

film-coated

tablet

Azifast Azithromycin

250mg

Swiss Pharm

India (Crowther)

Feb 2008

(812)

Jan. 2010 10.5 4.5 Film coated

round tablet

Nobaxin Azithromycin

250mg

Lek-Am Potland

(Jil Nig Ltd)

Nov. 2007

(171207)

Nov.

2009

11.5 4.0 Yellow, film

coated

caplet

Klabax Clarithromycin

500mg

Ranbaxy Pharm

(Ranbaxy)

August 2008

(1834824)

July

2010

19.0 6.5 Scored film

coated

caplet

Klatril Clarithromycin

500mg

Fredun Pharm

India (Reals)

May 2007

(T-7005)

April

2009

19.0 6.5 Yellow film

coated

caplet

Thromyc-

500

Clarithromycin

500mg

Stallion Lab

(Pharmabase)

Nov. 2008

Ex08177

Oct.

2011

19.0 6.5 Yellow film

coated

caplet

Acem- 500 Clarithromycin

500mg

Emcure Pharm

(Fidson)

Sept 2007

(01B07007)

Aug

2010

21.0 6.0 Yellow film

coated

caplet

Clariwin-

500

Clarithromycin

500mg

Micro Labs Ltd

(Strides)

Nov. 2007

(CLWH0043)

Oct 2011 16.5 6.0 Yellow film

coated

caplet

32

Pure Drug Samples

Pure drug samples of azithromycin and clarithromycin powders were obtained from

M.J. Biopharm pvt limited Taloja, Navi-Mumbai India, with certificate of analysis of the

samples attached in Appendix 1.

2.1.5 Dissolution Medium

Sodium phosphate buffer used for azithromycin and sodium acetate buffer for

clarithromycin respectively, as stated in the official monograph (43).

2.2 Methods

2.2.1 Evaluation of In Vitro Tablet Properties

2.2.1.1 Uniformity of Weight

The weights of different brands of azithromycin and clarithromycin tablets were

determined using an electronic weighing balance. Ten different tablets of each brand

were weighed individually. Their mean weight, standard deviations and coefficient of

variations were calculated.

2.2.1.2 Hardness Test (Crushing Strength) and Friability

The Monsanto hardness tester was used to assess the hardness of the tablet brands.

After the instrument was zeroed, one tablet was placed within the crushing chamber and

the screw wound up until the tablet was crushed. The force required to crush each tablet

was noted. This was repeated with five other tablets. The mean hardness and its standard

deviation was calculated for each tablet brand.

33

The friability of the batches was assessed using Roche Friabilator. Ten tablets

from each brand were weighed in an electronic balance. They were subjected to abrassive

shock for 4 minutes at 25 rpm. The tablets were dedusted, reweighed and the percentage

friability calculated from equation 4.

1

100

o

o

W

WWF ...................................................................... (4)

where F is friability (%), Wo is weight of dedusted tablets before friabilation and W is

weight of dedusted tablets.

2.2.1.3 Disintegration Time

Erweka disintegration test apparatus was used. One tablet was introduced into

each of six tubes of the disintegration apparatus containing distilled water and the time

taken for the tablet to completely disintegrate was recorded.

Results presented are mean and standard deviations of disintegration times of six

tablets from each tablet brand.

2.2.1.4 Drug Content

Five tablets from each of the brands were weighed and crushed, and a quantity of the

powder corresponding to 500mg of the pure drug was weighed, dissolved in 100ml of the

appropriate dissolution solvent (media) for azithromycin and clarithromycin to get

1mg/ml of the solution. The absorbances were taken at 215nm and 205nm respectively

using a UV spectrophotometer. The concentration of the sample withdrawn was

determined with reference to Standard Beer’s plot previously obtained.

34

2.2.1.5 Standard Beer-Lambert Plot

One hundred milligram of pure azithromycin and clarithromycin powder were

respectively weighed in a sensitive weighing balance. This was dissolved in 90ml of

sodium phosphate buffer of pH 6.0, and the volume made up to 100ml for azithromycin.

Serial dilution of the stock solution was done as described in the official monograph (43)

to get dilutions containing 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mg/ml respectively. Similar stock

solution and dilutions were obtained for clarithromycin pure drug sample as also

contained in the monograph using 0.1M sodium acetate buffer (45). The respective

absorbances of the various concentrations were read in shimadzu UV-visible

Spectrophotometer at Wavelength (λ) of 215nm for azithromycin and 205nm for

clarithromycin. The obtained absorbances were ploted against the corresponding

concentrations to get the calibration curve or Beer’s plot.

2.2.1.6 Dissolution Test

Erweka dissolution test apparatus was used. The dissolution media was as described

in the USP (27). The dissolution medium consisted of 900 ml sodium phosphate buffer or

sodium acetate for azithromycin and clarithromycin maintained at 37 ± 1oC. One tablet

was placed in the dissolution medium and the apparatus was operated at 50 rev min-1

. At

a predetermined time intervals, 5 ml of the dissolution medium was withdrawn and

replaced with 5 ml of fresh dissolution medium maintained at the same temperature. The

withdrawn samples were diluted appropriately and assayed for azithromycin and

clarithromycin at wavelengths of 215nm and 205nm respectively using the Shimadzu

UV-visible spectrophotometer. The absorbances of the withdrawn samples were

35

converted to concentration (in milligrams) using the Beer’s calibration curve of

azithromycin and clarithromycin respectively.

The dissolution efficiency (DE) is calculated from area under the dissolution time

curve at time ti (measured using the trapezoid rule) and expressed as a percentage of the

area of the rectangle described by 100% dissolution in the same time.

2.3.1 Preparation of media

2.3.1.1 Preparation of nutrient agar

A 28 gram quantity of nutrient agar was suspended in 1 litre of distilled water and

this was allowed to hydrate for 15 minutes. The suspension was heated to dissolve in an

autoclave at 100oC. Twenty milliliter (20 ml) of the molten nutrient agar was dispensed

into agar bottles, previously sterilized at 121oC for 15 minutes in an autoclave. After

sterilization, the sterile media was incubated at 37oC for 24 hours.

2.3.1.2 Preparation of Nutrient Broth

A 13 gram quantity of nutrient broth was dissolved in 1 litre of distilled water by

shaking. A 5 ml volume was dipensed into test tubes, corked and sterilized in an

autoclave at 121oC for 15 minutes. The sterile media was incubated at 37

oC for 24 hours.

36

2.3.2 Preparation of Drug Samples

2.3.2.1 Standard Concentration of Pure Drug Sample

A 5mg of pure drug sample of azithromycin, obtained using an electronic sensitive

balance, was dissolved in 10ml of sterile simulated gastric fluid (SGF) to obtain

0.5mg/ml which is equivalent to 500μg/ml. Further dilution were made from the

500μg/ml using 10 fold serial dilutions to obtain 5μg/ml as the stock solution. The

5μg/ml stock solution was further diluted using two fold serial dilutions to obtain three

other concentrations of 2.5μg/ml, 1.25μg/ml and 0.625μg/ml as standards S1, S2, S3 and

S4 respectively. The same method was also employed in obtaining S1, S2, S3 and S4 for

the clarithromycin pure drug sample.

2.3.2.2 Test Concentration of the Drug Brands

One tablet from each brand of azithromycin tablet corresponding to 250mg of the

drug was dissolved in 50ml of Simulated Gastric Fluid (SGF) to obtain 5mg/ml which is

equivalent to 500μg/ml. Using 10 fold serial dilution, further dilutions were made from

the 500μg/ml to obtain 5μg/ml as the stock solution. Using two fold serial dilution

technique, 1.25μg/ml and 0.65μg/ml were obtained as T1 and T2 for the test brands.

Similarly, one tablet from each brand of clarithromycin tablet corresponding to

500mg of the drug was dissolved in 100ml of SGF to obtain 5mg/ml equivalent to

500μg/ml. Using 10 fold and two fold serial dilutions respectively, 1.25μg/ml and

0.625μg/ml as T1 and T2 were obtained.

37

2.3.3 Standardization of the Micro-Organism

The micro-organisms, Staphylococcus aureus and Escherichia Coli were

standardized turbidimetrically.

2.3.4 One Point Microbiological Assay

The macrobroth diffusion method was used. The two test micro-organisms (S.

aureus and E. coli) were used seperately on the different brands of azithromycin and

clarithromycin being investigated.

A 0.1ml volume of the standardized test microorganisms was seeded in a 20 ml of

sterile molten nutrient agar. The culture plates were divided into five equal segments

using wax pencil. With the aid of a standard cork borer, holes of 8mm in diameter were

bored in each of the segments. Another hole of the same diameter was also bored in the

center (making it six holes in each plate) and each of these holes represents different

concentrations of both standard and the test: S1, S2 ,S3 ,S4 and T1, T2 respectively as

represented in Figs. 3 and 4.

Using a pasture pipette, one drop of each concentration was dropped in each

corresponding hole and was allowed to stand for 15 minutes to ensure proper diffusion

before incubation. The plates were incubated at 37oC for 48 hours.

The inhibition zone diameters (IZDs) were measured and the average taken for

each. A plot of the inhibition zone diameter (IZD) in mm against the log concentration

was obtained for the standard. The various IZDs of the test were traced in the graph to get

the concentration of the drug which was converted to the percentage using the standard.

38

Fig.3: A Cup plate illustration for Assay of Azithromycin Tablet Brands

S1

S4

S2

T2

S3

T1

S1

S3

T1

S2

T2

S4

Fig.4: A Cup plate illustration for Assay of Clarithromycin Tablet Brands

39

CHAPTER THREE

3.0 Results And Discussion

3.1 In Vitro Tablet Properties

3.1.1 Uniformity of Weight

The results of the weight uniformity tests of the tablet brands, clearly show that all

the brands passed the US weight variation limits, as all the brands of azithromycin and

clarithromycin tablets had coefficient of variation of less than 5% (Tables 2 and 3). This

shows that there was uniformity in die filling during the compression for all the tablet

brands..

Weight uniformity determination in tablet formulations indicate the pattern of die

filling as the granules pour into the die prior to compression (46). The amount of granule

compressed is the amount that fills the dies at each compression cycle.

3.1.2 Crushing Strength (Hardness)

The results of the hardness test on the tablet brands are presented in tables 2 and 3.

In the azithromycin brands, Zithromax showed the highest mean hardness value of

11.58 ± 0.58 kgf. Hardness value of 7.80 ± 0.22 kgf was obtained for Azith, 6.00 ± 0.38

kgf for Nobaxin and 5.83 ± 0.52 kgf forAzifast (Table 4).

In the clarithromycin brands, Clariwin showed the highest mean hardness of 12.10

± 0.67 kgf. Klabax, Thromyc, Klatril and Acem had hardness of 9.90 ± 0.58, 9.41 ±

0.38, 7.75 ± 0.28 and 7.67 ± 0.82 kgf respectively ( Table 5) .

The relatively high and varied hardness of the different tablet brands may be

attributed to the type and concentration of binder employed in the production of the

tablets. The method of granulation and compression force employed could also be

40

responsible (47). Generally, it is very crucial that tablets must posses some level of

hardness to enable them withstand mechanical stress during handling in manufacture,

packaging, shipping and distribution. Tablets with crushing strength of greater or equal to

5kgf are considered acceptable for normal release tablets (45). This implies that all the

brands have acceptable hardness.

3.1.3. Friability

The percentage friability for all the brands of both drugs were less than 1%. Though

friability has no upper or lower limit specified in the official monographs, conventionally

compressed normal release tablets with friability of 1 % or less are considered acceptable

(48). The results of the tablets friability in Tables 4 and 5 indicate that the tablets will

withstand the rigors of handling and transportation.

3.1.4 Disintegration Time Test

The results of the study as presented on Tables 4 and 5 show that all the tablet

brands disintegrated within 30 minutes. The longer disintegation time value of Clariwin

was consistent with its observed higher hardness value while Zithromax disintegrated

within a very short time despite its observed higher hardness in the azithromycin brand.

Differences in disintegration time of tablets could be attributable to the type and

concentrations of binder solution used. Also, the compressional pressure employed

during compaction affects the hardness of tablets and by extension, disintegration time.

Tsige and Alexander (49) reported that starch solution, which forms a thin film of starch

mucilage around the granules forms a viscous barrier between the granules and the water

which ultimately retards disintegration.

41

Disintegration of tablets into granules causes a relatively large increase in effective

surface area of the drug. The overall rate of tablet disintegration is influenced by several

interdependent factors which include the concentration and type of binder, disntegrant,

lubricant, as well as the compation pressure. USP specifies that film coated tablets

should have a standard disintegration time of 30 minutes.

3.2 Beer’s Plot

The Beer’s calibration curve for azithromycin and clarithromycin are shown in

Figures 5 and 6. Azithromycin and clarithromycin obeyed Beer’s law in the concentration

range of 0.1 to 1.0 mg/ml.

42

Table 2: Azithromycin weight Uniformity Analysis Result

Zithromax Azith Azifast Nobaxin

Mean Weight (X) (g) 0.4781 0.5663 0.3551 0.3907

Variance 0.00035222 0.0005243 0.0006796 0.0000266

Standard deviation (S.D) 0.0059 0.00724 0.00824 0.00517

Coefficient of variation (%) 1.255 1.279 2.32 1.32

Table 3: Clarithromycin (mg) Weight Uniformity Analysis Result

Klabax Klatril Thromyc Acem Clariwin

Mean Weight (X) (g) 0.8986 1.0356 0.9912 0.8719 0.6868

Variance 0.00002049 0.00008451 0.0001087 0.00004444 0.0001487

Standard Deviation (S.D) 0.00453 0.00919 0.01043 0.006666 0.01219

Coefficient of variation

(C.V) (%)

0.503 0.888 1.05 0.76 1.78

43

Table 4: Hardness, Friability and Disintegration Time of Azithromycin

Tablet Brands

Parameter Zithromax Azith Azifast Nobaxin

Hardness (Kgf) 11.58 + 0.58 7.80 + 0.22 5.83 + 0.52 6.0+ 0.38

Friability (%) 0.05 0.12 0.92 0.15

Disintegration

Time (min)

3.75 + 0.50 3.25 + 0.50 11.50 + 1.29 5.75+ .32

Table 5: Hardness, Friability and Disintegration Time of Clarithromycin

Tablet Brands

Parameter Klabax Klatril Thromyc Acem Clariwin

Hardness

(Kgf)

9.90 + 0.58 7.75 + 0.28 9.41 + 0.38 7.67+ 0.82 12.10 + 0.67

Friability (%) 0.03 0.07 0.0.6 0.06 0.10

Disintegration

Time (min)

4.50 + 1.29 2.25 + 0.95 7.50 + 1.00 4.25+ 0.96 16.50 + 0.58

44

Fig 5: Beer’s Plot of Azithromycin in Sodium Phosphate Buffer

of pH 6.0

45

Fig 6: Beer’s Plot of Clarithromycin in Sodium Acetate Buffer of

pH 5.0

46

3.3 Drug Content

The results of drug content (assay) of the brands of azithromycin and

clarithromycin tablets are presented in Tables 6 and 7. The results show that all the tablet

brands contain the correct amount of the active ingredients (45), and therefore can be

considered acceptable. Presence of active ingredient in the correct proportion in a dosage

form is essential to its therapeutic effectiveness. Globally, a dosage form that contains

no or insufficient quantity of active ingredient is considered fake or substandard (50).

47

Table 6: Absolute Drug Content of Azithromycin Tablet Brand

S/no Brand Drug Content (mg) %

1 Zithromax 252.08 100.83

2 Nobaxin 245.80 98.32

3 Azith-250 245.60 98.42

4 Azifast 245.13 98.05

Table 7: Absolute Drug Content of Clarithromycin Tablet Brands

S/no Brand Drug Content (mg) %

1 Thromyc 500 499.70 99.94

2 Klabax 491.20 98.24

3 Acem 500 486.15 97.23

4 Klatril 483.05 96.61

5 Clariwin 482.10- 96.42

48

3.4 Dissolution Profiles

The four brands of azithromycin and five brands of clarithromycin tablets

exhibited good release profiles. In the azithromycin brands, Zithromax, Azifast and

Nobaxin released above 70% of the active ingredient within 45 minutes while Azith

released about 68% within the same time. Similar trend was not observed with the

clarithromycin brands as it took slightly longer time to attain similar level of release

(Figures 7 and 8).

At 60 minutes, over 85% of the active ingredients from all the brands except

Klatril were released. This suggests that non of the tablet brands contains excipients that

have tendency to delay disintegration and /or dissolution. Furthermore, the relatively high

hardness of the tablets appeared not to have exerted any appreciable delay in the drug

dissolution from the tablets.

Dissolution rate test is an in vitro test designed to infer the in vivo availability of

orally administered tablets. Dissolution rate of tablets is usually considered the rate-

limiting step to drug bioavailability, because drugs are absorbed from solution. A number

of factors are known to influence the rate of tablet dissolution. These include: type and

volume of dissolution medium used, compressional force, type and concentration of

binder, disintegrant and other added excipients used, amount of lubricant, temperature of

the dissolution medium and the agitation rate (47).

49

Fig. 7: Dissolution Profile of Azithromycin Tablet Brands

50

Fig. 8: Dissolution Profile of Clarithromycin Brands

51

3.5 Microbiological Assay

The results of the microbiological assay of azithromycin and clarithromycin tablets

are shown in Tables 8 and 9. Microbiological assay of azithromycin tablets in the

presence of Staphylococcus aureus indicated drug content of 95 % for Zithromax, 93 %

for Azith, 85 % for Nobaxin and 82 % for Azifast. When E. coli was used as assay

organism, the drug content obtained were 117, 92, 94 and 98 % for Azifast, Nobaxin,

Zithromax and Azith respectively (Table 8).

In the presence of Staph. aureus, the assay results obtained for the clarithromycin

tablet brands show percentage of 98.0, 90.0, 116.0,100.8 and 103.0 % for Klatril, Klabax,

Acem, Thromyc and Clariwin respectively. Corresponding percentage assay values of

95.0, 91.0, 96.0, 103.3 and 98.0 % were obtained when E. Coli was used as assay

organism (Table 9).

The accuracy of assay of antibiotic via the microbiology method depends on the

micro-organism used, the medium, incubation temperature and on the care taken in the

preparation of the standard and test antibiotic solutions. It also depends on the sensitivity

of the micro-organism to the antibiotic (51). The results in tables 8 and 9 indicate that

E. coli was more sensitive to azithromycin than to Staph. aureus while Staph. aureus and

E. coli had almost the same level of sensitivity to clarithromycin. The results further

indicates that E. coli would be preferable to Staph. aureus as an organism for the

microbiological assay of azithromycin while E.coli or Staph. aureus could be used in the

microbiological assay of clarithromycin.

52

Table 8: Microbiological Assay Results of Azithromycin Tablet Brands

Brand Drug Content (%)

Staph aureus E.coli

Azifast 82.0 117.0

Nobaxin 85.0 92.0

Zithromax 95.0 98.0

Azith 93.0 94.0

Table 9: Microbiological Assay Results of Clarithromycin Tablet Brands

Brand Drug Content (%)

Staph aureus E.coli

Klatril 98.0 95.0

Klabax 90.0 91.0

Acem 116.0 96.0

Thromyc 100.8 100.3

clariwin 103.0 98.0

53

3.6.1 Bioequivalence Prediction (F1 Values)

The results obtained using Zithromax (R) and Klabax (R) as innovator brands for

azithromycin and clarithromycin tablet brands respectively are presented in Table 10 and

11.

The results show that all brands of azithromycin and clarithromycin show equivalent

dissolution profiles . This suggests that they could be bioequivalent in vivo. The Moore

and Flanner (17) model equation approved by the FDA (F1 similarity factor) stipulates

that F1 values in the range of 0-15% indicate bioequivalence while values above 15%

indicate inequivalence or difference.

3.6.2 Dissolution Efficiency (DE)

The results of the dissolution efficiency at 30 minnutes (DE30

) are shown in Table

12 and 13. There were no significant differences (p ≥ 0.5) in the DE30

of all tablet brands

of azithromycin or clarithromycin. Dissolution efficiency (DE) offers a suitable

alternative to the single point dissolution measurement for quality control of immediate

release products (52). It has been employed to predict the relative in vivo bioavailability

of different brands of ciprofloxacin and pefloxacin film coated tablets (53,54).

The DE parameter is claimed to offer a good theoritical correlation with in vivo data.

This is because the DE as well as the degree of absorption of a drug in vivo is

proportional to the concentration in solution and to the time this solution is in contact

with a suitable absorptive region of the gastro-intestinal tract (31,32).

In addition, DE is a comparative parameter which allows comparism to be

made between a large number of formulations (31).

54

3.6.3 Comparison of the Results of the Spectrophotometric and the Microbiological

assay methods

The assay results obtained with the two methods are in conformity with the

compendial requirements (45) for the content of active ingredient of tablets of

azithromycin and clarithromycin. Except for the Azifast brand of azithromycin (with E.

coli as assay organism), there are no significant differences between the assay results of

the other three brands of azithromycin (Nobaxin, Zithromax and Azith) using the two

assay methods. For clarithromycin tablet brands, a significant difference was obtained

only with Acem in the presence of Staph. aureus. Overall, microbiological assay using

E. coli as the organism gave results comparable to that obtained by the

spectrophotometric method for the two drugs.

3.6.4 Correlation Between F1 Values and Dissolution Efficiencies

The results of all the brands of azithromycin and clarithromycin show that there is no

significant differences (p > 0.5) between the F1 and dissolution efficiencies obtained. This

is as shown in the statistical analysis in which the results of the F1 of all the brands were

compared to the dissolution efficiency values.

55

Table 10: F1 Values of Azithromycin brands (%)

Nobaxin Azith Azifast

3.07 6.31 2.70

Table 11: F1 Values of Clarithromycin brands (%)

Clariwin Klatril Acem Thromyc

7.39 11.55 7.10 1.00

56

Table 12: Dissolution Efficiency (DE30

) of Azithromycin Tablet Brands

Brand Dissolution Efficiency

(%)

Zithromax 25.0

Azith 22.5

Azifast 24.5

Nobaxin 26.0

Table 13: Dissolution Efficiency (DE30

) of Clarithromycin Tablet Brands

Brand

Dissolution Efficiency

(%)

Thromyc 22.5

Klabax 21.5

Acem 20.5

Klatril 19.3

Clariwin 21.0

57

CHAPTER FOUR

Conclusion

The many factors and process involved in the production of pharmaceutical dosage

forms affect the bioavailability of formulated drugs in tablet and capsule dosage forms.

The various brands of azithromycin and clarithromycin exhibited fair hardness and

disintegration resulting in their good release profile. It is interesting to note that despite

the higher hardness of the innovator brand of azithromycin (Zithromax), the

disintegration time was low and the release profile very good.

The weight uniformly test indicates that the four brands of azithromycin and

clarithromycin conformed with the United States Pharmacopeias specification of not

more than 5% deviation for tablets weights of 250mg or more (55). Similarly all the

brands complied with the friability test specification of less than 1% loss in weight and

no tablet caps,laminates or breaks up in the course of the test.

In terms of absolute drug content, all the brands were within the Pharmacopea

range of ± 10% of the stated or labeled claim. This when compared with the innovator

brands of each drug have active drug content of between 98.5 % - 100.5 %. This satisfied

the specified limit of 99-101 % (56). This result was also corroborated by the

microbiological assay using two micro-organisms.

Further statistical analysis of the two results obtained from the two assay methods

showed that the difference between the obtained results were non-significant (P>0.05)

and the methods independent. The application of the FDA approved Moore and Flanner

equation in assessing bioequivalence showed that all the brands are similar,and could be

considered bioequivalent. The correlation between the similarity factor (F1) and the

58

dissolution efficiencies (DE) shows that the methods are different and the results were

significant (p< 0.05).

In conclusion, this work has shown that the dissolution efficiencies (DE) of the

brands of azithromycin and clarithromycin respectively are similar and are thus inter-

changeable with the innovator brands, of Zithromax and Klabax. It is also mandatory for

manufacturers and all other key players in drug distribution business including the

importers and industrial pharmacists to assure the final consumers of high quality and

efficacious products. This is only possible in an environment of high ethical and moral

standard.

59

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66

APPENDIX 1

67

68

69

70

71

APPENDIX 2

72

73

74

75

76

APPENDIX 3

Table 14: Azithromycin Pure Drug (mg/ml)

Concentration (mg/ml) Absorbance

0.1 0.151

0.2 0.302

0.4 0.604

0.6 0.906

0.8 1.208

1.0 1.512

Table 15: Clarithromycin Pure Drug (mg/ml)

Concentration (mg/ml) Absorbance

0.1 0.315

0.2 0.632

0.4 0.260

0.6 1.898

0.8 2.524

1.0 3.154

77

APPENDIX 4

Table 16: Uniformity of Weight for Azithromycin Tablet Brands (mg)

Tablet Zithromax Azith Azifast Nobaxin

1 471.4 565.4 353.2 390.2

2 472.3 580.4 353.5 395.9

3 474.6 554.5 374.1 387.7

4 480.9 564.2 354.9 395.4

5 469.2 561.0 356.3 393.0

6 467.9 574.5 346.7 383.1

7 482.1 565.9 352.8 389.7

8 479.0 569.7 343.9 393.0

9 470.0 561.7 353.7 382.2

10 464.0 565.8 361.4 397.1

Mean ( X ) 473.1 566.31 355.1 390.7

78

Table 17: Uniformity of Weight for Clarithromycin Tablet Brands (mg)

Tablet Klabax Klatril Thromyc Acem Clariwin

1 891.8 1029.9 986.8 872.5 679.7

2 899.7 1043.4 995.8 872.0 682.7

3 894.5 1025.2 976.4 869.4 675.6

4 900.5 1038.1 978.3 872.6 718.8

5 898.3 1047.2 1008.4 868.4 690.5

6 897.9 1025.9 984.7 874.4 683.6

7 899.1 1040.8 993.2 855.6 678.6

8 901.8 1031.1 1005.4 877.7 690.5

9 894.7 1025.6 989.6 878.9 684.8

10 908.1 1048.9 993.7 877.0 683.2

Mean ( X ) 898.6 1035.6 991.2 871.9 686.8

79

APPENDIX 5

Table 18. Weight Uniformity Summary of Azithromycin Tablet Brands

Zithromax Azith Azifast Nobaxin

0.4790 ± 0.0059 0.5660 ± 0.0072 0.3550 ± 0.0082 0.3910 ± 0.0052

Table 19. Weight Uniformity Summary of Clarithromycin Tablet Brands

Klabax Klatril Thromyc Acem Clariwin

0.8990 ± 0.0045 1.0360 ± 0.0092 0.9910 ± 0.0104 0.8720 ± 0.0067 0.6870 ± 0.0122

80

APPENDIX 6

Table 20: Azithromycin Tablet Brands Disintegration Time (min)

No Zithromax Azith Azifast Nobaxin

1 4 11 11 5

1 3 3 12 7

3 4 3 13 5

Means(x) 3.75 3.25 11.5 5.75

Table 21: Clarithromycin Brands Disintegration Time (min)

No Klabax Klatril Thromyc Acem Clariwin

1 3 1 8 5 17

2 4 3 8 5 16

3 6 2 8 4 16

4 5 3 6 3 17

Mean(x) 4.5 2.25 7.5 4.25 16.5

81

APPENDIX 7

Table 22: Analysis of Disintegration Time of

Azithromycin Tablet Brands

Zithromax Azith Azifast Nobaxin

Mean( X )disintegration time 3.75 3.25 11.5 5.75

Variance 0.25 0.25 1.6 0.917

Standard deviation (S.D) 0.5 0.5 1.29 0.957

Coefficient of variation (%) 133.3 15.38 11.22 5.5

Table 23: Clarithromycin Disintegration Time Analysis

Klabax Klatril Thromyc Acem Clariwin

Mean( X )disintegration time 4.5 2.25 7.5 4.25 16.5

Variance 1.67 0.92 1 0.92 0.33

Standard Deviation (S.D) 1.29 0.95 1 0.96 0.58

Coefficient of variation (C.V) (%) 28.7 42.5 13.3 22.5 3.4

82

APPENDIX 8

Table 24: FRIABILITY for Azithromycin Brands

Zithromax Azith Azifast Nobaxin

Weight of 10 tabs before (g) 4.705 5.652 3.587 3.944

Weight of 10 tabs after (g) 4.703 5.645 3.554 33.938

Difference (g) 0.002 0.007 0.033 0.006

Friability (%) 0.05 0.12 0.92 0.15

Table 25: FRIABILITY for Clarithromycin Brands

Klabax Klatril Thromyc Acem Clariwin

Weight of 10 tabs before (g) 8.970 10.356 9.937 8.733 6.861

Weight of 10 tabs after (g) 8.9677 10.349 9.931 8.728 6.854

Difference (g) 0.003 0.007 0.006 0.005 0.007

Friability (%) 0.03 0.07 0.06 0.06 0.10

83

APPENDIX 9

Table 26: Dissolution Result for Zithromax - 250

S/No Withdrawal

time (minutes)

Absorbance Concentration

(mg/ml)

Amount of

Drug released

(mg)

%

Released

1 5 0.069 0.0457 20.55 8.15

2 10 0.138 0.0913 41.10 16.31

3 15 0.207 0.1370 61.65 24.46

4 20 0.277 0.1833 82.50 32.72

5 30 0.415 0.2750 123.6 49.03

6 40 0.552 0.3653 164.40 65.22

7 45 0.621 0.4110 184.96 73.37

8 60 0.762 0.5043 226.95 90.02

9 75 0.762 0.5043 226.95 90.02

10 90 0.762 0.5043 226.95 90.02

84

Table 27: Dissolution Result for Nobaxin - 250

S/No Withdrawal

Time(mins)

Absorbance Concentration

(mg/ml)

Amount of Drug released

(mg)

%

Released

1 5 0.071 0.0470 21.15 8.60

2 10 0.142 0.0940 42.29 17.21

3 15 0.213 0.1410 63.44 25.81

4 20 0.284 0.1880 84.59 34.41

5 30 0.426 0.2820 126.88 51.62

6 40 0.568 0.3759 169.17 68.82

7 45 0.639 0.4229 190.32 77.43

8 60 0.781 0.5169 232.61 94.63

9 75 0.781 0.5169 232.61 94.63

10 90 0.781 0.5169 232.61 94.63

85

Table 28: Dissolution Result for Azith - 250

S/No Withdrawal time

(minutes)

Absorbance Concentration

(mg/ml)

Amount of Drug

released (mg)

%

Released

1 5 0.062 0.0410 18.47 7.52

2 10 0.123 0.0814 36.63 14.92

3 15 0.186 0.1231 55.40 22.56

4 20 0.248 0.1641 73.86 30.07

5 30 0.372 0.2462 110.80 45.11

6 40 0.496 0.3282 147.72 60.15

7 45 0.558 0.3693 166.19 67.67

8 60 0.744 0.4924 221.59 90.22

9 75 0.744 0.4924 221.59 90.22

10 90 0.744 0.4924 221.59 90.22

86

Table 29: Dissolution Result for Azifast - 250

S/No Withdrawal time

(minutes)

Absorbance Concentration

(mg/ml)

Amount of Drug

released (mg)

%

Released

1 5 0.067 0.0443 19.96 8.14

2 10 0.135 0.0894 40.21 16.40

3 15 0.204 0.1350 60.76 24.79

4 20 0.269 0.1780 80.12 32.68

5 30 0.405 0.2681 120.62 49.21

6 40 0.536 0.3548 159.64 65.13

7 45 0.603 0.3991 179.60 73.27

8 60 0.741 0.4904 220.69 90.33

9 75 0.741 0.4904 220.69 90.33

10 90 0.741 0.4904 220.69 90.33

87

Table 30: Summary of Azithromycin Tablet Brands Release Profile

S/No Withdrawal time (min) Zithromax Nobaxin Azith Azifast

1 5 8.15 8.60 7.52 8.14

2 10 16.31 17.21 14.92 16.40

3 14 24.46 25.81 22.56 24.79

4 20 32.72 34.41 30.07 32.68

5 30 49.03 51.62 45.11 49.21

6 40 65.22 68.82 60.15 65.13

7 45 73.37 77.43 67.67 73.27

8 60 90.02 94.63 90.22 90.03

9 75 90.02 94.63 90.22 90.03

10 90 90.02 94.63 90.22 90.03

88

Table 31: Dissolution Result for Clariwin - 500

S/No Withdrawal time

(minutes)

Absorbance Concentration

(mg/ml)

Amount of Drug

released (mg)

%

Released

1 5 0.116 0.0368 33.08 6.86

2 10 0.234 0.0742 66.74 13.84

3 15 0.350 0.1109 99.82 20.71

4 25 0.467 0.1480 133.19 27.63

5 30 0.701 0.2221 199.93 41.47

6 40 0.937 0.2969 267.24 55.43

7 45 1.054 0.3340 300.61 62.35

8 60 1.402 0.4443 399.86 82.94

9 75 1.518 0.4810 432.95 89.80

10 90 1.518 0.4810 432.95 89.80

89

Table 32: Dissolution Result for Klabax - 500

S/no Withdrawal time

(minutes)

Absorbance Concentration

(mg/ml)

Amount of drug

released (mg)

%

released

1 5 0.157 0.0498 44.78 9.12

2 10 0.285 0.0903 81.28 16.65

3 15 0.413 0.1309 117.79 23.98

4 20 0.541 0.1714 154.30 31.41

5 30 0.797 0.2526 227.31 46.28

6 40 1.053 0.3337 300.32 61.14

7 45 1.182 0.3746 337.12 68.63

8 60 1.437 0.4554 409.84 83.44

9 75 1.547 0.4902 441.22 89.82

10 90 1.547 0.4902 441.22 89.82

90

Table 33: Dissolution Result for Klatril - 500

S/no Withdrawal time

(minutes)

Absorbance Concentration

(mg/ml)

Amount of drug

released (mg)

%

released

1 5 0.108 0.0342 30.88 6.38

2 10 0.217 0.0688 61.89 12.81

3 15 0.326 0.1033 92.98 19.25

4 20 0.434 0.1375 123.78 25.62

5 30 0.652 0.2066 185.96 38.50

6 40 0.869 0.2754 247.85 51.31

7 45 0.978 0.3099 278.93 57.74

8 60 1.296 0.4107 369.62 76.52

9 75 1.522 0.4823 434.09 89.86

10 90 1.522 0.4823 434.09 89.86

91

Table 34 :Dissolution Result for Acem - 500

S/no Withdrawal time

(minutes)

Absorbance Concentration

(mg/ml)

Amount of drug

released (mg)

%

released

1 5 0.117 0.0371 33.37 6.86

2 10 0.230 0.0729 65.60 13.49

3 15 0.351 0.1112 100.11 20.59

4 20 0.468 0.1483 133.48 27.46

5 30 0.702 0.2225 200.22 41.18

6 40 0.936 0.2966 266.95 54.91

7 45 1.053 0.3337 300.32 61.78

8 60 1.404 0.4449 400.43 82.37

9 75 1.531 0.4852 436.65 89.81

10 90 1.531 0.4852 436.65 89.81

92

Table 35: Dissolution Result for Thromyc - 500

S/no Withdrawal time

(minutes)

Absorbance Concentration

(mg/ml)

Amount of drug

released (mg)

%

released

1 5 0.131 0.0415 37.36 7.48

2 10 0.262 0.0830 74.72 14.95

3 15 0.393 0.1245 112.09 22.43

4 20 0.524 0.1661 149.45 29.90

5 30 0.786 0.2491 224.17 44.86

6 40 1.048 0.3321 298.90 59.82

7 45 1.179 0.3736 336.26 67.29

8 60 1.572 0.4982 448.35 89.72

9 75 1.577 0.4998 449.77 90.08

10 90 1.577 0.4998 449.77 90.08

93

Table 36: Summary of Clarithromycin Tablet Brands Release Profile

S/no Withdrawal time (min) Thromyc Klabax Acem Klaril Clariwin

1 5 7.48 9.12 6.86 6.38 6.86

2 10 14.95 16.65 13.49 12.81 13.84

3 14 22.43 23.65 20.59 19.25 20.71

4 20 29.90 31.41 27.46 25.62 27.63

5 30 44.86 46.28 41.18 38.50 41.47

6 40 59.82 61.14 54.91 51.31 55.43

7 45 67.29 68.63 61.78 57.74 62.35

8 60 89.72 83.44 82.37 76.52 82.94

9 75 90.08 89.82 89.81 89.86 89.80

10 90 90.08 89.82 89.81 89.86 89.80

94

APPENDIX 10

Table 37: Inhibition Zone Diameter (IZD) of AZIFAST – 250mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 27 - 21 17 19 14

2 28 25 21 17 20 13

3 28 25 21 17 19 14

4 28 24 21 17 20 14

5 28 25 22 17 20 14

Average IZD (mm) 27.8 24.75 21.2 17 19.6 13.8

Table 38:Inhibition Zone Diameter (IZD) of Nobaxin - 250mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 28 24 21 17 20 14

2 28 25 21 17 21 14

3 28 25 20 17 21 13

4 26 - - - - -

5 27 25 22 17 20 14

Average IZD (mm) 27.4 24.75 21 17 20.5 13.75

95

Table 39:Inhibition Zone Diameter (IZD) of ZITHROMAX - 250mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 28 25 22 17 21 15

2 25 25 21 16 21 14

3 28 25 21 17 21 14

4 28 25 21 17 21 14

5 27 25 21 17 21 15

Average IZD(mm) 27.2 25 21.2 16.8 21 14.4

Table 40: Inhibition Zone Diameter (IZD) of AZITH – 250mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 28 24 21 17 20 15

2 28 24 22 17 19 15

3 28 25 21 17 20 16

4 28 25 21 17 20 15

5 28 25 20 17 19 15

Average IZD (mm) 28 24.6 21 17 19.6 15.2

96

Table 41: Inhibition Zone Diameter (IZD) of AZIFAST – 250mg Against

Escherichia coli

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 25 21 17 14 17 13

2 26 21 18 13 17 13

3 25 21 17 13 15 13

4 26 20 16 13 17 13

5 26 22 17 13 17 13

Average IZD (mm) 25.6 21 17 13.2 16.6 13

Table 42: Inhibition Zone Diameter (IZD) of NOBAXIN – 250mg Against

Escherichia coli

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 26 21 17 13 17 9

2 26 21 17 12 17 9

3 25 22 17 12 16 10

4 26 21 16 13 17 9

5 26 20 17 13 15 8

Average IZD (mm) 26 21 16.8 12.6 16.4 9

97

Table 43: Inhibition Zone Diameter (IZD) of ZITHROMAX – 250mg Against

Escherichia coli.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 26 21 18 13 17 12

2 26 21 17 14 17 13

3 25 22 17 13 16 12

4 26 21 17 13 15 13

5 26 21 17 12 17 13

Average IZD (mm) 26 21.2 17.2 13 16.4 12.6

98

Table 44: Inhibition Zone Diameter (IZD) of AZITH – 250mg Against Escherichia

coli.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 26 20 17 13 16 13

2 26 22 17 14 16 13

3 25 21 17 14 16 12

4 26 21 16 13 17 12

5 25 20 * 13 15 13

Average IZD (mm)

25.6 20.8 16.8 13.4 16 12.6

99

Table 45: Inhibition Zone Diameter (IZD) of KLATRIL– 500mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

5 2.5 1.25 0.625 1.25 0.625

IZD (mm) S1 S2 S3 S4 T1 T2

1 26 22 17 14 17 11

2 26 21 17 14 17 10

3 25 23 17 15 15 12

4 26 22 17 13 16 10

5 26 21 17 16 16 10

Average IZD (mm) 25.8 21.8 17 14.4 16.2 10.6

Table 46: Inhibition Zone Diameter (IZD) of KLABAX– 500mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 26 22 17 14 17 12

2 26 21 17 14 17 12

3 26 23 17 15 16 11

4 26 22 17 13 17 11

5 26 21 17 13 17 11

Average IZD (mm) 26 21.8 17 14.2 16.8 11.6

100

Table 47: Inhibition Zone Diameter (IZD) of ACEM – 500mg Against

Staphylococcus aureus

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 24 22 17 13 16 10

2 26 22 17 14 16 10

3 26 23 17 14 15 11

4 26 21 17 15 16 10

5 25 22 17 15 16 11

Average IZD (mm) 25.4 22 17 14 15.8 10.4

Table 48: Inhibition Zone Diameter (IZD) of THROMYC – 500mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 26 21 18 15 16 12

2 26 23 17 14 15.8 11

3 26 21 16 14 15 12

4 25 22 16 14 16 10

5 26 22 17 13 16 11

Average IZD (mm) 25.8 21.8 16.8 14 15.76 11.2

101

Table 49: Inhibition Zone Diameter (IZD) of CLARIWIN – 500mg Against

Staphylococcus aureus.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 26 23 17 13 15 12

2 26 22 17 13 16 12

3 26 21 17 14 16 12

4 25 21 17 15 15 12

5 25 22 17 14 15 10

Average IZD (mm) 25.8 21.6 17 13.8 15.4 11.8

Table 50: Inhibition Zone Diameter (IZD) of KLATRIL – 500mg Against

Escherichia coli

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 23 18 15 10 13.3 8

2 22 19 15 11 13 9

3 22 19 13 10 14 8

4 23 17 14 9 14 8

5 23 19 13 10 14 8

Average IZD (mm) 22.6 18.4 14 10 13.6 8.2

102

Table 51: Inhibition Zone Diameter (IZD) of KLABAX – 500mg Against

Escherichia coli.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 23 18 13 10 14 8

2 23 19 15 10 13 8

3 23 19 15 10 14 8

4 21 19 14 8 14 8

5 23 18 15 9 14 8

Average IZD (mm) 22.6 18.6 14.4 9.4 13.8 8

Table 52: Inhibition Zone Diameter (IZD) of ACEM – 500mg Against Escherichia

coli.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 23 19 14 8 14 8

2 23 18 15 10 14 8

3 23 18 15 10 14 8

4 23 19 15 10 - 8

5 23 19 12 10 14 9

Average IZD (mm) 23 18.6 14.2 9.6 14 8.2

103

Table 53: Inhibition Zone Diameter (IZD) of THROMYC – 500mg Against

Escherichia coli.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 24 18 15 10 13 8

2 23 19 13 10 14 9

3 23 18 13 9 14 7

4 23 19 15 9 13 -

5 23 19 15 10 14 -

Average IZD (mm) 23.2 18.6 14.2 9.6 13.6 8

Table 54: Inhibition Zone Diameter (IZD) of CLARIWIN – 500mg against

Escherichia coli.

Concentrations (µg/ml)

IZD (mm) S1 S2 S3 S4 T1 T2

1 23 19 14 10 15 9

2 23 19 15 9 15 9

3 23 19 15 10 15 9

4 23 18 15 10 12 8

5 23 20 13 9 14 9

Average IZD (mm) 23 19 14.4 9.6 14.2 8.8

104

APPENDIX 11

Summary Table for Staphylococcus aureus:

Table 55: Azifast against Staph. aureus

Table 56: Nobaxin Against Stap. aureus

NOBAXIN-250mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD

(T)(mm)

5 0.6990 27.4

2.5 0.3979 24.75

1.25 0.0969 21 T1 =20.5

0.625 -0.0241 17 T2 =13.75

AZIFAST - 250mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD

(T)(mm)

5 0.6990 27.8

2.5 0.3979 24.75

1.25 0.0969 21.2 T1 =19.6

0.625 -0.0241 17 T2 =13.8

105

Table 57: Zithromax Against Stap. aureus

ZITHROMAX – 250mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD

(T)(mm)

5 0.6990 27.2

2.5 0.3979 25

1.25 0.0969 21 T1 =21

0.625 -0.0241 16.8 T2 =14.4

Table 58: Azith Against Stap. aureus

AZITH – 250mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD(T) (mm)

5 0.6990 28

2.5 0.3979 24.6

1.25 0.0969 21 T1 =19.6

0.625 -0.0241 17 T2 =15.2

Table 59:Azifast Against Escherichia coli

AZIFAST – 250mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD(T)(mm)

5 0.6990 25.6

2.5 0.3979 21

1.25 0.0969 17 T1 =16.6

0.625 -0.0241 13.2 T2 =16

106

Table 60: Nobaxin Against Escherichia coli

NOBAXIN – 250mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD

(T)(mm)

5 0.6990 26

2.5 0.3979 21

1.25 0.0969 16.8 T1 =16.4

0.625 -0.0241 12.6 T2 =9

Table 61:Zithromax Against Escherichia coli

ZITHROMAX – 250

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD

(T)(mm)

5 0.6990 26

2.5 0.3979 21.2

1.25 0.0969 17.2 T1 =16.4

0.625 -0.0241 13 T2 =12.6

107

Table 62: Azith Against Escherichia coli

AZITH – 250

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD(T )(mm)

5 0.6990 25.6

2.5 0.3979 20.8

1.25 0.0969 16.8 T1 =16

0.625 -0.0241 13.4 T2 = 12.6

Table 63: Klatril Against Staph. aureus

KLATRIL - 500mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD

(T)(mm)

5 0.6990 25.8

2.5 0.3979 21.8

1.25 0.0969 17 T1 =16.2

0.625 -0.0241 14.4 T2 =10.6

Table 64: Klabax Against Staph. aureus

KLABAX – 500mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD (T)(mm)

5 0.6990 26

2.5 0.3979 21.8

1.25 0.0969 17 T1 =16.8

0.625 -0.0241 14.2 T2 =11.6

108

Table 65: Acem Against Staph. aureus

ACEM – 500mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD (T)(mm)

5 0.6990 25.4

2.5 0.3979 22

1.25 0.0969 17 T1 =15.8

0.625 -0.0241 14 T2 =10.4

Table 66: Thromyc Against Staph. aureus

Table 67: Clariwin Against Staph. aureus

CLARIWIN – 500mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD (T)(mm)

5 0.6990 25.8

2.5 0.3979 21.6

1.25 0.0969 17 T1 =15.4

0.625 -0.0241 13.8 T2 =11.8

THROMYC – 500mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD

(T)(mm)

5 0.6990 25.8

2.5 0.3979 21.8

1.25 0.0969 16.8 T1 =15.76

0.625 -0.0241 14 T2 =11.2

109

Table 68: Klatril Against Eschericia coli

KLATRIL – 500mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD(T)(mm)

5 0.6990 22.6

2.5 0.3979 18.4

1.25 0.0969 14 T1 =13.6

0.625 -0.0241 10 T2 =8.2

Table 69: Klabax Against Eschericia coli

KLABAX – 500mg

Concentrations

(μg/ml)

Log Concentrations

μg/ml

IZD (S)(mm) IZD (T) (mm)

5 0.6990 22.6

2.5 0.3979 18.6

1.25 0.0969 14.4 T1 =13.8

0.625 -0.0241 9.4 T2 =8.0

Table 70: Acem Against Eschericia coli

ACEM – 500mg

Concentrations (μg/ml) Log Concentrations μg/ml IZD (S)(mm) IZD(T)(mm)

5 0.6990 23

2.5 0.3979 18.6

1.25 0.0969 14.2 T1 =14

0.625 -0.0241 9.6 T2 =8.2

110

Table 71: Thromyc Against Eschericia coli

THROMYC – 500mg

Concentrations

(μg/ml)

Log

Concentrations

μg/ml

IZD

(S)(mm)

IZD

(T)(mm)

5 0.6990 23.2

2.5 0.3979 18.6

1.25 0.0969 14.2 T1 =13.6

0.625 -0.0241 9.6 T2 =8

Table 72: Clariwin Against Eschericia coli

CLARIWIN – 500mg

Concentrations

(μg/ml)

Log

Concentrations

μg/ml

IZD

(S)(mm)

IZD

(T)(mm)

5 0.6990 23

2.5 0.3979 19

1.25 0.0969 14.4 T1 =14.2

0.625 -0.0241 9.6 T2 =8.8

111

APPENDIX 12

Azithromycin brands

Table 73:F1 Pre-analysis for Nobaxin

S/No

Time (min)

Quantity dissolved (mg)

RT=TT Zithromax (R) Nobaxin

1 5 20.55 21.15 -0.60

2 10 41.10 42.29 -1.19

3 15 61.65 63.44 -1.79

4 20 82.50 84.59 -2.09

5 30 123.60 126.88 -3.28

6 40 164.40 169.17 -4.

7 45 184.96 190.32 -5.36

8 60 226.95 232.61 -5.66

9 75 226.95 232.61 -5.66

10 90 226.95 232.61 -5.66

∑ = 1359.61 ∑ = 41.72

112

Table 74: F1 Pre-analysis for Azith

S/No

Time (min)

Quantity dissolved (mg)

RT=TT Zithromax (R) Azith (T)

1 5 20.55 18.47 2.08

2 10 41.10 36.63 4.47

3 15 61.65 55.40 6.25

4 20 82.50 73.86 8.64

5 30 123.60 110.80 12.8

6 40 164.40 147.7 16.68

7 45 184.96 166.1 18.77

8 60 226.95 22.9 5.36

9 75 226.95 221.59 5.36

10 90 226.95 221.59 5.36

∑ = 1359.61 ∑=85.77

113

Table 75: F1 Pre-analysis for Azifast

S/No

Time (min)

Quantity dissolved (mg)

RT=TT Zithromax (R) Azifast (T)

1 5 20.55 19.96 0.59

2 10 41.10 40.21 0.89

3 15 61.65 60.76 0.89

4 20 82.50 80.12 2.38

5 30 123.60 120.62 2.98

6 40 164.40 159.64 4.76

7 45 184.96 179.60 5.36

8 60 226.95 220.59 6.26

9 75 226.95 220.59 6.26

10 90 226.95 220.59 6.26

∑ = 1359.61 ∑=36.63

114

Clariythromycin brands:

Table 76: F1 Pre-analysis for Klabax

S/No

Time (min)

Quantity dissolved (mg)

RT=TT Klabax (R) Clariwin (T)

1 5 44.78 33.08 11.70

2 10 81.28 66.74 14.54

3 15 117.79 99.82 17.97

4 20 154.30 133.19 21.11

5 30 227.31 199.93 27.38

6 40 300.32 267.24 33.08

7 45 337.12 300.61 36.51

8 60 409.84 399.86 9.98

9 75 441.22 432.95 8.27

10 90 441.22 432.95 8.27

∑ = 2555.18 ∑=188.81

115

Table 77: F1 Pre-analysis for Klatril

S/No

Time (min)

Quantity dissolved (mg)

RT=TT

Klabax (R) Katril(T)

1 5 44.78 30.80 13.98

2 10 81.28 61.89 19.39

3 15 117.79 92.98 24.81

4 20 154.30 123.78 30.52

5 30 227.31 185.96 41.35

6 40 300.32 247.85 52.47

7 45 337.12 278.93 58.19

8 60 409.84 368.62 40.22

9 75 441.22 434.09 7.13

10 90 441.22 434.09 7.13

∑ = 2555.18 ∑=295.19

116

Table 78: F1 Pre-analysis for Acem

S/No

Time (min)

Quantity dissolved (mg)

RT=TT Klabax (R) Acem(T)

1 5 44.78 33.37 11.41

2 10 81.28 65.60 15.68

3 15 117.79 100.11 17.68

4 20 154.30 133.48 20.82

5 30 227.31 200.22 27.09

6 40 300.32 266.95 33.37

7 45 337.12 300.32 36.8

8 60 409.84 400.43 9.41

9 75 441.22 436.65 4.57

10 90 441.22 436.65 4.57

∑ = 2555.18 ∑ = 181.4

117

Table 79: F1 Pre-analysis for Thromyc

S/No

Time (min) Quantity dissolved (mg)

RT=TT Klabax (R) Thromyc (T)

1 5 44.78 37.36 7.42

2 10 81.28 74.72 6.56

3 15 117.79 112.09 5.7

4 20 154.30 149.45 4.85

5 30 227.31 224.17 3.14

6 40 300.32 298.90 1.42

7 45 337.12 336.26 0.86

8 60 409.84 448.35 -38.51

9 75 441.22 449.77 -8.55

10 90 441.22 449.77 -8.55

∑ = 2555.18 ∑=-25.66

118

APPENDIX 13

The weight uniformity, hardness, friability and disintegration test analysis were done

using the formulas below:

Mean ( X ) = Σ(X)

n ……………………………………………………………….(5)

Variance = Σ (X-X)2 where (1<n<30) …………………………………….…(6)

n-1

Standard deviation (S.D) = √Variance ………………………………………(7)

Coefficient of variation = XMean

xSD %100……………………………………...(8)

119

FIGURES

Fig. 9: Dissolution profile of Zithromax

120

Fig. 10: Dissolution Profile of Nobaxin

121

Fig. 11: Dissolution Profile of Azith

122

Fig. 12: Dissolution Profile of Azifast

123

Fig. 13: Dissolution Profile of Thromyc

124

Fig. 14: Dissolution Profile of Klabax

125

Fig. 15: Dissolution Profile of Acem

126

Fig. 16: Dissolution Profile of Klatril

127

Fig. 17: Dissolution Profile of Clariwin

128

Fig. 18: IZD Vs Log Conc. of Azifast against S. aureus

129

Fig. 19: IZD Vs Log Conc. of Nobaxin against Staph. aureus

130

Fig. 20: IZD Vs Log Conc. of Zithromax against Staph. aureus

131

Fig. 21: IZD Vs Log Conc. of Azith against Staph. aureus

132

Fig. 22: IZD Vs Log Conc. of Azifast against E. coli

133

Fig. 23: IZD Vs Log Conc. of Nobaxin against E. coli

134

Fig. 24: IZD Vs Log Conc. of Zithromax against E. coli

135

Fig. 25: IZD Vs Log Conc. of Azith against E. coli

136

Fig. 26: IZD Vs Log Conc. of Klatril against Staph. aureus

137

Fig. 27: IZD Vs Log Conc. of Klabax against Staph. aureus

138

Fig. 28: IZD Vs Log Conc. of Acem against Staph. aureus

139

Fig. 29: IZD Vs Log Conc. of Thromyc against Staph. aureus

140

Fig. 30: IZD Vs Log Conc. of Clariwin against Staph. aureus

141

Fig. 31: IZD Vs Log Conc. of Klatril against E. coli

142

Fig. 32: IZD Vs Log Conc. of Klabax against E. coli

143

Fig. 33: IZD Vs Log Conc. of Acem against E. coli

144

Fig. 34: IZD Vs Log Conc. of Thromyc against E. coli

145

Fig. 35: IZD Vs Log Conc. of Clariwin against E. coli