t he is lam ia u niver sity of b ah awa lpu r

Formulation of Microemulsion Based

Aceclofenac Gel and Its In vitro In vivo Studies

A Critique Submitted

in

Partial fulfillment of the requirements for the degree

of

DOCTOR OF PHILOSOPHY (Pharmaceutics)

by

Muhammad Zubair Malik

B. Pharm., M. Phil. (Pharmaceutics)

Department of Pharmacy,

Faculty of Pharmacy & Alternative

Medicine, Khawaja Farid Campus,

The Islamia University of Bahawalpur

Pakistan (2009-2012)

Certificate

It is hereby certified that the research wok presented by Muhammad Zubair Malik s/o

Malik Muhammad Ramzan Azad in the dissertation entitled “Formulation of

Microemulsion Based Aceclofenac Gel and Its In vitro In vivo Studies” carried out under

my supervision for the fulfillment of the requirements for the degree of Doctor of

Philosophy (Pharmaceutics) in the Department of Pharmacy, Faculty of Pharmacy and

Alternative Medicine, the Islamia University of Bahawalpur.

Prof. Dr. Mahmood Ahmad

Supervisor,

Faculty of Pharmacy and Alternative Medicines,

The Islamia University of Bahawalpur.

Declaration

I, Muhammad Zubair Malik of the Department of Pharmacy, the Islamia University of

Bahawalpur, hereby declare that the research work entitled “Formulation of

Microemulsion Based Aceclofenac Gel and Its In vitro In vivo Studies” is done by me. I

also certify that this dissertation does not incorporate any material previously submitted

for a degree in any university without acknowledgement; and to the best of my

knowledge and belief it does not contain any material previously published or written by

another person where due reference is not made in the text.


II

To

“My Parents, especially to my father (who devoted

his whole life for the betterment of his children and

I am at this stage because of his never ending love

and effort), Teachers (especially Prof. Dr.

Mahmood Ahmad, Rasool Bakhsh sb., Abuzar sb.

and Abdu-Shakoor), Mother, Sister, Brothers

(especially Muhammad Farooq Malik), my

children and my wife for their never ending moral

support and prayers which always proved to be a

hand of blessing in my academic and social life

during hours of worry”

III

If oceans turn into ink and all of the wood becomes pens, even than, the praises of,

“Allah Almighty” cannot be expressed. He who created the Universe and Know

whatever is there in it, hidden or evident. He, the Lord of all the worlds, is the Most

Affectionate, The Merciful and The Master of the Day of Requital. I humbly thank

ALLAH Almighty, the Merciful and the Beneficent, who gave me strength, thoughts,

health and supportive people to enable me achieve this objective. And then all my

respects to Holy Prophet Muhammad (Sallal-ALLAH-o-Alaih-e-wa Aalih-e-

Wasallam) and his Companions who revolutionized the humanity with the teachings

of Islam and shown us the way to success in this world and hereafter.

I would like to express my respectful and sincere gratitude to my respectable

supervisor, Prof. Dr. Mahmood Ahmad (Dean, Faculty of Pharmacy & Alternative

Medicine, the Islamia University of Bahawalpur). I feel myself lucky to be gifted with

his scientific insight, passionate motivation, professional guidance, supportive attitude

and productive advices at the time when I was in any problem or made any mistake

which helped me not only in my research but will also act as a guide in my whole life.

It was only due to his committed efforts that now a days Department of Pharmacy, the

Islamia University of Bahawalpur is considered as one of the best institute in

Pakistan.

I would like to thank Dr. Naveed Akhtar (Chairman, Department of Pharmacy) for

his kind guidance and encouraging attitude. Special thanks for Mr. Muhammad

Usman (Assistant Professor) who helped in publishing article.

I am really thankful to Mr. M. Shafique for his moral and financial support and

encouragement throughout my research work. Special thanks are extended to all my

teachers and praiseworthy colleagues. It was their guidance and motivation which

helped me to achieve this goal. I am profoundly obliged to all my class fellows

especially Saleem Qureshi and Abu Bakar Munir who helped and supported me

throughout my research work. Last but not least I am also thankful to the Higher

Education Commission for successful completion of this huge task without the help of

which I would never think of it!


IV

ABBREVIATION MEANINGS

ACF Aceclofenac

ALI Air Liquid Interface

Approx. Approximately

AUC Area Under the Curve

ANOVA Analysis of variance

B.C. Bicontinuous

BCS Biopharmaceutical classification system

β-CD Beta cyclodextrin

CDNBD 4-carboxyl-2, 6-dinitrobenzenediazonium ion

CG Conventional gel

CHZ Chlorzoxazone

Cl Clearance

Cmax Maximum plasma concentration

Conc. Concentration

˚C Degree Centigrade

Cox Cyclooxygenase

Cryo-FESEM Cryo-field emission scanning electron microscopy

DDA Diclofenac diethyl ammonium

DHP Disodium hydrogen phosphate

DLS Dynamic light scattering

DMSO Dimethyl sulphoxide

DSC Differential Scanning Calorimetry

DS Diclofenac sodium

Er Enhancement ratio

F F relative value for Bioavailability

FTIR Fourier transform infra-red

GIT Gastrointestinal Tract

HCl Hydrochloride

HLD Hydrophilic, Lipophilic deviation

HPLC-UV High performance liquid chromatography with ultra

violet detector

V

HQC High concentration quality control solution

IBU Ibuprofen

IP Isopropyl alcohol

IPP Isopropyl palmitate

Jss Steady state flux

Kc Stability constant

Ke Elimination rate constant

Kg Kilogram

Kp Permeability coefficient

LCDP Lacidipine

LC/MS/MS Liquid chromatography-tandem mass spectrometry

LLP Light liquid paraffin

LOD Limit of detection

LOQ Limit of quantitation

LQC Low concentration quality control solution

ME Microemulsion

MEGs Microemulsion based gels

MHC Major histocompatibility complex

mg milligram

mL, ml milliliter

MQC Medium concentration quality control solution

NMR Nuclear magnetic resonace

NSAIDs Non steroidal anti inflammatory drugs

ODS Octadecyl silane

PARA Paracetamol

PEG Polyethylene glycol

PIT Phase inversion temperature

PLGA Poly(lactic-co-glycolic acid

PVP Poly vinyl pyrrolidone

Q24 Cumulative amount release

q. s Quantity sufficient

RI Refractive index

r.p.m. Revolutions per minute

VI

RSD Relative standard deviation

SA Surface area

SC Stratum corneum

SD Standard Deviation

S.E.M Standard Error of Mean

SEM Scanning electron microscope

ST Surface tension

TEC Triethyl citrate

TEM Transmission electron microscope

TGA Thermogravimetric analysis or thermal gravimetric

analysis

T½ Half life

Tmax Time to reach max concentration

USP-NF United States Pharmacopoeia-National Formulary

UV Ultra violet

Vd Volume of distribution

XRD X-Ray Diffraction

VII

Bismillah ................................................................................................................ I

Dedication ............................................................................................................. II

Acknowledgement .............................................................................................. III

Abbreviation ....................................................................................................... IV

TABLE OF CONTENTS ................................................................................. VII

LIST OF FIGURES ........................................................................................ XIV

LIST OF TABLES ........................................................................................ XXVI

LIST OF APPENDICES .......................................................................... XXXIII

ABSTRACT ................................................................................................ XXXIV

1. INTRODUCTION ................................................................................................. 3

2. LITERATURE REVIEW ............................................................................... 6

2.1 CHARACTERISTICS OF ACECLOFENAC ......................................... 6

2.1.1 Chemical name of Aceclofenac................................................................ 6

2.1.2 Physicochemical Characteristics of Aceclofenac ..................................... 7

2.2 ANATOMY AND PHYSIOLOGY OF SKIN ................................................... 7

2.2.1 Epidermis.................................................................................................. 8

2.2.1.1 Stratum basale .......................................................................................... 8

2.2.1.2 Stratum spinosum ..................................................................................... 9

2.2.1.3 Stratum granulosum ................................................................................. 9

2.2.1.4 Stratum corneum ...................................................................................... 9

2.2.1.5 Stratum lucidum ....................................................................................... 9

2.2.1.6 Dermoepidermal junction/basement membrane .................................... 10

2.2.2 Dermis .................................................................................................... 10

2.2.3 Hypodermis/Subcutaneous Subcutis ...................................................... 11

2.3 BLOOD AND LYMPHATIC VESSELS .............................................. 12

2.4 NERVE SUPPLY ................................................................................... 12

VIII

2.5 DERIVATIVE STRUCTURES OF SKIN ............................................. 12

2.5.1 Hair ......................................................................................................... 12

2.5.2 Nails ........................................................................................................ 13

2.5.3 Sebaceous glands .................................................................................... 13

2.5.4 Sweat glands ........................................................................................... 13

2.6 SKIN FUNCTIONS ............................................................................... 14

2.6.1 Barrier function and skin desquamation ................................................ 14

2.6.1.1 Lipids ...................................................................................................... 15

2.6.1.2 Shedding(desquamation)of skin cells ......................................................... 15

2.6.2 UV protection ......................................................................................... 15

2.6.3 Thermoregulation ................................................................................... 16

2.6.4 Immunological surveillance ................................................................... 16

2.7 SKIN PERMEABILITY STUDIES ....................................................... 20

2.8 SOLUBILITY STUDIES OF ACECLOFENAC AND EXCIPIENTS . 26

2.9 MICROEMULSION .............................................................................. 30

2.9.1 Types of Microemulsion ........................................................................ 32

2.9.2 Methods of microemulsion preparation ................................................. 32

2.9.2.1 Phase Titration Method .......................................................................... 32

2.9.2.2 Phase Inversion Method ......................................................................... 33

2.10 GEL ......................................................................................................... 42

2.10.1 Preparation of Gels ................................................................................. 42

2.10.1.1 Temperature effect .................................................................................. 42

2.10.1.2 Flocculation with salts and non solvents ............................................... 42

2.10.1.3 Chemical reaction .................................................................................. 43

2.11 WORK DONE ON ACECLOFENAC ....................................................... 46

2.12 DETERMINATION OF ACECLOFENAC ................................................ 49

2.13 MECHANISM OF ACTION ................................................................. 52

2.14 PHARMACOKINETICS ....................................................................... 53

2.14.1 Pharmacokinetics of aceclofenac through skin absorption .................... 53

3. MATERIALS AND METHODS .................................................................. 57

3.1 MATERIALS ..................................................................................................57

IX

3.1.1 Instruments............................................................................................... 57

3.1.2 Chemicals ................................................................................................ 57

3.2 METHODS ..................................................................................................... 58

3.2.1 Solubility studies for screening of Excipients ........................................ 58

3.2.2 Calibration Curve for Aceclofenac in Methanol, Ethanol, Isopropyl

Alcohol and n-Butanol ........................................................................... 58

3.2.3 Solubility of Aceclofenac in various oils ............................................... 58

3.2.4 In vitro Permeability Studies of aceclofenac in different oils ................ 59

3.2.5 Pseudo-ternary phase study to construct phase diagram for

microemulsion region ............................................................................. 59

3.2.5.1 Water Titration Method .......................................................................... 59

3.2.5.2 Construction of Pseudoternary phase Diagrams ................................... 60

3.3 SELECTION OF MICROEMULSION FORMULATIONS FOR

DETAILED STUDIES TO INVESTIGATE EFFECTS OF

SURFACTANTS AND CO-SURFACTANTS ON SKIN

PERMEATION ...................................................................................... 63

3.4 PREPARATION OF ACECLOFENAC-LOADED

MICROEMULSIONS.. .......................................................................... 63

3.5 PREPARATION 0F ACECLOFENAC MICROEMULSION Using

Different OIL PHASES .......................................................................... 64

3.5.1 Blank Microemulsion preparations containing oleic acid and almond oil

................................................................................................................ 64

3.6 PREPARATION OF GEL BASES AND ACECLOFENAC

MICROEMULSION BASED GELS ..................................................... 65

3.6.1 Preparation of Carbopol 934 and Carbopol 940 Gel bases ...................... 65

3.6.2 Preparation of Xanthan gum Gel bases ...................................................... 65

3.6.3 Preparation of Carbopol 934, Carbopol 940 and Xanthan gum based

Gels containing microemulsion without active drug ............................. 65

3.6.4 Preparation of Carbopol 934, Carbopol 940 and Xanthan gum based Gel

containing microemulsion with active drug ............................................... 66

X

3.6.5 Preparation of Carbopol 934, Carbopol 940 and Xanthan gum based gels

containing hydro-alcoholic solution ....................................................... 66

3.7 CHARACTERIZATION OF MICROEMULIONS AND

ACECLOFENAC MICROEMULSION BASED GEL ......................... 68

3.7.1 Visosity ................................................................................................... 68

3.7.2 Spreadability ........................................................................................... 68

3.7.3 Conductivity Measurements ................................................................... 68

3.7.4 pH Measurements ................................................................................... 68

3.7.5 Refractive Index measurements ............................................................. 68

3.7.6 % Transmittance measurements ............................................................. 69

3.7.7 Centrifugation (Phase separation test) ................................................... 69

3.7.8 Drug content ........................................................................................... 69

3.7.9 Homogeneity .......................................................................................... 69

3.7.10 SEM (Scanning Electron Microscope) ................................................... 69

3.7.11 Fourier Transform Infra red (FTIR) ...................................................... 69

3.7.12 X-Ray Diffraction (XRD) ...................................................................... 70

3.7.13 Thermo Gravimetric Analysis (TGA) and Differential Scanning

Calorimetry (DSC) ................................................................................. 70

3.7.14 Globule charge (Zeta Potential) and globule size distribution (Zeta Size)

................................................................................................................ 70

3.7.15 In-vitro Skin permeation release rate experiments of Aceclofenac from

Microemulsions and Microemulsion based Gel ...................................... 70

3.7.15.1 Skin Preparation .................................................................................... 70

3.7.15.2 Skin Barrier Integrity Checking ............................................................. 71

3.7.15.3 Franz Diffusion Cell ............................................................................... 71

3.7.16 Assay of Aceclofenac for Permeation Experiments............................... 72

3.7.16.1 Standard Preparation ............................................................................. 72

3.7.16.2 Sample preparation ................................................................................ 72

3.7.17 In-vitro data calculation ......................................................................... 72

3.7.17.1 Cumulative Amount of Drug Permeated per unit area (Qn) ................. 72

3.7.17.2 Steady State Flux (Jss) ........................................................................... 73

XI

3.7.17.3 Permeability Coefficient (Kp) ................................................................ 73

3.8 STABILITY STUDIES .......................................................................... 73

3.9 IN VIVO TRANSDERMAL STUDIES IN RABBITS .......................... 74

3.10 ANTI-INFLAMMATORY ACTIVITY STUDY IN RATS ................. 74

3.11 STUDY OF ANALGESIC EFFECT IN RATS ..................................... 75

3.12 SKIN IRRITATION STUDY ................................................................ 75

3.13 HPLC METHOD VALIDATION .......................................................... 76

3.13.1 Accuracy and Precision .......................................................................... 76

3.13.2 Specificity/Selectivity ............................................................................ 76

3.13.3 Detection limit and Quantitation limit ................................................... 76

3.13.4 Linearity and range ................................................................................. 76

3.13.5 Freeze thaw stability of aceclofenac in plasma ...................................... 76

3.13.6 Extraction yield/Recovery of aceclofenac ............................................. 76

3.14 APPROVAL OF THE STUDY.............................................................. 77

3.15 METHODS FOR IN-VIVO DETERMINATION .................................. 77

3.15.1 Inclusion criteria ..................................................................................... 77

3.15.2 Exclusion Criteria ................................................................................... 77

3.15.3 Administration of dugs ........................................................................... 78

3.15.4 Sample Collection .................................................................................. 78

3.15.5 Preparation of the Mobile Phase ............................................................ 78

3.15.6 Preparation of stock solutions and working standard solutions ............. 78

3.15.7 Preparation of plasma standards and samples ........................................ 79

3.15.8 Column ................................................................................................... 79

3.15.9 Flow rate ................................................................................................. 79

3.15.10 UV Detection Wavelength ..................................................................... 79

3.16 PHARMACOKINETIC PARAMETERS .............................................. 79

3.17 STATISTICAL ANALYSIS .................................................................. 79

4. RESULTS..................................................................................................... 80

XII

5. DISCUSSION ............................................................................................. 135

5.1 SOLUBILITY AND PERMEABILITY OF ACECLOFENAC IN

VARIOUS OILS .................................................................................. 135

5.2 MICROEMULSION FORMULATIONS OF ACECLOFENAC USING

DIFFERENT OIL PHASES ................................................................. 136

5.3 MICROEMULSION BASED GEL FORMULATION OF

ACECLOFENAC ................................................................................. 136

5.4 CHARACTERIZATION .......................................................................... 136

5.4.1 Viscosity ............................................................................................... 136

5.4.2 Spreadability ......................................................................................... 138

5.4.3 Conductivity Measurements ................................................................. 139

5.4.4 pH Measurements ................................................................................. 139

5.4.5 Refractive Index measurements ........................................................... 140

5.4.6 % Transmittance measurements ........................................................... 140

5.4.7 Centrifugation (Phase separation test) ................................................. 141

5.4.8 Drug content ......................................................................................... 141

5.4.9 Homogeneity.. ...................................................................................... 141

5.4.10 Scanning Electron Microscope (SEM) ................................................. 142

5.4.11 Fourier Transform Infra Red (FTIR) .................................................... 142


Calorimetry (DSC) ............................................................................ 143

5.4.13 X-Ray Diffraction (XRD) .................................................................... 144

5.4.14 Globule charge (Zeta Potential) and hydrodynamic size (Zeta Size) .. 144

5.4.15 Globule size/ hydrodynamic size (Zeta Size) ....................................... 144

5.5 IN-VITRO SKIN PERMEATION RELEASE RATEA EXPERIMENTS

OF ACECLOFENAC FROM MICROEMULSIONS AND

MICROEMULSION BASED ACECLOFENAC GEL ....................... 145

5.6 STABILITY STUDIES ........................................................................ 145

5.7 IN VIVO TRANSDERMAL STUDIES IN RABBITS ........................ 145

5.8 ANTI-INFLAMMATORY ACTIVITY STUDY IN RATS ............... 146

5.9 STUDY OF ANALGESIC EFFECT IN RATS ................................... 146

XIII

5.10 SKIN IRRITATION STUDY .............................................................. 147

5.11 HPLC METHOD VALIDATION.. ...................................................... 147

5.12 PHARMACOKINETIC PARAMETERS ............................................ 151

CONCLUSION ..................................................................................................... 152

6. REFRENCES ............................................................................................. 153

XIV

FIGURE DESCRIPTION PAGE

2.1 Structural Formula of aceclofenac 6

2.2 Typical Structure of the skin 8

2.3 Corneocyte lipid bilayers 11

2.4 Typical ternary phase diagram for a soybean oil, polyoxyethylene

ether surfactant and water system 31

3.1 Overall flow diagram of microemulsion based gel formulation

and its in vitro in vivo studies 56

3.2 Flow diagram of preparation of microemulsion based gel 67

4.1 Standard curve of Aceclofenac in Methanol 80

4.2 Standard curve of Aceclofenac in IPA 81

4.3 Standard curve of Aceclofenac in n-butanol 82

4.4 Standard curve of Aceclofenac in Ethanol 83

4.5 Solubility data of aceclofenac in various oils 85

4.6 Permeability data of aceclofenac in various oils 85

4.7 Solubility data of aceclofenac in various vehicles 86

4.8 Pseudoternary phase diagram of Almond oil, Tween 80-Isopropyl

alcohol (2:1) and water. 87

4.9 SEM image of aceclofenac pure drug 90

4.10 SEM image of blank micoromulsion 91

4.11 SEM image of aceclofenac micoromulsion 91

4.12 SEM image of blank microemulsion based gel 92

4.13 SEM image of aceclofenac microemulsion based gel

92

4.14

FTIR spectrum of aceclofenac and all excipients used in

microemulsion and microemulsion based aceclofenac gel

formulations

93

4.15 XRD of aceclofenac 94

4.16 XRD of Blank Microemulsion 94

4.17 XRD of Aceclofenac Microemulsion 95

XV


4.18 XRD of Blank Microemulsion based gel 95

4.19 XRD of Microemulsion based aceclofenac gel 96

4.20 TGA & DSC of Aceclofenac 96

4.21 TGA & DSC of Blank Microemulsion 97

4.22 TGA & DSC of Aceclofenac Microemulsion

97

4.23 TGA & DSC of Blank microemulsion based gel 98

4.24 TGA & DSC of Microemulsion based gel of Aceclofenac 98

4.25 Charge distribution of aceclofenac. 99

4.26 Charge distribution of blank Microemulsion

99

4.27 Charge distribution of aceclofenac Microemulsion 100

4.28 Charge distribution of Microemulsion based aceclofenac gel 100

4.29 Size distribution of aceclofenac pure drug. 101

4.30 Size distribution of blank Microemulsion 102

4.31 Size distribution of aceclofenac Microemulsion 103

4.32 Size distribution of blank Microemulsion based gel 104

4.33 Size distribution of microemulsion based aceclofenac gel 105

4.34 Permeation study of aceclofenac from microemulsion containing

aceclofenac 107

4.35 Permeation study of aceclofenac from microemulsion based

aceclofenac gel 107

4.36 Permeation study of aceclofenac from Alkeries gel 108

4.37

Plasma concentration verses time profile of Aceclofenac plotted

on rectangular co-ordinate graph, administered as a topical dose

of 2 mg (microemulsion based gel) in 12 rabbits of group AR

individually

111

4.38


on semi log graph, administered as a topical dose of 2 mg

(microemulsion based gel) in 12 rabbits of group AR individually

111

XVI


4.39



of 2 mg (conventional marketed gel) in 12 Rabbits of group R

individually

114

4.40



(conventional marketed gel) in 12 Rabbits of group R

individually

114

4.41 Percentage inhibition of oedema by ACF microemulsion, ACF

microemulsion based gel and ACF marketed gel 118

4.42

Percentage inhibition of writhes (analgesic effect) by ACF

microemulsion, ACF microemulsion based gel and ACF

marketed gel

119

4.43 Linearity curve of aceclofenac in plasma 122

4.44



of 20 mg (Marketed conventional gel) in 18 volunteers

125

4.45


on semi log graph, administered as a topical dose of 20mg

(Marketed conventional gel)in 18 volunteers

125

4.46



of 20 mg (Microemulsion based aceclofenac gel)in 18 volunteers

127

4.47



(Microemulsion based aceclofenac gel) in 18 volunteers.

127

4.48

Mean Plasma concentration verses time profile of Aceclofenac

from ME Gel and Conventional gel plotted on rectangular co-

ordinate graph, administered as a topical dose of 20 mg in 36

volunteers.

128

4.49


from ME Gel and Conventional gel plotted on semi log graph,

administered as a topical dose of 20 mg in 36 volunteers.

129

4.50




Rabbits.

130

4.51


from ME Gel and Conventional gel plotted on semi log graph,

administered as a topical dose of 2 mg in 24 Rabbits

130

A1 FTIR Spectrum of aceclofenac 167

A2 FTIR Spectrum of almond oil 167

A3 FTIR Spectrum of Carbopol 940 168

XVII


A4 FTIR Spectrum of Tweem 80 168

A5 FTIR Spectrum of Isopropyl alcohol 169

A6 FTIR Spectrum of dimethyl sulphoxide 169

A7 FTIR Spectrum of Triethyl amine 170

4.52



of 2 mg (microemulsion based gel) in Rabbit AR 1

171

4.53


on semi log graph, administered as a topical dose of 2 mg in

Rabbit AR 1

171

4.54




172

4.55



(microemulsion based gel) in Rabbit AR 2

172

4.56




173

4.57




173

4.58




174

4.59




174

4.60




175

4.61




175

XVIII


4.62




176

4.63




176

4.64




177

4.65




177

4.66




178

4.67




178

4.68




179

4.69




179

4.70




180

4.71




180

4.72




181

4.73




181

XIX


4.74




182

4.75




182

4.76



of 2 mg (marketed conventional gel) in Rabbit R 1

183

4.77



(marketed conventional gel) in Rabbit R 1

183

4.78




184

4.79




184

4.80




185

4.81




185

4.82




186

4.83




186

4.84




187

4.85




187

4.86




188

4.87




188

4.88




189

XX


4.89




189

4.90




190

4.91




190

4.92




191

4.93




191

4.94




192

4.95




192

4.96




193

4.97




193

4.98




194

4.99




194

4.100



of 20 mg marketed gel in volunteer 1

196

4.101



marketed gel in volunteer 1

196

4.102




198

4.103




198

XXI


4.104




200

4.105




200

4.106




202

4.107




202

4.108




204

4.109




204

4.110




206

4.111




206

4.112




208

4.113




208

4.114




210

4.115




210

4.116




212

4.117




212

4.118




214

XXII


4.119




214

4.120




216

4.121




216

4.122




218

4.123




218

4.124




220

4.125




220

4.126




222

4.127




222

4.128




224

4.129




224

4.130




226

4.131




226

4.132




228

4.133




228

XXIII


4.134




230

4.135




230

4.136



of 20 mg microemulsion based gel in volunteer 1

232

4.137



microemulsion based gel in volunteer 1

232

4.138




234

4.139




234

4.140




236

4.141




236

4.142




238

4.143




238

4.144




240

4.145




240

4.146




242

4.147




242

4.148




244

XXIV


4.149




244

4.150




246

4.151




246

4.152




248

4.153




248

4.154




250

4.155




250

4.156




252

4.157




252

4.158




254

4.159




254

4.160




256

4.161




256

4.162




258

4.163




258

XXV


4.164




260

4.165




260

4.166




262

4.167




262

4.168




264

4.169




264

4.170




266

4.171




266

XXVI

TABLE DESCRIPTION PAGE

2.1 Physicochemical properties of aceclofenac 7

2.2 Immune components of the skin 16

2.3 Problems found in different trials 47

3.1 Visual observations to record the addition of water 62

3.2 Smix compositions of selected microemulsion formulations 63

4.1 Standard curve of Aceclofenac in Methanol 80

4.2 Standard curve of Aceclofenac in IPA 81

4.3 Standard curve of Aceclofenac in Butanol 82

4.4 Standard curve of Aceclofenac in Ethanol 83

4.5 Solubility and permeability of aceclofenac in various oils (n=3) 84

4.6 Solubility of aceclofenac in various vehicles (n=3) 86

4.7 Characteristics of different formulations (n=3) 89

4.8 Size distribution of aceclofenac pure drug 101

4.9 Size distribution of blank microemulsion 102

4.10 Size distribution of aceclofenac microemulsion 103

4.11 Size distribution of blank microemulsion based gel 104

4.12 Size distribution of microemulsion based aceclofenac gel 105

4.13 Permeability of aceclofenac from different formulations (n=3) 106

4.14 Accelerated stability studies of different formulations at 40ºC ±

5°C/75% ± 5% RH(n=3). 108

4.15 Long term stability studies of different formulations at room

temperature 25ºC ± 5°C/65% ± 5% RH(n=3). 109

4.16 Concentration of ACF in rabbit plasma calculated from

chromatograms by forecasting method after administration of

Microemulsion based aceclofenac gel in group AR 110

4.17 Pharmacokinetics parameters of Aceclofenac in rabbits (Group

AR) after application of microemulsion based gel 112

XXVII


4.18 Concentration of ACF in rabbits (Group R) plasma calculated

from chromatograms by forecasting method after administration

of marketed aceclofenac gel 113

4.19 Pharmacokinetics parameters of Aceclofenac in rabbits (Group

R) after application of marketed gel 115

4.20 Anti-inflammatory activity study in rats (n=6 per group) 117

4.21 Study of analgesic effect in rats (n=6 per group) 118

4.22 Skin Irritations Study of ACF microemulsion 119

4.23 Skin Irritation study of ACF microemulsion based gel 120

4.24 Plasma Sample Concentration Data of Aceclofenac (Within-

Batch Precision and Accuracy) 120

4.25 Plasma Sample Concentration Data of Aceclofenac (Between-

Batch Precision and Accuracy) 121

4.26 Detection and Quantitation limit 121

4.27 Linearity curve of aceclofenac in plasma 121

4.28 Freeze thaw stability of aceclofenac in plasma 122

4.29 Extraction yield/Recovery of aceclofenac 123

4.30 Concentration of ACF in human plasma calculated from


marketed aceclofenac gel 124

4.31 Concentration of ACF in plasma calculated from


Microemulsion based aceclofenac gel 126

4.32 Comparison of mean plasma concentrations of aceclofenac from

ME gel and Conventional gel in volunteers. 128

4.33 Comparison of mean plasma concentrations of aceclofenac from

ME gel and Conventional gel in rabbits. 129

4.34 Comparison of Pharmacokinetic parameters of aceclofenac

microemulsion based gel and marketed conventional gel in

rabbits (t test) 131

4.35 Pharmacokinetics parameters of Aceclofenac in volunteers after

application of marketed gel 132

4.36 Pharmacokinetics parameters of Aceclofenac in volunteers after

application of microemulsion based gel 133

4.37 Comparison of mean plasma concentration of aceclofenac from

ME gel and Conventional gel in rabbits. (student t test) 134

4.38 Comparison of Pharmacokinetic parameters of aceclofenac

microemulsion based gel and conventional gel. (t test) 134

XXVIII


4.39 Plasma concentration (µg/ml) of Aceclofenac marketed gel A

administered as a topical dose of 2 mg Aceclofenac in Rabbit

AR1 171

4.40

Plasma concentration (µg/ml) of Aceclofenac marketed gel

A administered as a topical dose of 2 mg Aceclofenac in

rabbit AR 2

172

4.40



Rabbit AR 3

173

4.41



Rabbit AR 4

174

4.42



Rabbit AR 5

175

4.43



Rabbit AR 6

176

4.44



Rabbit AR 7

177

4.45



Rabbit AR 8

178

4.46



Rabbit AR 9

179

4.47



Rabbit AR 10

180

4.48



Rabbit AR 11

181

4.49



Rabbit AR 12

182

4.50



Rabbit R 1

183

4.51



Rabbit R 2

184

XXIX


4.52



Rabbit R 3

185

4.53



Rabbit R 4

186

4.54



Rabbit R 5

187

4.55



Rabbit R 6

188

4.56



Rabbit R 7

189

4.57



Rabbit R 8

190

4.58



Rabbit R 9

191

4.59



Rabbit R 10

192

4.60



Rabbit R 11

193

4.61



Rabbit R 12

194

4.62

Concentration of ACF in human plasma calculated from

chromatograms by forecasting method after administration

of marketed aceclofenac gel in volunteer 1

195

4.63




197

4.64




199

4.65




201

XXX


4.66




203

4.67




205

4.68




207

4.69




209

4.70




211

4.71




213

4.72




215

4.73




217

4.74




219

4.75




221

4.76




223

4.77




225

4.78




227

4.79




229

XXXI


4.80



of microemulsion based aceclofenac gel in volunteer 1

231

4.81




233

4.82




235

4.83




237

4.84




239

4.85




241

4.86




243

4.87




245

4.88




247

4.89




249

4.90




251

4.91




253

4.92




255

4.93




257

XXXII


4.94




259

4.95




261

4.96




263

4.97




265

XXXIII

LIST OF APPENDICES

APPENDIX I

FTIR spectra of active and excipients

FTIR Spectrum of aceclofenac---------------------------------167

FTIR Spectrum of almond oil----------------------------------167

FTIR Spectrum of Carbopol 940------------------------------168

FTIR Spectrum of Tweem 80----------------------------------168

FTIR Spectrum of Isopropyl alcohol--------------------------169

FTIR Spectrum of dimethyl sulphoxide-----------------------169

FTIR Spectrum of Triethyl amine------------------------------170

APPENDIX I I

Individual in vivo results of rabbits--------------------------171

APPENDIX III

In vivo studies of marketed conventional gel---------------195

APPENDIX I V

In vivo studies of microemulsion based gel-----------------231

APPENDIX V

Chromatograms of blank plasma and spiked plasma----267

XXXIV

ABSTRACT

The aim of this research project was to develop formulation of aceclofenac

microemulsion based gel and its in vitro in vivo studies. Excipients were

screened by solubility studies (almond oil with highest solubility was selected)

and phase diagram study for optimized formulations. For in vitro studies

spectrophotometric and HPLC analytical methods were developed and

validated under ICH guidelines. The different formulations were undergone

stability studies and stable formulations were subjected to study in vitro release

of aceclofenac from different formulations by Franz diffusion cell. The

formulation with highest permeation rate (i.e. almond oil containing

aceclofenac, Tween 80, isopropyl alcohol and water) was selected for further in

vitro characterization including rheological studies, Zeta size, Zeta potential,

XRD, SEM, FTIR, TGA and DSC. The in vivo studies were performed on rats,

rabbits and finally on human volunteers. The flux, Jss (µg/cm2/h) for

aceclofenac microemulsion, aceclofenac microemulsion based gel and

conventional marketed gel was 1.71 ± 0.06, 1.52 ± 0.07 and 0.91 ± 0.03,

respectively. The percentage inhibition of inflammation was 76.39%, 74.31%

and 70.14%, respectively for aceclofenac microemulsion, aceclofenac

microemulsion based gel and conventional marketed gel. The percentage

inhibition of analgesic effect was 80.16%, 75.05% and 70.96%, respectively

for aceclofenac microemulsion, aceclofenac microemulsion based gel and

conventional marketed gel. The pharmacokinetic parameters i.e. Cmax (µg/mL),

Tmax (h), T1/2(h) and AUC0-inf in rabbits for microemulsion based gel and for

conventional marketed gel were (6.56, 5.49); (5.88, 5.91); (4.53, 4.03) and

(65.21, 52.32), respectively. The pharmacokinetic parameters i.e. Cmax (µg/mL),

Tmax (h), T1/2(h) and AUC0-inf in healthy human volunteers for microemulsion

based gel and for conventional marketed gel were (8.30, 6.61); (5.50, 5.89);

(4.26, 4.08) and (57.62, 55.18), respectively. Therefore, it is concluded that

microemulsion based gel has greater bioavailability as compared to

conventional marketed brand and it has avoided GIT disturbances with

XXXV

enhanced patient’s compliance.

Key Words: Aceclofenac, Bioavailability, Pharmacokinetics, Microemulsion, Gel,

Conventional, almond oil, carbopol, tween 80.

1

A. NEED OF THE PROJECT

1) Aceclofenac (NSAIDs) is a poorly water soluble drug which creates

solubility problems in biological fluids and these problems result in less or

decreased bioavailability.

2) Aceclofenac produces serious gastrointestinal complications such as ulcer,

severe bleeding and perforation resulting in hospitalization and even death

(Baria et al., 2009). Therefore, these side effects result in patient’s non

compliance. So to meet the therapeutic goal there is a need to increase

solubility thereby preventing GIT problems.

3) Moreover, this drug is prescribed mostly for local effect in osteoarthritis,

ankylosing spondylitis and rheumatoid arthritis. Local application of this drug

will enhance its concentration locally thereby preventing GIT side effects.

B. HYPOTHESIS

An easy to administer formulation with enhanced in vitro and in vivo

performance characteristics is proposed to be formulated.

Null hypothesis is that the formulation may not be formulated.

C. AIMS AND OBJECTIVES

1. Various formulations of microemulsion based gels containing

aceclofenac with different oil phases and gel bases are prepared.

2. Bioavailability of best formulated gel and marketed gel is compared.

D. EXPECTED OUT COME

1. Solubility of aceclofenac will be enhanced and GIT problems

prevented due to local application of formulation.

2. The Microemulsion based gel formulation has improved bioavailability

and pharmacokinetic parameters with enhanced patient’s compliance.

E. ADVANTAGES TO THE INDUSTRY AND COMMUNITY

Industry can take benefit by formulating the cheaper microemulsion based gel

instead of tablet dosage form which requires sophisticated equipments as well

as large number of other excipients which increase the cost of solid dosage

form. In case of gel formulations, limited number of equipments as well as

2

very few excipients are required for the manufacturing of microemulsion

based gels.

The community can take benefit from this formulation because it is much easy

to apply drug on the affected area without any drug administration skills. So,

the patient’s compliance will enhance.

F. BACKGROUND OF THE STUDY

Aceclofenac is selected as a model drug. It is a phenyl acetic acid derivative

which is a drug of choice in the treatment of osteoarthritis, rheumatoid arthritis

and ankylosing spondylitis. Researchers have made several attempts to

develop oral drug delivery systems for aceclofenac. The chronic (prolong) oral

administration of aceclofenac tends to cause severe gastric irritation and

variation in oral bioavailability. Topical administration of aceclofenac in a gel

formulation offers the advantage of enhanced drug delivery to the affected

areas by-passing gastric irritation for extended time due to better spreadability

and greater viscosity of gel compared to other topical preparations.

G. METHODOLOGY

The proposed (new) method was successfully employed for formulation of

microemulsions and various formulations of microemulsions containing

aceclofenac were prepared with different oil phases. Different gel bases

prepared with different gelling agents and then microemulsions containing

aceclofenac were added to these gel bases to form microemulsion based

aceclofenac gels and selected formulation of aceclofenac microemulsion based

gel was subjected to characterization, stability studies, in vitro evaluation and

also compared in vivo with commercially available aceclofenac gel for

bioavailability and pharmacokinetic parameters evaluation.

3

1. INTRODUCTION

Usually topical dermatological products are used for local effects either on one

or more of skin layers. Topical application of drugs rapidly gaining

importance as a route for systemic administration of drugs previously used for

local effects in diseases of skin. The application of topical administration is to

deliver a drug at or immediately beneath the point of application (Mehta,

2000).

Previously, topical application of drugs used only for their local effects in

diseases of skin but now this site is rapidly becoming an important route of

drug administration (Alfonso, 2000).

The skin is an exceptionally effective barrier for most drugs for therapeutic

treatment. Very few drugs in therapeutic amount are permeated through skin

such as nitroglycerine, scopolamine, nicotine, clonidine, fentanyl, estradiol,

testosterone, lidocaine and oxybutinin (Prausnitz et al., 2004).

Consequently, the systems that make the skin more permeable and thereby

enhance transdermal delivery are of great formulation interest. The strategies

to deliver the medicament into the skin for systemic circulation have been

evolved. The extensive research has been reported on lipids as skin penetration

enhancers (Nishihata et al., 1987; Yokomizo et al., 1996a; Kirjavainen et al.,

1999).

Aceclofenac is selected as a model drug. It is a phenyl acetic acid derivative

which is drug of choice in the treatment of osteoarthritis, rheumatoid arthritis

and ankylosing spondylitis (Young et al., 2005). Researchers have attempted

development of oral drug delivery systems for aceclofenac. The chronic oral

administration of aceclofenac tends to cause severe gastric irritation (Luigi et

al., 1995). Topical administration of aceclofenac offers the advantage of

enhanced drug delivery to the affected areas by-passing gastric irritation. Luigi

et al. (1995) have evaluated clinical efficiency of topical aceclofenac cream

(1.5% w/w). The formulation showed improved therapeutic efficacy. Yang et

al. (2002) has formulated microemulsion containing aceclofenac (3%w/w) for

topical delivery. Microemulsions were prepared using different oil phases viz

oleic acid, linoleic acid, triacetin and labrafac. Labrasol was used as

surfactant. Transcutol was mixed as a co-surfactant for enhancing skin

permeability of aceclofenac. Microemulsion containing linoleic acid as oil

4

phase showed highest flux (Jss = 32.05 ± 9.17μg/cm2/hr) as compared to the

formulations prepared with other oil phase. However, microemulsions suffer

from the disadvantage that it requires large amounts of surfactant and co-

surfactant necessary for stabilizing the nanodroplets (Eccleston et al., 1994).

Poor viscosity and spreadability of microemulsions exhibit difficulty to

administer. On the other hand gels compared to microemulsions have high

viscosity and spreadability and hence can be administered to the skin with

much ease.

The regular use of aceclofenac through oral route causes ulcerogenic effect

(Sean, 2002). Fewer attempts have been made for subcutaneous absorption in

order to enhance bioavailability, the improvement of its solubility and

dissolution characteristics (Lugar et al., 1996).

Aceclofenac is practically insoluble in water leading to poor dissolution and

variable bioavailability upon oral administration (Hinz et al., 2003; Legrand et

al., 2004).

Transdermal drug administration generally refers to topical application of

agents to healthy intact skin either for localized treatment of tissues underlying

the skin or for systemic therapy. For transdermal products, the goal of dosage

design is to maximize the flux through skin into the systemic circulation and

simultaneously minimize the retention and metabolism of drug in skin (Misra,

1997). Transdermal drug delivery has many advantages over the oral route of

administration such as improving patient’s compliance in long term therapy,

bypassing first-pass metabolism, sustaining drug delivery, maintaining a

constant and prolonged drug level in plasma, minimizing inter and intra

patient variability and making it possible to interrupt or terminate treatment

when necessary (Keith et al., 1983).

Aceclofenac exhibits a multifactor mechanism of action which is mediated by

selective inhibition of prostaglandin E2. The most widely cited side-effect of

NSAIDs includes: gastrointestinal ulcer accompanied by anaemia due to

bleeding which is also true for aceclofenac. In order to avoid the gastric

irritation, minimize the systemic toxicity and achieve a better therapeutic

effect, one promising method is to administer the drug via skin (McNeill et al.,

1992).

5

In the present study, hydroalcoholic solution of aceclofenac was used as

standard formulation and excipients were selected on the basis of solubility

studies of aceclofenac. Skin penetration enhancers were selected on the basis

of their solubility and safety margin. Permeation studies were conducted by

using Chow method (Chow et al., 1984). Comparative in vivo studies

performed by using animal models (Albino rats and Rabbits) and human

volunteers. Aceclofenac was analyzed in plasma samples by a newly

developed and validated HPLC-UV method.

6

2. LITERATURE REVIEW

2.1 CHARACTERISTICS OF ACECLOFENAC

Aceclofenac chemically designated as 2-[2-[2-(2, 6-dichlorophenyl) aminophenyl] acetyl]

oxyacetic acid). Aceclofenac was developed in order to provide a highly effective pain

relieving therapy with a reduced side effect profile, especially gastrointestinal tract events

that are frequently experienced with NSAID therapy. Aceclofenac is practically insoluble

in water leading to poor dissolution and variable bioavailability upon oral administration

(Lugar et al., 1996; Hinz et al., 2003; Legrand et al., 2004). The chemical structure of

aceclofenac is presented below:

Figure 2.1: Structural Formula of aceclofenac

Aceclofenac is a potent analgesic, antipyretic and anti-inflammatory agent with side

effects affecting the gastrointestinal tract, liver, kidney and platelet functions (Goodman

and Gilman, 2001).

Aceclofenac is a highly lipophilic drug and its physiochemical properties suggest that it

has good potential for transdermal drug delivery (Shakeel et al., 2007).

2.1.1 Chemical name

Chemical name of aceclofenac is (2-(2,6 dichloroanalino) Phenylacetoxyacetic acid)

(European Pharmacopoeia 5).

7

2.1.2 Physicochemical characteristics

Physicochemical characteristics of aceclofenac as given in European Pharmacopoeia 5

are documented below:

Table 2.1: Physicochemical properties of aceclofenac

Molecular formula C16H13Cl2NO4

Molecular weight 354.2 Daltons

Color White or almost white

Solubility Practically insoluble in water, freely soluble in

acetone, soluble in alcohol.

Physical state Crystalline

2.2 ANATOMY AND PHYSIOLOGY OF SKIN

The skin, also called integument is the largest organ of body which makes up 16% of

body weight and its surface area is approximately 1.8 m2

(Gawkrodger, 2012; Ro and

Dawson, 2005). It has several functions, the most important being a physical barrier to

the environment, allowing and limiting the inward and outward passage of water,

electrolytes and various substances while providing protection against microorganisms,

ultraviolet radiations, toxic agents and mechanical insults. There are three structural

layers of the skin: the epidermis, the dermis and sub cutis. Hair, nails, sebaceous, sweat

and apocrine glands are regarded as derivatives of skin (Figure 2.1). Skin is a dynamic

organ in a constant state of change, as cells of the outer layers are continuously shed and

replaced by inner cells moving up to the surface. Although structurally consistent

throughout the body, skin varies in thickness according to anatomical site and age of the

individual. Outermost layer of the skin called stratum corneum provides the barrier

function of the skin. The typical structure of the skin is shown in Figure 2.1.

8

Figure 2.2: Typical Structure of the skin (Azeem et al., 2008)

The skin consists of following layers with their specific functions:

2.2.1 Epidermis

The epidermis is stratified squamous epithelium. The main cells of the epidermis are

keratinocytes, which synthesize a protein keratin. Protein bridges called desmosomes

connect the keratinocytes which are in a constant state of transition from deeper layers to

superficial. The epidermis varies in thickness from 0.05 mm on eyelids to 0.8- 1.5 mm on

soles of feet and palms of hand. The five separate layers of epidermis are formed by

different stages of keratin maturation. Moving from upper layers towards inside skin

following are five layers of epidermis:

stratum corneum (horny layer)

stratum lucidum (thin layer of translucent cells)

stratum granulosum (granular cell layer)

stratum spinosum (spinous or prickle cell layer)

stratum basale (basal or germinativum cell layer)

Malphigian layer consists of the stratum spinosum and stratum granulosum.

2.2.1.1 Stratum basale

The inner most layer of epidermis which lies adjacent to the dermis comprises mainly

dividing and non-dividing keratinocytes, wshich are attached to the basement membrane

by hemidesmosomes. As keratinocytes divide and differentiate, they move from this

deeper layer to the surface. Making up a small proportion of basal cell population is

E

p

i

d

e

r

m

i

s

9

pigment (melanin) producing melanocytes. These cells are characterized by dendritric

processes, which stretch between relatively large numbers of neighbouring keratinocytes.

Melanin accumulates in melanosomes that are transferred to the adjacent keratinocytes

where they remain as granules. Melanin pigment provides protection against ultraviolet

(UV) radiation; chronic exposure to light increases the ratio of melanocytes to

keratinocytes, some are found in facial skin compared to lower back and a greater

number on outer arm compared to inner arm. The number of melanocytes is same in

equivalent body sites in white and black skin but the distribution and rate of production

of melanin is different. Intrinsic ageing diminishes the melanocyte population. Large

numbers of Merkel cells are also found in the basal layer in touch-sensitive sites such as

the finger tips and lips. They are closely associated with cutaneous nerves and seem to be

involved in light touch sensation.

2.2.1.2 Stratum spinosum

As basal cells reproduce and mature, they move towards the outer layer of skin, initially

forming the stratum spinosum. Inter cellular bridges, the desmosomes, which appear as

prickles at a microscopic level, connect the cells. Langerhans cells are dendritic,

immunologically active cells derived from the bone marrow and are found on all

epidermal surfaces but are mainly located in the middle of this layer (Gawkrodger, 2012;

Ro and Dawson, 2005). They play a significant role in immune reactions of the skin,

acting as antigen-presenting cells.

2.2.1.3 Stratum granulosum

Continuing their transition to the surface, the cells continue to attend, lose their nuclei

and their cytoplasm appears granular at this level.

2.2.1.4 Stratum lucidum

It is present superficially to the stratum granulosum and is seen most clearly in relatively

thick skin specimens, such as from the load-bearing areas of the body (soles of feet and

palms) and usually absent in the thin skin (Laiq, 2008).

2.2.1.5 Stratum corneum

The final outcome of keratinocytes maturation is found in the stratum corneum, which is

made up of layers of hexagonal-shaped, nonviable cornified cells known as corneocytes.

10

In most areas of the skin, there are different layers of stacked corneocytes with palms and

soles having the most. Each corneocyte is surrounded by a protein envelope and is filled

with water-retaining keratin proteins. The cellular shape and orientation of keratin

proteins add strength to stratum corneum. Surrounding the cells in extracellular space are

stacked layers of lipid bilayers as shown in Figure 2.2. The resulting structure provides

natural physical and water-retaining barrier of skin. The corneocyte layer can absorb

three times its weight in water but if its water content drops below10%, it no longer

remains pliable and cracks. The movement of epidermal cells to this layer usually takes

about 28 days and is known as the epidermal transit time.

2.2.1.6 Dermoepidermal junction/basement membrane

This is a complex structure composed of two layers. Abnormalities here result in the

expression of rare skin diseases such as bullous pemphigoid and epidermolysis bullosa.

The structure is highly irregular, with dermal papillae from the papillary dermis

projecting perpendicular to the skin surface. It is via diffusion at this junction that the

epidermis obtains nutrients and disposes of waste. The dermoepidermal junction flattens

during ageing which accounts in part for some of the visual signs of ageing.

2.2.2 Dermis

The dermis varies in thickness ranging from 0.6 mm on eyelids to 3 mm on back, palms

and soles. It is found below epidermis and is composed of a tough, supportive cell matrix.

Two layers comprise dermis:

a) A thin papillary layer

b) A thicker reticular layer.

The papillary dermis lies below and connects with the epidermis. It contains thin loosely

arranged collagen fibers. Thicker bundles of collagen run parallel to the skin surface in

deeper reticular layer, which extends from the base of papillary layer to subcutis tissue.

The dermis is made up of fibroblasts, which produce collagen, elastin and structural

proteoglycans, together with immuno-competent mast cells and macrophages. Collagen

fibers make up 70% of the dermis, giving it strength and toughness. Elastin maintains

normal elasticity and flexibility while proteoglycans provide viscosity and hydration.

11

Embedded within the fibrous tissue of dermis are dermal vasculature, lymphatics,

nervous cells and fibers, sweat glands, hair roots and small quantities of striated muscle.

Figure 2.3: Corneocyte lipid bilayers (Gawkrodger, 2012; Ro and

Dawson, 2005)

2.2.3 Hypodermis/Subcutaneous Sub cutis

This is made up of loose connective tissue and fat, which can be up to 3 cm thick on the

abdomen.

12

2.3 BLOOD AND LYMPHATIC VESSELS

Dermis receives a rich blood supply. A superficial artery plexus is formed at the papillary

and reticular dermal boundary by branches of subcutis artery, branches from this plexus

form capillary loops in papillae of dermis, each with a single loop of capillary vessels,

one arterial and one venous. The veins drain into mid-dermal and subcutaneous venous

networks. Dilatation or constriction of these capillary loops plays a direct role in

thermoregulation of the skin. Lymphatic drainage of the skin occurs through abundant

lymphatic meshes that originate in papillae and feed into larger lymphatic vessels that

drain into regional lymph nodes.

2.4 NERVE SUPPLY

The skin has rich innervations with hands, face and genitalia having highest density of

nerves. All cutaneous nerves have their cell bodies in the dorsal root ganglia and both

myelinated and non-myelinated fibers are found. Free sensory nerve endings lie in the

dermis where they detect pain, itch and temperature. Specialized corpuscular receptors

also lie in the dermis allowing sensations of touch to be received by Meissner's

corpuscles and pressure and vibration by Pacinian corpuscles. The autonomic nervous

system supplies the motor innervations of the skin: adrenergic fibers innervate blood

vessels, hair erectormuscles and apocrine glands while cholinergic fibers innervate

eccrine sweat glands. The endocrine system regulates the sebaceous glands, which are not

innervated by autonomic fibers.

2.5 DERIVATIVE STRUCTURES OF THE SKIN

2.5.1 Hair

Hair can be found in varying densities of growth over the entire surface of body,

exceptions being on the palms, soles and glans penis. Follicles are most dense on the

scalp and face and are derived from epidermis and the dermis. Each hair follicle is lined

by ger minative cells, which produce keratin and melanocytes, which synthesize pigment.

The hair shaft consists of an outer cuticle, a cortex of keratinocytes and an inner medulla.

The root sheath, which surrounds hair bulb, is composed of an outer and inner layer. An

erectorpili muscle is associated with hair shaft and contracts with cold, fear and emotion

to pull the hair erect, giving the skin goose bumps.

13

2.5.2 Nails

Nails consist of a dense plate of hardened keratin between 0.3 and 0.5 mm thick

(Gawkrodger, 2012; Ro and Dawson, 2005). Finger nails function to protect the tip off

the fingers and to aid grasping. The nail is made up of a nail bed, nail matrix and a nail

plate. The nail matrix is composed of dividing keratinocytes, which mature and keratinize

into the nail plate. Underneath the nail plate lays nail bed. The nail plate appears pink due

to adjacent dermal capillaries and white lunula at the base of plate is distal, visible part of

matrix. The thickened epidermis which underlies free margin of nail at proximal end is

called hyponychium. Finger nails grow at 0.1 mm per day whereas the toe nails grow

more slowly.

2.5.3 Sebaceous glands

These glands are derived from epidermal cells and are closely associated with hair

follicles especially those of the scalp, face, chest and back; they are not found in hairless

areas. They are small in children, enlarging and becoming active at puberty, being

sensitive to androgens. They produce an oily sebum by holocrine secretion in which the

cells breakdown and release their lipid cytoplasm. The full function of sebum is unknown

at present but it does play a role in the following:

Maintaining the epidermal permeability barrier, structure and differentiation skin-specific

hormonal signaling transporting antioxidants to the skin surface protection from UV

radiation (Gawkrodger, 2012; Ro and Dawson, 2005).

2.5.4 Sweat glands

There are thought to be over 2.5 million sweat glands on skin surface and they are present

over majority of body. They are located within dermis and are composed of coiled tubes,

which secrete a watery substance. They are classified into two different types: eccrine

and apocrine.

Eccrine glands are found all over the skin especially on palms, soles, axillae and

forehead. They are under psychological and thermal control. Sympathetic (cholinergic)

nerve fibers innervate eccrine glands. The watery fluid they secrete contains chloride,

lactic acid, fatty acids, urea, glyco proteins and mucopolysaccharides.

14

Apocrine glands are larger, the ducts of which empty out into the hair follicles. They are

present in axillae, anogenital region, areolae and are under thermal control. They become

active at puberty, producing an odorless protein-rich secretion which when acted upon by

skin bacteria gives out a characteristic odor. These glands are under the control of

sympathetic (adrenergic) nerve fibers.

2.6 SKIN FUNCTIONS

The skin is a complex metabolically active organ, which performs important

physiological functions that are summarized below:

Provides a protective barrier against mechanical, thermal and physical injury and noxious

agents.

Prevents loss of moisture.

Reduces the harmful effects of UV radiation.

Acts as a sensory organ.

Helps in temperature regulation.

Plays a role in immunological surveillance.

Synthesizes vitamin D3 (cholecalciferol).

Have cosmetic, social and sexual associations.

2.6.1 Barrier function and skin desquamation

As the viable cells move towards stratum corneum, they begin to clump proteins into

granules in the granular layer. The granules are filled with protein fillagrin which

becomes complexed with keratin to prevent breakdown of fillagrin by proteolytic

enzymes. As degenerating cells move towards the outer layer, enzymes breakdown the

keratin-fillagrin complex. Fillagrin forms on the outside of corneocytes while water-

retaining keratin remains inside. When moisture content of skin reduces, fillagrin is

further broken down into free amino acids by specific proteolytic enzymes in the stratum

corneum. The breakdown of fillagrin only occurs when the skin is dry in order to control

the osmotic pressure. In healthy skin, water content of stratum corneum is normally

around 30%. The free amino acids, along with other components such as lactic acid, urea

and salts are known as natural moisturizing factors and are responsible for keeping the

skin moist and pliable due to their ability to attract and hold water.

15

2.6.1.1 Lipids

The major factor in the maintenance of a moist, pliable skin barrier is the presence of

intercellular lipids. These form stacked bilayers that surround corneocytes and

incorporate water into stratum corneum. The lipids are derived from lamellar granules,

which are released into extracellular spaces of degrading cells in the granular layer; the

membranes of these cells also release lipids. Lipids include cholesterol, free fatty acids

and sphingolipids. Ceramide, a type of sphingolipid, is mainly responsible for generating

the stacked lipid structures that trap water molecules in their hydrophilic region. These

stacked lipids surround corneocytes and provide an impermeable barrier by preventing

movement of water and natural moisturizing factors out of surface layers of skin. After

the age of 40, there is a sharp decline in skin lipids thus increasing our susceptibility to

dry skin conditions.

2.6.1.2 Shedding (desquamation) of skin cells

Shedding cells of stratum corneum is an important factor in maintaining skin integrity

and smoothness. Desquamation involves enzymatic process of dissolving protein bridges,

desmosomes, between corneocytes and eventual shedding of these cells. The proteolytic

enzymes responsible for desquamation are located intra cellular and function in presence

of a well-hydrated stratum corneum. In the absence of water, cells do not desquamate

normally and skin becomes roughened, dry, thickened and scaly. In normal healthy skin

there is a balance in production and shedding of corneocytes. In diseases such as psoriasis

in which increased corneocyte production and decrease in shedding occurs as a result of

which skin becomes dry, rough due to accumulation of cells on the skin.

2.6.2 UV protection

Melanocytes, located in basal layer and melanin have important roles in skin's barrier

function by preventing damage by UV radiation. In the inner layers of epidermis, melanin

granules form a protective shield over the nuclei of keratinocytes; in the outer layers, they

are more evenly distributed. Melanin absorbs UV radiation, thus protecting the cell's

nuclei from DNA (deoxyribonucleic acid) damage. UV radiation induces keratinocytes

proliferation, leading to thickening of epidermis.

16

2.6.3 Thermoregulation

The skin plays an important role in maintaining a constant body temperature through

changes in blood flow in cutaneous vascular system and evaporation of sweat from the

surface.

2.6.4 Immunological surveillance

Acting as a physical barrier, skin also plays an important immunological role. It normally

contains all the elements of cellular immunity, with the exception of B cells (Group of

white blood cells WBCs). Immune components of the skin are given in table 2.1

Table 2.2 Immune components of the skin (Gawkrodger, 2012; Ro and Dawson, 2005)

Defense Type Component Immune action

Structural Skin Impenetrable barrier to most external organisms

Cellular

Blood and lymphatic

vessels

Provision of transport network for cellular

Defense

Langerhans cells Antigen presentation

T Lymphocytes Facilitate immune reactions. Self-regulating

through the action of T suppressor cells.

Mast cells Facilitate inflammatory skin reactions.

Keratinocytes Secrete inflammatory cytokines; have ability to

express surface immune reactive molecules.

Systemic

Cytokines and

eicosanoids

Cytokines: cell mediation chemicals produced

by components of the cellular defense system.

Eicosanoids: non-specific inflammatory

mediators produced by mast cells, macrophages

and keratinocytes.

Adhesion molecules Increase the number of cellular defense

facilitators in an area by binding to T cells.

Complement cascade Activation of this initiates a host of destructive

mechanisms, including opsonisation, lysis,

chemotaxis and mast cell degranulation.

Immunogenetic

Major

Histocompatibility

Complex (MHC)

Enables immunological recognition of antigens

Flexibility and protective function of stratum corneum is related with its moisture level

and it depends basically on three factors: (1) the rate at which water in dermis reaches

stratum corneum, (2) the rate at which water is eliminated by evaporation and (3) ability

of stratum corneum to retain water. This is tightly linked with the role of surface lipid

17

film, natural moisturizing factors and polar lipids e.g. glycolipids, phospholipids and free

fatty acids which make up the well known “lamellae” in the intercellular spaces of

stratum corneum (Potts and Francoeur, 1991). Dryness of skin causes roughness and

itching. As a result of dilated peripheral blood capillaries in dermis, the surface of severe

dry skin becomes cracked and reddened (erythema). Therefore, to keep the skin surface

supple and healthy, moisture level of stratum corneum is crucial.

Skin wrinkling is disturbing to many individuals and is a prime reminder of

disappearance of youth. Areas of skin exposed to sun and tough environmental conditions

and chemicals readily show the signs of ageing (Jung, 2008). Unfortunately, majority of

effective moisturizing actives cannot be applied without some modification because they

are difficult to apply and can leave the skin feel tacky. For this reason, they are

formulated into microemulsions which can be stabilized with an appropriate emulsifier

system. Cosmetic microemulsion formulations can be used to protect the skin against

harmful environmental factors, to replace the loss of natural skin oils and moisture and if

damage has occurred to promote the restoration of skin functions. With the introduction

of new raw materials and advances in microemulsion technology, products with good

functionality and aesthetic appeal can be developed. Furthermore, microemulsions can be

used to enhance skin permeation of the loaded ingredients.

Several plausible mechanisms of skin permeation enhancement property of

microemulsions have been proposed. A large amount of active principle can be

incorporated in the formulation due to high solubilizing capacity that might increase

thermodynamic activity towards the skin.

The surfactant and co-surfactant in microemulsions may reduce the diffusional barrier of

stratum corneum by acting as penetration enhancers (Rhee, 2001). The percutaneous

absorption of active also increases due to hydration effect of stratum corneum if water

content in microemulsion is high enough. The small droplets provide better adherence to

skin and have large surface area thereby providing high concentration gradient and

improved active agent permeation.

If the aim is to provide sustained release of lipophilic active then it is incorporated into

the inner oil phase in o/w microemulsion so that active will partition from the inner

compartment to the outer aqueous compartment and then it releases to skin. Since

18

microemulsions act as super-solvent of highly lipophilic compounds, they can

incorporate a large amount of the agent. When the aim is immediate absorption and

shorter duration of action, then w/o type can be formulated. The situation shall be

reversed when the active is hydrophilic. Depending on the requirement, the desired type

of microemulsion can be formulated.

Gulten et al. (2007) tested formulations (w/o microemulsion of diclofenac) by placing

samples (0.1 g) with or without Diclofenac Sodium (DS) on gauze dressing (1x1cm2)

fixed with stretch adhesive tape on the inner arms of 9 human volunteers for 8 hours. The

application areas were separated at least 1-cm distance from each other on each arm.

MexameterTM

was used to measure erythema after 8 hours. The skin of inner arms was

also measured before application of the formulations as a control. The results of skin

irritation study were evaluated using repeated measures analysis of variance (ANOVA),

with P<0.05 taken as the level of significance. The results of skin irritation study were

analyzed according to repeated measures by one-way ANOVA. It was seen that the

difference between erythema values of formulations and control was insignificant. There

was also no significant difference among erythema values of any of formulations

(P>0.05). Consequently, addition of DS and different co-surfactants in the formulations

has no effect on skin irritation features of microemulsions.

Many investigations have been carried out on permeability of skin. Because of many

technical difficulties involved in in vivo studies, little quantitative information has been

published. Moreover, much of literature on the subject is contradictory and confused. As

skin has low oxygen consumption, it would appear to be particularly suitable for in vitro

studies. Therefore, the penetration of a series of 14

C and 35

S labeled non-electrolytes

through excised skin has been measured in an attempt to determine some of the

physicochemical factors involved in the penetration of skin. It was found from the

experimental results that barrier to diffusion through skin lies in epidermis, since the rate

of diffusion through the dermis is at least two orders faster than through whole skin. The

permeability constants for whole skin, which are effectively those for epidermis alone,

have been shown to parallel the ether/water partition coefficient of penetrating molecules.

This coefficient has been used as a lipoid-water model which corresponds well with

permeability of various plant cells. Molecular size appears to have little effect on the rate

19

of penetration, for example ethyl iodide with a molecular volume of 25-23 penetrated at

least thirty times more rapidly than urea with a molecular volume of 13-67. Thus, the

rate-limiting process in the passage of substances through skin appears to be diffusion

through some kind of lipoid layer, or layers, in the epidermis. Hence, it has been

suggested that the lipoid solubility of penetrating substance is an important factor in the

mechanism of skin permeability, but no quantitative demonstration of this principle

seems previously to have been made. The initial delay period in the penetration of skin is

related to the permeability constant of diffusing substance, slowly penetrating substances

showing long delay periods. A thick homogeneous lipoid barrier would yield a delay

period, but this delay would tend to be independent of the permeability. An alternative

system, resulting in a similar type of diffusion curve to that observed, would be an

aqueous layer sandwich between two thin lipoid layers. Considering diffusion from a

concentration Co through an aqueous layer of area A and finite thickness b, bounded by

two thin lipoid membranes with permeability constants P1 and P2, then from Fick's Law

the rate of transfer through outer lipoid membrane will be

dS1

= AP1 (Co-Cm) (1)

dt

Where, Cm is concentration in aqueous layer. If concentration beneath second lipoid layer

is small compared with Cm then the rate of transfer of material through second membrane

can be written

dS2 = AP2Cm (2)

dt

The concentration in the aqueous layer at any time is

Cm = Sl –S2 /Ab (3)

Equations (1), (2) and (3) yield on integration

S2 = PACo (t-td + td e-t/td

) (4)

20

S2 =PACo (t-td + td e-t/td

) (5)

Where, P= P1P2/ P1+ P2 and

td= b/ P1+ P2 (6)

td can be described as a delay period and, as t increases, S2 tends to rise linearly with time

(Treherne, 1956).

2.7 SKIN PERMEABILITY STUDIES

Das and Ahmad (2008) studied the enhancing effect of ascorbic acid and triethyl citrate

(TEC) on in vitro skin permeation of rofecoxib across rat epidermis. The skin which was

treated with ascorbic acid and TEC at different concentrations, followed by application of

rofecoxib gel, showed higher permeation flux than skin without treatment. Skin pre-

treatment with ascorbic acid and TEC at different concentrations followed by rofecoxib

gel was found to increase rofecoxib retention in skin. These pre-treatment experiments

did not show any significant change in lag time as compared to control. With the help of

FTIR spectra of ascorbic acid and TEC treated rat epidermis, they found that these

enhancers act by interacting with lipid alone or both lipid and protein of rat epidermis in a

dose-dependent manner. They suggested that a rapid percutaneous absorption at effective

therapeutic level is possible when using ascorbic acid and TEC as permeation enhancers

for faster anti-inflammatory activity.

Desai (2004) prepared microemulsion gels containing rofecoxib and rofecoxib solid

dispersion with polyethylene glycol (PEG) 4000 for the study of rapid percutaneous

absorption. Topical microemulsion gels (MEGs) were prepared by using pure rofecoxib

as well as its solid dispersion to compare the efficacy of individual MEG with

conventional gel (CG). MEGs showed better spreadability than CG and also showed

increased globular size with increasing concentration of oil phase. The release of

rofecoxib through dialysis membrane and excised rat abdominal skin was affected by the

size of oil globule in MEGs. Rofecoxib release was higher for MEGs when compared to

CG. MEGs containing rofecoxib-PEG 4000 solid dispersion exhibited higher cumulative

drug permeation when compared to MEG containing pure rofecoxib. MEGs containing

rofecoxib-PEG 4000 solid dispersion exhibited faster anti-inflammatory activity than CG.

21

Barakat et al. (2011) prepared microemulsion formulations with different surfactant and

co-surfactant ratios Smix i.e. F1-F6, 1:1, 2:2, 3:1, 4:1, 1:2 and 3:2 w/w, respectively by

spontaneous emulsification method and characterized for morphology, droplet size and

rheological characteristics. The in vitro skin permeation studies were performed using

Franz diffusion cell with rabbit skin as a permeation membrane. A significant increase in

the steady-state flux (Jss), permeability coefficient (Kp) and enhancement ratio (Er) was

observed in microemulsion formulations compared with conventional indomethacin gel.

The anti-inflammatory effects of microemulsion formulations showed a significant

increase in percentage edema inhibition value after 4 hours. The optimized formulation

showed a significant increase in the steady-state flux (Jss) and permeability coefficient

(Kp). The enhancement ratio (Er) was found to be 8.939 in optimized formulation F1

compared with IND gel.

Idrees et al. (2011) prepared different surfactant and co-surfactant mixtures with different

ratios and phase diagrams were constructed. 2:1 (Smix) with oleic acid (oil) was selected.

Six microemulsions each containing 5% drug, 5% oil, 56% Smix and 34% water, were

prepared and compared for their permeation and phase behaviors. In vitro Transdermal

permeation through rabbit skin of all microemulsions was higher than saturated hydro

alcoholic drug solution. Tween 20 and ethanol as Smix produced the highest flux amongst

all the Smix and were used to prepare formulations with different values of oil and Smix.

While the type of surfactant did not affect droplet size, propylene glycol as co-surfactant

produced the largest droplets and highest viscosity. Decrease in oil or Smix concentration

resulted in decrease of droplet size and increase in permeation flux while decrease in

viscosity also increased the permeation flux of microemulsions. Finally, the selected

microemulsion formulation comprising 5% flurbiprofen, 5% oleic acid, 46% Tween 20 :

ethanol (2:1) and 44% water, showed the highest Transdermal flux and caused no skin

irritation.

Li et al. (2006) examined Transdermal permeation of two types of NSAIDs, [3H]

flurbiprofen and [14

C] indomethacin, by use of Ussing-type chamber method. It was

found that the Transdermal permeability in absorptive direction (Pabs) of [3H] flurbiprofen

was significantly higher than that of [14

C] indomethacin. A lower pH (5.0) on the

epidermal side increased the accumulation and Pabs of [3H] flurbiprofen (18-fold and 50-

22

fold, respectively) and [14

C] indomethacin (18-fold and 22-fold, respectively) compared

with pH 7.4. Co-administration of unlabeled flurbiprofen and indomethacin increased Pabs

of [3H] flurbiprofen and [

14C] indomethacin, respectively, in a concentration-dependent

manner. Similar high-affinity transport was also observed in the uptake of [3H]

flurbiprofen by human epidermal keratinocytes.

Cui et al. (2011) prepared microemulsion containing ligustrazine phosphate for its

Transdermal delivery in a convenient, efficient and safe administration. The prepared

microemulsions had average diameters ranging from 32.1 to 108.7 nm with mild pH

values and suitable stability. The optimized microemulsion with permeation flux of 41.01

µg/cm2/h across rat skin in vitro and showed no obvious irritation on back skin of rabbits.

On the basis of results it was concluded that the studied microemulsion system might be a

promising vehicle for Transdermal delivery of ligustrazine phosphate.

Naoui et al. (2011) prepared 3 microemulsions of different microstructure, o/w, w/o and

bicontinuous at skin temperature (32°C) having same oil and water contents and

containing same ingredients which were selected using Kahlweit fish phase diagrams

method. The microemulsions were quaternary mixtures of the Polysorbate 21

(Tween®21) and Sorbitan monolaurate (Span®20) surfactants, isononyl isononanoate oil

and water. The Franz cell method was used to monitor skin absorption of caffeine loaded

in microemulsions over 24 h exposures to the excised pig skin. The Transdermal flux of

caffeine was in the order aqueous solution ≈ w/o < bicontinuous < o/w microemulsion.

The o/w microemulsion allows permeation of 50% of applied dose within 24 h. These

results suggested that structure of microemulsions is of relevance for skin absorption and

water-continuous structures allow faster transport of hydrophilic drugs.

Chandra et al. (2009) screened almond oil, olive oil, linseed oil and nutmeg oil as the oil

phase. A microemulsion-based system was chosen due to its good solubilizing capacity

and skin permeation capabilities. The pseudo ternary phase diagrams for microemulsion

regions were constructed using various oils, egg lecithin as the surfactant, isopropyl

alcohol (IPA) as co-surfactant and distilled water as aqueous phase. Microemulsion gel

formulations were prepared using Carbopol and filled into a reservoir-type Transdermal

system. The ability of various microemulsion formulations to deliver dexamethasone

through the rat skin was evaluated in vitro using Keshary Chien diffusion cells. In order

23

to enhance permeation, the skin was treated with an abrading gel (apricot seed powder in

hydrogel base). The in vitro permeation data showed that microemulsions increased the

permeation rate of dexamethasone compared with control. The optimum formulation

consisting of 0.1% dexamethasone, 10% olive oil, 70% egg lecithin:IPA (2:1) and water

showed a permeation rate of 54.9 µg/cm 2

/h. The studied microemulsion-based hydrogel

was stable toward centrifugation test and was non-irritating to the skin. The

pharmacodynamic studies indicated that microemulsion based on nutmeg oil

demonstrated a significantly (P < 0.05) higher anti-inflammatory potential. The nutmeg

oil-based Transdermal microemulsion gel system demonstrated 73.6% inhibition in rat

paw edema. Thus, microemulsion-based Transdermal systems are a promising

formulation for dermal delivery of dexamethasone.

An outstanding barrier against external environment is provided by outermost layer of

skin, stratum corneum (SC), which is also responsible for skin impermeability toward

most solutes. This barrier function is related to unique composition of SC lipids and their

complex structural arrangement. Therefore, penetration enhancers target lipoidal matrix

of SC. The data obtained from infrared, thermal and fluorescence spectroscopic

examinations of SC and its components showed that enhancer has improved permeation

of solutes through SC is associated with alterations involving the hydrocarbon chains of

SC lipid components. Data obtained from electron microscopy and X-ray diffraction

reveals that disordering of lamellar packing is also an important mechanism for increased

permeation of drugs induced by penetration enhancers (Marjukka et al., 1999).

One long-standing approach for improving Transdermal drug delivery uses penetration

enhancers (also called sorption promoters or accelerants) which penetrate into skin to

reversibly decrease the barrier resistance. Numerous compounds have been evaluated for

penetration enhancing activity, including sulphoxides (such as dimethylsulphoxide,

DMSO), Azones (e.g. laurocapram), pyrrolidones (for example 2-pyrrolidone, 2P),

alcohols and alkanols (ethanol, or decanol), glycols (for example propylene glycol, a

common excipient in topically applied dosage forms), surfactants (also common in

dosage forms) and terpenes. Many potential sites and modes of action have been

identified for skin penetration enhancers; the intercellular lipid matrix in which the

accelerants may disrupt the packing design, the intracellular keratin domains or through

24

increasing drug partitioning into the tissue by acting as a solvent for the permeant within

the membrane. Further potential mechanisms of action, for example with the enhancers

acting on desmosomal connections between corneocytes or altering metabolic activity

within skin or exerting an influence on thermodynamic activity/solubility of drug in its

vehicle are also feasible and are also considered in this review (Williams and Barry,

2004).

Surfactants are found in many existing therapeutic, cosmetic and agro-chemical

preparations. In recent years, surfactants have been employed to enhance the permeation

rates of several drugs via Transdermal route. The application of Transdermal route to a

wider range of drugs is limited due to significant barrier to penetration across the skin

which is associated with outermost stratum corneum layer. Surfactants have effects on

permeability characteristics of several biological membranes including skin. They have

potential to solubilize lipids within stratum corneum. The penetration of the surfactant

molecule into lipid lamellae of stratum corneum is strongly dependent on the partitioning

behavior and solubility of surfactant. Surfactants ranging from hydrophobic agents such

as oleic acid to hydrophilic sodium lauryl sulfate have been tested as permeation

enhancer to improve drug delivery. The effect of surfactants on the enhancement of drug

permeation through skin has been well reviewed. Research in this area has proved the

usefulness of surfactants as chemical penetration enhancer in the Transdermal drug

delivery. In many instances they have been found to be more effective than other

enhancers. Focus should be on skin irritation and toxicity with a view to select from a

wide range of surfactants (Som et al., 2012).

Kweon et al. (2004) developed a Transdermal preparation containing diclofenac diethyl

ammonium (DDA) using an o/w microemulsion system. Lauryl alcohol was chosen as

the oil phase of the microemulsion as it showed a good solubilizing capacity and

excellent skin permeation rate of the drug. The optimum formulation of the

microemulsion consisted of 1.16% of DDA, 5% of lauryl alcohol, 60% of water in

combination with the 34.54% of labrasol (surfactant)/ethanol (co-surfactant) (1:2). The

efficiency of formulation in the percutaneous absorption of DDA was dependent

upon the contents of water and lauryl alcohol as well as labrasol ethanol mixing

ratio. It was concluded that the percutaneous absorption of DDA from

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term=%20Som%2BI%5bauth%5d

25

microemulsions was enhanced with increasing lauryl alcohol and water contents, and

with decreasing the labrasol-ethanol mixing ratio in formulation.

The objective of study was to prepare saturated solutions of ibuprofen with different

concentrations and to investigate their effect on permeation of ibuprofen across rat

epidermis. Ibuprofen saturated solutions were prepared using 0.1, 0.2, 0.3 and 0.4 M

disodium hydrogen phosphate solution (DHP). The solubility of ibuprofen in DHP

increased as the molarity of DHP increased. Thus, the four saturated solutions of

ibuprofen (0.1M-DHP-IBU, 0.2M-DHP-IBU, 0.3M-DHP-IBU and 0.4M-DHP-IBU) have

different concentrations of the same drug and showed same pH (pH7.0 F1). The

permeability study was also carried out using human epidermis and silastic membrane.

Permeation rate of ibuprofen across rat epidermis and human epidermis from 0.4M-DHP-

IBU was much greater than from 0.1M-DHP-IBU. The magnitudes of increase in the

drug flux were 46.4-fold with rat epidermis and 9.4-fold with human epidermis. Such a

great increase in drug flux was not observed with silastic membrane as it was only 1.4-

fold. This suggests that the increased drug flux is likely due to drug–skin interaction and

not due to the increased concentration of ibuprofen as such. Surface tension (ST)

measurements of DHP versus ibuprofen concentration showed ST reduction of DHP from

72 to 27.9 dyne/cm. This is an indication that ibuprofen acted as ionic surfactant and the

observed skin permeability enhancement is attributed to disruption of stratum corneum

barrier. Results of DSC study supported this assumption. DSC of untreated rat stratum

corneum samples showed lipid transitions at 41.9 ºF (0.08 ºC) (T1), 55.1 ºF (1.68 ºC)

(Tx), 70.2 ºF (0.18 ºC) (T2) and 77.5 ºF (0.18 ºC) (T3), while those pretreated with 0.4M-

DHP-IBU did not show the first three lipid transitions. Also, pretreatment of rat

epidermis with 0.4M-DHP-IBU enhanced permeation of diclofenac sodium greater than

1250-fold. This corroborates that ibuprofen not only enhances its own permeation but

also that of other drugs, such as diclofenac sodium (Al-Saidan, 2004).

Gannu et al. (2010) developed and optimized the microemulsion based Transdermal

therapeutic system for lacidipine (LCDP), a poorly water soluble and low bioavailable

drug. The pseudo-ternary phase diagrams were developed for various microemulsion

formulations composed of isopropyl myristate, tween 80 and labrasol. The

microemulsion was optimized using a three-factor, three-level Box–Behnken design, the

26

independent variables selected were isopropyl myristate, surfactant mixture (tween 80

and labrasol) and water; dependent variables (responses) were cumulative amount

permeated across rat abdominal skin in 24h (Q24; Y1), flux (Y2) and lag time (Y3).

Mathematical equations and response surface plots were used to relate the dependent and

independent variables. The regression equations were generated for responses Y1, Y2

and Y3. The statistical validity of polynomials was established and optimized formulation

factors were selected by feasibility and grid search. Validation of the optimization study

with 10 confirmatory runs indicated high degree of prognostic ability of response surface

methodology. The gel of optimized formulation showed a flux of 43.7µg cm−2

h−1

which

could meet the target flux (12.16 µg cm−2

h−1

). The bioavailability studies in rabbits

showed that about 3.5 times statistically significant (p<0.05) improvement in

bioavailability, after Transdermal administration of microemulsion gel compared to oral

suspension. The in vitro–in vivo correlation was found to have biphasic pattern and

followed type A correlation.

2.8 SOLUBILITY STUDIES OF ACECLOFENAC AND EXCIPIENTS

One of the most important physicochemical properties of a drug/drug candidate is its

solubility. Therefore, the knowledge of solubility is required from the earliest stages of

drug discovery to the latest stage of drug formulation. The solubility in organic solvent is

required in developing synthesize/extraction media, the solubility in water is needed to

make a solution of drug to be tested for its pharmacological/toxicological activities and

also to further proceed with the biopharmaceutical requirements and at the final stage of

drug development, i.e. in its formulation as an oral or parenteral solution. Solubility is

defined as the maximum quantity of a drug dissolved in a given volume of a

solvent/solution, depends on the solubility expression unit.

The solubility of a drug/drug candidate could be determined by experimental procedures

mainly classified in two groups, namely the thermodynamic and kinetic solubility

determination methods. (Abolghasem et al., 2008).

According to Biopharmaceutical Classification System (BCS) there are two main

indicators of drug bioavailability; 1) the aqueous solubility and 2) the ability of drug

molecules to permeate biologic membranes (Amidon et al., 1995).

27

Determination of solubility by allowing a solid compound to equilibrate with an aqueous

medium is usually too time consuming and requires large amount of sample to be feasible

for high throughput screening. Instead, the kinetic solubility is measured in which

dimethyl sulfoxide solution of the compound is gradually added to an aqueous media and

solubility is determined as the concentration at which precipitation is formed as detected

by light scattering. The advantages of the kinetic method are that it is relatively rapid,

requires only small sample and that it is easily automated (Dehring et al., 2004). The

presence of dimethyl sulfoxide in the final medium (frequently 0.5%-5% v/v) and

potential formation of supersaturated solutions are the disadvantages of this method.

Bergström et al., 2002; Glomme et al., 2005; Yalkowsky, 1999; Delaney, 2005;

Bergstrom, 2005 have developed automated and miniaturized methods for determination

of solubility of solid compounds but these methods require equilibration time that can be

several days or weeks for slowly dissolving drugs. Inadequate equilibration time can

result in significant underestimation of the solubility. Alternatively, aqueous drug

solubility can be estimated from melting point, the octanol-water partition coefficient, the

hydrogen-bonding capacity of the molecule and its non polar surface area. However, such

computational methods for solubility estimation are not accurate. The drug training sets

used to create the methods tend to be over represented by low molecular weight drugs

and uncharged drugs that are somewhat soluble in water and the sets are subject to an

unknown degree of experimental error (Delaney, 2005). Drug-like molecules, especially

those that possess ionizable moieties, are ill-represented in these training sets. Training

sets containing drug-like compounds of wide molecular diversity might allow better

methods to be developed (Delaney, 2005; Bergstrom, 2005).

Loftsson and Hreinsdottir (2006) used a modified shake-flask solubility method and

shorten the equilibration time through heating prior to equilibration at desired

temperature. In this method the equilibrium solubility is approached from super

saturation and accelerated precipitation through addition of the original solid compound

after cooling to room temperature. Here they reported solubility of 48 different drugs and

pharmaceutical excipients in pure water at room temperature.

Sreenivasa et al. (2010) have prepared and characterized inclusion complexes of

aceclofenac with β-CD and HP-β-CD to enhance its solubility. The phase solubility

28

analysis indicated the formation of 1:1 molar inclusion complex of aceclofenac with β-

CD and HP-β-CD. Apparent stability constant (KC) was 42.003 M-1

and 48.477 M-1for β-

CD and HP-β-CD complexes, respectively. The inclusion complexes were prepared by

three different methods viz. physical, kneading and co-precipitation method. The

prepared complexes were characterized using FT-IR and differential scanning

calorimetry. The inclusion complex prepared with HP-β-CD by kneading method

exhibited greatest enhancement in solubility and fastest dissolution (98.61% aceclofenac

release in 60 min) of aceclofenac.

Tiwari et al. (2011) determined the solubility of aceclofenac in distilled water,

hydrotropic solutions (30% urea and 30% sodium citrate) and solutions containing

different concentrations of hydrotropic agents (urea and sodium citrate). It was found

from the results that the aqueous solubility of aceclofenac was increased more than 250

times in hydrotropic blends, 5 and 25 times in 30% sodium citrate and 30% urea,

respectively. Therefore, it was concluded that the solubility of aceclofenac has increased

synergistically by mixed hydrotropy.

Maha et al. (2009) determined the solubility of aceclofenac in distilled water and in

Sorensen’s buffer solutions of pH 4, pH 5, pH 6 and pH 7.4 by equilibrating an excess

amount of drug with each solvent in a thermostatically controlled shaker water bath at

37°C for 24 hours. The mixtures were then filtered and suitably diluted with a respective

solvent and analyzed spectrophotometrically for aceclofenac concentrations at 275 nm

with reference to a corresponding calibration curve. The equilibrium solubility was taken

as the average value of each experiment (n=3). The solubility of aceclofenac in water,

Sorensen’s buffer solutions of pH 4, pH 5, pH 6 and pH 7.4 were 0.105, 0.139, 0.474,

1.317 and 5.786 mg/ml, respectively.

Vinnakota et al. (2011) employed mixed hydrotropic solubilisation phenomenon by using

the solution of 30% urea and 20% of sodium citrate to estimate poorly water-soluble drug

aceclofenac from fine powder and its tablet dosage forms. The solubility of aceclofenac

in distilled water was found to be 0.225 mg/ml, whereas in the mixture of 30% urea and

20% sodium citrate, the solubility was found to be 19.64 mg/ml. The increase in

solubility of aceclofenac in the mixture was more than 100 folds. Aceclofenac showed

maximum absorbance at 274.5nm. Beer’s law was obeyed in the concentration range of

29

5-40 µg/ml. The estimated label claim was found to be 100.30 ± 1.252 mg. The recovery

studies revealed that any small change in the drug concentration in the solution could be

accurately determined by the proposed method. The co-efficient of variation were not

more than 1.0% which confirmed good intermediate precision for the proposed method.

The low values of LOD and LOQ indicated good sensitivity of proposed method. Thus

the proposed method is new, simple, environmentally friendly, accurate and cost-

effective which can be successfully employed in routine analysis of aceclofenac in

tablets.

Hyun-Jong et al. (2012) developed a microemulsion system for its intranasal delivery

to achieve rapid onset of action and improved bioavailability of udenafil. It was

characterized by phase behavior, particle size, transmission electron microscope

(TEM) images, and the drug solublisation capacity of the microemulsion. A single

isotropic region was found in pseudo-ternary phase diagrams developed at various

ratios with Cap-Mul MCM L8 as an oil, labrasol as a surfactant, and transcutol

or its mixture with ethanol (1:0.25, v/v) as a co-surfactant. Optimized

microemulsion formulations with a mean diameter of 120–154 nm achieved enhanced

solubility of udenafil (>10 mg/ml) compared with its aqueous solubility (0.02

mg/ml). An in vitro permeation study was performed in human nasal epithelial

(HNE) cell mono layers cultured by the air–liquid interface (ALI) method and the

permeated amounts of udenafil increased up to 3.41-fold versus that of pure

udenafil. According to the results of an in vivo pharmacokinetic study in rats,

intranasal administration of udenafil-loaded microemulsion had a shorter Tmax value

(1 min) compared with oral administration and improved bioavailability (85.71%)

compared with oral and intranasal (solution) administration. The microemulsion

system developed for intranasal administration may be a promising delivery system

of udenafil, with a rapid onset of action and improved bioavailability.

Kapil et al. (2011) investigated piperine, an amide alkaloid of black pepper, for

Transdermal enhancer activity using human cadaver skin in vitro with aceclofenac as the

model drug. Furthermore, FT-IR studies were conducted to understand to possible

enhancement mechanism. Piperine, at all three concentrations tested, significantly

increased flux of the drug compared to control (p<0.05). Similarly permeability

30

coefficient (Kp), cumulative amount release (Q24) and enhancement ratio (ER) shown

significant increase over control sample whereas skin content of aceclofenac and lag-time

of enhancer treated epidermal membrane shown proportionate reduction over control.

FT-IR studies reveal that piperine reduces peak area by 19.17 % and 16.87 % for

symmetric and asymmetric stretching peaks. In addition, piperine significantly reduces

percentage of secondary structures of keratin at amide I band. These results indicated that

piperine enhances Transdermal permeation of aceclofenac by biphasic mechanism

involving partial extraction of stratum corneum (SC) lipid and interaction with SC

keratin.

2.9 MICROEMULSION

The concept of microemulsions was first introduced by Hoar and Schulman during

1940s. Microemulsions are optically clear, thermodynamically stable and usually low

viscous solutions (Hoar and Schulman, 1943). It is defined as a system of water, oil and

amphiphile which is an optically isotropic and thermodynamically stable liquid micro-

dispersion (Danielson and Lindmann, 1981; Tenjarla, 1999; Lawrence and Rees, 2012).

Particle size and stability is the essential distinction between normal emulsion and

microemulsion because normal emulsions are kinetically stable while microemulsions are

thermodynamically stable. The stability of the microemulsion can be disturbed by

addition of salt, other additives, pressure or temperature. Normal emulsions undergo

aging by coalescence of droplets and Ostwald ripening (transfer of material from small

droplets to larger ones). As a result of these processes a decrease in the free energy of

dispersion occurs. Ruckenstein and Chi (1975) had proposed that thermodynamic

stability of microemulsions was due to interfacial free energy, interaction energy between

droplets and entropy of dispersion. Microemulsions offer several advantages such as

enhanced drug solubility, good thermodynamic stability, ease of manufacturing and

enhancing effect on Transdermal delivery compared to conventional formulations

(Lawrence and Rees, 2012; Gasco, 1997). Water insoluble drugs may be delivered

through oil-in-water (o/w) microemulsions (Jeppson and Ljunberg, 1975; Mizushima et

al., 1982; Kronevi and Ljunberg, 1983) while water soluble drugs may be delivered

through water-in-oil (w/o) microemulsions. These systems may also be used for sustained

release of drugs by formulating intramuscular preparations (Gasco and Lattanzi, 1990).

31

Microemulsions are thermodynamically stable, transparent isotropic solutions with

particle sizes ranging from 5 to 100 nm and arise from the spontaneous self-assembly of

the hydrophobic or hydrophilic parts of surfactant molecules. Numerous studies have

been conducted on microemulsions, researching their use in a wide variety of systems,

including pharmaceuticals, cosmetics, food, oil recovery, as models for biological

membranes and as reaction media. Moreover, new applications are constantly being

reported.

Microemulsions are typically formed with exact concentrations of water, oil, surfactant

and possibly co-surfactant and are deemed oil-in-water (o/w) or water-in-oil (w/o)

emulsion depending on the continuous phase. The concentrations at which

microemulsions form are normally mapped out on ternary phase diagrams, similar to that

shown in Fig 2.3.

Figure 2.4: Typical ternary phase diagram for a soybean oil, polyoxyethylene ether

surfactant and water system at 5°C, 20°C, 30°C and 37°C, showing areas of

micro emulsion formation (Flanagan et al., 2006).

32

Primarily, microemulsions differ from normal, coarse emulsions in that micro emulsions

normally form spontaneously (no energy addition required), have very small particle

sizes (<100 nm), are transparent/translucent and are thermodynamically stable. The oil

type normally used in microemulsion formation is a hydrocarbon, or short and medium

chain triglyceride. Long chain triglycerides are more difficult to make soluble as they are

semi-polar compared to hydrocarbon oils and they are too bulky to penetrate the

interfacial film to assist in the formation of an optimal curvature (Gaonkar and Bagwe,

2003).

2.9.1 Types of Microemulsion

Winsor identified four general types of phase equilibria: Type I (o/w), II (w/o), III (B.C.)

and IV (isotropic micellar solution). Type I and II are two-phase systems, Type III a

three-phase system and Type IV a single-phase system. Depending on surfactant type and

sample environment, Types I, II, III or IV form preferentially, the dominant type being

related to the molecular arrangement at the interface. Conductivity measurement is a

simple method to determine different microstructures of microemulsions for an ionic

surfactant system, but cannot be applied to a nonionic surfactant system.

2.9.2 Methods of microemulsion preparation

Microemulsions are usually prepared by following methods:

2.9.2.1 Phase Titration Method

In this method, microemulsions are prepared by the spontaneous emulsification method

also called phase titration method and can be depicted from phase diagrams. As

quaternary phase diagram (four component system) is time consuming and difficult to

interpret, pseudo ternary-phase diagram is often constructed to find the different zones

including microemulsion zone, in which each corner of the diagram represents 100% of

the particular component. Metastable systems should not be included in observations.

(Shafiq et al., 2007). Schematic representation of pseudo ternary phase diagram showing

microemulsion region is given in Figure 2.3.

33

2.9.2.2 Phase Inversion Method

When excess of the dispersed phase is added, phase inversion of microemulsions occurs

or in response to temperature. Drastic physical changes occur including changes in

particle size during phase inversion. Curvature of the surfactant is changed by these

methods spontaneously. However, for non-ionic surfactants, it can be achieved by

changing the temperature of the system which forces a transition from an o/w

microemulsion at low temperatures to a w/o microemulsion at higher temperatures

(transitional phase inversion). During cooling, the system crosses a point of zero

spontaneous curvature and minimal surface tension, promoting the formation of finely

dispersed oil droplets. This method is referred to as phase inversion temperature (PIT)

method. Other parameters such as salt concentration or pH value instead of the

temperature alone may be considered as well. Moreover, a transition in the spontaneous

radius of curvature can be obtained by changing the water volume fraction. By

successively adding water into oil, initially water droplets are formed in a continuous oil

phase. Increasing the water volume fraction changes the spontaneous curvature of the

surfactant from initially stabilizing a w/o microemulsion to an o/w microemulsion at the

inversion locus. Short-chain surfactants form flexible monolayer at the o/w interface

which results in a bicontinuous microemulsion at the inversion point (Azeem et al.,

2008).

Malakar et al. (2011) developed insulin-loaded microemulsions for Transdermal delivery

containing the oil phase i.e. isopropyl myristate or oleic acid, the surfactant i.e. tween 80

and isopropyl alcohol as the co-surfactant. The compositions of microemulsions were

determined by constructing pseudo ternary phase diagrams. The permeation flux of

insulin-microemulsions containing oleic acid as oil phase through mouse skin and goat

skin (in vitro) was comparatively higher than that of microemulsions containing

isopropyl myristate as oil phase. The insulin-loaded microemulsion containing 10% oleic

acid, 38% aqueous phase and 50% surfactant and co-surfactant phase with 2% dimethyl

sulfoxide (DMSO) as permeation enhancer showed greater permeation flux (4.93 ±

0.12μg/cm2/hour) through goat skin. The in vitro insulin permeation from these

microemulsions followed the Korsmeyer-Peppas model (R2=0.923 to 0.973) for 24 hours

34

with non-Fickian, “anomalous “mechanism. This preliminary data indicated that

microemulsions were promised vehicle for transdermal delivery of insulin.

Microemulsions are widely used as vehicle for cosmetic active ingredients due to their

numerous advantages i.e. solubilizing both hydrophilic and lipophilic ingredients, in

addition to the drug delivery. They are well employed in cosmetics as moisturizing and

soothing agents, as sunscreens, as antiperspirants and as body cleansing agents (Azeem et

al., 2008).

Pakpayat et al. (2009) developed ascorbic acid microemulsions for topical application.

These microemulsions were prepared using HLD (hydrophilic lipophilic deviation)

concept to optimize the formulation. From this optimal formulation, the realization of

dilution ternary diagrams leads to obtain microemulsion zones. The effects of

composition variables on the physicochemical characteristics of each system were also

investigated. Ascorbic acid was loaded in the formulations after optimization of the

microemulsion systems. The surface properties and the structure of the microemulsions

were characterized by surface tension and small angle neutron scattering. Bicontinuous

structure microemulsions were identified and the influence of ascorbic acid localization

at the interface leading to modifications of the microemulsion structure was pointed out.

The in vitro transdermal penetration of ascorbic acid microemulsions were studied by

Franz cells. Three different microemulsions were envisaged. The results confirmed that

these microemulsion systems present a real interest for formulation and protection of

ascorbic acid. A major location of ascorbic acid found in the epidermis where

decomposition of melanin occurred indicates that microemulsion could be considered as

a suitable carrier system for application of ascorbic acid as a whitening agent. In addition,

a good passage of drug in dermis could be interesting for relative oxygen matrix damage.

Lee et al. (2005) developed an o/w microemulsion system to enhance skin permeability

of aceclofenac. Labrafil M 1944 CS was used as oil phase of the microemulsion due to its

good solubilizing capacity. The concentration ranges of oil, surfactant, Cremophor ELP

and co-surfactant, ethanol, for microemulsion formation were obtained by constructing

pseudo ternary phase diagrams. 18 formulations with various values of oil of 6-30%,

water of 0-80% and the mixture of surfactant and co-surfactant (at the ratio of 2) of 14-

70% were selected. Franz diffusion cells mounted with rat skin were used to evaluate in

35

vitro transdermal permeability of aceclofenac from the microemulsions. HPLC and

Zetasizer Nano-ZS were used to evaluate the level of aceclofenac permeated and droplet

size of microemulsions was characterized, respectively. The skin permeation of

aceclofenac was investigated and terpenes were added to the microemulsions at a level of

5% to study their effects. The mean diameters of microemulsions ranged between

approximately 10-100 nm and skin permeability of aceclofenac incorporated into the

microemulsion systems was 5-fold higher than that of ethanol vehicle. Among the

terpenes added, limonene had the best enhancing ability.

Yang et al. (2002) prepared microemulsion to develop novel transdermal formulation for

increasing skin permeability of aceclofenac. On the basis of solubility and phase

studies, oil and surfactant were selected and their composition was determined.

Microemulsion was spontaneously prepared by mixing ingredients and the

physicochemical properties were investigated. The mean diameters of microemulsion

were approximately 90 nm and the system was physically stable at room

temperature at least for 3 months. In addition, the in vitro and in vivo

performance of microemulsion formulation was evaluated. Aceclofenac was released

from microemulsion in acidic aqueous medium and dissolved amounts of aceclofenac

was approximately 30% after 6 hours. Skin permeation of aceclofenac from

microemulsion formulation was higher than that of cream. Following transdermal

application of aceclofenac preparation to delayed onset muscle soreness, serum

creatinine phosphokinase and lactate dehydrogenase activity was significantly

reduced by aceclofenac. Aceclofenac in microemulsion was more potent than cream

in the alleviation of muscle pain. Therefore, the microemulsion formulation of

aceclofenac appear to be a reasonable transdermal delivery system of drug with

enhanced skin permeability and efficacy for treatment of muscle damage.

Jadhav et al. (2011) conducted a study to investigate the microemulsion based topical

drug delivery system of antifungal drug fluconazole in order to bypass its gastrointestinal

adverse effects and to improve patient compliance. The pseudoternary phase diagrams

were developed for combinations of isopropyl palmitate (IPP) or light liquid paraffin

(LLP) as the oil phase, aerosol OT as surfactant and sorbitan mono oleate as co-surfactant

using water titration method. Microemulsions obtained were analyzed for transdermal

36

permeability of fluconazole using Keshary-Chien diffusion cell through an excised rat

skin. Higher in vitro permeation was observed from IPP based microemulsion. Thus, it

was selected for further formulation studies. The developed microemulsion was

characterized for optical birefringence, globule size and polydispersibility index. The

average globule size of microemulsion was found be less than100 µm. Centrifugation

studies were carried out to confirm the stability of developed formulation. The

formulation was thickened with a gelling agent carbopol 940, to yield a gel with desirable

properties facilitating the topical application. The developed microemulsion based gel

was characterized for pH, spreadability, refractive index and viscosity. Optimized

formulation was then subjected to in vitro antifungal screening in comparison to currently

available marketed gel formulation of fluconazole (Flucos gel). Optimized

microemulsion based gel formulation was found to exhibit significant antifungal activity

as compared to marketed formulation. The safety of gel formulation for topical use was

evaluated using skin irritation test. Thus, this study indicates that microemulsion can be a

promising vehicle for topical delivery of fluconazole.

Chudasama et al. (2011) developed new oil-in-water microemulsion-based (ME) gel

containing 1% itraconazole for topical delivery. Potential excipients were selected on the

basis of solubility in oils and surfactants. Pseudoternary phase diagrams were constructed

to define the microemulsion existence ranges. The best microemulsion was characterized

for its morphology and particle size distribution. The best microemulsion was

incorporated into polymeric gels of lutrol F127, xanthan gum and Carbopol 934 for

convenient application and evaluated for pH, drug content, viscosity and spreadability. In

vitro drug permeation of ME gels was determined across excised rat skins. Furthermore,

in vitro antimycotic inhibitory activity of gels was conducted using agar-cup method and

Candida albicans as a test organism. The droplet size of the optimized microemulsion

was found to be <100 nm. The optimized lutrol F 127 ME gel showed pH in the range of

5.68 ± 0.02 and spreadability of 5.75 ± 1.396 g cm/s. The viscosity of ME gel was found

to be 1805.535 ± 542.4 mPa. The permeation rate (flux) of prepared ME gel was found to

be 4.234 μg/cm/h. The release profile exhibited diffusion controlled mechanism of drug

release from ME gel. The developed ME gels were non irritant and there was no

erythema or edema. The antifungal activity showed widest zone of inhibition with lutrol

37

F127 ME gel. These results indicate that the studied ME gel may be a promising vehicle

for topical delivery of itraconazole.

Lee et al. (2010) prepared microemulsion-based hydrogel for skin delivery of

itraconazole. Microemulsion prepared with transcutol as a surfactant, benzyl alcohol as

an oil and the mixture of ethanol and phosphatidyl choline (3:2) as a co-surfactant were

characterized by solubility, phase diagram and particle size. Microemulsion based

hydrogels were prepared using 0.7 % of xanthan gum (F1-1) or carbopol 940 (F1-2) as

gelling agents and characterized by viscosity studies. The in vitro permeation data

obtained by using Franz diffusion cells and hairless mouse skin showed that the

optimized microemulsion (F1) consisting of itraconazole (1% w/w), benzyl alcohol

(10%w/w), transcutol (10% w/w) and the mixture of ethanol and phospahtidylcholine

(3:2) (10% w/w) and water (49%w/w) showed significant difference in flux (~1

µg/cm2/h) with their corresponding microemulsion based hydrogels (0.25-0.64

µg/cm2/h). However, the in vitro skin drug content showed no significant difference

between F1 and F1-1, while F1-2 showed significantly low skin drug content. The effect

of the amount of drug loading (0.02, 1 and 1.5% w/w) on the optimized microemulsion

based hydrogels (F1-2) showed that the permeation and skin drug content increased with

higher drug loading (1.5%). The in vivo study of optimized microemulsion based

hydrogels (F1-2 with1.5% w/w drug loading) showed that this formulation could be used

as a potential topical formulation for itraconazole.

Rupali et al. (2010) prepared a microemulsion based gel using capmul MCM C8, jojoba

oil, Brij 96 V and ethanol as excipients. From rheological measurement, it was found that

microemulsion gel was highly stable, viscoelastic, having good flow properties, good

spreadability and applicability. The increase in percutaneous penetration of drug

indicated that the excipients i.e. Capmul MCM C8, jojoba oil, Brij 96 V and ethanol help

to improve drug penetration and controlled drug permeation through skin compared to

other control and marketed gel. A significant increase in the anti-inflammatory effect as

compared with marketed gel was observed during in vivo studies because of

incorporation of jojoba oil. From in vitro and in vivo data it can be concluded that the

developed microemulsion and microemulsion gel have great potential for transdermal

drug delivery.

38

Badawi et al. (2009) incorporated different concentrations of salicylic acid in an ME base

composed of isopropyl myristate, water and Tween 80: propylene glycol in the ratio of

15:1. 3 ME systems of concentrations S2%, S5% and S10% containing 2%, 5% and 10%

of salicylic acid, respectively were prepared. Microemulsions were evaluated by

examination under cross-polarizing microscope, measuring of percent transmittance, pH

measurement, determination of specific gravity, assessment of rheological properties and

accelerated stability study. The data showed that the addition of salicylic acid markedly

affected the physical properties of base. All systems were not affected by accelerated

stability tests. Stability study for 6 months under ambient conditions was carried out for

S10%. No remarkable changes were recorded except a decrease in the viscosity value

after 1 month. The results suggested that ME could be a suitable vehicle for topical

application of different concentrations of salicylic acid.

Eskandar et al. (2012) prepared microemulsion formulations by mixing appropriate

amount of surfactant including tween 80 and labrasol, co-surfactant such as propylene

glycol and oil phase including isopropyl myristate– transcutol P (10:1 ratio). The

prepared microemulsions were evaluated regarding their particle size, zeta potential,

conductivity, stability, viscosity, differential scanning calorimetry (DSC), scanning

electron microscopy (SEM), refractory index (RI) and pH. The results showed that

maximum oil was incorporated in microemulsion system that contained surfactant to co-

surfactant ratio (Km) of 4:1. The mean droplets size range of microemulsion formulation

was in the range of 14.1 to 36.5 nm and its refractive index (RI) and pH were 1.46 and

6.1, respectively. Viscosity range was 200-350 cps. Drug release profile showed 49% of

the drug released in first 8 hours of experiment belongs to ME-7. Also, hexagonal and

cubic structures were seen in the SEM photograph of microemulsions. It was concluded

that physicochemical properties and in vitro release were dependent upon the contents of

S/C, water and oil percentage in formulations. Also, ME-7 may be preferable for topical

tretinoin formulation.

Pradip et al. (2006) developed an oral microemulsion formulation for enhancing the

bioavailability of acyclovir. A labrafac-based microemulsion formulation with labrasol as

surfactant and Plurol Oleique as co-surfactant was developed for oral delivery of

acyclovir. Phase behavior and solubilizing capacity of microemulsion system were

39

characterized and in vivo oral absorption of acyclovir from microemulsion was

investigated in rats. A single isotropic region, which was considered to be a bicontinuous

microemulsion, was found in the pseudo ternary phase diagrams developed at various

Labrasol: Plurol Oleique: Labrafac ratios. With increase of Labrasol concentration, the

microemulsion region area and the amount of water and Labrafac solublized into the

microemulsion system increased; however, the increase of Plurol Oleique percentage

produced opposite effects. The microemulsion system was also investigated in terms of

other characteristics, such as interfacial tension, viscosity, pH, refractive index, diffusion

and bioavailability. Acyclovir, a poorly soluble drug, displayed high solubility in a

microemulsion formulation using Labrafac (10%), Labrasol (32%), Plurol Oleique (8%)

and water (50%). The in vitro intraduodenal diffusion and in vivo study revealed an

increase of bioavailability (12.78 times) after oral administration of the microemulsion

formulation as compared with commercially available tablets.

Baboota et al. (2007) developed and evaluated microemulsion formulations for

Terbinafine (TB) with a view to enhance its permeability through skin and provide

release for 24 h. Various o/w microemulsions were prepared by spontaneous

emulsification method. On the basis of solubility studies, oleic acid was chosen as oil

phase, caprylo caproyl macrogol-8- glyceride (labrasol S) and purified diethylene glycol

monoethyl ether (Transcutol P) were used as surfactant and co-surfactant, respectively.

Pseudoternary phase diagrams were constructed to obtain the concentration range of oil,

surfactant, co-surfactant and water for microemulsion formulation. The optimized

microemulsion consisted of 2% w/w, 8% w/w oleic acid, 31% w/w labrasol S, 31% w/w

transcutol P and 30% w/w distilled water. Permeability parameters like Jss and Kp were

found to be significantly higher for formulation F4 as compared to other formulations (P

< 0.05). Microbiological studies of microemulsion showed better anti-fungal activity

against Candida albicans and Aspergillus flavus as compared to marketed product (P <

0.05).

Madan et al. (2009) aimed to prepare, characterize and in vitro evaluation of a Winsor-IV

type microemulsion based drug delivery system incorporating celecoxib as BCS class–II

model drug. Attempts were made to prepare cost effective o/w microemulsion using

tween 80, glycerol, sun-flower oil and water. The existence of microemulsion zone was

40

investigated using phase diagrams. The systems were characterized by polarized light

microscopy, viscosity, refractive index, droplet size of dispersed phase by dynamic light

scattering technique, thermal and centrifugal stability and drug release profile. The

obtained microemulsion was found optically isotropic with non-Newtonian behavior. The

average droplet size was 100-300 nm. Microemulsions showed reversibility of

transparency at ambient temperature after storage at 5 °C. The solubility enhancement of

formulated products was apparent from higher release rate from microemulsion as

compared to commercial product. The drug release profile was demonstrated to be

promising for oral delivery of celecoxib.

Boonme et al. (2006) prepared and characterized microemulsion systems of isopropyl

palmitate (IPP), water and 2:1 Brij 97 and 1-butanol by different experimental

techniques. A pseudoternary phase diagram was constructed using water titration method.

At 45% wt/wt surfactant system, microemulsions containing various ratios of water and

IPP were prepared and identified by electrical conductivity, viscosity, differential

scanning calorimetry (DSC), cryo-field emission scanning electron microscopy (cryo-

FESEM) and nuclear magnetic resonance (NMR). The results from conductivity and

viscosity suggested a percolation transition from water-in-oil (w/o) to oil-in-water (o/w)

microemulsions at 30% wt/wt water. From DSC results, the exothermic peak of water

and the endothermic peak of IPP indicated that the transition of water/oil to oil/water

microemulsions occurred at 30% wt/wt water. Cryo-FESEM photomicrographs revealed

globular structures of microemulsions at higher than 15% wt/wt water. In addition, self-

diffusion coefficients determined by NMR reflected that the diffusability of water

increased at higher than 35% wt/wt water, while that of IPP was in reverse. Therefore, the

results from all techniques are in good agreement and indicate that the water/oil and

oil/water transition point occurred in the range of 30% to 35% wt/wt water.

Arun et al. (2009) designed two novel O/W microemulsions of ketoprofen for improving

transdermal absorption and prepared these formulations by constructing the pseudo-

ternary phase diagrams using oleic acid, polysorbate-80, propylene glycol and water in

different ratios and were gelled by incorporating cab-o-sil. Oleic acid was screened as the

oil phase due to good solubilizing capacity and excellent skin permeation rate of

ketoprofen. In vitro diffusion study was carried out using artificial semi permeable

41

membrane. Formulation-2 showed higher diffusion rate than the formulation-1. The

formulation-2 consisted of 3% ketoprofen, 5% menthol, 31.61% oleic acid, 0.5%

tocopheryl acetate, 23.71% polysorbate-80, 23.71% propylene glycol, 0.18% methyl

paraben, 0.02% propyl paraben, 6% cab-o-sil, triethanolamine (qs) and 6.29% water.

Formulation-1 consisted of 3% ketoprofen, 33.45% oleic acid, 0.5% tocopheryl acetate,

25.08% polysorbate-80, 25.08% propylene glycol, 0.18% methyl paraben, 0.02% propyl

paraben, 6% cab-o-sil, triethanolamine (qs) and 6.69% water. Diffusion was increased

when the formulation was incorporated with 5% menthol. The diffusion rate of

ketoprofen from formulation was fast and rapid than marketed sample. Cab-o-sil was

used for improving the viscosity and stability of the system. The percentage of drug

release across membrane from marketed product, formulation-1 and formulation-2 were

found to be 64.65%, 84.64% and 90.20% respectively in 8 hrs.

Ozguney et al. (2006) evaluated and compared the in vitro and in vivo transdermal

potential of w/o microemulsion (M) and gel (G) bases for diclofenac sodium. The effect

of dimethyl sulfoxide (DMSO) as a penetration enhancer was also examined when it was

added to the M formulation. Franz diffusion cells with excised dorsal rat skin were used

to study the in vitro permeation potential of these formulations. A carrageenan-induced

rat paw edema model was used to investigate in vivo performance of these formulations.

As a reference formulation, commercial formulation of diclofenac sodium was used.

Analysis of variance was used to analyze results of in vitro permeation studies and the

paw edema tests by repeated measures. The in vitro permeation studies found that M was

superior to G and commercial formulation of DS (C) and that adding DMSO to M

increased the permeation rate. The permeability coefficients (Kp) of DS from M and M +

DMSO were higher (Kp = 4.9 × 10−3 ± 3.6 × 10−4cm/h and 5.3 ×10−3 ± 1.2

×10−3cm/h, respectively) than Kp of DS from C (Kp = 2.7 × 10−3 ± 7.3 × 10−4 cm/h)

and G (Kp = 4.5 × 10−3 ± 4.5×10−5cm/h). In the paw edema test, M showed the best

permeation and effectiveness and M + DMSO had nearly the same effect as M. The in

vitro and in vivo studies showed that M could be a new, alternative dosage form for

effective therapy.

42

2.10 GEL

Gels are defined as semisolid systems consisting of dispersions made up of either small

inorganic particles or large organic molecules enclosing and interpenetrated by a liquid.

Gels in which the macromolecules are distributed throughout the liquid in such a manner

that no apparent boundaries exist between them and the liquids are called single-phase

gels. In instances in which the gel mass consists of floccules of small distinct particles,

the gel is classified as a two-phase gel and frequently called a magma or a milk. Gels and

magmas are considered as colloidal dispersions each contains particles of colloidal

dimensions (Ansel, 1990).

2.10.1 Preparation of Gels

Gels can be prepared by different methods which are given below:

2.10.1.1 Temperature effect

The solubility of most lyophilic colloids e.g. gelatin, agar, sodium oleate is reduced on

lowering the temperature, so that cooling a concentrated hot solution will often produce a

gel. In contrast to this, some materials such as the cellulose ethers owe their water

solubility to hydrogen bonding with the water. Raising the temperature of these sols will

disrupt hydrogen bonding and reduced solubility will cause gelation (Rawlins, 1992).

2.10.1.2 Flocculation with salts and non-solvents

Gelation is produced by adding just sufficient precipitant to produce the gel state but

insufficient to bring about complete precipitation. It is necessary to ensure rapid mixing

to avoid local high concentrations of precipitant. Solutions of ethyl cellulose, polystyrene,

etc. in benzene can be gelled by rapid mixing with suitable amounts of a non-solvent such

as petroleum ether.

The addition of salts to hydrophobic sols brings about coagulation and gelation is rarely

observed. However, the addition of suitable proportions of salts to moderately

hydrophilic sols such as aluminum hydroxide, ferric hydroxide and betonies, produces

gels. As a general rule, the addition of about half of the amount of electrolyte needed for

complete precipitation is adequate. With positively charged hydroxide sols, divalent ions

such as SO4-2

are more effective than univalent ions such as Cl-. The gels formed are

frequently thixotropic in behaviour. Such hydrophilic colloids as gelatin, proteins and

43

acacia are only affected by high concentrations of electrolytes, while the effect is to ‘salt

out’ the colloid and gelation does not occur (Rawlins, 1992).

2.10.1.3 Chemical reaction

In the preparation of sols by precipitation from solution, e.g. Aluminium Hydroxide [Al

(OH)3] sol prepared by interaction in aqueous solution of an aluminum salt and sodium

carbonate and increased concentration of reactants will produce a gel structure. Silica gel

is another example and is produced by the interaction of sodium silicate and acids in

aqueous solution (Rawlins, 1992).

Gaikwad et al. (2012) studied the effect of various carbopols (934 and 940) on drug

release from fluconazole gel formulation developed for topical application. Full factorial

design has been applied to study the effect of type of carbopols on quality attributes of

fluconazole gel formulation. Fluconazole topical gel formulation batches (F1 to F9) have

been formulated as per runs obtained in factorial design and further evaluated for pH,

drug content, viscosity and in vitro drug release kinetics, etc. Fourier transform infrared

spectroscopy (FTIR) study indicated no chemical or structural changes in fluconazole

during formulation studies. Drug diffusion studies have shown time required for 90% of

total drug release (t 90%) from all formulations in between 28.7 ± 2.3 to 208.4 ± 3.9 min.

Batch F9 showed maximum t 90% attributed to highest viscosity resulted in slower drug

release amongst all batches (F1-F9), however, opposite results have been observed with

batch F1. These results are in accordance with concept of inverse relationship between

drug release and viscosity of formulation. It has been observed that drug release from all

gel formulation batches obeyed korsmeyer-peppas model. Permeation of drug through

formed gel depends on viscosity and pH of gel. From present study it can be concluded

that topical gel formulations of fluconazole with desirable drug diffusion pattern can be

successfully prepared by using carbopol 934 and carbopol 940, where both carbopols

have extended the drug release at their respective higher concentrations.

Kumar et al. (2009) extracted mucilage from Anacardium occidentale which was

subjected to toxicity studies for its safety and preformulation studies for its suitability as a

gelling agent. The gum was extracted by using water as solvent and precipitated using

acetone as non-solvent. Physicochemical characteristics such as solubility, ash values,

Pre-compression parameters, swelling index, loss on drying and pH were studied. 8

44

batches of aceclofenac gels were prepared with different concentration of mucilage i.e.

2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% and 5.5%. The gels were evaluated for drug

content, viscosity determination and in vitro permeation across dialysis membrane, skin

irritation and stability tests. The gels prepared with 5.0% of mucilage were found to be

ideal and comparable with a commercial preparation. The prepared gels did not produce

any dermatological reactions and were well tolerated by the guinea pig. The gels were

found to be stable with respect to viscosity, drug content and physical appearance at all

temperature conditions for 3 months. Studies indicated that the extracted mucilage may

be a good source as a pharmaceutical adjuvant specifically as a gelling agent.

Najmuddin et al. (2010) designed and evaluated gels for topical delivery of water

insoluble antifungal agent Ketoconazole with an aim to increase its penetration through

skin and thereby its flux. The solubility of Ketoconazole is increased by complexation

with ß-cyclodextrin which was prepared by solvent evaporation technique with 1:1 and

then incorporated into gels. The complex was characterized by infrared spectroscopy.

There was no interaction between drug and carrier. The Ketoconazole gel formulations

were made with different polymers like carbopol 940, hydroxy propyl methyl cellulose,

methyl cellulose and sodium carboxymethylcellulose, containing various permeation

enhancers namely sodium lauryl sulphate (0.5-1.0%) and dimethyl sulfoxide (5-20%) in

different proportions. The formulated gels were evaluated for various physicochemical

parameters like, drug content, pH, viscosity, spreadability, extrudability, in-vitro drug

release. The in-vitro drug release study were carried out using pH 7.4 phosphate buffer,

All the formulated topical preparations showed pH in the range of 6.5 to 7.4 and also

showed good spreadability and extrudability. The carbopol 940 with 15% of dimethyl

sulfoxide (KCD3) showed best in-vitro drug release 98.07% at the end of 6 hrs.

El-Megrab et al. (2006) prepared microemulsion gels and lipogels containing either ethyl

oleate or oleic acid as an oil phase for topical administration of meloxicam (MLX). In

addition, Hydrogel and hydroalcoholic gels containing carbopol 940 as a gelling agent

were also prepared. In-vitro drug release through cellophane membrane and permeation

through the excised rabbit skin in Sorensen’s phosphate buffer (pH 7.4) containing 1%

w/v sodium lauryl sulphate were performed .The influence of initial drug concentration

(0.5, 0.65, 1% w/w) was studied. The permeation properties of ME from ethyl oleate

45

microemulsion which is the best formula achieved was studied in comparison to the

commercially available piroxicam gel. Moreover, the anti-inflammatory activity of MLX

after oral and topical administration in rats was studied and compared to that of

piroxicam gel. The results of an in-vitro drug release and its percutaneous permeation

revealed that the ethyloleate microemulsion gel showed the highest results. Meloxicam

gel (ethyl oleate microemulsion gel 1%) showed good protection against inflammation as

compared to Feldene® gel in rats.

Aejaz et al. (2010) prepared various compositions of aceclofenac solid dispersions by

physical mixing, fusion and solvent evaporation methods. PVP, PEG 6000, mannitol and

urea as carrier to enhance the solubility of drug. The formulations were evaluated for drug

content, in vitro dissolution study and also characterized by IR and DSC studies. There

was no interaction between drug and carrier. The general trend indicated that there was

an increase in in vitro drug release for solid dispersion prepared in the following order

Urea > PEG600 > PVP > Mannitol. Based on in vitro drug release pattern, 1:3 drug carrier

ratios were selected as ideal dispersion for gels. Carbopol 940 selected as ideal gel base

for preparation of gels and dispersions are incorporated to gel bases by trituration. The

in vitro release of aceclofenac solid dispersion incorporated gel was significantly

improved when compared to pure drug incorporated gel.

Kashyap et al. (2010) formulated aceclofenac gels by using different concentration of

Poloxamer 407 for topical drug delivery with an objective to increase transparency and

spreadability. These preparations were further compared with marketed Hifenac® gel.

Spreadability and consistency of poloxamer 407 gel containing aceclofenac (A9) were

12.4 g.cm/sec and 8mm as compared to 13.2 g.cm/sec and 11 mm respectively of

marketed gel, indicating good spreadability and consistency of the prepared gel (A9). The

transparency of prepared batch A9 was good as compared to the marketed gel. The percent

drug release was 97.11 and 98.66 from A9 and marketed gel respectively in 120 min. No

irritation was observed by skin irritation test. Stability studies under accelerated condition

showed satisfactory results. It can be concluded that poloxamer 407 gel containing

aceclofenac showed good consistency, homogeneity, spreadability and stability and has

wider prospect for topical preparations.

46

Atul et al. (2010) investigated and evaluated the potential of ethosomes to increase

transdermal transport of aceclofenac. Effects of different concentrations of lecithin (2, 3,

4, 5 and 6% w/w) and ethanol (20, 30 and 40% w/w) on different properties of ethosomes

(EF‐1 to EF‐7) were studied. The size of vesicles was found to have increased with

increasing lecithin concentration (2‐6%). Also it was observed that the size of vesicles

decreased significantly with increasing ethanol concentration (20‐40%). EF‐2 ethosomes

with 3% lecithin and 20% ethanol were found to have shown highest aceclofenac release

(92.72 ± 1.04%). In final phase of formulation development, EF‐2 Ethosomes containing

aceclofenac were converted to gel using three different carbopol concentrations (1, 1.5 and

2% w/w). The gel containing aceclofenac encapsulated in EF‐2 ethosomes in 1.5% gel

was found to be the optimized formulation (G‐2). G‐2 was found to have shown excellent

in vitro drug release and in vivo activity comparing gel containing free aceclofenac drug

and marketed gel (Hifenac®).

2.11 WORK DONE ON ACECLOFENAC

Shah et al. (2010) prepared a topical preparation containing aceclofenac using an o/w

microemulsion system. Isopropyl myristate was chosen as the oil phase as it showed a

good solubilizing capacity. Pseudo-ternary phase diagrams were used to obtain the

concentration ranges of the oil, surfactant (labrasol) and co-surfactant (plurol oleique) for

microemulsion formation. Five different formulations were formulated with various

amounts of the oil (5-25%), water (10-50%) and the mixture of surfactant and co-

surfactant at the ratio of 4:1 (45-65%). In vitro, permeability of aceclofenac from the

microemulsions was evaluated using Keshary Chien diffusion cells with 0.45-μm

cellulose acetate membrane. The amount of aceclofenac permeated was analyzed by

HPLC and the droplet size and zeta potential of microemulsions was determined using a

Zetasizer Nano-ZS. The mean diameters of microemulsion droplets approximately ranged

between 154 - 434 nm and the permeability of aceclofenac incorporated into the

microemulsion systems was 3 folds higher than that of the marketed formulation. These

results indicate that the microemulsion system studied is a promising tool for

percutaneous delivery of aceclofenac.

Debnath et al. (2009) formulated topical gel containing 1.5% aceclofenac, 1% benzyl

alcohol, 3% linseed oil, 10% methyl salicylate, 0.01% capsaicin, 5% menthol and

47

characterized this formulation. They prepared five trials and selected trial no. 5 which

had no significant problems. These problems are given in following table 2.2.

Table 2.3: Problems found in different trials (Debnath et al., 2009)

Trial: 1 Trial: 2 Trial: 3 Trial: 4 Trial: 5

1.Dehydration

problem

2.Fine particles are

observed

3.Aceclofenac is not

dissolved properly

4.Viscosity problem

1.More creaming

2.Phase separation

3.Viscosity problem

4.Aceclofenac is not

dissolve properly

1.Consistency is

not good

2.Viscosity

problem

3.Spreadibility is

poor

Spreadability

is poor

Nil

Shaikh et al. (2009) prepared organogels; the components and their concentration

necessary for organogels formation were evaluated using phase diagram. Solubility of

aceclofenac was determined. The in vitro skin permeability of aceclofenac from ethyl

oleate based lecithin organogels [EO/lecithin organogel] and hydrogel was investigated.

The in vivo characterization of ethyl oleate based organogel study was compared with

that of hydrogel. The alterations in microstructure of organogels during diffusion study

were elucidated. Viscosity and micellar size of the organogel sample were estimated. The

safety of optimized organogel was determined using histopathological investigation. The

flux calculated for skin permeability of aceclofenac was in the order EO/lecithin

organogel > hydrogel. The In vivo results also demonstrated that organogels are more

effective in faster drug release as compared to hydrogels. It was observed that viscosity

of gels decreased with increasing stress. The size of micellar aggregation increased with

water addition and has been revealed in dynamic light scattering (DLS) study. The

histopathological data showed that EO/lecithin organogel were safe enough for topical

purpose.

Dua et al. (2010) have prepared ointments, creams and gels containing 1% (m/m)

aceclofenac. They were tested for physical appearance, pH, spreadability, extrudability,

drug content uniformity, in vitro diffusion and in vitro permeation. Gels prepared using

carbopol 940 (AF2, AF3) and macrogol bases (AF7) were selected due to best results.

They were evaluated for acute skin irritancy, anti-inflammatory and analgesic effects

using the carrageenan-induced thermal hyperalgesia and paw edema method. AF2 was

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shown to be significantly (p< 0.05) more effective in inhibiting hyperalgesia associated

with inflammation, compared to AF3 and AF7. Hence, AF2 may be suggested as an

alternative to oral preparations.

Modi and Patel (2011) investigated the potential of a nanoemulsion formulation for

topical delivery of aceclofenac. Various oil-in-water nanoemulsions were prepared by

spontaneous emulsification method. The nanoemulsion area was identified by

constructing pseudoternary phase diagrams. The prepared nanoemulsions were subjected

to different thermodynamic stability tests. The nanoemulsion formulations that passed

thermodynamic stability tests were characterized for viscosity, droplet size, transmission

electron microscopy and refractive index. Topical permeation of aceclofenac through rat

abdominal skin was determined by Franz diffusion cell. The in vitro skin permeation

profile of optimized formulations was compared with that of aceclofenac conventional

gel and nanoemulsion gel. A significant increase in permeability parameters such as

steady-state flux (Jss), permeability coefficient (Kp) and enhancement ratio (Er) was

observed in optimized nanoemulsion formulation consist of 2% wt/wt of aceclofenac, 10

% wt/wt of Labrafac, 45% wt/wt surfactant mixture (Cremophor EL: Ethanol) and 43 %

wt/wt of distilled water. The anti inflammatory effects of formulation showed a

significant increase percent inhibition value after 24 hours when compared with

aceclofenac conventional gel and nanoemulsion gel on carrageenan-induced paw edema

in rats. These results suggested that nanoemulsions are potential vehicles for improved

transdermal delivery of aceclofenac.

Bhardwaj et al. (2010) made an attempt to prepare fast dissolving tablets of aceclofenac

using various super disintegrates sodium starch glycolate following by direct

compression technique. Aceclofenac (anti-inflammatory and analgesic) which was

selected as the model drug has poor aqueous solubility that results in variable dissolution

rate and hence poor bioavailability. These tablets were evaluated for hardness, friability,

weight variation, disintegration time, water absorption ratio and wetting time, in vitro

dissolution studies. All the formulation showed disintegration time in range of 12.2 to

27.5 second along with rapid in vitro dissolution. It was concluded that the fast dissolving

tablets of the poor soluble drug can be made by direct compression technique using

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selective super disintegrants showing enhanced dissolution, taste masking and hence

better patient compliance and effective therapy.

Patel et al. (2011) developed a gel formulation of aceclofenac using four types of gelling

agents namely carbopol, hydroxypropylmethylcellulose, carboxymethylcellulose sodium

and sodium alginate. Effect of penetration enhancer (propylene glycol) on the release was

also studied. The gels were evaluated for physical appearance, rheological behavior,

stability and drug release from all gelling agents through a standard cellophane

membrane using Keshary-Chien diffusion cell. All gels showed acceptable physical

properties concerning color, homogeneity, consistency, spreadability and pH value.

Carbopol showed superior drug release than followed by carboxymethylcellulose

sodium, hydroxypropylmethylcellulose and sodium alginate among all the gel

formulations. Increase in polymer concentration decreased the release of drug. The

release of drug was not linearly proportional with the concentration of penetration

enhancer or co-solvents. Storage at ambient conditions for two months showed that

the physical appearance, rheological properties and drug release remained

unchanged.

2.12 DETERMINATION OF ACECLOFENAC

Kothapalli et al. (2009) developed a reverse phase high performance liquid

chromatographic method (HPLC) for the simultaneous estimation of aceclofenac,

Chlorzoxazone (CHZ) and paracetamol (PARA) in the pharmaceutical formulation using

RP-C8 column. The mobile phase (acetonitrile and double distilled water) was pumped at

a flow rate of 1 ml/min in the ratio of 60:40 and the eluents were monitored at 230.0 nm.

Linearity was obtained in the concentration range of 1-60 μg/ml for aceclofenac, 1-50

μg/ml for both PARA and CHZ. The method was statistically validated and RSD was

found less than 2% indicating high degree of accuracy and precision of the proposed

HPLC method. Due to its simplicity, rapidness, high precision and accuracy, the

proposed HPLC method may be used for determining aceclofenac, chlorzoxazone and

paracetamol in bulk drug samples or in pharmaceutical dosage form.

Godse et al. (2009) developed a simple, rapid and selective HPLC method for

quantitation of aceclofenac and paracetamol from bulk drug and pharmaceutical

formulations using a mobile phase consisting mixture of methanol and water (70:30 v/v)

50

at the flow rate of 1mL/min. An ODS C-18 (Intersile 25 cm x 4.6 mm, 10 μm) column

was used as stationary phase. The retention time of aceclofenac and paracetamol were 1.8

min. and 2.7 min., respectively. Linearity was observed in the concentration range of 2-

50 μg/mL for aceclofenac and 5-50 μg/mL for paracetamol. Percent recoveries obtained

for aceclofenac and paracetamol were 100.6% and 100.7%, respectively. The proposed

method is precise, accurate, selective and rapid for the simultaneous determination of

aceclofenac and paracetamol.

Pawar et al. (2009) developed a simple, fast and precise reversed phase high performance

liquid chromatographic method for the simultaneous determination of aceclofenac,

paracetamol and chlorzoxazone. Chromatographic separation of the three drugs was

performed on an intersil C18 column (250 mm x 4.6 mm, 5 μm) as stationary phase with

a mobile phase comprising of 10 mM potassium dihydrogen phosphate (pH adjusted to

5.55 with ammonia): acetonitrile in the ratio of 60:40 (v/v) at a flow rate of 1.0 mL/min

and UV detection at 205 nm. The linearity of aceclofenac, paracetamol and

chlorzoxazone were in the range of 5.00-15.00 μg/μL, 25.00-75.00 μg/μL and 25.00-

75.00 μg/μL, respectively. The limit of detection for aceclofenac, paracetamol and

chlorzoxazone was found to be 18.0 ng/mL, 22.0 ng/mL and 9.0 ng/mL, respectively

whereas, the limit of quantification was found to be 55 ng/mL, 65 ng/mL and 27.0

ng/mL, respectively. The recovery was calculated by standard addition method. The

average recovery was found to be 99.04%, 99.57% and 101.63% for aceclofenac,

paracetamol and chlorzoxazone, respectively. The proposed method was found to be

accurate, precise and rapid for the simultaneous determination of aceclofenac,

paracetamol and chlorzoxazone.

Kang and Kim (2008) developed a new LC/MS/MS-based method which allows

simultaneous determination of aceclofenac and its three metabolites (4-OH-aceclofenac,

diclofenac and 4-OH-diclofenac) in plasma. After acetonitrile-induced precipitation of

proteins from the plasma samples, aceclofenac, 4-OH-aceclofenac, diclofenac, 4-OH-

diclofenac and flufenamic acid (an internal standard) were chromatographed on a

reverse-phase C18 analytical column. The isocratic mobile phase of acetonitrile/0.1%

formic acid(aq) [80:20 (v/v)] was eluted at 0.2 mL/min. Quantification was performed on

a triple quadruple mass spectrometer employing electrospray ionization and the ion

51

transitions were monitored in multiple reaction-monitoring mode. The monitored

transitions for aceclofenac, diclofenac, 4-OH-diclofenac, 4-OH-aceclofenac and

flufenamic acid were m/z 352.9→74.9, 296.1→251.7, 311.8→267.7, 368.9→74.9 and

279.9→235.9, respectively. The coefficient of variation of the assay precision was less

than 6.5% and the accuracy ranged from 93% to 103%. The limits of detection were 2

ng/mL for aceclofenac and 0.2 ng/mL for both diclofenac and 4-OH-diclofenac. This

method was used successfully to measure the concentrations of aceclofenac and its three

metabolites in plasma from healthy subjects after administration of a single 100-mg oral

dose of aceclofenac. This analytic method is a very simple, sensitive and accurate way to

determine the pharmacokinetics of aceclofenac and its metabolites.

A liquid–liquid extraction-based reversed phase HPLC method with UV detection was

validated and applied for analysis of aceclofenac and three of its metabolites (4-hydroxy-

aceclofenac, diclofenac, 4-hydroxy-diclofenac) in human plasma by Hinz et al. (2003).

The analytes were separated using an acetonitrile–phosphate buffer gradient at a flow rate

of 1 mL/min. and UV detection at 282 nm. The retention times for aceclofenac,

diclofenac, 4-hydroxy-aceclofenac, 4-hydroxy-diclofenac and ketoprofen (internal

standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min., respectively. The validated

quantitation range of the method was 10–10,000 ng/mL for aceclofenac, 4-

hydroxyaceclofenac and diclofenac and 25–10,000 ng/mL for 4-hydroxy-diclofenac. The

developed procedure was applied to assess the pharmacokinetics of aceclofenac and its

metabolites following administration of a single 100 mg oral dose of aceclofenac to three

healthy male volunteers.

Bhinge et al. (2009) developed a stability-indicating assay method for the determination

of aceclofenac after being subjected to different International Conference on

Harmonization prescribed stress conditions, such as hydrolysis, oxidation, heat and

photolysis. Aceclofenac is decomposed under hydrolytic stress (neutral, acidic and

alkaline) and also on exposure to light (in solution form). The compound is stable to

oxidative stress, heat and photolytic stress (in solid form). The major degradation product

is diclofenac, which is confirmed through comparison with the standard. Separation of

drug from major and minor degradation products is achieved on a C18 column using

methanol and 0.02% of ortho phosphoric acid in a ratio of 70:30. The method is linear

http://bhinge.jr.lib.bioinfo.pl/auth:Bhinge,JR

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over the concentration range of 17-100 µg/mL (r2= 0.9988). The detection wavelength is

275 nm. The method is validated for linearity, range, precision, accuracy, specificity and

selectivity.

Segun et al. (2012) developed a sensitive spectrophotometric method for determination of

aceclofenac following azo dye formation with 4-carboxyl-2, 6-dinitrobenzenediazonium

ion (CDNBD). Spot test and thin layer chromatography revealed the formation of a new

compound distinct from CDNBD and aceclofenac. Optimization studies established a

reaction time of 5 min at 30°C after vortex mixing the drug/CDNBD for 10 s. An

absorption maximum of 430 nm was selected as analytical wavelength. A linear response

was observed over 1.2-4.8 µg/mL of aceclofenac with a correlation coefficient of 0.9983

and the drug combined with CDNBD at stoichiometric ratio of 2: 1. The method has a

limit of detection of 0.403 µg/mL, limit of quantitation of 1.22 µg/mL and is reproducible

over a three day assessment. The method gave Sandell’s sensitivity of 3.279 ng/cm2. Intra

and inter-day accuracies (in terms of errors) were less than 6% while precisions in the

order of 0.03-1.89 % (RSD). The developed spectrophotometric method is of equivalent

accuracy (p > 0.05) with British Pharmacopoeia (2010) potentiometric method. It has the

advantages of speed, simplicity, sensitivity and more affordable instrumentation and

could found application as a rapid and sensitive analytical method of aceclofenac. It is the

first described method by azo dye derivatization for the analysis of aceclofenac in bulk

samples and dosage forms.

2.13 MECHANISM OF ACTION

The mode of action of aceclofenac is largely based on the inhibition of prostaglandin

synthesis. Aceclofenac is a potent inhibitor of the enzyme cyclooxygenase (Cox), which

is involved in the production of prostaglandins. In vitro data indicate inhibition of Cox-1

and Cox-2 by aceclofenac in whole blood assays, with selectivity for Cox-2 being

evident. Aceclofenac has shown stimulatory effects on cartilage matrix synthesis that

may be linked to the ability of drug to inhibit IL-1 activity. In vitro data indicate

stimulation of synthesis of glycosaminoglycan in osteoarthritic cartilage by drug. The

duration of morning stiffness and pain intensity are reduced and spinal mobility

improved, by aceclofenac in patients with ankylosing spondylitis (Blot et al., 2000).

Aceclofenac is metabolized to a major metabolite, 4'-hydroxy aceclofenac and to a

53

number of other metabolites including 5-hydroxy aceclofenac, 4'-hydroxydiclofenac,

diclofenac and 5-hydroxydiclofenac (Wood et al., 2001; Blot et al., 2000; Hinz et al.,

2003).

2.14 PHARMACOKINETICS

Aceclofenac is well absorbed from gastrointestinal tract and peak plasma concentrations

(Cmax) reached 1-3 hours after an oral dose. The drug is more than 99% bound to plasma

proteins and the volume of distribution (Vd) is approximately 25 liters. The presence of

food reduced rate of absorption (increased Tmax) but not the extent of absorption (Cmax or

AUC). In patients with knees pain and synovial fluid effusion, the plasma concentration

of aceclofenac was twice that in synovial fluid after multiple doses of the drug.

Aceclofenac is metabolized mainly to 4’ hydroxy-aceclofenac. The drug is eliminated

primarily through renal excretion with 70-80% of administered dose found in urine as

glucoronides and rest being excreted in feces. The plasma elimination half life of

aceclofenac is approximately 4 hours (Sean Sweetman, 2002; Goodman and Gilman,

2001).

Pharmacokinetic parameters of aceclofenac tablets, aceclofar (test) and Bristaflam

(reference) in 24 human volunteers (mean standard deviation; n=24) are given as follows:

Pharmacokinetic parameter of Aceclofar (test) and Bristaflam (reference) are AUC0-t

(mg/ml.h) 22.65 ± 4.48, 21.88 ± 3.91; AUC0-1(mg/ml.h) 24.02 ± 4.74, 23.17 ± 4.28 ;

Cmax(mg/ml) 8.64 ± 1.86, 9.36 ± 2.20; Tmax(h) 1.99 ± 0.80, 1.91 ± 0.75; T1/2(h) 3.30 ±

0.68, 3.36 ± 0.90; Lz(/h) 0.2207 ± 0.0560, 0.2254 ± 0.0811, respectively (Najib et al.,

2004).

2.14.1 Pharmacokinetic of aceclofenac through skin absorption

Shakeel et al. (2009) prepared and characterized nanoemulsion formulation of

aceclofenac by the method of Shakeel et al., 2007. Nanoemulsion (F1), nanoemulsion

based gel (NG1) and marketed tablets were administered to male Wistar rats and plasma

concentration of aceclofenac from formulations F1, NG1 and marketed tablet at different

time intervals was determined by reported HPLC method described by Hinz et al., 2003.

The graph between plasma aceclofenac concentration and time was plotted for each

formulation. It was found that the plasma concentration profile of aceclofenac for F1 and

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http://www.omicsonline.org/ArchiveJBB/2009/May/03/JBB1.13.php#Hinz

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NG1 showed greater improvement of drug absorption than the oral tablet formulation.

Peak (maximum) plasma concentration (Cmax) of aceclofenac in F1, NG1 and tablet was

9.4 ± 1.1, 8.8 ± 0.89 and 10.2 ± 3.4 μg/ml respectively whereas Tmax was 6 ± 0.31, 6 ±

0.34 and 2 ± 0.27 h respectively. AUC0→t and AUC0→ω in formulations F1, NG1 and

tablet were 61.4 2.98, 54.2 ± 2.58 and 20.8 ± 3.5 μg.h/ml respectively and 77.5 ± 3.1,

69.4 ± 2.85 and 29.1 ± 4.2 μg.h/ml respectively. These pharmacokinetic parameters

(Cmax, Tmax, AUC0→t and AUC0→ω) obtained with formulations F1 and NG1 were

significantly different from those obtained with oral tablet formulation (P<0.05). This

indicated that transdermal application can significantly modify pharmacokinetic profile

of aceclofenac. The significant AUC values observed with F1 and NG1 also indicated

increased bioavailability of aceclofenac from F1 and NG1 in comparison with oral tablet

formulation (P<0.05). The Ke and T1/2 for F1, NG1 and tablet were found 0.154, 0.152

and 0.159 h-1

, respectively & 4.50, 4.55 and 4.35 h, respectively. There was no significant

variation in Ke and T1/2 for F1, NG1 when compared with tablet formulation (P≥0.05).

This indicated that transdermal application could not change intrinsic pharmacokinetic

parameters such as Ke and T1/2. The formulations F1 and NG1 were found to enhance the

bioavailability of aceclofenac by 2.95 and 2.60 folds (percent relative bioavailability 295

and 260) with reference to oral tablet formulation. This increased bioavailability from

transdermal formulations (F1 and NG1) may be due to enhanced skin permeation and

avoidance of hepatic first pass metabolism of aceclofenac in the form of transdermal

formulations.

Tabassum et al. (2010) evaluated and compared the in vitro and in vivo transdermal

potential of gel (G) and patch formulation (P) for aceclofenac (AC). The effects of

different penetration enhancers were also examined. Franz diffusion cells using excised

dorsal rat skin were employed to study the in vitro potential of these formulations. A

carrageenan-induced rat paw edema model was used to investigate the in vivo

performance of gel and patch formulations containing aceclofenac. The commercial

formulation of aceclofenac (C) was used as a reference formulation. The in vitro

permeation studies found that G was superior to P and C and that adding permeation

enhancer to the formulations increased the permeation rate. The permeability coefficients

(Kp) of AC from G and P were higher (Kp = 0.3465 x 10−2 cm/h and 0.228 x 10−2 cm/h

55

respectively than the Kp of AC from C = 0.1314 x 10−2cm/h. In the paw edema test, G

showed the best permeation and effectiveness. The in vitro and in vivo studies showed

that G could be a new, alternative dosage form for effective therapy.

56

SOLUBILITY STUDIES

OF ACECLOFENAC

SELECTION OF EXCIPIENTS

SURFACTANTS OILS CO-SURFACTANTS

2:1 Smix (TWEEN 80 + IPA)

PHASE DIAGRAM STUDIES BY WATER TITRATION METHOD

MICROEMULSION

WATER PENETRATION

ENHANCER

CARBOPOL 940 WAS SELECTTED ON

THE BASIS OF STABILITY STUDIES

(GELLING AGENT + WATER)

GEL BASE

MICROEMULSION

BASED GEL

CHARACTERIZATION

IN VITRO FRANZ DIFFUSION CELL STUDIES

IN VIVO STUDIES

RATS RABBITS

HUMAN VOLUNTEERS

FORMULATION OF MICROEMULSION BASED ACECLOFENAC GEL

AND ITS IN VITRO IN VIVO STUDIES

ALMOND OIL TWEEN 80 IPA

PERMEABILITY STUDIES OF ACECLOFENAC IN

DIFFERENT OILS WITHTOUT PENETRATION

RHEOLOGICAL STUDIES

FTIR

XRD

THERMAL ANALYSIS

SEM

Figure 3.1: OVERALL FLOW DIAGRAM OF MICROEMULSION BASED GEL

FORMULATION AND ITS IN VITRO IN VIVO STUDIES

GELLING AGENT +

WATER

CARBOPOL

940

XANTHAN

GUM

CARBOPOL

934

STABILITY STUDIES

ANTI INFLAMMATORY EFFECT

ANALGESIC EFFECT

TRNSDERMAL

STUDIES

PHARMACOKINETIC & BIOEQUIVALENCE STUDIES

STABILITY

STUDIES

57

3. MATERIALS AND METHODS

3.1 MATERIALS:

3.1.1 Chemicals

The following materials were used:

Aceclofenac (Sami pharmaceuticals, Pakistan); Carbopol (BDH Chemicals Ltd,

Poole, UK); Mineral oil (Acros Organics, USA); Tween 80 & Tween 20 (Fisher

Scientific, Germany); Span 85, PLGA and Glacial acetic acid (Sigma, Germany).

Methanol, Ethanol, Propylene glycol, Acetone, Oleic acid, Palmitostearyl, n-hexane

Isopropyl alcohol, Cyclohexane, Potassium dihydrogen phosphate, Sodium hydroxide

pellets, Triethylamine, Acetonitrile, Ammonium acetate, Ethyl acetate, Ethyl alcohol,

n-butane, Methyl and propyl paraben (Merck, Germany).

3.1.2 Instruments

The following instruments were used during the practical work.

Sykam GmbH HPLC system (Germany) was used which consisted of HPLC Pump

Sykam S2100 solvent delivery system, Column thermostate Sykam 4011 thermo

controller, Detector Sykam S3210 UV/VIS, Operating system Clarity operating

software (MS-Windows) with Microsoft Windows XP Professional and 20 µl

Rheodyne Injector; Hot plate magnetic stirrer (Velp Scienifca, Germany); UV-

Spectrophotometer double beam (Shimadzu 1601, Japan); pH meter (Inolab,

Germany); Conductometer (WTW, Germany); Digital weighing balance (Precisa,

Switzerland); Vacuum pump (ILMVAC-Germany);

Automatic dissolution apparatus USP (Pharma Test, Germany); Oven (Mammert,

Germany); Optical microscope (Nikon, Japan); Whatman Filter Paper (Whatman,

Germany); Vortex Mixer (Seouline BioScirnce-Korea); Centrifuge machine (Heltich,

Germany); Centrifuge tubes (pyrex France); Disposable Syringes (BD pakistan);

Eppendorf tubes (Greiner lavortechnik-Germany); FTIR (Bruker, Tenser 27,

Germany); SEM ( Hitachi, S3400N); DSC & TGA (DuPont thermal analyzer with

2010 DSC194 module); Zeta potential & Zeta size (Zetasizer Nano series ZEN 3600

Malvern Software DTS (Nano) United Kingdom); XRD (Philips Analytical XRD

Model: PW 3710, Holland); Franz diffusion cell (PermeGear, USA); Sonicator (Elma,

Germany); Peristaltic Pump (Heidolph, Germany); Ultra Low temperature freezer

http://www.malvern.com/labeng/products/zetasizer/zetasizer_nano/zetasizer_nano_zs.htm


58

(Sanyo, Japan); Mexameter (Courage and Khazaka, Germany); Tewameter (Courage

and Khazaka, Germany); Syringe filter unit (Millipore, UK); Cellulose acetate

membrane filters (Sartorius, Germany); Water distillation apparatus (IRMECO

GmbH, Germany); SpectraMax 340 microplate reader (Molecular Devices, USA);

Water deionizer (Elga, UK); Programmable Rheometer (Brookfield); Electric balance

Percia XB 120A (Japan); Abbey Refractometer HEDAO (China).

3.2 METHODS:

3.2.1 Solubility studies for screening of Excipients

First of all the solubility of oils, surfactants and co-surfactants were determined in

methanol, ethanol, isopropyl alcohol and n-butanol. 2 ml each of oil, surfactant, co-

surfactant and water were taken in a stoppered 20 ml vial separately containing

magnetic bar. 100 mg of aceclofenac was added in increment in each of the vial

covered with the stopper and stirred at 200 rpm on a thermostatic magnetic stirrer for

72 hours at 25°C. The resultant solutions were centrifuged at 6000 rpm for 10

minutes. The cleared portions were separated in a 20 ml glass tubes. The cleared

portions of each of oils, surfactant and co-surfactant containing aceclofenac were

suitably diluted in their respective solvents and filtered through 0.45µ filter paper.

The UV absorption of aceclofenac in respective filtrates was determined at ƛmax 276

nm using respective solvent oil filtrates as blank.

3.2.2 Calibration curve for aceclofenac in methanol, ethanol, isopropyl alcohol

and n-butanol

The different oils such as almond, oleic acid, castor, cinnamon, canola, clove,

paraffin, isopropyl myristate, sesame, sunflower, eucalyptus oil, corn and coconut

were investigated for their solubility in methanol, ethanol, isopropyl alcohol and n-

butanol to construct the calibration curves in respective solvent for oil solubility

because aceclofenac is freely soluble in these solvents. The stock solutions of

aceclofenac were prepared in these solvents. Serial dilutions comprising of 0.312,

0.625, 1.25, 2.5, 5, 10 and 20 µg/ml were made from the respective stock solutions.

3.2.3 Solubility of aceclofenac in various oils

Shafiq et al. (2007) determined the solubility of aceclofenac in distilled water and

nanoemulsion by UV spectrophotometer at the wavelength of 276 nm. Excess amount

of aceclofenac in all sample matrices were added in 20 ml stoppered glass vials in

triplicate. These stoppered glass vials were kept in a mechanical shaker water bath

59

(Memmert, Germany) at the temperature of 25 ± 1°C for 72 h to reach equilibrium.

After 72 h, solutions were filtered through 0.45µ filter and diluted suitably with

respective solvent and subjected for quantification of aceclofenac by UV

spectrophotometric method at the wavelength of 276 nm.

3.2.4 In vitro permeability studies of aceclofenac in different oils

After solubility studies of aceclofenac, it was further subjected to permeability studies

in different oils without the use of solubility enhancer/surfactant. 2 mg/ml of

aceclofenac in each of oil was applied to the 0.45µ cellulose acetate filter paper in the

donor compartment of Franz diffusion cell containing Phosphate buffer solution of pH

7.4 at 32 ± 1 °C. The donor compartment was covered with aluminum foil with soft

white paraffin to prevent drying of the oil. Samples (300 µl) were withdrawn at

regular intervals (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h) and replaced with fresh

phosphate buffer pH 7.4. The drug content was determined by UV

spectrophotometer method at the wavelength of 276 nm (Shafiq et al., 2007).

3.2.5 Pseudo-ternary phase study to construct phase diagram for

microemulsion region:

On the basis of solubility studies oils i.e. almond oil and oleic acid, surfactants i.e.

Tween 80 and Tween 20 and co-surfactants ethanol and isopropyl alcohol were

selected for construction of phase diagram to select optimum formulation of

microemulsion.

3.2.5.1 Water Titration Method

To define large existence area of microemulsion without drug, pseudo-ternary phase

diagrams were constructed by Shafiq et al. (2007a) to obtain the components and their

concentration ranges. Water titration method is mostly used to construct phase

diagrams for microemulsions in which water is added drop wise to the mixture of oil,

surfactant and co-surfactant (Junyaprasert et al., 2006; Yong et al., 2005; Correa et

al., 2005 & 2007; Sheu et al., 2004; Murthy et al., 2006; Trotta et al., 2003).

For selection of microemulsion formulations from phase diagrams in the least

possible time, pseudoternary phase diagram was constructed to select best formulation

(Shafiq et al., 2007a).

At the ratio of surfactant to co-surfactant of 2:1, oily mixtures of oil, surfactant and

co-surfactant were prepared. This ratio of surfactant and co-surfactant mixture (Smix)

60

was selected because it has been successfully used to prepare microemulsions of

similar NSAID with similar surfactants and co-surfactants (Chen et al., 2004 & 2006).

Almond oil and oleic acid were optimized as an oil phase on the basis of solubility

study. For each phase diagram, oil and surfactant + co-surfactant mixture (Smix) were

combined in different weight ratios ranging from 1:9 to 9:1 in separate 20-ml

stoppered glass vials. Sixteen different combinations of oil and Smix (1:9, 1:8, 1:7, 1:6,

1:5, 1:4, 1:3.5, 1:3, 1:2.3, 1:2, 1:1.5, 1:1, 1:0.7, 1:0.43, 1:0.25, and 1:0.11) were made

so that maximum ratios were covered for the study to delineate boundaries of phases

precisely formed in phase diagrams.

The oily phase (1.0 g) containing oil and surfactant + co-surfactant mixture (Smix) was

constantly stirred slowly avoiding bubble formation with a small Teflon-coated

magnetic bar; while micropipette was used to add the aqueous phase. The aqueous

phase was filtered through 0.45 μm membrane filter. The varying amount of water

was added to produce a water concentration in the range of 5% to 95% of total weight

at around 5% increments.

3.2.5.2 Construction of Pseudoternary phase Diagrams

5% aqueous phase was added to the oil- Smix mixture. After each addition it was

allowed to mix and equilibrate. In case of any bubble formation, the mixture was

degassed by sonication. It was then assessed visually and observations were recorded

which are given in table 3.1. Through visual observation, the following classes were

categorized:

1. Transparent, single-phase and easily flow able oil/water microemulsions

2. Clear and highly viscous mixtures that did not show a change in the meniscus after

tilted to an angle of 90o, called microemulsion gel.

3. Milky or cloudy mixture i.e. emulsion (macroemulsion)

4. Milky gel i.e. emulgel

The physical state marked in table 3.1 was plotted on a pseudoternary phase diagram

with one axis representing the oil phase (O), the second representing the aqueous

phase (W), and the third representing the mixture of surfactant and co-surfactant

(Smix) at a fixed weight ratio i.e., 2:1. The phase diagrams were constructed by ProSim

Ternary Diagram software. For each surfactant and co-surfactant mixture (Smix), a

separate phase diagram was developed and for each phase diagram, table 3.1 was used

61

to record the visual observations. In the phase diagrams, only microemulsion points

were plotted.

The pseudoternary phase diagrams were constructed and from these constructed

pseudoternary phase diagrams; microemulsion formulations were selected and

prepared according to the composition given in table 3.2. The formulations

MET20IPA, MET20ETH and MET80ETH were excluded from the study due to

stability problems and formulation MET80IPA was selected for further studies and it

was used in the formulation of microemulsion based gels of various gelling agents.

62

ME: Oil/water Microemulsion: Transparent, single-phase and easily flowable.

E: Emulsion (macroemulsion): Milky or cloudy mixture

Table 3.1: Visual observations to record addition of water

Oil = Almond oil Surfactant = Tween80

Co-Surfactant = IPA Smix Ratio = 2:1

Sr. No.

Observations made After Each Addition of Aqueous Phase

Sum after each

addition 0.025 0.05 0.075 0.1 0.15 0.2 0.25 0.4 0.6 0.8 1.1 1.45 1.95 2.6 3.6 5.1 8.6 18.6

Ratio

Oil: Smix

Water

added 0.025

g

0.025

g

0.025

g

0.05

g

0.0

5 g

0.05

g

0.1

5 g

0.20

g

0.2

0 g

0.30

g

0.35

g

0.50

g

0.65

g

1.00

g

1.50

g

3.50

g

10.0

g

1 1:9 E E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME




5 1:5 E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME

6 1:4 (2:8) E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME

7 1:3.5 E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME

8 1:3 E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME

9 1:2.33 (3:7) E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME

10 1:2 E E E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME

11 1:1.5 (4:6) E E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME

12 1:1 (5:5) E E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME

13 1:0.67 (6:4) E E E E ME ME ME ME ME E E E E E E E E E

14 1:0.43 (7:3) E E ME ME ME ME ME E E E E E E E E E E E

15 1:0.25 (8:2) E E E E E E E E E E E E E E E E E E

16 1: 0.11 (9:1) E E E E E E E E E E E E E E E E E E

63

3.3 SELECTION OF MICROEMULSION FORMULATIONS FOR

DETAILED STUDIES TO INVESTIGATE EFFECTS OF

SURFACTANTS AND CO-SURFACTANTS ON SKIN PERMEATION

For further studies, from the constructed pseudoternary phase diagram,

microemulsion formulations were selected and prepared according to the composition

given in table 3.2. Amounts of drug (2% w/w), oil (10% w/w), Smix i.e., surfactant +

co-surfactant 2:1 (50% w/w) penetration enhancer (2%) and water (36% w/w) used in

various formulations were the same. The variables selected were the type of surfactant

and co-surfactant.

Table 3.2: Smix compositions of selected microemulsion formulations

Microemulsion Code

Smix (2:1)b

Surfactant Co-surfactant

MET20IPA Tween 20 Isopropyl Alcohol

MET80IPA Tween 80 Isopropyl Alcohol

MET20ETH Tween 20 Ethanol

MET80ETH Tween 80 Ethanol

b = Smix is surfactant + co-surfactant mixture (2:1)

3.4 PREPARATION OF ACECLOFENAC-LOADED MICROEMULSIONS

A known amount of aceclofenac was dissolved in almond oil. This almond oil

containing aceclofenac was mixed vigorously under magnetic stirring with Smix

(surfactant + co-surfactant, 2:1). Now a suitable amount of filtered deionized water

was added with slowly under constant stirring (1200 rpm) at ambient temperature.

The prepared microemulsions containing aceclofenac were stored at ambient

temperature (Chen et al., 2006).

The microemulsion formulations were compared with hydroalcoholic solution of drug

for transdermal drug delivery potential. The hydroalcoholic solution of aceclofenac

64

was prepared by dissolving known amount of aceclofenac in hydroalcoholic solution

and filtering the solution through 0.45µ membrane filter.

3.5 PREPARATION OF ACECLOFENAC MICROEMULSION USING

DIFFERENT OIL PHASES

3.5.1 Blank Microemulsion preparations containing oleic acid and almond oil

Surfactant mixture was prepared by manual mixing tween 20 or tween 80 and ethanol

or isopropyl alcohol surfactant and co-surfactant, respectively in 2:1 ratio. 5g of

surfactant mixture was added to 1g of oleic acid (oil) or almond oil and was mixed

vigorously under magnetic stirring. Then 3.8 g filtered de-ionized water was added

gradually under constant stirring (1200 rpm) at ambient temperature to form 10 g

microemulsion. 0.2 g of dimethylsulfoxide was added to microemulsion as a skin

penetration enhancer.

A) Tween 20 and ethanol (2:1)

I) Microemulsion containing oleic acid

Surfactant mixture was prepared by manual mixing tween 20 and ethanol surfactant

and co-surfactant, respectively in 2:1 ratio. 5g of surfactant mixture was added to 1g

of oleic acid (oil) and was mixed vigorously under magnetic stirring. About 0.2 g of

aceclofenac was then mixed to oil-surfactant mixture until completely dissolved. Then

3.6 g filtered de-ionized water was added gradually under constant stirring (1200 rpm)

at ambient temperature to form 10 g microemulsion. 0.2 g of dimethylsulfoxide was

added to microemulsion as a skin penetration enhancer.

II) Microemulsion containing almond oil

Surfactant mixture was prepared by manual mixing tween 20 and ethanol surfactant

and co-surfactant respectively in 2:1 ratio. 5 g of surfactant mixture was added to 1g

of almond oil and was mixed vigorously under magnetic stirring. About 0.2g of

aceclofenac was then mixed to oil-surfactant mixture until completely dissolved. Then

3.6 g filtered de-ionized water was added gradually under constant stirring (1200 rpm)

at ambient temperature to form 10 g microemulsion. 0.2 g of dimethylsulfoxide was

added to microemulsion as a skin penetration enhancer.

65

B) Tween 20 and isopropyl alcohol (2:1)

Microemulsions containing oleic acid and almond oil were prepared by the same

method as A.

C) Tween 80 and ethanol (2:1)


method as A.

D) Tween 80 and isopropyl alcohol (2:1)


method as A.

E) Preparation of hydroalcoholic solution

1) 0.4 g aceclofenac was dissolved in 14.6 g ethanol and mixed well until completely

dissolved.

2) 5 g deionized water was added to alcoholic drug solution to form 20 g hydro-

alcoholic solution under gentle mixing.

3.6 PREPARATION OF GEL BASES AND ACECLOFENAC

MICROEMULSION BASED GELS

3.6.1 Preparation of carbopol 934 and carbopol 940 gel bases

1) Carbopol 934 and carbopol 940 based gels were prepared by gradually dissolving

1 g each of carbopol 934 and 940 in 17 g of deionized water separately at room

temperature under continuous stirring at about 600 rpm for 2 hours.

2) Triethylamine was added until gel formed and pH was adjusted in the range 4-7.

Different compositions of carbopol 934 and 940 were used to get best gel base.

3.6.2 Preparation of xanthan gum gel bases

1) Xanthan gum based gel was prepared by first dissolving 1 g of xanthan gum in 17

g of deionized water at room temperature under continuous stirring at 600 rpm.

2) The methyl and propyl parabens were added separately in small amount of de-

ionized water at room temperature under continuous stirring at 300 rpm for a time

until methyl and propyl paraben were dissolved.

3) This solution was then gradually dissolved in xanthan gum base. Different

compositions of xanthan gum were prepared to get the best base.

66

3.6.3 Preparation of carbopol 934, carbopol 940 and xanthan gum based gels

containing microemulsion without active drug

82 g microemulsion without aceclofenac was thoroughly mixed with 18 g carbopol

934 or carbopol 940 or xanthan gum gel bases at 2500, 1000 and 500 rpm each

formulation for 5, 10 and 15minutes, respectively at ambient temperature to prepare

100 g of polymer based gel containing microemulsions without drug.

3.6.4 Preparation of carbopol 934, carbopol 940 and xanthan gum based gel

containing microemulsion with active drug

82 g microemulsion of aceclofenac was thoroughly mixed with 18 g carbopol 934 or

carbopol 940 or xanthan gum gel bases at 2500, 1000 and 500 rpm each formulation

for 5, 10 and 15 minutes, respectively at ambient temperature to prepare 100 g of

polymer based gel containing aceclofenac microemulsions.

3.6.5 Preparation of carbopol 934, carbopol 940 and xanthan gum based gels

containing hydroalcoholic solution

82 g hydro-alcoholic solution of aceclofenac was thoroughly mixed with 18 g

carbopol 934, carbopol 940 or xanthan gum gel bases at 2500, 1000 and 500 rpm for

5, 10 and 15 minutes, respectively at ambient temperature to prepare 100 g of polymer

based gel containing hydroalcoholic solution.

67

Figure 3.1: Flow diagram of preparation of microemulsion based gel

Surfactant

+

Co-surfactant

Oil Drug

Gel Base

Water

Gelling

Agent

Water

Microemulsion

Microemulsion

based Gel

Penetration

enhancer

Triethylamine

68

3.7 CHARACTERIZATION OF MICROEMULSIONS & ACECLOFENAC

MICROEMULSION BASED GEL

3.7.1 Viscosity

Brookfield RVDV III ultra, Programmable Rheometer with spindle CP41 (Brookfield

Engineering Laboratories, Middleboro, MA) was used to determine viscosities of

different formulations at 25ºC in triplicate.

3.7.2 Spreadability

Spreadability of all formulations was measured in terms of diameter. In this method,

weighed quantity of formulations i.e. 500 mg was placed on one glass slide (10g) and

another glass slide (10g) was placed over the first slide. As a result of compression

between two slides, a circle of the formulation is produced and its diameter was

measured (Desai, 2004).

Kalra et al. (2010) determined the spreadability of the gel using the following

technique adopted by Joshi et al. (2006). 0.5 g gel was placed within a circle of 1 cm

diameter premarked on a glass plate over which a second glass plate was placed. A

weight of 500 g was allowed to rest on the upper glass plate. The increase in diameter

due to spreading of gels was noted.

3.7.3 Conductivity Measurements

Conductometer WTW cond 197i (Weilhein, Germany) was used to determine the

conductivities (σ) of blank microemulsion, microemulsion containing aceclofenac,

blank microemulsion based gel and microemulsion based gel containing aceclofenac

at 25ºC. Experiments were repeated in triplicate.

3.7.4 pH Measurements

pH meter (WTW inolab, Germany) was used to measure the pH values of Blank

microemulsion, microemulsion containing aceclofenac, blank microemulsion based

gel and microemulsion based gel containing aceclofenac at 25ºC.

3.7.5 Refractive Index measurements

Abbe refractometer was used to measure the refractive indices of Blank

microemulsion, blank microemulsion based gel, microemulsion containing

aceclofenac and aceclofenac microemulsion based gel at 25ºC.

69

3.7.6 % Transmittance measurements

Shimadzu double beam spectrometer was used to determine the transparency of

formulations. The transmittance was measured for blank microemulsion,

microemulsion containing drug, blank microemulsion based gel, microemulsion based

gel containing drug and marketed conventional gel at 25ºC.

3.7.7 Centrifugation (Phase separation test)

The formulations were subjected to centrifugation test to determine the phase

separation. 5 g of the formulations were placed in centrifuge tubes and these tubes

were placed in centrifuge machine which was run at 3000 rpm for 30 minutes. After

30 minutes, tubes were inspected for the phase separation of formulations at 25ºC

(Jadhav et al., 2010).

3.7.8 Drug content

The assay of aceclofenac was done by HPLC method as described under methods for

in vivo determination.

3.7.9 Polydispersity Index (PDI) and Homogeneity

All developed gels were tested for homogeneity by visual inspection after the gels

have been set in the container at 25ºC. They were tested for their appearance and

presence of any aggregates (Kashyap et al., 2010).

To determine Polydispersity index of all formulations, Zetasizer Nano series ZEN

3600 Malvern Software DTS (Nano) United Kingdom was used. Measurements were

made in triplicate at 25ºC and mean was reported.

3.7.10 Scanning Electron Microscope (SEM)

Hitachi S4000N was used for imaging at variable pressure without coating under

vacuum. Thin films of liquids samples were prepared and dried at 105 ºC for imaging.

3.7.11 Fourier Transform Infra Red (FTIR)

IR spectrums of all formulations were obtained by FTIR (Bruker, Tensor 27,

Germany) at 25ºC.



70

3.7.12 X-Ray Diffraction (XRD)

Crystallinity of aceclofenac, for pure active drug, blank microemulsion,

microemulsion containing drug, blank microemulsion based gel microemulsion based

gel containing active drug were evaluated by X-ray diffractometer (Bruker D8

Discover, Germany) using Ni-filtered CuK alpha radiation source. The tube voltage of

35KV, current of 35 mA and scanning rate of 5° min-1

, over a range of 8°-60°

diffraction angle (2θ) range.


Calorimetry (DSC)

An amount (4-5 mg) of crushed active drug, 2-4 ml of blank microemulsion,

microemulsion, 4-5 mg blank microemulsion based gel and microemulsion based gel

were placed in aluminum pans and sealed prior to test. Measurements were performed

at a rate 10°C per minute,

under nitrogen flow of 25 ml per minute,

over a temperature

range of 0°C to 500

°C. Indium was used for the equipment calibration.

3.7.14 Globule charge (Zeta Potential) and globule size distribution (Zeta Size)

To determine charge and size distribution of active and all formulations, Zetasizer

Nano series ZEN 3600 Malvern Software DTS (Nano) United Kingdom was used.

Measurements were made at 25ºC.

3.7.15 In vitro skin permeation release rate experiments of aceclofenac from

microemulsions and microemulsion based gel

The rabbit skin was used instead of human skin because of difficulty in availability of

later. Hydrophobic drugs like aceclofenac was used for permeation release rate study

on rabbit skin by using Franz diffusion cell (Maghraby et al., 2008; Hu et al., 2006;

Ogiso et al., 2001). The Department of Pharmacy, the Islamia University of

Bahawalpur, Pakistan provided the male white rabbits from its animal house.

3.7.15.1 Skin Preparation

Long hairs were removed by the use of scissors and comb from dorsal region. Then

electric hair clipper was used to shave the short hairs carefully to avoid any scratch to

skin. Then the hair removing cream was applied carefully to the very short hairs and

then after 10 minutes, hairs were removed and cleaned with soaked tissue paper. In

order to achieve normal functions of the skin, the hair removal process was done one



71

day before the start of permeations release rate study. Demarcate concerned area of

skin which had to remove from rabbit skin (Shah et al., 2006).

Dissection box was used to sacrifice the rabbit and to take the demarcated hairless

skin. Heat separation was used to remove epidermis and scalpel (blade) was used to

remove subcutaneous fat. Careful separation of epidermis from the dermis was done

after soaking of removed hairless skin at 60ºC in water for one minute (Pellet et al.,

1997; Chevalier et al., 2008). Micrometer gauge was used to determine the thickness

of epidermis (Mitchell et al., 2004). The epidermis was preserved at -50ºC after

soaking in distilled water and covering with aluminium foil (Chi et al., 2001;

Ozguney et al., 2006).

3.7.15.2 Skin Barrier Integrity Checking

Transepidermal water loss study (physical method) was used to check the skin barrier

activity (OECD 2004a & b). Visual inspection was done to determine the integrity of

skin qualitatively by Mitchell et al. (2004). Before the removal and after storage of

skin, trans-epidermal water loss study was done by TeewameterTM

(Courage and

Khazaka, Germany). The normal value of trans-epidermal water loss is 4.5 g/m2/h

(Maibach et al., 2000). The pieces having trans-epidermal water loss less than 15g/

m2/h were used by Sintov et al. (2006).

3.7.15.3 Franz Diffusion Cell

Diffusion cells of vertical Franz type with diffusional surface area of 1.767cm2 were

used.

The receptor compartment contained the phosphate buffer solution with capacity of

12 ml at pH 7.4. Phosphate buffer pH 7.4 was used for the study of NSAIDs like

flurbiprofen (Chi et al., 2001; Fang et al., 2003; Ozguney et al., 2006; Seki et al.,

2004).

Phosphate buffer solution of pH 7.4 was used to soak the skin by using Franz

diffusion cell and equilibrate the skin at 4ºC for 12 hour (Ogiso et al., 2001).

Phosphate buffer solution of pH 7.4 was used to fill the receptor medium and skin was

placed between the donor and receptor compartments of the cell. The stratum

corneum side faced towards the donor compartment. Carefully, skin was fixed

between donor and receptor compartment and adjusted by clamp (Roessler et al.,

2001). Horizontal tilting of Franz diffusion cell was done to remove any formed

72

bubbles from the port of sample. Water bath and a peristaltic pump were used to

maintain the temperature of receptor solution at 32ºC. Teflon-coated magnet bar was

used to stir the solution at 500 rpm. The concentration of the test formulation was 1g

containing about 20 mg aceclofenac and was applied to the skin in donor

compartment and aluminum foil was used to cover the donor compartment.

Sampling was done by using long needle syringe after 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,

5, 5.5, 6, 7, 8, 10, 12, 14, 18, and 24 hour by taking 1 ml of the sample and was

diluted up to 10 ml with phosphate buffer solution of pH 7.4 and absorbance was

measured by UV-visible spectrophotometer at wavelength of 276 nm taking

phosphate buffer as a blank. The removed amount of solution was refilled with same

amount of phosphate buffer pH 7.4. Reading was repeated in triplicate (Shakeel et al.,

2009).

3.7.16 Assay of aceclofenac for permeation experiments

3.7.16.1 Standard Preparation

Equivalent to 100 mg of Standard aceclofenac was weighed in 100 ml volumetric

flask and the volume was made up to 100 ml with phosphate buffer pH 7.4. 1 ml was

taken from this solution into another 100 ml volumetric flask and volume was made

up to 100 ml with phosphate buffer pH 7.4. It was filtered and absorbance of the

filtrate was measured at 276 nm using filtered phosphate buffer as a blank.

3.7.16.2 Sample preparation

1.7 ml of sample was taken and diluted up to 100 ml with phosphate buffer solution of

pH 7.4, filtered and absorbance of filtrate was measured by UV-visible

spectrophotometer at wavelength of 276 nm taking filtered phosphate buffer pH 7.4 as

a blank. The removed amount of solution was refilled with same amount of phosphate

buffer pH 7.4. Readings was repeated in triplicate (Shakeel et al., 2009).

3.7.17 In vitro data calculation

3.7.17.1 Cumulative Amount of Drug Permeated per unit area (Qn)

In the in vitro study the samples were taken from sample port after specified intervals

and were replaced with fresh phosphate buffer pH 7.4. So the solution of receptor

compartment was constantly diluted. The equation of Hayton and Chen (1982) was

73

used for correction of sample removal from receptor compartment for concentration

of aceclofenac (Singh et al., 2001).

C’n = Cn (Vt/Vt-Vs) (C’n-1/Cn-1)----------------------------------------(1)

Where,

C’n….. Concentration of drug corrected in the nth sample

Cn…… Concentration of drug measured in the nth sample

C’n-1... Concentration of drug corrected in the (n-1) the sample

Cn-1… Concentration of drug measured in the (n-1) the sample

Vt…… The total volume of receptor compartment solution

Vs….... Sample volume

Data was expressed as the cumulative drug permeation per unit of skin surface area:

Qn = C’n/S--------------------------------------------------------------------(2)

Where, S = 1.767 cm2

3.7.17.2 Steady State Flux (Jss)

Cumulative amount of drug permeated per unit area (Qn) was plotted as function of

time in the receptor compartment and its unit is µg/cm2. Steady State Flux (Jss) was

determined from the linear plot by the slope of curve and its unit is µg/h/cm2

(Kreilgaard et al., 2000; Ozguney et al., 2006).

3.7.17.3 Permeability Coefficient (Kp)

Permeability coefficient (Kp) values were determined by the following equation and

its unit is cm/h:

Kp = Jss/Cd-------------------------------------------------------------------(3)

Where,

Cd = Concentration of drug in the donor compartment

It was considered that drug concentration was negligible in the receptor compartment

under sink conditions as compared with the donor compartment where it was 5.0%

w/w or 5.0 x 104 µg/ml (Sintov et al., 2006).

3.8 STABILITY STUDIES

Physical stability of microemulsion and polymer based gel formulations were

determined by centrifugation at 10000 rpm for 15 minutes. These tests were used to

determine stability of selected formulations (Chen et al., 2006).

74

The stability parameters were studied by keeping microemulsion and microemulsion

based gel formulations at 40ºC ± 5°C/75% ± 5% RH for a period of 6 months and

long term stability studies were performed at ambient temperature (room temperature)

i.e. 25°C for a period of one year. Following parameters were studied for the stability

of aceclofenac microemulsion, microemulsion based aceclofenac gel and marketed

conventional gel (alkeries gel):

Phase separation

Visual clarity

Drug assay

pH

Viscosity

3.9 IN VIVO TRANSDERMAL STUDIES IN RABBITS

Transdermal studies of aceclofenac microemulsion based gel selected on the basis of

in vitro permeability studies were performed on 24 rabbits which were divided into

two groups each consisting of 12 rabbits for each formulation i.e. conventional

marketed and test gel. The hair from dorsal region of the rabbits were shaved off with

hair clipper and further it was freed from small hair by the application of hair

removing cream one day before the application of test and marketed conventional gel.

0.5 g of microemulsion and gel containing 10 mg aceclofenac were applied on the

dorsal skin in separate groups. Blood samples of 0.5 ml from jugular vein of rabbits

were withdrawn at 0, 1, 2, 3, 6, 12, 24 hours in heparinized centrifuge test tubes.

Plasma was separated at 6000 rpm for 10minutes and precipitated with methanol by

vortexing. The HPLC method was used to analyze this sample which is described

under methods for in vivo determination.

3.10 ANTI-INFLAMMATORY ACTIVITY STUDY IN RATS

Albino male rats were used to study the anti-inflammatory effect of aceclofenac

microemulsion; aceclofenac microemulsion based gel and marketed gel. First the

inflammation was produced in the right hind paw by injecting 0.05 ml of 10%

formalin. The volume of right paw was noted by micrometer over a period till there

was no further increase in volume due to inflammation. 0.1g of microemulsion based

gel was applied on the right paw and reduction in swollen paw was noted. The anti-

75

inflammatory activity was noted as percentage inhibition which was measured by the

following formula (Arun, 2009):

Percentage inhibition = (1-Vt/Vc) X 100-------------------------------------------(4)

Where,

Vt and Vc=Volume of hind paw after application of test formulation and control.

3.11 STUDY OF ANALGESIC EFFECT IN RATS

Mice of either sex were used to determine analgesic effect of aceclofenac

microemulsion; aceclofenac microemulsion based gel and marketed gel. 0.6% acetic

acid was injected intra-peritoneal to produce pain which was observed by a

characteristic stretching behavior called writhing. The mice are placed individually

into glass beaker and observed for a period of 30 minutes. The number of writhes was

recorded for each animal. A writhe is indicated by a contraction of abdomen with

simultaneous stretching of at least one hind limb (Omeh Yusuf and Ezeja Maxwell,

2010).

3.12 SKIN IRRITATION STUDY

MexameterTM

(Courage and Khazaka, Germany) was used to measure erythema and

edema level before and after the application of gel. Before application of

formulations, the measured value of skin was used as control value. 1g of each of

aceclofenac microemulsion, aceclofenac microemulsion based gel and aceclofenac

marketed gel was applied to the marked inner forearms and spread uniformly with the

help of applicator and a gauze dressing (1x1cm2) was wrapped on the inner forearms.

Stretch adhesive tape was used to fix the formulations at Inner forearms. After 48

hours, visual observation of forearms was done for any skin irritation or skin lesion by

an expert dermatologist using MexameterTM

. The visual scoring of irritation and

edema was quantified and evaluated through an arbitrary numeric scale as follows:

No erythema = 0, Very light erythema = 1, Light erythema = 2, Moderate erythema =

3 and severe erythema/dark pink (crossing the marked circle) = 4; No edema = 0, very

light edema = 1, Light edema = 2, Moderate edema = 3 and Strong edema (crossing

the marked circle) = 4. Average irritation index was classified by modified Draize

system i.e. non irritating = 0.5-2.0, slightly irritating = 2.1-5.0, moderately irritating =

5.1-8.0 (Gulten et al., 2007).

76

3.13 HPLC METHOD DEVELOPMENT AND VALIDATION

FDA/ICH guidelines were followed for bio-analytical method validation. Following

parameters were validated for HPLC bio-analytical method namely accuracy,

precision (Intra day and inter day), specificity/selectivity, detection limit, quantitation

limit, linearity and range.

3.13.1 Accuracy and precision

According to FDA and ICH guidelines, accuracy and precision were determined by

injecting 3 concentrations and 3 replicates of each concentration into chromatograph

and the response was recorded. Further 6 different samples of aceclofenac were

analyzed on HPLC to confirm the accuracy and precision of the method.

3.13.2 Specificity/Selectivity

To determine the specificity/selectivity of the method, solutions of the blank

formulations and blank plasma were injected and response was recorded for any

interference with analyte to rectify the interference.

3.13.3 Detection limit and Quantitation limit

Detection limit of the method was determined by sufficiently diluting the spiked

plasma sample and these diluted samples were injected into HPLC to check any

response on the detector and concentration at which the detector showed deflection

was the detection limit of method. Similarly, the concentration of plasma sample

spiked with aceclofenac which was determined with precision and accuracy, denoted

as quantitation limit.

3.13.4 Linearity and range

The linearity of method was determined by spiking known amount of analyte i.e.

aceclofenac covering range of analyte in the target sample matrix.

3.13.5 Freeze thaw stability of aceclofenac in plasma

Known concentrations i.e. LQC and HQC of aceclofenac were spiked in plasma,

extracted and analyzed by newly developed HPLC method.

3.13.6 Extraction yield/recovery of aceclofenac

The % extraction yield/recovery of aceclofenac from plasma was determined by

spiking known concentrations (LQC & HQC) of aceclofenac in plasma and were

determined by the developed HPLC method.

77

3.14 APPROVAL OF THE STUDY

The study on human and rabbits was approved by the advanced studies and research

board (AS & RB), the Islamia University of Bahawalpur, Pakistan and the

Institutional Ethics Committee, Faculty of Pharmacy and Alternative Medicine, the

Islamia University of Bahawalpur, Pakistan.

3.15 METHODS FOR IN VIVO DETERMINATION

Experimental work was performed at the Department of Pharmacy, the Islamia

University of Bahawalpur. Eighteen Healthy Human subjects were participated in the

study. These volunteers were divided into two groups. Group first was named as G1,

and the group two was named as G2. Group one (G1) received the standard drug

(Marketed gel) and the second group (G2) received the formulation of aceclofenac

microemulsion based gel. This single dose drug regimen was applied at 08:00 AM.

After one week of washed out period the subjects were given the second dosage;

group first (G1) received formulation of aceclofenac microemulsion based gel and

second group (G2) received standard drug (Marketed gel).

3.15.1 Inclusion criteria

The selection of volunteers was carried out carefully. Subjects participated in study

were of:

Age from 18 – 40 years

Body weight in the range of 50 -70 kg

Considering in good health based on medical history, physical examination,

routine serum and urine chemistries.

3.15.2 Exclusion Criteria

Subject was excluded if:

Abnormal findings upon medical histories, physical examinations and screening

tests.

Histories of kidney disease or an estimated creatinine clearance was less than 50

ml/minutes, liver or cardiovascular diseases or a hematocrit of <36% at screening.

Any condition known to interfere with absorption of drugs was present.

Known positive human immunodeficiency virus (HIV) serology, AIDS.

History of hypersensitivity to aceclofenac or any member of NSAIDs.

78

Weight greater than 130% of ideal body weight, pregnant or nursing or had

donated blood within 30 days prior to study.

Have taken any other prescription or non-prescription drugs for one week before

study and during entire study period.

Serious mental and physical illness within a year before study.

Limited mental capacity to extent, the subject was unable to provide legal consent

and information regarding side effects or tolerance to study drug.

3.15.3 Administration of drugs

Before application of test and reference drug product, the specific area of volunteer

skin was washed with washing soap and dried. A specified amount of reference and

test product was applied to the specific area on the bodies of the 18 volunteers. The

area was covered with the help of tape to avoid loss of product during test period.

3.15.4 Sample Collection

A 20-gauge venous cannula was inserted into forearm for collection of blood

samples. A blood sample was collected before drug was given (zero time) and then

at 0.50, 1, 2, 3, 4, 5, 6, 8, 10, 12 and 24 hours after application of aceclofenac

microemulsion based gel. A 3 ml blood sample was collected each time in

heparinized syringe. Blood samples were centrifuged at 6000 rpm for 10 minutes and

plasma was collected. The plasma samples were then frozen at -50°C in the ultra-low

refrigerator until assay.

3.15.5 Preparation of mobile phase

The mobile phase consists of a mixture of 20 mM Potassium dihydrogen

Phosphate:Acetonitrile (60:40, v/v) adjusted to pH 7.0 by 2M KOH. The mobile

phase was filtered through a 0.45µm membrane filter, sonicated and degassed before

use.

3.15.6 Preparation of stock solutions and working standard solutions

Stock solutions of aceclofenac (100 mg/ml) were prepared monthly by dissolving

100 mg of drug in 100 ml methanol and storing at 8°C. Aceclofenac concentrations

in the working standard solutions chosen for calibration curve were 0.039, 0.078,

0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, and 20 µg/ml. These working solutions were

made by further dilution of the stock solution in methanol. They were prepared fresh

79

daily. A stock solution of the internal standard (100 mg/ml each) was prepared by

dissolving 10 mg of Diclofenac potassium in 100 ml methanol and was stored at 8°C.

3.15.7 Preparation of plasma standards and samples

Frozen human plasma samples were left on the bench to melt naturally and were

vortexed prior to use. 1 ml of plasma was taken and added 3 ml of Acetonitrile to

precipitate proteins and vortex for 1 minute. Then 0.5 ml of methanol was added and

vortex for 2 minutes again. It was centrifuged for 10 minutes at 6000 rpm. After

centrifugation, the layer was transferred to epindorf tube and evaporated to dryness

under nitrogen flux. It was then reconstituted with mobile phase. Quality control

samples were prepared by spiking drug free human plasma with different

concentrations of working standard solutions of aceclofenac while Diclofenac was

added at 0.1 mg/ml throughout.

3.15.8 Column

Hypersil ODS (C18) reversed phase column (250mm x 4.6mm I.D, 5µm).

3.15.9 Flow rate

Flow rate was 1 ml/min.

3.15.10 UV Detection Wavelength

λmax = 276 nm

3.16 PHARMACOKINETIC PARAMETERS

Maximum plasma concentration (Cmax), Time of peak plasma concentration (Tmax),

Area under curve (AUC0-∞), Area under the first moment curve (AUMC0-∞), Mean

residence time (MRT), Half Life (t1/2), Elimination rate constant (Ke), Volume of

distribution (Vd), Total body clearance (ClT) and absorption rate constant were

determined.

3.17 STATISTICAL ANALYSIS

Two-way analysis of variance (ANOVA) was used to measure skin permeation

release rate by statistical data. For study of skin irritation, statistical paired sample t-

test was used at the level of P=0.05. For in vivo studies student t was used for

comparison of Pharmacokinetic parameters. These tests were performed by SPSS

12.0 software.

80

4. RESULTS

4.1 CALIBRATION CURVE FOR ACECLOFENAC IN METHANOL,

ETHANOL, ISOPROPYL ALCOHOL AND N-BUTANOL

Aceclofenac is freely soluble in methanol, ethanol, isopropyl alcohol and n-butanol.

Therefore, to determine the solubility of aceclofenac in different oils, surfactants, co-

surfactants, solubility of oils as well as surfactants and co-surfactants was determined

in methanol, ethanol, isopropyl alcohol and n-butanol. For this purpose, standard

curves of aceclofenac in methanol, ethanol, isopropyl alcohol and n-butanol were

constructed separately in triplicate which are given in Tables 4.1 to 4.4 and Figures

4.1 to 4.4

Table 4.1 Standard curve of aceclofenac in methanol (n=3)

Aceclofenac concentration

(µg/ml) Absorbance

0.3125 16

0.625 37

1.25 40

2.5 106

5 203

10 399

20 765

Figure 4.1 Standard curve of aceclofenac in methanol

y = 38.177x + 7.2644

R² = 0.998

0

100

200

300

400

500

600

700

800

900

0 5 10 15 20 25

Abso

rban

ce

Aceclofenac concentration (µg/ml)

81

Table 4.2 Standard curve of aceclofenac in IPA (n=3)


(µg/ml) Absorbance

0.3125 13

0.625 26

1.25 66

2.5 103

5 194

10 411

20 876

Figure 4.2 Standard curve of aceclofenac in IPA

y = 43.415x - 4.8621

R² = 0.9981

0

100

200

300

400

500

600

700

800

900

1000

0 5 10 15 20 25

Abso

rban

ce


82

Table 4.3 Standard curve of aceclofenac in n-butanol (n=3)


(µg/ml) Absorbance

0.3125 25

0.625 55

1.25 122

2.5 240

5 467

10 944

20 1742

Figure 4.3 Standard curve of aceclofenac in n-butanol

y = 87.669x + 16.517

R² = 0.9982

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 5 10 15 20 25

Abso

rban

ce

Aceclofenac oncentration (µg/ml)

83

Table 4.4 Standard curve of aceclofenac in Ethanol (n=3)


Absorbance

0.3125 10

0.625 20

1.25 40

2.5 80

5 151

10 327

20 711

Figure 4.4 Standard curve of aceclofenac in ethanol

y = 35.418x - 9.523

R² = 0.9977

0

100

200

300

400

500

600

700

800

0 5 10 15 20 25

Abso

rban

ce


84

4.2 SOLUBILITY AND IN VITRO PERMEABILITY STUDIES OF

ACECLOFENAC IN DIFFERENT OILS

With the help of standard curves, solubility of aceclofenac was determined in

different excipients and results of solubility studies are given in Table 4.5, Table 4.6,

Figure 4.5 and Figure 4.7. Moreover, the permeation study was conducted on the oils,

the results of which are given in Table 4.5 and Figure 4.6.

Table 4.5 Solubility and permeability of aceclofenac in various oils (n=3)

Sr.

No. OIL

Solubility

mg/ml

Flux, Jss

(µg/cm2/h)

Permeability

Coefficient

Kp × 10-3

(cm/h)

1 Almond oil 9.16 1.45± 0.04 0.072 ± 0.006

2 Oleic acid 8.56 1.21 ± 0.06 0.061 ± 0.003

3 Castor oil 1.33 0.997 ± 0.08 0049 ± 0.002

4 Cinnamon oil 1.06 1.067 ± 0.04 0.053 ± 0.003

5 Clove oil 0.83 1.080 ± 0.02 0.054 ± 0.004

6 Canola oil 2.05 0.968 ± 0.06 0.048 ± 0.003

7 IPM 4.10 1.095 ± 0.01 0.055 ± 0.003

8 Sesame oil 4.35 1.029 ± 0.02 0.051 ± 0.004

9 Sunflower oil 1.10 1.080 ± 0.03 0.054 ± 0.006

10 Corn oil 0.30 1.048 ± 0.04 0.052 ± 0.005

11 Coconut oil 0.36 1.061 ± 0.05 0.053 ± 0.007

12 Paraffin oil 0.76 0.935 ± 0.08 0.0457± 0.004

13 Eucalyptus oil 1.83 0.955 ± 0.02 0.048 ± 0.006

14 Hydroalcoholic

solution 150.65 14.91± 0.05 0.746± 0.04

n= triplicate analysis for solubility and permeability

85

Figure 4.5 Solubility data of aceclofenac in various oils

Figure 4.6 Permeability data of aceclofenac in various oils

0

10

20

30

40

50

60

70

80

90

100

Solu

bil

ity m

g/m

l

Oils

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60

Hydroalcoholic soln

Almond oil

Oleic acid

IPM

Clove oil

Sunflower oil

Cinnamom oil

Coconut oil

Corn oil

Sesame oil

Caster oil

Canola oil

Euclyptus oil

Paraffin oil

Permeability Flux, Jss (µg/cm2/h)

Oils

86

Table 4.6 Solubility of aceclofenac in various vehicles (n=3)

Sr. No. Name

(Excipients/Vehicles)

Solubility

(mg/ml)

1 PG 12.18

2 Span 85 56.41

3 Tween 20 407.23

4 Tween 80 476.16

5 PEG 600 433.24

6 Water 0.02

Figure 4.7 Solubility data of aceclofenac in various vehicles

0

100

200

300

400

500

600

Tween 80 PEG 600 Tween 20 Span 85 PG Water

Solu

bil

ity m

g/m

l

Excipients/vehicles

87

4.3 CONSTRUCTION OF PSEUDO TERNARY PHASE DIAGRAMS

Water titration method was used to define large existence area of microemulsion

without drug at the ratio of surfactant to co-surfactant of 2:1. The large existence area

of microemulsion without drug is shown in Figure 4.8.

Figure 4.8: Pseudo ternary phase diagram of Almond oil, Tween 80-Isopropyl

alcohol (2:1) and water.

4.4 MICROEMULSION FORMULATIONS OF ACECLOFENAC

Formulae of various preparations are given below:

4.4.1 Tween 80 and isopropyl alcohol (2:1)

I) Blank microemulsion containing almond oil

Smix (g) Oil (g) Water (g) Dimethyl Sulfoxide (DMSO) (g)

50 10 38 2

II) Aceclofenac microemulsion containing almond oil

Smix (g) Oil (g)

Drug

(aceclofenac)

(g)

Water

(g)

Dimethyl Sulfoxide (DMSO)

(g)

50 10 2 36 2

88

4.4.2 Microemulsion based aceclofenac gel formulation

The carbopol 940 gel base was prepared with water in which almond microemulsion

containing aceclofenac was incorporated. The formulae of Carbopol 940 gel base,

microemulsion based blank gel and microemulsion based aceclofenac gel are given

below:

Carbopol 940 gel base

Carbopol 940 (g) Water (g)

1 18

I) Microemulsion based blank gel

Smix (g) Oil

(g)

Water

(g)

Dimethyl Sulfoxide

(DMSO)

(g)

Carbopol 940

gel base

50 10 19 2 19

II) Microemulsion based aceclofenac gel

Smix

(g)

Oil

(g)

Drug

(aceclofenac)

(g)

Water

(g)

Dimethyl

Sulfoxide

(DMSO)

(g)

Carbopol 940

gel base

50 10 2 17 2 19

4.5 CHARACTERIZATION OF MICROEMULSIONS AND

MICROEMULSION BASED GELS

4.5.1 Rheological studies of formulations

Rheological studies of each formulation were done in triplicate. The results are given

in Table 4.7

89

Table 4.7: Characteristics of different formulations (n=3)

Sr.

No. Formulations

Viscosity

(cP) Spreadability

(cm) Conductivity

(µS/cm) pH R I % T

Phase

separation %age Homogeneity

Polydispersity

Index

1

Blank

microemulsion

containing

Almond oil

15.08 5.5 30.4 4.97 1.411 98.0 No ----- Good 0.343

2

Almond oil

microemulsion

containing

aceclofenac

45.24 5.1 41.5 4.39 1.418 98.1 No 99.09 Good 0.599

3

Blank

microemulsion

based Gel

557.95

4.5 79.1 5.35 1.415 98.0 No ------ Good 0.197

4

Aceclofenac

microemulsion

based Gel

588.11

4.9 150.7 4.78 1.424 97.8 No 99.14 Good 0.786

5 Marketed Gel 611.12 3.2 1.8 4.57 1.445 6.5 No 99.11 Good 1.200

90


Scanning Electron Microscope is also used for imaging of the surface of

microspheres, microcapsules as well as micro globules by preparing thin film after

drying on glass slide. It shows the surface morphology. The SEM images of pure

aceclofenac, blank microemulsion and aceclofenac microemulsion, blank

microemulsion based gel and aceclofenac microemulsion based gel are given below in

Figure 4.9 to 4.13.

Figure 4.9: SEM image of aceclofenac pure drug

91

Figure 4.10: SEM image of blank microemulsion

Figure 4.11: SEM image of aceclofenac microemulsion

92

Figure 4.12: SEM image of blank microemulsion based gel

Figure 4.13: SEM image of aceclofenac microemulsion based gel

93

4.5.3 Fourier Transform Infra Red (FTIR)

Individual separate FTIR spectra of aceclofenac, almond oil, tween 80, isopropyl

alcohol, dimethyl sulfoxide, carbopol 940 and Triethylamine are given in appendix I

from Figure: A1 to A7, respectively. FTIR spectra of aceclofenac, almond oil, test

formulations and marketed gel formulation are given below in Figure: 4.14.

Figure 4.14: FTIR spectra of aceclofenac and all excipients used in

microemulsion and microemulsion based aceclofenac gel

formulations

Tra

nsm

itta

nce

[%

]

Wave number (cm-1

)

94


XRD technique is used to determine the crystal and amorphous nature of the

compound. The results of active compound and different formulations are given

below in Figure 4.15 to 4.19.

Figure 4.15: XRD of aceclofenac

Figure 4.16: XRD of Blank microemulsion

c

o

u

n

t

s

c

o

u

n

t

s

c

95

Figure 4.17: XRD of aceclofenac microemulsion

Figure 4.18: XRD of Blank microemulsion based gel

c

o

u

n

t

s

c

o

u

n

t

s

96

Figure 4.19: XRD of microemulsion based aceclofenac gel


Calorimetry (DSC)

Thermo grams of active drug and its various formulations are given below in Figure

4.20 to 4.24.

Figure 4.20: TGA and DSC of aceclofenac

c

o

u

n

t

s

97

Figure 4.21: TGA and DSC of blank microemulsion

Figure 4.22: TGA and DSC of aceclofenac microemulsion

98

Figure 4.23: TGA and DSC of Blank microemulsion based gel

Figure 4.24: TGA and DSC of microemulsion based gel of aceclofenac

99

4.5.6 Globule charge (Zeta Potential)

The charge on globule i.e. Zeta potential was measured for active drug, its

microemulsion, microemulsion based gel formulations and blank formulations. Zeta

potential of aceclofenac microemulsion and microemulsion based aceclofenac gel

were found to be -63.85 mV and -8.58 mV, respectively. Results are given in Figures

from 4.25 to 4.28.

Figure 4.25: Charge distribution of aceclofenac.

Figure 4.26: Charge distribution of blank microemulsion

0

50000

100000

150000

200000

250000

-200 -100 0 100 200

Tota

l C

ounts

Zeta potential (mV)

Zeta Distribution Data

0

5000

10000

15000

20000

25000

30000

35000

40000

-200 -100 0 100 200

Tota

l C

ounts

Zeta potential (mV)


100

Figure 4.27: Charge distribution of aceclofenac microemulsion

Figure 4.28: Charge distribution of microemulsion based aceclofenac gel

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

-200 -100 0 100 200

Tota

l C

ounts

Zeta potential (mV)


0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

-200 -100 0 100 200

Tota

l C

ounts

Zeta potential (mV)


101

4.5.7 Globule size (hydrodynamic size)

The globule size was determined for pure drug, blank formulations and formulations

containing active drug. The results are given in Tables 4.8 to 4.12 and Figures 4.29 to

4.33.

Table 4.8: Size distribution of aceclofenac pure drug

Size classes

(nm)

Number Distribution Data

(%)

122.42 18.23

141.77 43.23

164.18 31.77

190.14 6.77

Figure 4.29: Size distribution of aceclofenac pure drug.

0

5

10

15

20

25

30

35

40

45

50

1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04

% S

ize

dis

trib

uti

on

size (nm)

102

Table 4.9: Size distribution of blank microemulsion

Size classes

(nm)


(%)

2.70 5.87

3.12 19.32

3.62 26.82

4.19 22.44

4.85 13.92

5.61 7.00

6.50 2.98

7.53 1.10

8.72 0.36

10.10 0.11

11.70 0.04

13.54 0.02

15.69 0.01

18.17 0.01

21.04 0.0026

24.36 0.0011

28.21 0.0004

32.67 0.0001

Figure 4.30: Size distribution of blank microemulsion

0

5

10

15

20

25

30

1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04

% S

ize

dis

trib

uti

on

size (nm)

103

Table 4.10: Size distribution of aceclofenac microemulsion

Size classes

(nm)


(%)

4.85 3.25

5.61 14.07

6.50 25.39

7.53 25.82

8.72 17.50

10.10 8.93

11.70 3.59

13.54 1.14

15.69 0.27

18.17 0.04

21.04 0.0023

24.36 0.0000

28.21 0.0003

32.67 0.0011

37.84 0.0015

43.82 0.0013

50.75 0.0009

58.77 0.0005

Figure 4.31: Size distribution of aceclofenac microemulsion

0

5

10

15

20

25

30

1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04

% S

ize

dis

trib

uti

on

size (nm)

104

Table 4.11: Size distribution of blank microemulsion based gel

Size classes

(nm)


(%)

37.84 5.18

43.82 20.36

50.75 35.00

58.77 29.64

68.06 9.82

Figure 4.32: Size distribution of blank microemulsion based gel

0

5

10

15

20

25

30

35

40

1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04

% S

ize

dis

trib

uti

on

size (nm)

105

Table 4.12: Size distribution of microemulsion based aceclofenac gel

Size classes

(nm)


(%)

43.82 7.07

50.75 23.92

58.77 33.18

68.06 24.48

78.82 9.75

91.28 1.60

Figure 4.33: Size distribution of microemulsion based aceclofenac gel

0

5

10

15

20

25

30

35

1.0E-01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04

% S

ize

dis

trib

uti

on

size (nm)

106

4.5.8 In vitro skin permeation release rate experiments of aceclofenac from

microemulsion, microemulsion based gel and marketed gel

In vitro skin permeation release rate experiments of aceclofenac were performed

using rabbit’s dorsal skin in Franz diffusion cell. Results are given in Table 4.13

Figures 4.34 to 4.36.

Table 4.13: Permeability of aceclofenac from different formulations (n=3)

Sr.

No. Formulation

Flux, Jss

(µg/cm2/h)

Permeability

Coefficient

Kp × 10-3

(cm/h)

Enhancement

ratio

P-

Value Importance

1 Aceclofenac

microemulsion 1.73 ± 0.06 0.085 ± 0.008 3.53 0.007 Significant

2

Microemulsion

based

aceclofenac gel

1.52 ± 0.07 0.076 ± 0.005 3.10 0.005 Significant

3 Marketed gel 0.91 ± 0.03 0.055 ± 0.002 1.86 No

significant

4

Aceclofenac in

phosphate

buffer pH 7.4

without

penetration

enhancer

0.49± 0.04 0.023 ± 0.003 Control ------- --------------

107

Figure 4.34: Permeation study of aceclofenac from microemulsion

containing aceclofenac

Figure 4.35: Permeation study of aceclofenac from microemulsion based

aceclofenac gel

0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30

Per

mea

tio

n (µ

g/c

m2/h

)

Time (hour)

-10

0

10

20

30

40

50

0 5 10 15 20 25 30

Per

mea

tio

n (

µg

/cm

2/h

)

Time (hour)

108

Figure 4.36: Permeation study of aceclofenac from Marketed gel

4.5.9 Stability studies

The stability of aceclofenac microemulsion, microemulsion based aceclofenac gel and

marketed gel (Alkeries) was determined in accelerated storage condition for a period

of six months. The results are given in Table 4.14. Long term stability studies of

different formulations at room temperature were also carried out and formulations are

still under room temperature storage conditions. The results after one year of storage

of formulations at room temperature are given in Table 4.15.

Table 4.14: Accelerated stability studies of different formulations at 40ºC ± 5°C/75%

± 5% RH (n=3)

Sr.

No. Formulation

Phase

separation

Visual

clarity

Viscosity cp

pH

Drug

Assay

%

1 Aceclofenac

microemulsion No Clear 46.25 4.41 98.05

2

Microemulsion

based

aceclofenac

gel

No Clear 590.16 4.80 98.07

3 Marketed gel No Translucent 615.21 4.61 98.03

-5

0

5

10

15

20

25

30

0 5 10 15 20 25 30

Per

mea

tio

n (µ

g/c

m2/h

)

Time (hour)

109

Table 4.15: Long term stability studies of different formulations at room temperature

25ºC ± 5°C/65% ± 5% RH(n=3)

Sr.

No. Formulation

Phase

separation

Visual

clarity

Viscosity

cp pH

Drug

Assay

%

1 Aceclofenac

microemulsion No Clear 47.25 4.45 97.95

2

Microemulsion

based

aceclofenac

gel

No Clear 593.24 4.77 97.88

3 Marketed gel No Translucent 614.51 4.59 97.76

4.6 IN VIVO TRANSDERMAL STUDIES OF MICROEMULSION IN

RABBITS.

In vivo transdermal studies of aceclofenac microemulsion based gel (selected on the

basis of in vitro permeability studies) and marketed gel were performed on 24 rabbits

which were divided into two groups of 12 rabbits for each formulations. A dose of 2

mg aceclofenac was used for each formulation. Results are given in Tables 4.16,

4.18 and Figures 4.37 to 4.40. Pharmacokinetic parameters are given in Tables

4.17 and 4.19. However, plasma concentrations and respective graphs of

individual rabbits are given in appendix II.

110

Table 4.16: Concentration of aceclofenac in rabbit plasma calculated from chromatograms by forecasting method after

administration of microemulsion based aceclofenac gel in group AR

" CONCENTRATION IN PLASMA" calculated form chromatogram by forecasting aceclofenac microemulsion based gel in Rabbits (group AR)

TIM

E (H

ou

rs)

Rabbits 1 2 3 4 5 6 7 8 9 10 11 12 SUM MEAN SD S.E.M

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0.9407 0.9326 0.4493 0.7916 1.0348 0.51514 0.51085 0.46959 0.51644 0.81051 0.79339 0.678 8.44 0.70 0.21 0.05

2 2.2244 2.3597 1.1496 2.2077 2.6324 1.93534 1.79444 2.35592 1.97041 2.22648 2.64752 1.46 24.96 2.08 0.45 0.10

3 3.5728 3.6077 2.4754 3.5539 4.1348 3.34434 3.34434 3.49165 3.3778 3.33378 4.22738 2.605 41.07 3.42 0.51 0.11

6 6.6076 6.3747 6.2155 6.4565 6.8732 6.63224 6.09541 6.43654 6.71226 6.83633 6.97382 6.545 78.76 6.56 0.26 0.06

12 3.0767 2.374 2.4188 2.9022 2.3743 1.16392 2.32423 2.69755 2.16539 1.6659 1.88225 2.585 27.63 2.30 0.53 0.12

24 0.3165 0.6865 0.5026 0.6817 0.3243 0.32556 0.16353 0.66601 0.13296 0.42918 0.49072 0.268 4.99 0.42 0.19 0.04

SUM 16.74 16.34 13.2 16.6 17.37 13.92 14.23 16.12 14.88 15.3 17.02 14.14 185.85 15.49 2.15 0.47

Mean 2.391 2.334 1.89 2.37 2.482 1.988 2.033 2.302 2.125 2.186 2.431 2.02 26.55 2.21 0.31 0.07

SD 2.163 2.139 2.21 2.41 2.344 2.172 2.236 2.37 2.344 2.469 2.251 2.251 27.20 2.27 0.20 0.04

S.E.M 0.472 0.467 0.48 0.53 0.512 0.474 0.488 0.518 0.512 0.539 0.491 0.491 5.94 0.49 0.04 0.01

111

Figure 4.37: Plasma concentration verses time profile of aceclofenac plotted


of 2 mg (microemulsion based gel) in 12 Rabbits of group AR

individually



(microemulsion based gel) in 12 Rabbits of group AR

individually

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

con

c. (

µg/m

l)

Time (Hour)

AR1

AR2

AR3

AR4

AR5

AR6

AR7

AR8

AR9

AR10

AR11

AR12

0.1

1

10

0 5 10 15 20 25

Pla

sma

conc.

(µ

g/m

l)

Time (Hour)

AR1

AR2

AR3

AR4

AR5

AR6

AR7

AR8

AR9

AR10

AR11

AR12

112

Table 4.17: Pharmacokinetics parameters of aceclofenac in rabbits (Group AR) after application of microemulsion based gel

Rabbit

No.

PHARMACOKINETICS OF ACECLOFENAC AFTER APPLICATION OF MICROEMULSION BASED GEL (20mg/g) IN

RABBITS GROUP AR

Cmax

µg/mL

Tmax

h

t1/2

h

Lz (Ke) 1/h

MRT

h

AUC0-t

µg/mL*h

AUC0-inf

µg/mL*h

Clearance

mL/h

Vz

L

Vss

L

Ka

1/h

1 6.61 5.56 4.03 0.17 8.95 69.636 71.575 27.943 0.163 0.250 1.53

2 6.38 6.10 5.74 0.12 10.40 64.685 70.078 28.540 0.236 0.297 1.53

3 6.22 4.90 5.01 0.14 10.02 59.310 62.862 31.816 0.230 0.319 1.42

4 6.46 6.00 5.58 0.12 10.51 69.370 74.799 26.738 0.215 0.281 1.53

5 6.87 6.00 4.10 0.17 8.47 66.176 68.073 29.381 0.174 0.249 1.58

6 6.63 6.00 4.37 0.16 8.08 51.414 53.170 37.616 0.237 0.304 1.56

7 6.10 6.00 3.40 0.20 8.24 58.321 59.168 33.802 0.166 0.279 1.53

8 6.44 6.00 5.56 0.12 10.44 67.055 72.268 27.675 0.222 0.289 1.53

9 6.71 6.00 3.15 0.22 7.90 59.729 60.357 33.136 0.151 0.262 1.58

10 6.836 6 4.68 0.148 8.770 58.035 60.646 32.979 0.223 0.289 1.57

11 6.97 6.00 4.87 0.14 8.92 63.162 66.311 30.161 0.212 0.269 1.58

12 6.55 6.00 3.87 0.18 8.85 61.674 63.214 31.639 0.177 0.280 1.56

Mean 6.56 5.88 4.53 0.16 9.13 62.380 65.210 30.952 0.200 0.281 1.54

SD 0.26 0.34 0.86 0.03 0.96 5.362 6.401 3.149 0.032 0.021 0.04

S.E.M 0.06 0.07 0.19 0.01 0.21 1.171 1.398 0.688 0.007 0.005 0.01

Sum 78.76 70.56 54.36 1.90 109.55 748.565 782.520 371.423 2.404 3.367 18.52

113

Table 4.18: Concentration of aceclofenac in rabbits (Group R) plasma calculated from chromatograms by forecasting method after

administration of marketed aceclofenac gel in group R.

" CONCENTRATION IN PLASMA" calculated form chromatogram by forecasting aceclofenac Marketed gel in Rabbits (Group R)

TIM

E (H

ou

rs)

Rabbit 1 2 3 4 5 6 7 8 9 10 11 12 SUM MEAN SD S.E.M

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0.291 0.621

0.669 0.521 0.778 0.278 0.118 0.277 0.438 0.877 0.294 5.16 0.47 0.24 0.05

2 1.553 1.469 1.108 1.691 1.107 1.691 1.087 1.553 1.759 1.13 1.808 1.159 17.12 1.43 0.29 0.06

3 2.851 2.397 2.86 2.287 2.393 2.882 2.196 2.683 2.759 2.687 2.28 2.096 30.37 2.53 0.29 0.06

6 3.898 3.361 3.736 3.386 2.955 3.685 3.044 3.643 3.415 3.273 3.416 4.103 41.91 3.49 0.34 0.07

12 1.771 1.649 1.607 1.838 1.022 1.643 1.793 1.372 1.016 1.749 1.495 1.725 18.68 1.56 0.28 0.06

24 0.323 0.112 0.096 0.141 0.368 0.189 0.365 0.351 0.122 0.206 0.093 0.033 2.40 0.20 0.12 0.03

SUM 10.69 9.609 9.41 10 8.366 10.87 8.763 9.72 9.348 9.483 9.969 9.41 115.64 9.68 1.55 0.34

Mean 1.527 1.373 1.57 1.43 1.195 1.553 1.252 1.389 1.335 1.355 1.424 1.344 16.52 1.38 0.22 0.05

SD 1.233 1.497 1.23 1.09 1.367 1.135 1.379 1.355 1.269 1.221 1.471 1.471 15.51 1.29 0.12 0.03

S.E.M 0.269 0.327 0.27 0.24 0.298 0.248 0.301 0.296 0.277 0.267 0.321 0.321 3.39 0.28 0.03 0.01

114

Figure 4.39: Plasma concentration verses time profile of aceclofenac

plotted on rectangular co-ordinate graph, administered as a

topical dose of 2 mg (conventional marketed gel) in 12 Rabbits

of group R individually



(conventional marketed gel) in 12 Rabbits of group R

individually

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 5 10 15 20 25

Pla

sma

con

c. (

µg/m

l)

Time (Hour)

R1

R2

R3

R4

R5

R6

R7

R8

R9

R10

R11

R12

0.01

0.1

1

10

0 5 10 15 20 25

Pla

sma

conc.

(µ

g/m

l)

Time (Hour)

R1

R2

R3

R4

R5

R6

R7

R8

R9

R10

R11

R12

115

Table 4.19: Pharmacokinetics parameters of aceclofenac in rabbits (Group R) after application of marketed gel

Rabbit

No.

PHARMACOKINETICS OF ACECLOFENAC MICROEMULSION GEL (20mg/g) IN RABBITS (GROUP R)

Cmax

µg/mL

Tmax

h

t1/2

h

Lz (Ke) 1/h

MRT

h

AUC0-t

µg/mL*h

AUC0-inf

µg/mL*h

Clearance

mL/h

Vz

L

Vss

L

Ka

1/h

1 3.89 6.00 4.23 0.16 8.84 65.964 68.027 29.400 0.179 0.260 1.51

2 3.36 6.00 3.12 0.22 7.99 61.522 62.094 32.209 0.145 0.257 1.48

3 3.73 4.90 3.04 0.23 7.04 55.141 55.570 35.991 0.158 0.253 1.42

4 3.38 6.00 3.40 0.20 7.97 50.559 51.271 39.008 0.191 0.311 1.48

5 2.95 6.00 5.10 0.14 8.63 43.518 45.841 43.629 0.321 0.376 1.41

6 3.68 6.00 3.92 0.18 6.71 44.737 45.592 43.868 0.248 0.294 1.49

7 3.04 6.00 4.54 0.15 8.76 55.082 57.335 34.883 0.229 0.306 1.51

8 3.64 6.00 4.67 0.15 8.20 52.885 55.014 36.355 0.245 0.298 1.48

9 3.41 6.00 3.37 0.21 6.87 45.798 46.340 43.159 0.210 0.297 1.48

10 3.27 6.00 4.85 0.18 8.07 51.148 52.287 38.250 0.213 0.309 1.44

11 3.41 6.00 4.59 0.23 7.43 47.630 48.046 41.626 0.184 0.309 1.46

12 4.10 6.00 3.50 0.28 6.26 40.332 40.444 49.451 0.179 0.310 1.45

Mean 5.49 5.91 4.03 0.19 7.73 51.193 52.322 38.986 0.208 0.298 1.47

SD 0.32 0.32 0.72 0.04 0.86 7.503 7.796 5.644 0.048 0.033 0.03

S.E.M 0.07 0.07 0.16 0.01 0.19 1.638 1.702 1.232 0.010 0.007 0.01

Sum 65.92 70.90 48.33 2.32 92.78 614.313 627.860 467.830 2.501 3.580 17.61

116


To study and compare anti-inflammatory activity of microemulsion based aceclofenac

gel with marketed gel (Alkeries), 12 rabbits were selected and divided into two groups

each consisting of 6 rabbits. One group received test formulation and other marketed

or standard formulations. Results are given in Table 4.20 and Figure 4.41.

117

Table 4.20: Anti-inflammatory activity study in rats (n=6 per group)

Name of

Formulation

Volume of the

paw before

application of test

formulations and

standard

formulation

ml (Mean ±

S.E.M)

Volume of the paw after application of test

formulations and standard formulation

ml (Mean ± S.E.M)

Difference Inhibition Percentage

Inhibition

1h 2h 4h 6h

aceclofenac

microemulsion 0.765 ± 0.08 0.615 ± 0.008 0.415 ± 0.01 0.33 ± 0.03 0.34 ± 0.02 0.236 0.764 76.39

aceclofenac

microemulsion based

gel

0.798 ± 0.06 0.623 ± 0.004 0.425 ± 0.03 0.43 ± 0.06 0.34 ± 0.04 0.257 0.743 74.31

Marketed gel 0.788 ± 0.01 0.644 ± 0.008 0.525 ± 0.01 0.44 ± 0.03 0.46 ± 0.02 0.299 0.701 70.14

Control 0.775 ± 0.02 0.88 ± 0.03 0.97 ± 0.06 1.13 ± 0.05 1.44 ± 0.07 -------------------------------------------------

118

Figure 4.41: Percentage inhibition of oedema by aceclofenac microemulsion,

aceclofenac microemulsion based gel and aceclofenac marketed

gel


To study and compare the analgesic effect of microemulsion based aceclofenac gel

with marketed gel (Alkeries), the formulations were applied to each of rat who

received 0.6% acetic acid. Results are given in Table 4.21 and Figure 4.42.

Table 4.21: Study of analgesic effect in rats (n=6 per group)

Name of

Formulation

No. of writhes within

0.5 h

(Mean ± S.E.M)

Difference from

–ve control

(Mean ±

S.E.M)

Percentage

Inhibition

(Mean ± S.E.M)

aceclofenac

microemulsion 11.0 ± 0.45 65.33 ± 2.64 80.16 ± 3.24

Aceclofenac

microemulsion based

gel

13.67 ± 0.71 61.17 ± 3.18 75.05 ± 3.90

Marketed gel 16 ± 0.82 57.83 ± 3.76 70.96 ± 4.62

Negative Control 81.5 ± 0.76 -------- --------

62

64

66

68

70

72

74

76

78

80

ACF Microemulsion ACF Microemulsion based

gel

Marketed gel

Per

centa

ge

Inhib

itio

n

119

Figure 4.42: Percentage inhibition of writhes (analgesic effect) by Aceclofenac

microemulsion, Aceclofenac microemulsion based gel and

Aceclofenac marketed gel

4.9 SKIN IRRITATIONS STUDY OF FORMULATIONS

To evaluate the skin sensitivity for test formulations, patch of microemulsion gel base

and blank microemulsion was applied on the forearm of each volunteer and observed

for Erythma and edema during 24 hours. Results are given in Tables 4.22 and 4.23.

Table 4.22: Skin Irritations Study of aceclofenac microemulsion

Volunteers Control After 48 hours

Edema Erythma Edema Erythma

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

6 0 0 0 0

7 0 0 0 0

8 0 0 0 0

9 0 0 0 0

10 0 0 0 0

12 0 0 0 0

13 0 0 0 0

14 0 0 0 0

15 0 0 0 0

16 0 0 0 0

17 0 0 0 0

18 0 0 0 0

0

10

20

30

40

50

60

70

80

90

ACF microemulsion ACF microemulsion

based gel

ACF Marketed gel

% I

nh

ibit

ion

120

Table 4.23: Skin Irritation study of aceclofenac microemulsion

based gel

Volunteers Control After 48 hours

Edema Erythma Edema Erythma

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

4 0 0 0 0

5 0 0 0 0

6 0 0 0 0

7 0 0 0 0

8 0 0 0 0

9 0 0 0 0

10 0 0 0 0

12 0 0 0 0

13 0 0 0 0

14 0 0 0 0

15 0 0 0 0

16 0 0 0 0

17 0 0 0 0

18 0 0 0 0

4.10 HPLC METHOD VALIDATION

A new HPLC-UV method was developed and validated in human plasma. Results of

validation are given in Tables 4.24-4.29. The chromatograms of blank plasma and

spiked plasma are given in appendix V.

Table 4.24: Plasma Sample Concentration Data of aceclofenac (Within-Batch

Precision and Accuracy)

Batch No. LQC

0.5μg/ml MQC

10μg/ml LQC

20μg/ml Batch-01 0.49 9.95 20.02

0.50 9.96 19.98

0.48 9.94 19.88

Batch-02 0.49 9.98 19.74

0.50 10.02 19.95

0.49 10 20.08

Batch-03 0.48 9.98 19.89

0.50 10 19.98

0.49 9.99 20.08

Mean 0.4911 9.980 19.956

SD 0.0078 0.0260 0.1077

N 18 16 15

Nominal 0.50 10 20

%CV 1.5918 0.2603 0.5398

%Accuracy 98.22 99.80 99.78

121

Table 4.25: Plasma Sample Concentration Data of aceclofenac (Between-Batch

Precision and Accuracy)

Batch No. LQC

0.5μg/ml MQC

10μg/ml LQC

20μg/ml Batch 01 0.49 9.95 20.11

Batch 02 0.49 9.96 19.97

Batch 03 0.48 9.93 19.78

Batch 04 0.49 9.98 19.69

Batch 05 0.50 10.01 19.98

Batch 06 0.49 10 20.1

Mean 0.490 9.972 19.938

SD 0.006 0.031 0.170

N 6 6 6

Nominal 0.50 10 20

%CV 1.2907 0.3069 0.8543

%Accuracy 98.00 99.72 99.69

Table 4.26: Detection and Quantitation limit

Detection limit Quantitation limit

0.050 µg/ml 0.250 µg/ml

Table 4.27 Linearity curve of aceclofenac in plasma

Conc.

(µg/ml)

Peak Height of

STD aceclofenac

Peak Height of

ISTD Diclofenac

Peak Height

Ratio

0 0 0 0

0.312 4.169 6.624 0.629

0.625 8.353 6.613 1.263

1.25 16.934 6.711 2.523

2.5 30.603 6.629 4.617

5 58.93 6.666 8.840

10 116.31 6.634 17.532

20 206.753 6.644 31.119

122

Figure 4.43: Linearity curve of aceclofenac in plasma

Table 4.28: Freeze thaw stability of aceclofenac in plasma

Curve Code

Cycle

Cycle 0 Cycle 1 Cycle 2 Cycle 3

0.5 µg 20 µg 0.5 µg 20 µg 0.5 µg 20 µg 0.5 µg 20 µg

LQC HQC LQC HQC LQC HQC LQC HQC

ACF-06

0.493 20.001 0.490 19.987 0.486 19.841 0.484 19.743

0.492 19.998 0.492 19.958 0.489 19.912 0.487 19.811

0.486 19.962 0.486 19.942 0.481 19.816 0.479 19.791

Mean 0.4903 19.9870 0.4893 19.9623 0.4853 19.8563 0.4833 19.7817

SD 0.0038 0.0217 0.0031 0.0228 0.0040 0.0498 0.0040 0.0349

N 3 3 3 3 3 3 3 3

Nominal 0.50 20 0.50 20 0.50 20 0.50 20

%CV 0.7721 0.1086 0.6243 0.1143 0.8327 0.2508 0.8362 0.1767

y = 1.5626x + 0.6656

R² = 0.9946

0

5

10

15

20

25

30

35

0 5 10 15 20 25

Conc.

(

µg/m

l)

Peak height ratio

123

Table 4.29: Extraction yield/Recovery of aceclofenac

Curve

Code

LQC HQC

0.5µg 20 µg

EXTRACTED

%

Extraction EXTRACTED

%

Extraction

ACF-04

0.48 96 19.3 96.5

0.47 94 19.5 97.5

0.48 96 19.20 96

Mean 0.4767 95.33 19.333 96.67

SD 0.0058 1.1547 0.1528 0.76

N 3 3 3 3

Nominal 0.5 0.5 20 20

%CV 1.2112 1.2112 0.7901 0.7901

4.11 CONCENTRATION OF ACECLOFENAC IN HUMAN PLASMA

Plasma concentration of aceclofenac in each volunteer was calculated by forecasting

formula. Results are given in Tables (4.30 to 4.31) and Figures (4.44 to 4.47).

Comparison of mean plasma concentrations of aceclofenac from microemulsion based

aceclofenac gel and marketed conventional gel in volunteers is given in Table 4.32

and Figures 4.48 to 4.49 and comparison of mean plasma concentrations of

aceclofenac from ME gel and marketed conventional gel in rabbits is given in Table

4.33 and Figures 4.50 and 4.51. Comparison of Pharmacokinetic parameters of

aceclofenac microemulsion based gel and marketed conventional gel in rabbits is

given in Table 4.34. Pharmacokinetic parameters of conventional gel and

microemulsion based gel are given in Tables 4.35 and 4.36 respectively. Comparison

of mean plasma concentration of aceclofenac from ME gel and conventional gel in

rabbits after t test is given in Table 4.37. Comparison of Pharmacokinetic parameters

of aceclofenac microemulsion based gel and conventional gel in human volunteers

after t test is given in Table 4.38. The individual plasma concentrations in human

volunteers after application of marketed conventional gel and microemulsion based

gel are given in appendix III and IV, respectively.

124

Table 4.30: Concentration of aceclofenac in human plasma calculated from chromatograms by forecasting method after administration of a

topical dose of 20 mg (Marketed conventional gel)

" CONCENTRATION IN PLASMA" calculated form chromatogram by forecasting (Aceclofenac marketed gel)

TIM

E (

ho

urs)

Vntrs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 SUM MEAN SD S.E.M

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.5 0.1 0.06 0.5 0.67 0.35 0.44 0.6 0.78 0.5 0.43 0.22 0.29 0.22 0.28 0.38 0.37 0.22 0.37 6.79 0.38 0.19 0.04

1 0.53 0.54 1.36 0.89 0.57 0.68 1.26 1.04 0.86 0.86 0.63 0.66 0.92 1.06 1.08 0.64 0.58 0.88 15.03 0.84 0.25 0.05

1.5 2.97 0.83 3.11 1.69 1.12 1.87 1.69 1.31 1.68 1.27 1.32 1.48 1.61 2.31 2.35 1.58 0.99 1.23 30.40 1.69 0.63 0.14

2 3.83 1.5 3.98 2.99 2.12 2.33 3.01 2.27 2.62 1.75 2.15 2.55 1.9 3.03 3.07 2.51 1.64 1.87 45.13 2.51 0.71 0.15

3 4.65 2.65 5.11 5.03 3.29 3.32 4.15 3.73 3.32 2.51 3.46 3.99 2.69 3.77 4.14 3.56 2.78 3.11 65.27 3.63 0.78 0.17

4 5.23 3.43 5.74 5.44 4.3 4.93 4.83 4.09 5.16 4.06 4.31 4.6 3.88 4.53 5.36 4.72 4.03 3.68 82.33 4.57 0.65 0.14

5 6.19 5.92 6.51 5.62 5.44 4.9 5.73 5.3 5.92 5.03 5.06 5.49 5.35 5.1 5.65 5.73 5.09 5.04 99.06 5.50 0.44 0.10

6 6.84 6.74 6.81 6.66 6.96 5.93 6.53 7.07 6.43 6.32 6.04 5.94 5.91 5.95 5.91 6.04 5.77 5.83 113.66 6.31 0.44 0.10

8 3.22 3.08 5.45 2.48 4.99 2.72 4.59 3.05 4.59 4.92 4.06 4.3 3.91 4.29 4.38 4.15 3.78 3.86 71.82 3.99 0.82 0.18

12 0.93 1.25 2.79 1.07 0.94 1.22 2.31 1.45 0.56 1.86 1.44 2.33 2.26 2.13 2.24 2.1 1.16 1.62 29.64 1.65 0.62 0.14

24 0.23 0.21 0.46 0.16 0.31 0.18 0.26 0.18 0.3 0.31 0.24 0.39 0.26 0.25 0.32 0.25 0.25 0.23 4.78 0.27 0.08 0.02

125



of 20 mg (Marketed conventional gel) in 18 volunteers.



(Marketed conventional gel) in 18 volunteers.

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

con

c. (

µg/m

l)

Time (Hour)

Vounteer 1Vounteer 2Vounteer 3Vounteer 4Vounteer 5Vounteer 6Vounteer 7Vounteer 8Vounteer 9Vounteer 10Vounteer 11Vounteer 12Vounteer 13Vounteer 14Vounteer 15Vounteer 16Vounteer 17Vounteer 18

0.1

1

10

0 5 10 15 20 25

Pla

sma

conc.

(µ

g/m

l)

Time (Hour)


126

Table 4.31: Concentration of aceclofenac in plasma calculated from chromatograms by forecasting method after administration of

microemulsion based aceclofenac gel

" CONCENTRATION IN PLASMA" calculated from peak height ratios by forecasting ( Aceclofenac microemulsion based gel)

TIM

E (

hou

rs)

Vlntrs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 SUM MEAN SD S.E.M

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.5 0.27 0.2 0.45 0.09 0.69 0.59 0.40 0.42 0.45 0.36 0.36 0.32 0.22 0.3 0.11 0.378 0.2 0.165 5.96 0.33 0.16 0.03

1 1.08 0.42 1.73 1.34 1.01 1.28 0.90 0.63 0.86 0.85 0.72 0.65 1.09 0.9 1.04 0.745 0.94 0.918 17.06 0.95 0.30 0.07

1.5 1.51 1.58 2.35 2.32 1.08 2.29 1.81 1.45 1.67 2.28 2.03 1.15 2.22 2.6 2.3 1.511 1.91 1.986 34.03 1.89 0.44 0.10

2 2.23 2.32 3.59 3.51 1.81 2.94 2.94 2.28 2.24 3.29 2.57 3.23 3.46 3.4 2.68 2.628 3.1 2.984 51.17 2.84 0.52 0.11

3 3.35 4.06 5.91 4.8 2.85 4.1 4.02 3.83 3.19 4.17 3.59 4.52 4.25 5.4 3.3 3.895 4.03 4.012 73.30 4.07 0.75 0.16

4 4.8 6.24 6.19 6.7 5.32 5.44 5.17 4.74 4.71 4.49 4.31 5.71 5.77 6.5 4.15 5.496 4.6 5.116 95.46 5.30 0.76 0.17

5 5.23 7.38 7.59 7.68 6.56 7.28 6.31 6.09 5.38 5.84 6.42 7.06 6.54 6.6 5.04 6.559 5.61 6.734 115.86 6.44 0.79 0.17

6 9.23 9 8.97 8.73 8.54 8.87 8.63 8.41 7.85 8.11 7.92 8.17 8.5 8.6 8.27 7.669 7.54 8.48 151.49 8.42 0.47 0.10

8 4.32 5.26 2.67 3.95 3.35 6.24 2.69 3.45 1.99 4.2 1.13 5.25 2.87 2.3 2.25 3.867 2.16 4.5 62.47 3.47 1.34 0.29

12 2.03 2.61 1.59 2.11 1.39 3.55 1.2 1.03 0.98 1.47 0.79 2.63 0.85 0.9 1 1.934 1.37 2.508 29.92 1.66 0.78 0.17

24 0.39 0.34 0.23 0.28 0.21 0.38 0.23 0.27 0.16 0.3 0.08 0.37 0.29 0.1 0.18 0.246 0.14 0.31 4.55 0.25 0.09 0.02

127


on rectangular co-ordinate graph, administered as a topical

dose of 20 mg (Microemulsion based aceclofenac gel) in 18

volunteers



(Microemulsion based aceclofenac gel) in 18 volunteers.

0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

con

c. (

µg/m

l)

Time (Hour)

Vounteer 1

Vounteer 2

Vounteer 3

Vounteer 4

Vounteer 5

Vounteer 6

Vounteer 7

Vounteer 8

Vounteer 9

Vounteer 10

Vounteer 11

Vounteer 12

Vounteer 13

Vounteer 14

Vounteer 15

Vounteer 16

Vounteer 17

Vounteer 18

0.01

0.1

1

10

0 5 10 15 20 25

Pla

sma

conc.

(µg/m

l)

Time (Hour)


128

Table 4.32: Comparison of mean plasma concentrations of aceclofenac from

microemulsion based aceclofenac gel and marketed conventional gel in

volunteers.

Mean plasma conc. of aceclofenac (µg/ml) in Volunteers

Sr.

No. Time (hr)

Microemulsion

based Gel S.E.M

Marketed conventional

Gel S.E.M

0 0 0.000 0.000 0.000 0.000

1 0.5 0.38 0.041 0.331 0.034

2 1 0.84 0.055 0.948 0.065

3 1.5 1.69 0.138 1.890 0.097

4 2 2.51 0.154 2.843 0.115

5 3 3.63 0.170 4.072 0.165

6 4 4.57 0.142 5.303 0.167

7 5 5.50 0.097 6.437 0.173

8 6 6.31 0.096 8.416 0.103

9 8 3.99 0.178 3.471 0.293

10 12 1.65 0.136 1.662 0.170

11 24 0.27 0.016 0.253 0.019

Figure 4.48: Mean plasma concentration verses time profile of aceclofenac from

ME Gel and Conventional gel plotted on rectangular co-ordinate

graph, administered as a topical dose of 20 mg in 36 volunteers.

0

2

4

6

8

0 5 10 15 20 25

Pla

sma

con

c.

(µg/m

l)

Time (Hour)

Mean Plasma Conc.of ACF

ME Gel

Mean plasma Conc. of ACF

Conventional Gel

129

Figure 4.49: Mean plasma concentration verses time profile of aceclofenac from

ME Gel and Conventional gel plotted on semi log graph, administered

as a topical dose of 20 mg in 36 volunteers.

Table 4.33: Comparison of mean plasma concentrations of aceclofenac from ME

gel and marketed conventional gel in rabbits.

Mean plasma conc. of aceclofenac ( µg / ml) in rabbits

Sr. No. Time (hr) ME Gel S.E.M Marketed Gel S.E.M

0 0 0.000 0.000 0.000 0.000

1 1 0.704 0.15 0.469 0.10

2 2 2.080 0.45 1.426 0.31

3 3 3.422 0.75 2.531 0.55

4 6 6.563 1.43 3.493 0.76

5 12 2.303 0.50 1.557 0.34

6 24 0.416 0.09 0.200 0.04

0

1

10

0 5 10 15 20 25 30

Pla

sma

conc.

(µ

g/m

l)

Time (Hour)

Mean Plsma Conc. of ACF

ME Gel

Mean Plasma conc. of ACF

Conventional Gel

130

Figure 4.50: Mean Plasma concentration verses time profile of aceclofenac



Rabbits.

Figure 4.51: Mean Plasma concentration verses time profile of

aceclofenac from ME Gel and Conventional gel plotted

on semi log graph, administered as a topical dose of 2

mg in 24 Rabbits.

0

2

4

6

8

0 5 10 15 20 25

Pla

sma

con

c. (

µg/m

l)

Time (Hour)

Mean Plasma Conc.of

ACF ME Gel

Mean plasma Conc. of

ACF Conventional Gel

0

1

10

0 5 10 15 20 25

Pla

sma

conc.

(µ

g/m

l)

Time (Hour)

Mean Plsma Conc. of

ACF ME Gel

Mean Plasma conc. of

ACF Conventional Gel

131

Table 4.34: Comparison of Pharmacokinetic parameters of aceclofenac

microemulsion based gel and marketed conventional gel in rabbits

(t test)


Pharmacokinetic parameters calculated by Kinetica 4.4.1 software using

plasma concentration found by forecasting formula are given in Tables 4.35 to

4.36.

Pharmacokinetic

Parameter

Aceclofenac

microemulsion

based gel

Conventional gel P-Value Result

Cmax µg/mL 6.56 ± 0.12 5.49 ± 0.18 0.01 Significant

Tmax h 5.88 ± 0.11 5.889 ± 0.071 0.48 No significant

t1/2 h 4.53 ± 0.05 4.082 ± 0.046 0.18 No significant

Lz (Ke) 1/h 0.16 ± 0.01 0.17 ± 0.002 0.05

No significant

MRT h 7.92 ± 0.15 8.248 ± 0.130 0.28 No significant

AUC0-t µg/mL*h 56.11 ± 2.79 53.693 ± 1.785 0.27

No significant

AUC0-inf µg/mL*h 57.62 ± 2.87 55.187 ±1.884 0.13 No significant

Clearance mL/h 365.21 ± 19.01 369.864 ± 11.188 0.26

No significant

Vz L 2.25 ± 0.12 2.172 ± 0.062 0.67 No significant

Vss L 2.84 ± 0.10 3.032 ± 0.073 0.22 No significant

Ka 1/h 1.624 ± 0.01 1.551± 0.011 0.02

Significant

132

Table 4.35: Pharmacokinetics parameters of aceclofenac in volunteers after application of marketed conventional gel.

SBJECT

PHARMACOKINETICS OF ACECLOFENAC MARKETED GEL (Aceclofenac 20mg)

Cmax

µg/mL

Tmax

h

t1/2

h

Lz (Ke)

1/h

MRT

h

AUC0-t

µg/mL*h

AUC0-inf

µg/mL*h

Clearance

mL/h

Vz

L

Vss

L

Ka

1/h

1 6.94 6 3.91 0.17 7.27 49.68 50.77 393.93 2.22 2.865 1.588

2 7.17 6.00 4.20 0.16 8.18 45.05 46.26 432.38 2.62 3.536 1.600

3 7.71 6.00 4.51 0.15 8.94 75.84 78.79 253.84 1.65 2.269 1.631

4 7.62 5.00 4.08 0.17 7.06 48.41 49.31 405.61 2.38 2.864 1.544

5 7.58 6.00 4.00 0.17 8.03 51.33 52.77 379.02 2.18 3.045 1.628

6 6.90 5.00 4.12 0.17 7.70 44.74 45.77 436.93 2.59 3.363 1.497

7 7.94 6.00 3.85 0.18 8.30 65.30 66.75 299.63 1.66 2.487 1.651

8 7.07 6.00 3.93 0.18 7.85 48.82 49.83 401.33 2.27 3.150 1.596

9 6.43 6.00 4.14 0.17 7.56 47.52 48.82 409.70 2.44 3.095 1.549

10 6.32 6.00 4.12 0.17 8.87 55.16 56.95 351.20 2.09 3.114 1.541

11 6.04 6.00 3.90 0.18 8.22 49.71 50.95 392.53 2.21 3.226 1.523

12 5.94 6.00 4.63 0.15 9.26 60.00 62.60 319.48 2.13 2.957 1.505

13 5.91 6.00 4.02 0.17 8.89 54.99 56.53 353.81 2.05 3.1457 1.511

14 5.95 6.00 3.92 0.17 8.46 57.85 59.29 337.29 1.98 2.856 1.516

15 5.91 6.00 4.28 0.16 8.69 61.20 63.19 316.51 1.95 2.749 1.508

16 6.04 6.00 3.96 0.17 8.53 57.00 58.46 342.13 1.96 2.919 1.522

17 5.77 6.00 4.02 0.17 8.26 44.73 45.99 434.88 2.52 3.591 1.500

18 5.83 6.00 3.89 0.18 8.40 49.11 50.33 397.34 2.23 3.339 1.506

Mean 6.615 5.889 4.082 0.17 8.248 53.693 55.187 369.864 2.172 3.032 1.551

SD 0.752 0.323 0.213 0.008 0.594 8.177 8.629 51.242 0.283 0.335 0.051

S.E.M 0.164 0.071 0.046 0.002 0.130 1.785 1.884 11.188 0.062 0.073 0.011

Sum 119.062 106.000 73.477 3.064 148.462 966.470 993.360 6657.546 39.102 54.571 27.917

133

Table 4.36: Pharmacokinetics parameters of aceclofenac in volunteers after application of microemulsion based gel

SBJECT

PHARMACOKINETICS OF ACECLOFENAC MICROEMULSION BASED GEL (Aceclofenac 20mg)

Cmax

µg/mL

Tmax

h

t1/2

h

Lz (Ke)

1/h

MRT

h

AUC0-t

µg/mL*h

AUC0-inf

µg/mL*h

Clearance

mL/h

Vz

L

Vss

L

Ka

1/h

1 9.29 5 4.68 0.15 8.88 61.99 64.54 309.85 2.095 2.753 1.628

2 9.00 6.00 4.04 0.17 8.62 72.71 74.67 267.86 1.560 2.308 1.707

3 8.97 5.00 4.49 0.15 7.51 58.86 60.38 331.22 2.145 2.489 1.617

4 8.68 5.00 4.13 0.17 8.12 63.65 65.30 306.26 1.826 2.487 1.605

5 8.54 6.00 4.07 0.17 7.85 52.67 53.86 371.31 2.183 2.914 1.683

6 8.87 6.00 3.96 0.18 8.94 83.87 86.11 232.25 1.327 2.077 1.701

7 8.60 5.00 4.63 0.15 7.72 50.11 51.61 387.55 2.587 2.991 1.595

8 8.41 6.00 4.70 0.15 7.95 50.49 52.18 383.26 2.598 3.048 1.670

9 7.39 5.00 4.46 0.16 7.48 40.53 41.56 481.22 3.099 3.599 1.525

10 8.11 6.00 4.39 0.16 8.14 57.01 58.80 340.14 2.157 2.769 1.656

11 7.42 5.00 4.09 0.17 6.69 33.76 34.26 583.73 3.443 3.905 1.532

12 8.17 6.00 4.19 0.17 8.75 72.02 74.24 269.41 1.629 2.357 1.661

13 7.54 5.00 4.18 0.17 7.48 46.54 47.89 417.64 2.522 3.122 1.538

14 8.55 5.00 4.13 0.17 6.70 48.09 48.92 408.87 2.436 2.740 1.598

15 8.24 5.00 4.48 0.15 7.50 42.88 44.00 454.51 2.935 3.408 1.577

16 7.67 6.00 4.03 0.17 8.19 59.29 60.72 329.39 1.914 2.699 1.633

17 7.54 6.00 4.00 0.17 7.46 47.68 48.54 412.05 2.376 3.076 1.626

18 8.48 6.00 4.11 0.17 8.59 67.78 69.64 287.20 1.701 2.466 1.679

Mean 8.30 5.50 4.26 0.16 7.92 56.11 57.62 365.21 2.25 2.84 1.624

SD 0.582 0.514 0.250 0.009 0.675 12.763 13.218 87.084 0.560 0.471 0.056

S.E.M 0.127 0.112 0.055 0.002 0.147 2.787 2.886 19.014 0.122 0.103 0.012

Sum 149.408 99.000 76.763 2.935 142.581 1009.928 1037.231 6573.699 40.531 51.209 29.230

134

4.13 STATISTICAL ANALYSIS

Statistically mean plasma concentrations of aceclofenac from microemulsion based

gel (ME) and conventional gel in rabbits were compared by “t” test. Moreover,

pharmacokinetic parameters of aceclofenac ME and conventional gel were also

compared by “t” test. Results are given in following Tables.

Table 4.37: Comparison of mean plasma concentration of aceclofenac from ME

gel and conventional gel in rabbits. (t test)

Time

Hour

Mean plasma conc. of

aceclofenac in Rabbits

group AR administered

topically as ME Gel

Mean plasma conc. of

aceclofenac in Rabbits group

R administered topically as

Conventional Gel

P-Value Result

6 6.56 3.49 0.04 Significant

P<0.05 Significant difference; P>0.05 Non significant difference.

Table 4.38: Comparison of Pharmacokinetic parameters of aceclofenac

microemulsion based gel and conventional gel. (t test)

Pharmacokinetic

Parameter

Aceclofenac

microemulsion

based gel

Marketed

Conventional gel P-Value Result

Cmax µg/mL 8.30 ± 0.127 6.615 ± 0.164 0.001 Significant

Tmax h 5.50 ± 0.112 5.889 ± 0.071 0.163 No significant

t1/2 h 4.26 ± 0.055 4.082 ± 0.046 0.256 No significant

Lz (Ke) 1/h 0.16 ± 0.002 0.17 ± 0.002 0.141 No significant

MRT h 7.92 ± 0.147 8.248 ± 0.130 0.176 No significant

AUC0-t µg/mL*h 56.11 ± 2.787 53.693 ± 1.785 0.533 No significant

AUC0-inf µg/mL*h 57.62 ± 2.886 55.187 ±1.884 0.544 No significant

Clearance mL/h 365.21 ± 19.014 369.864 ± 11.188 0.852 No significant

Vz L 2.25 ± 0.122 2.172 ± 0.062 0.632 No significant

Vss L 2.84 ± 0.103 3.032 ± 0.073 0.190 No significant

Ka 1/h 1.624 ± 0.012 1.551± 0.011 0.0004 Significant

135

5. DISCUSSION

5.1 SOLUBILITY AND PERMEABILITY OF ACECLOFENAC IN VARIOUS

OILS

The solubility studies indicated that aceclofenac is soluble with varying proportion in

different oils, surfactants and co-surfactants. The solubility (mg/ml) of aceclofenac in

almond oil (9.16), oleic acid (8.56), Tween 80 (476.16), Tween 20(407.23) and PEG

600 (433.24) was significant (P<0.01) as compared to its hydroalcoholic solubility

(150.65). Highest solubility of aceclofenac was found in almond oil among the

studied oils. The flux (Jss) and permeability coefficient (Kp) of aceclofenac in different

oils was highest in almond oil (1.45µg/cm2/h and 0.073 cm/h) after 24 h as shown in

Table 4.5. This indicates that the presence of oils can significantly enhance the

permeability of a poorly soluble drug aceclofenac.

Shakeel et al. (2009) found solubility of aceclofenac in distilled water, nanoemulsion,

solid lipid nanosuspension (SLN) and polymeric nanosuspension (PN) at 25°C as

0.015 ± 0.002, 198.53 ± 4.21, 104.23 ± 3.05 and 83.73 ± 2.89 mg/ml, respectively.

The solubility of aceclofenac in all three nano carriers was highly significant as

compared to its aqueous solubility (P<0.001). Highest solubility of aceclofenac was

found in nanoemulsion formulation as compared to SLN and PN. The solubility of

aceclofenac in nanoemulsion was significant (P<0.05) as compared to its solubility in

SLN and PN. The highest solubility of aceclofenac in nanoemulsion could be due to

the presence of surfactant (Tween-80) and co-surfactant (Transcutol-P). The higher

solubility in Tween 80, Tween 20 and PEG 600 is due to their solubilizing nature

because these are also used as solubilizers.

Shakeel et al. (2007) found the solubility of aceclofenac in various oils as Triacetin

(8.22 ± 1.12 SD mg/mL), Labrafac (6.31 ± 0.52 SD mg/mL), oleic acid (4.01 ± 0.92

SD mg/ml), Labrafil (32.56 ± 2.43 SD mg/ml), IPM (2.97 ± 1.01 SD mg/ml) and

olive oil (1.69 ± 0.35 SD mg/ml), surfactants as Labrasol (386.45 ± 3.28 SD mg/ml),

Tween 80 (398.21 ± 2.89 SD mg/ml) and Cremophor EL (272.32 ± 2.94 SD mg/ml).

In our study the solubility of aceclofenac in oleic acid is 8.56 mg/ml while in above

study solubility was found as 4.01 ± 0.92 SD mg/mL which is less than our finding.

This may be due to difference of source of oleic acid or aceclofenac. Moreover, the

stirring time in previous study was 24 hours while in current study it was 72 hours and

this may increase the solubility. However, in our study the solubility of aceclofenac in

136

Tween 80 was 476.16 mg/mL. Again, the difference may be due to stirring time and

speed as well as source of Tween 80 and aceclofenac.

Kassem et al.(2009) determined solubility of aceclofenac in distilled water,

Sorenson’s buffers solutions of pH 4, pH 5, pH 6 and pH 7.4 in thermostatically

controlled water bath at 37°C as 0.105 mg/ml, 0.139 mg/ml, 0.474 mg/ml, 1.317

mg/ml and 5.786 mg/ml. From the above results it is clear that highest solubility of

aceclofenac was observed in pH 7.4. In our in vitro skin permeation studies,

Sorenson’s buffer pH 7.4 was used as medium in Franz diffusion cell due to higher

solubility of aceclofenac in this buffer.

5.2 MICROEMULSION FORMULATIONS OF ACECLOFENAC USING

DIFFERENT OIL PHASES

Various formulations of aceclofenac microemulsion were prepared using different oil

phases and ternary phase diagrams were constructed to define the boundary of phases.

The formulations with large boundary of phases defined by ternary phase diagram

were chosen and these were subjected to thermodynamic stability test. On the basis of

physical stability (without phase separation), one formulation of aceclofenac

microemulsion containing almond oil was selected and evaluated in vitro.

5.3 MICROEMULSION BASED GEL FORMULATION OF ACECLOFENAC

On the basis of solubility and in vitro studies, almond oil microemulsion was selected

for incorporation in different gel bases of xanthan gum, carbopol 934 and carbopol

940 based gels. Among these, carbopol 940 based gels showed best results with

respect to its stability and consistency, it was selected as the gel base in the final

formulation of aceclofenac microemulsion based gel.

5.4 CHARACTERIZATION

Almond oil based aceclofenac microemulsion and almond oil microemulsion based

aceclofenac gel were characterized by following tests:

5.4.1 Viscosity

To characterize microemulsions and gels viscosity is an important factor because it

affects spreadability and release of drug form the formulation. Viscosities of blank

microemulsion, microemulsion containing aceclofenac, microemulsion based blank

gel; microemulsion based aceclofenac gel and marketed aceclofenac gel (Alkeries)

were 15.08 cP, 45.24 cP, 557.95 cP, 588.11 cP and 611.12 cP, respectively. The

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results showed that microemulsions both blank and containing aceclofenac have less

effect on spreadability and release of drug from the formulation than gels. Among the

gels it is clear that microemulsion based aceclofenac gel is more easy to spread and

release of drug is faster than marketed gel.

Narendra and Prakash (2011) prepared aceclofenac nanoemulsion for transdermal

delivery. The viscosity of F1 (10% wt/wt of Labrafil, 5% wt/wt of Triacetin, 2%

wt/wt of aceclofenac, 35.33% wt/wt of Tween 80, 32% wt/wt of distilled water

and17.66% wt/wt of Transcutol P) was 92.20 cP ± 1.41cP (Mean ± SD). But in our

study the viscosity of microemulsion is less than the previous study because

combination of two oils i.e. Labrafil + Triacetin (2:1) is 15% w/w, Smix is 53% and

proportion of water is 32% and all these components has increased viscosity. Owing

to this, our formulation became less viscous than the previous one. Less viscous

formulation has greater spreadability as compared to highly viscous formulation.

Rohit et al. (2009) prepared aceclofenac topical microemulsion and evaluated

viscosity as 78.3 cP which is greater than our microemulsion because they use higher

proportion of Smix and oil phase.

Moghimipour et al. (2013) designed and characterized microemulsion systems for

Naproxen and found viscosity in the range of 253.73 cPs to 802.63cP which is much

higher than our microemulsion system. This is because the proportion of surfactant to

co-surfactant in Smix is 4:1 and 6:1 while in our microemulsion this is 2:1. Higher the

amount of Smix, higher will be the viscosity (Moghimipour et al.). Moreover, the

percentage of water in previous study is 5-10% while in our formulation it is 36-38%

which also decreases the viscosity of the system.

Aijaz et al. (2011) prepared aceclofenac gel with different gel bases like carbopol

974P, hydroxy propylmethyl cellulose and sodium carboxy methyl cellulose and

found viscosity in the range of 27000 cP to 32000 cP which is much higher than our

microemulsion based aceclofenac gel. As we used only one gelling agent or viscosity

builder i.e. Carbopol 940 with proportion of 19% while in previous study they used

high viscosity grade gelling agent i.e. Carbopol 974P along with two more viscosity

builders i.e. hdroxy propyl methyl cellulose and carboxy methyl cellulose due to

which viscosity is greater than viscosity of our microemulsion based gel which

reduces the spreadability.

Modi and Patel (2011) prepared and evaluated nanoemulsion based aceclofenac gel

for topical delivery. They found viscosity in the range of 105 x 105cP– 154 x 10

5 cP

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which is much higher than our formulation. This is due to high proportion of

surfactant to co-surfactant i.e. 3:1 and 4:1 while in current microemulsion we used 2:1

surfactant to co-surfactant. Moreover, we used one gelling agent without viscosity

builders while in previous study 10% polyethylene glycol and 10% propylene glycol

were also used in nanoemulsion based aceclofenac gel which may increase the

viscosity of the system. Thus it is concluded that higher the proportion of surfactant to

co-surfactant, higher will be viscosity of the system and lower will be spreadability.

Thus, our microemulsion based gel is easy to spread which enhances patient

spreadability.

Moreover, the microemulsion based aceclofenac gel was also compared with

conventional aceclofenac gel and it is clear from values that microemulsion based

aceclofenac is slightly less viscous than that of conventional gel which may be due to

microemulsion as well as less quantity of carbopol 940 gelling agent.

5.4.2 Spreadability

Spreadability is defined as the quality of being easy to spread or apply on a surface.

As the patient spreads topical drug formulation in an even layer to administer a

standard dose, efficacy of a topical therapy increases. Therefore, spreadability is

responsible for correct dosage transfer to the target site which is an important

characteristic of these formulations, ease of extrudability from the package, ease of

application on the substrate and most important, patient preference& compliance

(Garg et al., 2002). Easy spreadability is one of important parameter for

characterization of microemulsion and gels. This test is used to test applicability of

gels on skin (Waghmare et al., 2011). The spreadability values of blank

microemulsion, microemulsion containing aceclofenac, microemulsion based blank

gel; microemulsion based aceclofenac gel and marketed aceclofenac gel (Alkeries)

were 5.5, 5.1, 4.5, 4.1 and 3.2cm, respectively. The high spreadability value indicates

easy spreadability. Larger diameter indicates better spreadability (Kalra et al., 2010).

Desai (2004) prepared topical microemulsion based gel of rofecoxib and compared

spreadability of conventional gel and microemulsion based gel which was 0.85 cm

and 1.95 to 2.2 cm, respectively. Spreadability depends on viscosity i.e. higher the

viscosity less will be the spreadability. Our microemulsion based gel of aceclofenac

has larger spreadability suggesting that our formulation is more convenient to patient

than the previously studied formulations.

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5.4.3 Conductivity Measurements

Conductivity is defined as a measure of the ability of water/substance to pass

electrical current. Conductivity is used to determine the nature of continuous phase

and phase inversion phenomena. Conductivity is affected by the presence of inorganic

dissolved solids, organic compounds like oil, phenol, alcohol etc. and it is also

affected by temperature (APHA, 1992). The conductivity values for blank

microemulsion, aceclofenac microemulsion and blank microemulsion based gel and

aceclofenac microemulsion based gel shows that the continuous phase of the systems

is water because conductivity values range from 30.4 µS/cm to 150.7 µS/cm while for

marketed gel it is 1.7 which indicates that the gel base/continuous phase is oily.


for topical delivery. The conductivity of the resultant formulations was in the range of

0.0867 µS/cm to 0.149 µS/cm which showed that the microemulsion system is of

water in oil (w/o).


naproxen. The average conductivity of these systems was in the range of 0.046µS/cm

to 0.136µS/cm. This shows that microemulsion systems are of w/o type.

From above conductivity values of different formulations, it is clear that our

microemulsion formulations are of o/w type because these showed higher

conductivity values. The high conductivity of microemulsions and microemulsion

based gel also indicated the stability of formulations.

5.4.4 pH Measurements

pH of blank microemulsion and aceclofenac microemulsion was 4.97 and 4.39,

respectively. pH of aceclofenac microemulsion based gel and marketed gel were 4.78

and 4.57, respectively. The pH of aceclofenac microemulsion based gel was in good

agreement with the marketed gel.

Shah et al. (2010) prepared and evaluated microemulsions of aceclofenac having pH

in the range of 2.89 to 3.41. Moghimipour et al. (2013) designed and characterized

microemulsion systems for naproxen. The pH of these microemulsions was in the

range of 6.6 to 6.89.


for topical delivery. The pH of formulations was in the range of 7.38 to 7.67.

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Skin pH values are variable as reported in literature with a broad range of pH 4.0 to

7.0 (Lambers et al., 2006). pH of our formulations are well within the reported range

of skin pH.

5.4.5 Refractive Index measurements

It is dimensionless number which is defined as the ratio of speed of light in vacuum to

the speed of light in the substance. Since refractive index is a fundamental physical

property of a substance, it is often used to identify a particular substance, confirm its

purity, or measure its concentration. The mean values of refractive index of blank

microemulsion, aceclofenac microemulsion, blank microemulsion based gel,

aceclofenac microemulsion based gel and marketed gel were 1.411, 1.418, 1.415,

1.424 and 1.445 which are almost similar. Therefore, it can be concluded that

microemulsions and microemulsion based gels were thermodynamically and

chemically stable as well as remained isotropic and there were no interactions

between excipients and aceclofenac. Moreover, most transparent media when viewed

under visible light have refractive indices between 1 and 2 (Katakam and Narendra,

2011).


naproxen. The refractive index was in the range 1.4449 to 1.4561. Katakam and

Narendra (2011) prepared aceclofenac nanoemulsion for transdermal delivery. The

refractive index values were in the range 1.401 to 1.411. These studies show that our

formulation is in good agreement with previous studies regarding refractive index

measurements.

5.4.6 % Transmittance measurements

% transmittance test is used to check dilutability and clarity of the sample. The

transparency of a sample shows that there are no traces of undissolved drug or other

solid ingredients. The high value of % transmittance indicates that the system is

optically clear (Thakkar et al., 2011). The % transmittance of blank microemulsion,

aceclofenac microemulsion, blank microemulsion based gel, aceclofenac

microemulsion based gel and marketed aceclofenac gel are 98.0%, 98.1%, 98.0%,

97.8% and 6.5%, respectively.

Srinivas et al. (2012) prepared simvastatin microemulsion and measured %

transmittance at 238nm using UV spectrophotometer and found % transmittance in

the range from 98.7 to 99.9%.

http://www.ncbi.nlm.nih.gov/pubmed?term=Lambers%20H%5BAuthor%5D&cauthor=true&cauthor_uid=18489300

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The % transmittance test shows that all formulations of microemulsions and

microemulsion based gels were transparent except marketed gel which was opaque.

Moreover, transmittance test also determines stability of formulation with respect to

dilution at specific wavelength with a UV spectrophotometer. A formulation with

refractive index greater than 95% will remain stable after dilution with suitable

solvent. Also, the % transmittance data proves the transparency of microemulsions

and microemulsion based gels (Srinivas et al., 2012).

5.4.7 Centrifugation (Phase separation test)

This technique helps to determine physical stability of the system in terms of phase

separation. When blank microemulsion, aceclofenac microemulsion, blank

microemulsion based gel, aceclofenac microemulsion based gel and marketed

aceclofenac gel were subjected to centrifugation test at 3000 rpm for 30 minutes, there

were no sign of phase separation. This test proves that the systems are stable. Jadhav

et al. (2011) subjected microemulsion systems to centrifugation at 3000 rpm for 30

minutes and found no signs of phase separation which showed that microemulsion

system were stable. Chandra et al. (2009) prepared microemulsion based hydrogel

formulation for transdermal delivery of dexamethasone and determined its physical

stability by subjecting microemulsion system to centrifugation 3000 rpm for 15

minutes and found no signs of phase separation. These findings justified our results of

phase separation.

5.4.8 Drug content

The results of all formulations i.e. aceclofenac microemulsion, aceclofenac

microemulsion based gel and marketed aceclofenac gel were 99.09%, 99.14% and

99.11%. From results it is clear that the contents of microemulsion based aceclofenac

gel are in good agreement with that of conventional aceclofenac gel which fulfill the

pharmacopoeial requirement.

5.4.9 Polydispersity Index (PDI) and Homogeneity

Polydispersity is the ratio of standard deviation to mean droplet size, so it

indicates the uniformity of droplet size within the formulation (Rupali et al.,

2010). All the formulations i.e. blank microemulsion based gel, aceclofenac

microemulsion based gel and marketed aceclofenac were found without any aggregate

and appeared homogenous. Kashyap et al. (2010) prepared aceclofenac gel using

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Poloxamer 407 and observed good homogeneity for all prepared formulations and

marketed conventional gel with absence of lumps. Visual inspection under strong

light provides proof of uniformity and homogeneity of liquid dosage forms.

Polydispersity index is used to measure the uniformity and homogeneity of

particle/globule size in a system. Its value ranges from 0 to 1 which indicates how

much a system consists of uniform particle/globule size. A value of PDI close to 0

indicates higher uniformity between particles.

Polydispersity Indices of blank microemulsion, aceclofenac microemulsion and blank

microemulsion based gel, aceclofenac microemulsion based gel and marketed

conventional aceclofenac gels are 0.343, 0.599, 0.197, 0.786 and 1.200, respectively.

From above results it is clear that microemulsions and microemulsion based gels have

higher uniformity and homogeneity between particles than marketed conventional gel.

Modi and Patel (2011) prepared nanoemulsion based gel formulation of aceclofenac

for topical delivery and found PDI in range from 0.134 to 0.394 showing high

uniformity and homogenous.

Shinde et al. (2012) prepared microemulsion gel systems of nadifloxacin and

evaluated for Polydispersity index which was in the range of 0.854 to 1.254 indicating

homogenous systems. However, systems with PI greater than 1 are less homogenous

with globules of varying size.


The structure of micro globules of all formulations were also tested with SEM image

and it was found that size and shape of micro globules of all formulations has been

changed in terms of globules size and shape i.e. increased size and irregular shaped

globules due to agglomeration of micro globules. This is because of sample

preparation procedure in which each sample was spread on a glass slide and dried at

100°C due to which micro globules coalesce and by heating, micro globules ruptured.

The images showed the residual of active drug as well as excipients.

Patel et al. (2012) also performed SEM analysis of their gel formulation and

explained the same phenomenon which supports our findings.

5.4.11 Fourier Transform Infra-Red (FTIR)

Fourier transform infrared (FT-IR) spectroscopy is a physico-chemical method based

on measurement of vibration of a molecule excited by IR radiation at a specific

wavelength range. FT-IR spectroscopy is a reliable, rapid and economic technique

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which could be explored as a routine diagnostic tool for bacterial analysis by the food

industry, diagnostic laboratories and public health authorities (Davis and Mauer,

2010).The release of drug from formulation is influenced by the excipients and

sometimes there are interactions between drug and excipients which play a vital role

with respect to release of drug from formulation. Therefore, FTIR techniques were

used to study physical and chemical interactions between drug and excipients as well

as identification of aceclofenac in formulations. The spectrum of aceclofenac showed

major peaks at 3319.91, 2972.82, 1715.50, 1591.53 and 1279.86 cm-1

which

correspond to C-H (Stretching), O-H (Stretching), C=O (Stretching), N-H (Bending)

and C-N (Aromatic amine), respectively (Figure: 4.14). Yadav et al. (2009) also

described the spectrum of aceclofenac having major peaks at 3319.3, 2970.2, 1716.5,

1589.2 and 1280.6cm-1

. The spectrum was compared with reference spectrum of

aceclofenac for its identification and purity. FTIR spectra of blank microemulsion and

blank microemulsion based gel showed major peaks at 3393.2, 2925.9, 1647.4,

1457.9, 1299.04 cm-1

and 3389.2, 2972.7, 1646.2, 1458.3, 1299.6cm-1

, respectively. It

is clear from above mentioned major peaks of blank formulations that major peaks of

pure aceclofenac drug have been modified due to complex formation of aceclofenac

with excipients. This complex formation shows compatibility of aceclofenac with

excipients.


Calorimetry (DSC)

The DSC studies were performed to understand the compatibility of pure aceclofenac,

aceclofenac microemulsion and aceclofenac microemulsion based gel. Aceclofenac

exhibits a sharp endothermic peak at 150ºC which corresponds to melting point of

aceclofenac. In thermogram of aceclofenac microemulsion, sharp endothermic peak is

present at 150ºC showing crystalline nature of aceclofenac. However, in

microemulsion based gel, three endothermic peaks are present at 150ºC, 135ºC and

125ºC which shows that aceclofenac is present in crystalline as well as in amorphous

form. Phatak and Chaudhri (2012) worked on formulation of aceclofenac nanogel and

showed a sharp endothermic peak at 158.9ºC. DSC curves of selected formulations

were observed at about 125ºC. The thermogram showed the shifting of melting

endotherm of aceclofenac, which may indicate amorphization of drug as well as

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loss of its crystalline nature. However, decrease in crystallinity of aceclofenac does

not alter the pharmacological properties of the drug. Above study justify our findings.


The signs of crystallinity of aceclofenac for pure aceclofenac, aceclofenac

microemulsion and aceclofenac microemulsion based gel were found but were not

present in blank microemulsion and blank microemulsion based gel. The XRD

patterns of pure aceclofenac drug were sharp and in formulations reduction in both

number and intensity of peaks compared to pure aceclofenac indicating decreased

crystallinity or partial amorphization of active drug.

Yadav et al. (2009) also found deceased crystallinity of aceclofenac by improving its

solubility and confirmed by XRD study. This study justifies reduction in crystallinity

in aceclofenac microemulsion and microemulsion based aceclofenac gel.

5.4.14 Globule charge (Zeta Potential)

Zeta potential of aceclofenac microemulsion and microemulsion based aceclofenac

gel were found to be -63.85 mV and -8.58 mV, respectively. The large values of Zeta

potential indicate stability of formulations which seem to suggest micellar and

bicontinuous structures. Rupali et al. (2010) also suggested that higher value of zeta

potential indicates stable nature of microemulsion formulation and thus no

chances of aggregation of particles. However, Moghimipour et al. (2013) suggested

that lower values of Zeta potential seem to indicate reverse hexagonal and micellar

structures.

5.4.15 Hydrodynamic Size (Zeta Size)

The particle size of pure drug was in the range of 122.42 nm to 190.14 nm while for

aceclofenac microemulsion and microemulsion based aceclofenac gel was in the

range of 4.84 nm to 68.06 nm and 43.82 nm to 91.28 nm, respectively. The size of

drug particles decreases in microemulsion formulation which increases the surface

area. Hence, microemulsion has greater solubility of drug but the size of particles

increased in microemulsion based gel formulation and this is due to coalescence of

particles during incorporation of microemulsion into gel. Moghimipour et al. (2013)

characterize microemulsion system of naproxen and found particle size in the range of

7 nm to 79 nm. Rupali et al. (2010) developed and characterize microemulsion

formulation of aceclofenac and found particle size in the range of 15.85 nm to 50.14

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nm. Above studies justify our results of microemulsions. Modi and Patel (2011)

prepared and characterized nanoemulsion based gel of aceclofenac for topical delivery

and found particle size in the range of 17.52 nm to 99.43 nm which justifies our

results of microemulsion based aceclofenac gel.

5.5 IN VITRO SKIN PERMEATION RELEASE RATE

Average steady state flux, Jss (µg/cm2/h) of aceclofenac microemulsion,

microemulsion based aceclofenac gel and marketed conventional gel are 1.73 ± 0.06,

1.52 ± 0.07 and 0.91 ± 0.03, respectively. From results it is clear that microemulsion

has highest skin permeation release rate as compared to gel formulations. However,

microemulsion based aceclofenac gel has higher skin permeation release rate as

compared to marketed conventional gel which suggests that microemulsion has

decreased the particle size and higher viscosity has increased contact time which

enhanced skin permeation rate. Moreover, certain excipients and penetration

enhancers like tween 80, isopropyl alcohol, dimethyl Sulfoxide also aided in

permeability enhancement. Katakam and Narendra (2011) prepared aceclofenac

nanoemulsion formulation and found significantly increased permeability release rate

parameters like steady state flux, permeability coefficient and enhancement ratio.

They suggested that this is because of excipients and penetration enhancers.

5.6 STABILITY STUDIES

Stability of formulations was estimated by centrifugation, water dilution method and

storage at high temperature. Stability studies confirmed that microemulsion,

microemulsion based aceclofenac gel and marketed gel are stable at accelerated

stability conditions (40ºC ± 5°C/75% ± 5%RH) for a period of 6 months as well as at

long term stability conditions (Room temperature 25ºC ± 5°C/65% ± 5% RH) for a

period of one year. The results of drug assay indicate that there is no chemical

reaction occurring between drug and excipients and formulations are stable. Modi and

Patel (2011) found no significant change in particle size, phase separation and

degradation of aceclofenac observed up to 3 months. The centrifuged tests revealed

that nanoemulsion and nanoemulsion base gel were remained homogenous without

any phase separation throughout the test, indicates good physical stability. The above

study also justifies our stability study results.

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5.7 IN VIVO TRANSDERMAL STUDIES IN RABBITS

Transdermal studies in rabbits were performed to study the permeation of drug across

skin. The results showed that microemulsion based aceclofenac gel have shown

higher plasma concentration than the conventional aceclofenac gel. This may be due

to penetration enhancing effect of oil, penetration enhancer (dimethyl Sulfoxide) and

surfactant and co-surfactant which improved the solubility. Moreover, surfactants

have opened the water channel across the cell membrane. However, intrinsic

pharmacokinetic parameters i.e. Ke and T1/2 did not change significantly. Shakeel et

al. (2009) performed a comparative pharmacokinetic study of aceclofenac from oral

and Transdermal application and found increased bioavailability which may be due to

the enhanced skin permeation and avoidance of hepatic first pass metabolism of

aceclofenac in the form of topical formulations. The above study is in agreement with

our findings.


The anti-inflammatory activity of each formulation was measured as percentage

inhibition of hind paw volume in rats. From the results it is found that microemulsion

has highest percentage inhibition as compared to microemulsion based aceclofenac

gel and marketed conventional gel. However, microemulsion based aceclofenac gel

has higher activity as compared to conventional gel. Tabassum et al. (2010) evaluated

and compared the in vitro and in vivo transdermal potential of gel and patch

formulation for aceclofenac and concluded that aceclofenac Eudragit gel formulation

gives maximum inhibition of edema than the patch formulation. Patel et al. (2012)

also reported that nanostructured lipid carriers based gel has better anti-inflammatory

activity than other formulations. The above studies justify our results.


The analgesic effect of formulations was measured in rats as percentage inhibition of

writhes. Percentage inhibition of writhes by aceclofenac microemulsion,

microemulsion based aceclofenac gel and marketed gel was 80.16 ± 3.24%, 75.05 ±

3.90% and 70.96 ± 4.62%, respectively. From the results it is clear that

microemulsion has highest percentage inhibition as compared to other two

formulations. However, microemulsion based aceclofenac gel has higher percentage

inhibition of writhes than conventional gel.

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Mutalik et al. (2008) studied enhancement of dissolution rate and bioavailability of

aceclofenac by chitosan-based solvent approach and found that higher percentage

inhibition (82.11 ± 6.55%) as compared to pure aceclofenac (65.15 ± 7.25%) was

achieved due to improved solubility and dissolution rate of aceclofenac, which in turn

improved its rate of absorption. The above study is in good agreement with our results

in terms of percentage inhibition of writhes and justifies our results because we also

improved the solubility of aceclofenac by formulating its microemulsion and then

incorporation into carbopol 940 based gel. Dua et al. (2010) also suggested that

among semisolid formulations, carbopol gel base was most suitable dermatological

base for aceclofenac semisolid formulation with maximum release of aceclofenac.

These findings also support our microemulsion gel of aceclofenac for topical use.

5.10 SKIN IRRITATION STUDY

A major determining factor in choosing a surfactant is its safety because a large

amount of surfactants may cause skin irritation. Therefore, tween 80 which is non-

ionic surfactant was selected in aceclofenac microemulsion because it is less toxic

than ionic surfactants. Moreover, for o/w microemulsion, a surfactant with high HLB

value is required and HLB value of Tween 80 is 15 which fulfilled the purpose.

Before the application of formulations, all volunteers were screened through patch test

using blank formulations. MexameterTM

(Courage and Khazaka, Germany) was used

to measure Erythma and edema level before and after the application of formulations.

Before the application of formulations, the measured value of skin was used as a

control value. Modi and Patel (2011) also developed nanoemulsion based gel of

aceclofenac by using non-ionic surfactant and evaluated formulations for skin

irritation. They found that their formulations were non-irritant to skin. This study

supports our results.

5.11 HPLC METHOD DEVELOPMENT AND VALIDATION

A new HPLC-UV method was developed and validated according to FDA/ICH

guidelines. The precision and accuracy of the system was in the range of 98.22% to

99.80%. The method is specific and selective because it does not interfere with

excipients having detection limit of 0.050 µg/ml and quantitation limit of 0.250

µg/ml. The linearity of the system was between 0.320 µg/ml and 20 µg/ml. Freeze

thaw stability was performed by three freeze thaw cycles and the formulation was

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found stable. Extraction procedure was also validated by spiking drug in plasma and

recovered with validated HPLC method.

Calibration curves of aceclofenac were constructed in plasma and replicate assays

were done in three different periods and selectivity/specificity, linearity, precision,

accuracy, limit of quantitation, limit of detection and percent extraction yield were

evaluated.

5.11.1 Development of Assay Method

In developmental process of HPLC method, a mobile phase with several combinations

of buffer and organic phase as well as different mobile phases were investigated for

best resolution and rapid elution. Buffer, methanol and Acetonitrile in varying

percentages were tested and a mobile phase consisting of 20 mM potassium

dihydrogen phosphate-Acetonitrile in a molar ratio 60:40 (%v/v) which provided high

resolution and separation with sharp peak of aceclofenac. After different trials, pH

and molarity of mobile phase was selected and adjusted to pH 7.0 with 2M

Phosphoric acid. To determine optimal flow rate for pumping the mobile phase in

HPLC system, different flow rates were tested and 1ml/min. flow rate was found

appropriate.

Different stationary phases with suitable dimensions were also selected to improve

retention and separation. The run time was short requiring only 12 minutes. The

retention time for aceclofenac was 6.0 minutes.

5.11.2 Standardization/validation of HPLC Method

Following validation parameters were evaluated for validity of HPLC-UV method.

5.11.2.1 Accuracy and precision

The closeness of mean test results obtained by method to the true value

(concentration) of the analyte is called accuracy. Triplicate analysis of low, medium

and high concentrations of samples containing known amounts of analytes were done

to determine accuracy of method. A minimum of three concentrations in the range of

expected concentrations (intra-day) was measured to find accuracy in present method.

The measure of accuracy is the deviation of mean from true value.

The mean value of accuracy was 98.22%, 99.80% and 99.78% at low, medium and

high concentrations for aceclofenac, respectively. These values were found well

within the range as described by FDA for bioanalytical methods. The precision of an

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analytical method describes the closeness of individual measures of an analyte when

the procedure is applied repeatedly to multiple aliquots of a single homogenous

volume of biological fluid (inter-day). Precision was measured in triplicates with three

concentrations in the range of expected concentrations. The percent coefficient of

variation (%CV) in both intra-day and inter-day was less than 2% which is well

within the range of 15% for lowest and 20% for highest concentration of FDA criteria

for biological fluids.

5.11.2.2 Specificity/Selectivity

To determine the specificity/selectivity of the method, solutions of the blank

formulations and blank plasma were injected and response was recorded for any

interference with analyte to rectify the interference. There was no interference of

mobile phase or solvent or plasma residues with the analyte peak of interest. This

proves that the developed HPLC-UV method is specific and selective for the analyte

i.e. aceclofenac.

5.11.2.3 Detection limit and Quantitation limit

Gupta et al. (2011) defined limit of detection and limit of quantitation as “the lowest

level of drug that can be detected in sample is called limit of detection (LOD)”. The

lowest concentration at which the coefficient of variation (CV) and deviation from the

nominal concentration are less than 20% is called limit of quantification (LOQ).

Detection limit of the method was determined by sufficiently diluting the spiked

plasma sample and these diluted samples were injected into HPLC to check any

response on the detector and concentration at which the detector showed deflection

was the detection limit of method. Similarly, the concentration of plasma sample

spiked with aceclofenac which was determined with precision and accuracy, denoted

as quantitation limit. The detection limit and quantitation limit of current HPLC-UV

method are 0.050 µg/ml and 0.250 µg/ml, respectively.

5.11.2.4 Linearity and Range

With the help of calibration (standard) curve, linearity of the assay method was

determined and it was used to find out the relationship between instrument response

and known concentrations of the analytes. The calibration curves were prepared in the

expected same biological matrix i.e. plasma by spiking plasma matrix with known

concentrations of the analytes. The drug concentration in three replicates was run in

150

the HPLC system and the data was plotted and parameters of standard curve were

calculated. The slope, intercept and r-square have the values (mean ± SD) for

aceclofenac as 1.56 ± 0.03 (%CV of 1.8), 0.7 ± 0.011 (%CV of 1.7) and 0.988 ± 0.005

(%CV of 0.506), respectively. The method is linear over a range from 0.312 µg /ml to

20 µg/ml for plasma. These values of parameters were consistent with FDA

guidelines of bioanalytical method validation. Gupta et al. (2011) developed and

validated a new RP-HPLC method with UV detection for the determination of

aceclofenac in plasma. The method was linear from 0.5 to 12.0 µg /ml for plasma.

The retention time of aceclofenac was 7.20 minutes. The plasma method was found to

be precise (total coefficient of variation ranged from 0.82 to 4.63%), accurate and

specific during the study. Parameters shown in our study are comparable with the

previous reported values.

5.11.2.5 Freeze Thaw Stability of aceclofenac in plasma

In the current method, stability of analyte was determined after three freeze and thaw

cycles. Three aliquots at each of low, medium and high concentrations were stored at

-20˚C for 24 hours and thawed at room temperature. When completely thawed, the

samples were refrozen for 24 hours under the same conditions. The freeze-thaw cycle

should be repeated for three times, analyzed on each cycle for the determination of

drug in each aliquot. The method showed its stability for aceclofenac. Percent

difference for aceclofenac was 0.624 (cycle 1), 0.833 (cycle 2), 0.836 (cycle 3) for

low plasma concentration and for high concentrations it was 0.114 (cycle 1), 0.251

(cycle 2) and 0.177 (cycle 3). The values for low and high concentration for cycle 1,

cycle 2 and cycle 3 for aceclofenac (table 4.28) were found in acceptable range of

stability. The value of Percent Difference was less than 3 for all concentrations

included for stability testing. The method was found to be stable for testing

aceclofenac in human plasma.

5.11.2.6 Extraction yield/recovery of aceclofenac

The Extraction yield (recovery) of an analyte in an analysis method is the detector

response obtained from an amount of the analyte added to and extracted from the

plasma, compared to detector response obtained for known concentration of sample.

In current method, the extraction efficiency found to be consistent, precise and

reproducible. Recovery experiments were performed by comparing the analytical

results for extracted samples at two concentration levels with un-extracted standards

151

that represent 100% recovery. The percent extraction yield for aceclofenac at two

concentration levels i.e. low and high were 95.33% and 96.67%.The extraction yields

found in the present method were better than previously published method where %

recovery of 85-95.75% was reported by Gupta et al. (2011).

5.11.2.7 Application of method

The new validated HPLC-UV method was successfully applied for quantifying

aceclofenac in formulations, plasma samples of rabbits as well as volunteers.


Transdermal study of formulations was done on rabbits and from concentration found

in rabbit’s plasma; pharmacokinetic parameters were also determined using Kinetia

4.4.1 software. Pharmacokinetic parameters were determined from the concentration

of aceclofenac in human plasma and analyzed statistically for significance.

Significant difference was found between the plasma concentration of microemulsion

based aceclofenac gel and marketed conventional gel. This is because microemulsion

aceclofenac gel provided larger surface area for absorption as well as surfactants and

penetration enhancer also aided in transdermal absorption of aceclofenac.

In human volunteers, significant difference was found in plasma concentration of

aceclofenac Cmax and absorption rate constant Ka. This difference is due to high surface

area, greater penetration through skin by penetration enhancer and surfactants present

in formulation which also enhances solubility of drug in lipids content of skin as well

as widens the pores of stratum corneum.

However, difference between other pharmacokinetic parameters like time of peak

plasma concentration (Tmax), area under curve (AUC0-∞), area under the first moment

curve (AUMC0-∞), mean residence time (MRT), half life (t1/2), elimination rate

constant (Ke), Volume of distribution (Vd), total body clearance (ClT) and absorption

rate constant for both formulations were insignificant.

152

CONCLUSIONS

It is concluded that aceclofenac has higher solubility in almond oil and the flux,

Jss (µg/cm2/h) for aceclofenac microemulsion is higher than aceclofenac

microemulsion based gel. The percentage inhibition of inflammation and analgesic

effect was higher for aceclofenac microemulsion among all formulations. However,

between gels, microemulsion based aceclofenac gel has better anti-inflammatory and

analgesic effects than conventional marketed gel due to higher permeability release

rate. On the basis of pharmacokinetic analysis, it is concluded that microemulsion

based gel has greater bioavailability as compared to conventional marketed brand and

it has avoided GIT disturbances which enhanced patient compliance. Thus, the

hypothesis made under synopsis was completed successfully.

153

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167

APPENDIX I

FTIR spectra of active and excipients

Figure A1: FTIR of aceclofenac

Figure A2: FTIR of Almond oil

168

Figure A3: FTIR of Carbopol 940

Figure A4: FTIR of Tween 80

169

Figure A5: FTIR of Isopropyl alcohol

Figure A6: FTIR of Dimethyl Sulfoxide

170

Figure A7: FTIR of Triethylamine

171

APPENDIX II

Individual in vivo results of rabbits

Individual results of plasma concentration of aceclofenac in rabbits are given

in tables 4.38 to 4.61.

Table 4.38: Plasma conc. (µg/ml) of aceclofenac microemulsion based

gel administered as a topical dose of 2 mg ACF in Rabbit

AR1

Sr. No. Time

(hour) Plasma conc. aceclofenac µg/ml

1 0 0

2 1 0.941

3 2 2.224

4 3 3.573

5 6 6.608

6 12 3.077

7 24 0.316


plotted on rectangular co-ordinate graph, administered as a

topical dose of 2 mg (microemulsion based gel) in Rabbit AR 1


plotted on semi log graph, administered as a topical dose of

2 mg (microemulsion based gel) in Rabbit AR 1

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

172



AR 2

Sr. No. Time

(hour)

Plasma conc. aceclofenac

µg/ml

1 0 0

2 1 0.933

3 2 2.360

4 3 3.608

5 6 6.375

6 12 2.374

7 24 0.687


plotted on rectangular co-ordinate graph, administered as

a topical dose of 2 mg (microemulsion based gel) in Rabbit

AR 2


plotted on semi log graph, administered as a topical dose


0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6

Pla

sma

co

nc.

µg

/ml

Time (Hour)

173



AR 3

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.449

3 2 1.150

4 3 2.475

5 6 6.216

6 12 2.419

7 24 0.503




AR 3



of 20mg (microemulsion based gel) in Rabbit AR 3

0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6

Pla

sma

co

nc.

µg

/ml

Time (Hour)

174



AR 4

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.792

3 2 2.208

4 3 3.554

5 6 6.456

6 12 2.902

7 24 0.682



a topical dose of 2 mg (microemulsion based gel)in Rabbit

AR 4



of 2mg (microemulsion based gel) in Rabbit AR 4

0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6

Pla

sma

co

nc.

µg

/ml

Time (Hour)

175



AR 5

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 1.035

3 2 2.632

4 3 4.135

5 6 6.873

6 12 2.374

7 24 0.324



a topical dose of 2 mg (microemulsion based gel)in Rabbit

AR 5




0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

176



AR 6

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.515

3 2 1.935

4 3 3.344

5 6 6.632

6 12 1.164

7 24 0.326




AR 6




0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

177



AR 7

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.511

3 2 1.794

4 3 3.344

5 6 6.095

6 12 2.324

7 24 0.164




AR 7




0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6

Pla

sma

co

nc.

µg

/ml

Time (Hour)

178



AR 8

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.470

3 2 2.356

4 3 4.544

5 6 6.437

6 12 2.698

7 24 0.666




AR 8




0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6

Pla

sma

co

nc.

µg

/ml

Time (Hour)

179



AR 9

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.516

3 2 1.970

4 3 3.378

5 6 6.712

6 12 2.165

7 24 0.133




AR 9




0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

180



AR 10

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.811

3 2 2.226

4 3 3.334

5 6 6.836

6 12 1.666

7 24 0.429




AR 10




0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

181



AR 11

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.793

3 2 2.648

4 3 4.227

5 6 6.974

6 12 1.882

7 24 0.491




AR 11




0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

182



AR 12

Sr. No. Time

(hour)


µg/ml

1 0 0

2 1 0.678

3 2 1.460

4 3 2.605

5 6 6.545

6 12 2.585

7 24 0.268




AR 12




0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6

Pla

sma

co

nc.

µg

/ml

Time (Hour)

183

Table 4.50: Plasma concentration (µg/ml) of aceclofenac marketed gel

administered as a topical dose of 2 mg aceclofenac in

Rabbit R 1

Sr. No. Time

(hour) Plasma conc. aceclofenac µg/ml

1 0 0

2 1 0.291

3 2 1.553

4 3 2.851

5 6 3.898

6 12 1.771

7 24 0.323



a topical dose of 2 mg in Rabbit R 1



of 2 mg in Rabbit R 1

0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

184



Rabbit R 2

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.621

3 2 1.469

4 3 2.397

5 6 3.361

6 12 1.649

7 24 0.112







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

185



Rabbit R 3

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.462

3 2 1.108

4 3 2.86

5 6 3.736

6 12 1.607

7 24 0.096







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

186



Rabbit R 4

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.669

3 2 1.691

4 3 2.287

5 6 3.386

6 12 1.838

7 24 0.141







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

187



Rabbit R 5

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.921

3 2 1.107

4 3 2.393

5 6 2.955

6 12 1.022

7 24 0.368







0

1

2

3

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6

Pla

sma

co

nc.

µg

/ml

Time (Hour)

188


administered as a topical dose of 2 mg aceclofenac in Rabbit

R 6

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.778

3 2 1.691

4 3 2.882

5 6 3.685

6 12 1.643

7 24 0.189







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

189



Rabbit R 7

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.278

3 2 1.887

4 3 2.196

5 6 3.044

6 12 1.793

7 24 0.365







0

1

2

3

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

190



Rabbit R 8

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.118

3 2 1.553

4 3 2.683

5 6 3.643

6 12 1.372

7 24 0.351







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

100.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

191



Rabbit R 9

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.277

3 2 1.759

4 3 2.759

5 6 3.415

6 12 1.016

7 24 0.122







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

100.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

192



Rabbit R 10

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.938

3 2 1.13

4 3 2.687

5 6 3.273

6 12 1.749

7 24 0.206







0

1

2

3

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

193



Rabbit R 11

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.877

3 2 1.808

4 3 2.28

5 6 3.416

6 12 1.495

7 24 0.093







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

194



Rabbit R 12

Sr. No. Time

(hour)


(µg/ml)

1 0 0

2 1 0.294

3 2 1.159

4 3 2.096

5 6 4.103

6 12 1.725

7 24 0.033







0

1

2

3

4

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5

Pla

sma

co

nc.

µg

/ml

Time (Hour)

195

APPENDIX III

In-vivo determination of aceclofenac in humans (Marketed conventional

gel):

Individual plasma concentration of aceclofenac in volunteers from marketed

conventional aceclofenac gel are given in Table 4.62 to 4.79.

Table 4.62: Concentration of ACF in human plasma calculated from



Sample

No.

Time

(hour) Peak Height Ratio

Plasma conc.

aceclofenac

µg/ml

1 0 0 0

2 0.5 0.267 0.103

3 1 0.781 0.526

4 1.5 3.749 2.971

5 2 4.792 3.830

6 3 5.790 4.652

7 4 6.491 5.229

8 5 7.660 6.191

9 6 8.571 6.942

10 8 4.055 3.223

11 12 1.274 0.932

12 24 0.425 0.233

196



a topical dose of 20 mg marketed gel in volunteer 1



of 20mg marketed gel in volunteer 1

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

197




Sample No. Time

(hour)

Peak Height

Ratio

Plasma conc.

aceclofenac

µg/ml

1 0 0 0

2 0.5 0.213 0.059

3 1 0.797 0.540

4 1.5 1.149 0.829

5 2 1.967 1.503

6 3 3.358 2.649

7 4 4.310 3.433

8 5 7.326 5.916

9 6 8.852 7.174

10 8 3.888 3.085

11 12 1.655 1.246

12 24 0.391 0.205

198







0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

199




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.747 0.498

3 1 1.788 1.356

1.5 3.916 3.108

5 2 4.979 3.983

6 3 6.349 5.112

7 4 7.107 5.737

8 5 8.043 6.507

9 6 9.506 7.712

10 8 6.761 5.451

11 12 3.533 2.793

12 24 0.698 0.457

200

Figure 4.104 : Plasma concentration verses time profile of aceclofenac






0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9

Pla

sma

co

nc.

µg

/ml

Time (Hour)

201




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.954 0.669

3 1 1.226 0.893

4 1.5 2.195 1.691

5 2 3.774 2.991

6 3 6.250 5.030

7 4 7.957 6.437

8 5 9.391 7.618

9 6 7.014 5.659

10 8 3.149 2.477

11 12 1.447 1.075

12 24 0.331 0.156

202







0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

203




Sample No. Time (hour) Peak Height Ratio Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.571 0.353

3 1 0.839 0.574

4 1.5 1.503 1.121

5 2 2.710 2.115

6 3 4.141 3.294

7 4 5.368 4.304

8 5 6.748 5.440

9 6 9.343 7.577

10 8 6.200 4.989

11 12 1.278 0.936

12 24 0.519 0.311

204


plotted on rectangular co-ordinate graph, administered

as a topical dose of 20 mg marketed gel in volunteer 5


plotted on semi log graph, administered as a topical

dose of 20mg marketed gel in volunteer 5

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

205




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.674 0.438

3 1 0.968 0.681

4 1.5 2.414 1.871

5 2 2.976 2.334

6 3 4.177 3.323

7 4 6.133 4.934

8 5 8.520 6.900

9 6 6.122 4.925

10 8 3.443 2.718

11 12 1.619 1.217

12 24 0.357 0.177

206







0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

207




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.875 0.604

3 1 1.675 1.262

4 1.5 2.188 1.685

5 2 3.800 3.012

6 3 5.182 4.151

7 4 6.008 4.831

8 5 7.098 5.729

9 6 8.072 6.531

10 8 5.710 4.585

11 12 2.949 2.312

12 24 0.456 0.259

208







0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

209




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 1.090 0.781

3 1 1.401 1.037

4 1.5 1.738 1.315

5 2 2.904 2.275

6 3 4.671 3.730

7 4 5.108 4.090

8 5 6.583 5.304

9 6 8.722 7.066

10 8 3.849 3.053

11 12 1.900 1.448

12 24 0.360 0.180

210







0

1

2

3

4

5

6

7

8

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9 10

Pla

sma

co

nc.

µg

/ml

Time (Hour)

211




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.744 0.496

3 1 1.184 0.858

4 1.5 2.178 1.677

5 2 3.328 2.624

6 3 4.175 3.321

7 4 6.402 5.156

8 5 7.328 5.918

9 6 7.949 6.429

10 8 5.712 4.587

11 12 0.820 0.558

12 24 0.501 0.296

212







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

213



marketed aceclofenac gel in volunteer 10

Sample No. Time

(hour)

Peak Height

Ratio

Plasma conc.

aceclofenac

µg/ml

1 0 0 0

2 0.5 0.663 0.429

3 1 1.182 0.857

4 1.5 1.682 1.268

5 2 2.263 1.747

6 3 3.193 2.513

7 4 5.077 4.065

8 5 6.252 5.032

9 6 7.815 6.319

10 8 6.113 4.917

11 12 2.396 1.856

12 24 0.524 0.315

214







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

215




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.413 0.223

3 1 0.908 0.631

4 1.5 1.743 1.318

5 2 2.749 2.147

6 3 4.339 3.457

7 4 5.379 4.313

8 5 6.281 5.056

9 6 7.472 6.037

10 8 5.073 4.061

11 12 1.889 1.439

12 24 0.432 0.239

216







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9 10

Pla

sma

co

nc.

µg

/ml

Time (Hour)

217




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.498 0.294

3 1 0.938 0.656

4 1.5 1.940 1.481

5 2 3.243 2.554

6 3 4.990 3.993

7 4 5.726 4.599

8 5 6.802 5.485

9 6 7.357 5.942

10 8 5.364 4.301

11 12 2.971 2.330

12 24 0.616 0.390

218







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

219




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.411 0.222

3 1 1.256 0.917

4 1.5 2.099 1.612

5 2 2.449 1.900

6 3 3.403 2.686

7 4 4.858 3.884

8 5 6.644 5.355

9 6 7.318 5.910

10 8 4.888 3.909

11 12 2.891 2.264

12 24 0.457 0.260

220







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

221




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.484 0.282

3 1 1.426 1.058

4 1.5 2.942 2.306

5 2 3.815 3.025

6 3 4.720 3.771

7 4 5.638 4.526

8 5 6.340 5.104

9 6 7.368 5.951

10 8 5.349 4.289

11 12 2.729 2.131

12 24 0.450 0.254

222







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9

Pla

sma

co

nc.

µg

/ml

Time (Hour)

223




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.609 0.384

3 1 1.459 1.085

4 1.5 2.990 2.345

5 2 3.868 3.069

6 3 5.163 4.135

7 4 6.652 5.361

8 5 7.000 5.648

9 6 7.318 5.910

10 8 5.462 4.382

11 12 2.858 2.237

12 24 0.534 0.323

224







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9 10

Pla

sma

co

nc.

µg

/ml

Time (Hour)

225




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.590 0.369

3 1 0.922 0.642

4 1.5 2.059 1.579

5 2 3.185 2.507

6 3 4.465 3.561

7 4 5.875 4.721

8 5 7.098 5.729

9 6 7.473 6.038

10 8 5.185 4.153

11 12 2.688 2.097

12 24 0.451 0.254

226







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

227




Sample No. Time


Plasma conc.

aceclofenac µg/ml

1 0 0 0

2 0.5 0.403 0.215

3 1 0.851 0.584

4 1.5 1.348 0.994

5 2 2.136 1.642

6 3 3.522 2.784

7 4 5.038 4.032

8 5 6.322 5.089

9 6 7.146 5.768

10 8 4.726 3.776

11 12 1.546 1.156

12 24 0.439 0.245

228







0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

229





aceclofenac µg/ml

1 0 0 0

2 0.5 0.596 0.374

3 1 1.209 0.879

4 1.5 1.636 1.231

5 2 2.411 1.868

6 3 3.918 3.110

7 4 4.612 3.682

8 5 6.263 5.041

9 6 7.219 5.828

10 8 4.832 3.863

11 12 2.105 1.617

12 24 0.418 0.228

230






dose of 20mg marketed gel in volunteer 18.

0

1

2

3

4

5

6

7

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

231

APPENDIX IV

In-vivo determination of aceclofenac in humans (Microemulsion based

aceclofenac gel):

Individual plasma concentration of aceclofenac in volunteers from

microemulsion based aceclofenac gel are given in table 4.80 to 4.97.





aceclofenac µg/ml

1 0 0 0

2 0.5 1.053 0.267

3 1 2.321 1.077

4 1.5 3.005 1.515

5 2 4.121 2.228

6 3 5.879 3.352

7 4 8.145 4.801

8 5 15.071 9.229

9 6 11.012 6.634

10 8 7.399 4.324

11 12 3.816 2.033

12 24 1.241 0.387

232



as a topical dose of 20 mg microemulsion based gel in

volunteer 1



dose of 20mg microemulsion based gel in volunteer 1

0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9

Pla

sma

co

nc.

µg

/ml

Time (Hour)

233





aceclofenac µg/ml

1 0 0 0

2 0.5 0.947 0.199

3 1 1.299 0.424

4 1.5 3.104 1.578

5 2 4.269 2.323

6 3 6.989 4.062

7 4 10.394 6.239

8 5 12.186 7.385

9 6 14.717 9.003

10 8 8.864 5.261

11 12 4.719 2.610

12 24 1.162 0.336

234




volunteer 2




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

235





aceclofenac µg/ml

1 0 0 0

2 0.5 0.769 0.445

3 1 2.534 1.729

4 1.5 3.386 2.348

5 2 5.090 3.586

6 3 8.292 5.913

7 4 10.602 7.592

8 5 12.500 8.971

9 6 8.677 6.193

10 8 3.824 2.666

11 12 2.349 1.594

12 24 0.475 0.232

236



a topical dose of 20 mg microemulsion based gel in

volunteer 3



of 20mg microemulsion based gel in volunteer 3

0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

237





aceclofenac µg/ml

1 0 0 0

2 0.5 0.286 0.094

3 1 2.005 1.344

1.5 3.350 2.321

5 2 4.979 3.505

6 3 6.762 4.801

7 4 9.372 6.698

8 5 12.100 8.681

9 6 8.041 5.731

10 8 5.587 3.947

11 12 3.057 2.109

12 24 0.536 0.276

238




volunteer 4




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9

Pla

sma

co

nc.

µg

/ml

Time (Hour)

239





aceclofenac µg/ml

1 0 0 0

2 0.5 1.105 0.690

3 1 1.542 1.007

4 1.5 1.645 1.082

5 2 2.640 1.805

6 3 4.073 2.847

7 4 7.472 5.317

8 5 9.177 6.556

9 6 11.906 8.540

10 8 4.766 3.350

11 12 2.066 1.388

12 24 0.442 0.208

240




volunteer 5




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

241


chromatograms by forecasting method after

administration of microemulsion based aceclofenac gel in

volunteer 6


aceclofenac µg/ml

1 0 0 0

2 0.5 0.965 0.588

3 1 1.915 1.278

4 1.5 3.305 2.288

5 2 4.205 2.943

6 3 5.798 4.101

7 4 7.644 5.442

8 5 10.170 7.278

9 6 12.364 8.873

10 8 8.742 6.240

11 12 5.047 3.555

12 24 0.682 0.382

242




volunteer 6




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9 10

Pla

sma

co

nc.

µg

/ml

Time (Hour)

243





aceclofenac µg/ml

1 0 0 0

2 0.5 0.706 0.399

3 1 1.394 0.899

4 1.5 2.641 1.806

5 2 4.197 2.937

6 3 5.691 4.023

7 4 7.268 5.169

8 5 11.986 8.598

9 6 8.841 6.312

10 8 3.857 2.690

11 12 1.814 1.205

12 24 0.473 0.231

244




volunteer 7




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

245





aceclofenac µg/ml

1 0 0 0

2 0.5 0.729 0.416

3 1 1.026 0.632

4 1.5 2.148 1.448

5 2 3.298 2.283

6 3 5.428 3.832

7 4 6.683 4.744

8 5 8.531 6.087

9 6 11.724 8.408

10 8 4.900 3.448

11 12 1.579 1.034

12 24 0.532 0.273

246




volunteer 8




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

247





aceclofenac µg/ml

1 0 0 0

2 0.5 0.772 0.447

3 1 1.346 0.865

4 1.5 2.450 1.667

5 2 3.232 2.235

6 3 4.541 3.187

7 4 6.640 4.713

8 5 10.316 7.385

9 6 6.835 4.854

10 8 2.898 1.993

11 12 1.506 0.981

12 24 0.379 0.162

248




volunteer 9




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

249





aceclofenac µg/ml

1 0 0 0

2 0.5 0.651 0.360

3 1 1.323 0.848

4 1.5 3.292 2.279

5 2 4.683 3.290

6 3 5.892 4.169

7 4 6.332 4.488

8 6 8.189 5.838

9 8 11.316 8.111

10 10 5.940 4.204

11 12 2.175 1.467

12 24 0.568100358 0.299379867

250

Figure 4.154: Plasma concentration verses time profile of

aceclofenac plotted on rectangular co-ordinate graph,

administered as a topical dose of 20 mg microemulsion

based gel in volunteer 10

Figure 4.155: Plasma concentration verses time profile of

aceclofenac plotted on semi log graph, administered as

a topical dose of 20mg microemulsion based gel in

volunteer 10

0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9 10

Pla

sma

co

nc.

µg

/ml

Time (Hour)

251





aceclofenac µg/ml

1 0 0 0

2 0.5 0.648 0.357

3 1 1.141 0.716

4 1.5 2.944 2.026

5 2 3.690 2.569

6 3 5.096 3.590

7 4 6.088 4.311

8 5 10.364 7.419

9 6 4.547 3.192

10 8 1.709 1.128

11 12 1.240 0.787

12 24 0.269 0.082

252




volunteer 11




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

253





aceclofenac µg/ml

1 0 0 0

2 0.5 0.600 0.323

3 1 1.052 0.651

4 1.5 1.734 1.147

5 2 4.595 3.226

6 3 6.376 4.521

7 4 8.018 5.714

8 5 9.868 7.059

9 6 11.390 8.165

10 8 7.382 5.252

11 12 3.772 2.628

12 24 0.664 0.369

254




volunteer 12




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

255





aceclofenac µg/ml

1 0 0 0

2 0.5 0.453 0.216

3 1 1.655 1.090

4 1.5 3.215 2.223

5 2 4.919 3.462

6 3 6.005 4.251

7 4 8.089 5.765

8 5 10.527 7.538

9 6 7.075 5.028

10 8 4.101 2.867

11 12 1.319 0.845

12 24 0.551 0.287

256




volunteer 13




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9 10

Pla

sma

co

nc.

µg

/ml

Time (Hour)

257





aceclofenac µg/ml

1 0 0 0

2 0.5 0.573 0.303

3 1 1.328 0.851

4 1.5 3.733 2.600

5 2 4.824 3.393

6 3 7.595 5.407

7 4 9.108 6.506

8 6 11.923 8.552

9 8 7.861 5.600

10 10 3.357 2.327

11 12 1.352 0.869

12 24 0.356 0.145

258




volunteer 14




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7

Pla

sma

co

nc.

µg

/ml

Time (Hour)

259





aceclofenac µg/ml

1 0 0 0

2 0.5 0.314 0.115

3 1 1.590 1.042

4 1.5 3.317 2.297

5 2 3.842 2.679

6 3 4.698 3.301

7 4 5.861 4.147

8 6 11.499 8.244

9 8 7.129 5.068

10 10 3.252 2.250

11 12 1.536 1.003

12 24 0.402 0.179

260




volunteer 15




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9

Pla

sma

co

nc.

µg

/ml

Time (Hour)

261





aceclofenac µg/ml

1 0 0 0

2 0.5 0.676 0.378

3 1 1.181 0.745

4 1.5 2.235 1.511

5 2 3.772 2.628

6 3 5.515 3.895

7 4 7.718 5.496

8 5 9.180 6.559

9 6 10.707 7.669

10 8 5.476 3.867

11 12 2.817 1.934

12 24 0.494 0.246

262




volunteer 16




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

263





aceclofenac µg/ml

1 0 0 0

2 0.5 0.429 0.199

3 1 1.451 0.941

4 1.5 2.781 1.908

5 2 4.418 3.097

6 3 5.706 4.034

7 4 6.488 4.602

8 6 7.880 5.614

9 8 10.533 7.542

10 10 3.127 2.159

11 12 2.035 1.366

12 24 0.354 0.144

264




volunteer 17




0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8

Pla

sma

co

nc.

µg

/ml

Time (Hour)

265





aceclofenac µg/ml

1 0 0 0

2 0.5 0.383 0.165

3 1 1.420 0.918

4 1.5 2.888 1.986

5 2 4.262 2.984

6 3 5.676 4.012

7 4 7.194 5.116

8 5 9.421 6.734

9 6 11.823 8.480

10 8 6.348 4.500

11 12 3.607 2.508

12 24 0.583 0.310

266




volunteer 18


plotted on semi log graph, administered as a topical dose of

20 mg microemulsion based gel in volunteer 18

0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25

Pla

sma

co

nc.

µg

/ml

Time (Hour)

0.1

1.0

10.0

1 2 3 4 5 6 7 8 9 10

Pla

sma

co

nc.

µg

/ml

Time (Hour)

153

APPENDIX V

Chromatograms of blank plasma and spiked plasma

267

t he is lam ia u niver sity of b ah awa lpu r

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