t he is lam ia u niver sity of b ah awa lpu r
TRANSCRIPT
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)
1
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.
Muhammad Zubair Malik
I
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!
Muhammad Zubair Malik
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
5.4.12 Thermo Gravimetric Analysis (TGA) and Differential Scanning
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
FIGURE DESCRIPTION PAGE
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
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in 12 rabbits of group AR individually
111
XVI
FIGURE DESCRIPTION PAGE
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
114
4.40
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(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
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg (Marketed conventional gel) in 18 volunteers
125
4.45
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
(Marketed conventional gel)in 18 volunteers
125
4.46
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg (Microemulsion based aceclofenac gel)in 18 volunteers
127
4.47
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
(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
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.
129
4.50
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 2 mg in 24
Rabbits.
130
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
130
A1 FTIR Spectrum of aceclofenac 167
A2 FTIR Spectrum of almond oil 167
A3 FTIR Spectrum of Carbopol 940 168
XVII
FIGURE DESCRIPTION PAGE
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
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 Rabbit AR 1
171
4.53
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg in
Rabbit AR 1
171
4.54
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 Rabbit AR 2
172
4.55
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 2
172
4.56
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 Rabbit AR 3
173
4.57
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
(microemulsion based gel) in Rabbit AR 3
173
4.58
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 Rabbit AR 4
174
4.59
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2mg
(microemulsion based gel) in Rabbit AR 4
174
4.60
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 Rabbit AR 5
175
4.61
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 5
175
XVIII
FIGURE DESCRIPTION PAGE
4.62
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 Rabbit AR 6
176
4.63
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 6
176
4.64
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 Rabbit AR 7
177
4.65
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 7
177
4.66
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 Rabbit AR 8
178
4.67
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 8
178
4.68
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 Rabbit AR 9
179
4.69
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 9
179
4.70
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 Rabbit AR 10
180
4.71
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 10
180
4.72
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 Rabbit AR 11
181
4.73
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 11
181
XIX
FIGURE DESCRIPTION PAGE
4.74
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 Rabbit AR 12
182
4.75
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(microemulsion based gel) in Rabbit AR 12
182
4.76
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 1
183
4.77
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 1
183
4.78
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 2
184
4.79
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 2
184
4.80
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 3
185
4.81
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 3
185
4.82
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 4
186
4.83
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 4
186
4.84
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 5
187
4.85
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 5
187
4.86
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 6
188
4.87
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 6
188
4.88
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 7
189
XX
FIGURE DESCRIPTION PAGE
4.89
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 7
189
4.90
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 8
190
4.91
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 8
190
4.92
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 9
191
4.93
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 9
191
4.94
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 10
192
4.95
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 10
192
4.96
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 11
193
4.97
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 11
193
4.98
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (marketed conventional gel) in Rabbit R 12
194
4.99
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(marketed conventional gel) in Rabbit R 12
194
4.100
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 1
196
4.101
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 1
196
4.102
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 2
198
4.103
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 2
198
XXI
FIGURE DESCRIPTION PAGE
4.104
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 3
200
4.105
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 3
200
4.106
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 4
202
4.107
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 4
202
4.108
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 5
204
4.109
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 5
204
4.110
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 6
206
4.111
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 6
206
4.112
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 7
208
4.113
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 7
208
4.114
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 8
210
4.115
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 8
210
4.116
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 9
212
4.117
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 9
212
4.118
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 10
214
XXII
FIGURE DESCRIPTION PAGE
4.119
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 10
214
4.120
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 11
216
4.121
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 11
216
4.122
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 12
218
4.123
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 12
218
4.124
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 13
220
4.125
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 13
220
4.126
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 14
222
4.127
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 14
222
4.128
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 15
224
4.129
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 15
224
4.130
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 16
226
4.131
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 16
226
4.132
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 17
228
4.133
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 17
228
XXIII
FIGURE DESCRIPTION PAGE
4.134
Plasma concentration verses time profile of Aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg marketed gel in volunteer 18
230
4.135
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
marketed gel in volunteer 18
230
4.136
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 1
232
4.137
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 1
232
4.138
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 2
234
4.139
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 2
234
4.140
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 3
236
4.141
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 3
236
4.142
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 4
238
4.143
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 4
238
4.144
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 5
240
4.145
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 5
240
4.146
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 6
242
4.147
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 6
242
4.148
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 7
244
XXIV
FIGURE DESCRIPTION PAGE
4.149
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 7
244
4.150
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 8
246
4.151
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 8
246
4.152
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 9
248
4.153
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 9
248
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
250
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
250
4.156
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 11
252
4.157
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 11
252
4.158
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 12
254
4.159
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 12
254
4.160
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 13
256
4.161
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 13
256
4.162
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 14
258
4.163
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 14
258
XXV
FIGURE DESCRIPTION PAGE
4.164
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 15
260
4.165
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 15
260
4.166
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 16
262
4.167
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 16
262
4.168
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 17
264
4.169
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
microemulsion based gel in volunteer 17
264
4.170
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 18
266
4.171
Plasma concentration verses time profile of Aceclofenac plotted
on semi log graph, administered as a topical dose of 20 mg
microemulsion based gel in volunteer 18
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
TABLE DESCRIPTION PAGE
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
chromatograms by forecasting method after administration of
marketed aceclofenac gel 124
4.31 Concentration of ACF in plasma calculated from
chromatograms by forecasting method after administration of
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
TABLE DESCRIPTION PAGE
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
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 3
173
4.41
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 4
174
4.42
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 5
175
4.43
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 6
176
4.44
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 7
177
4.45
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 8
178
4.46
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 9
179
4.47
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 10
180
4.48
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 11
181
4.49
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit AR 12
182
4.50
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 1
183
4.51
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 2
184
XXIX
TABLE DESCRIPTION PAGE
4.52
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 3
185
4.53
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 4
186
4.54
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 5
187
4.55
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 6
188
4.56
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 7
189
4.57
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 8
190
4.58
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 9
191
4.59
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 10
192
4.60
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
Rabbit R 11
193
4.61
Plasma concentration (µg/ml) of Aceclofenac marketed gel
A administered as a topical dose of 2 mg Aceclofenac in
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
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 2
197
4.64
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 3
199
4.65
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 4
201
XXX
TABLE DESCRIPTION PAGE
4.66
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 5
203
4.67
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 6
205
4.68
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 7
207
4.69
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 8
209
4.70
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 9
211
4.71
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 10
213
4.72
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 11
215
4.73
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 12
217
4.74
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 13
219
4.75
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 14
221
4.76
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 15
223
4.77
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 16
225
4.78
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 17
227
4.79
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 18
229
XXXI
TABLE DESCRIPTION PAGE
4.80
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 1
231
4.81
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 2
233
4.82
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 3
235
4.83
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 4
237
4.84
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 5
239
4.85
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 6
241
4.86
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 7
243
4.87
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 8
245
4.88
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 9
247
4.89
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 10
249
4.90
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 11
251
4.91
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 12
253
4.92
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 13
255
4.93
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 14
257
XXXII
TABLE DESCRIPTION PAGE
4.94
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 15
259
4.95
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 16
261
4.96
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 17
263
4.97
Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 18
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
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
48
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
49
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
52
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
54
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
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
2 1:8 E E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME
3 1:7 E E E ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME
4 1:6 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)
Microemulsions containing oleic acid and almond oil were prepared by the same
method as A.
D) Tween 80 and isopropyl alcohol (2:1)
Microemulsions containing oleic acid and almond oil were prepared by the same
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.
3.7.13 Thermo Gravimetric Analysis (TGA) and Differential Scanning
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)
Aceclofenac concentration
(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
Aceclofenac concentration (µg/ml)
82
Table 4.3 Standard curve of aceclofenac in n-butanol (n=3)
Aceclofenac concentration
(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)
Aceclofenac concentration (µg/ml)
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
Aceclofenac concentration (µg/ml)
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
4.5.2 Scanning Electron Microscope (SEM)
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
4.5.4 X-Ray Diffraction (XRD)
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
4.5.5 Thermo Gravimetric Analysis (TGA) and Differential Scanning
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)
Zeta Distribution Data
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)
Zeta Distribution Data
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
-200 -100 0 100 200
Tota
l C
ounts
Zeta potential (mV)
Zeta Distribution Data
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)
Number Distribution Data
(%)
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)
Number Distribution Data
(%)
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)
Number Distribution Data
(%)
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)
Number Distribution Data
(%)
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
on rectangular co-ordinate graph, administered as a topical dose
of 2 mg (microemulsion based gel) in 12 Rabbits of group AR
individually
Figure 4.38: Plasma concentration verses time profile of aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(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
Figure 4.40: Plasma concentration verses time profile of aceclofenac plotted
on semi log graph, administered as a topical dose of 2 mg
(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
4.7 ANTI-INFLAMMATORY ACTIVITY STUDY IN RATS
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
4.8 STUDY OF ANALGESIC EFFECT IN RATS
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
Figure 4.44: Plasma concentration verses time profile of aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical dose
of 20 mg (Marketed conventional gel) in 18 volunteers.
Figure 4.45: Plasma concentration verses time profile of aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
(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)
Vounteer 1Vounteer 2Vounteer 3Vounteer 4Vounteer 5Vounteer 6Vounteer 7Vounteer 8Vounteer 9Vounteer 10Vounteer 11Vounteer 12Vounteer 13Vounteer 14Vounteer 15Vounteer 16Vounteer 17Vounteer 18
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
Figure 4.46: Plasma concentration verses time profile of aceclofenac plotted
on rectangular co-ordinate graph, administered as a topical
dose of 20 mg (Microemulsion based aceclofenac gel) in 18
volunteers
Figure 4.47: Plasma concentration verses time profile of aceclofenac plotted
on semi log graph, administered as a topical dose of 20mg
(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)
Vounteer 1Vounteer 2Vounteer 3Vounteer 4Vounteer 5Vounteer 6Vounteer 7Vounteer 8Vounteer 9Vounteer 10Vounteer 11Vounteer 12Vounteer 13Vounteer 14Vounteer 15Vounteer 16Vounteer 17Vounteer 18
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
from ME Gel and Conventional gel plotted on rectangular co-
ordinate graph, administered as a topical dose of 2 mg in 24
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)
4.12 PHARMACOKINETIC PARAMETERS
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.
Modi and Patel (2011) prepared and evaluated nanoemulsion based aceclofenac gel
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).
Moghimipour et al. (2013) designed and characterized microemulsion systems for
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.
Modi and Patel (2011) prepared and evaluated nanoemulsion based aceclofenac gel
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).
Moghimipour et al. (2013) designed and characterized microemulsion systems for
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%.
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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.
5.4.10 Scanning Electron Microscope (SEM)
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.
5.4.12 Thermo Gravimetric Analysis (TGA) and Differential Scanning
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.
5.4.13 X-Ray Diffraction (XRD)
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.
5.8 ANTI-INFLAMMATORY ACTIVITY STUDY IN RATS
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.
5.9 STUDY OF ANALGESIC EFFECT IN RATS
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.
5.12 PHARMACOKINETIC PARAMETERS
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
Figure 4.52: 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 Rabbit AR 1
Figure 4.53: Plasma concentration verses time profile of aceclofenac
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
Table 4.39: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
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
Figure 4.54: 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 Rabbit
AR 2
Figure 4.55: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit AR 2
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
Table 4.40: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 3
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.56: 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 Rabbit
AR 3
Figure 4.57: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
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
Table 4.41: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 4
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.58: 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 Rabbit
AR 4
Figure 4.59: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
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
Table 4.42: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 5
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.60: 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 Rabbit
AR 5
Figure 4.61: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as 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
Table 4.43: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 6
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.62: 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 Rabbit
AR 6
Figure 4.63: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit 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
Table 4.44: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 7
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.64: 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 Rabbit
AR 7
Figure 4.65: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit 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
Table 4.45: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 8
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.66: 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 Rabbit
AR 8
Figure 4.67: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit 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
Table 4.46: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 9
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.68: 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 Rabbit
AR 9
Figure 4.69: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit 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
Table 4.47: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 10
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.70: 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 Rabbit
AR 10
Figure 4.71: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit 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
Table 4.48: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 11
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.72: 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 Rabbit
AR 11
Figure 4.73: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit 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
Table 4.49: Plasma conc. (µg/ml) of aceclofenac microemulsion based
gel administered as a topical dose of 2 mg ACF in Rabbit
AR 12
Sr. No. Time
(hour)
Plasma conc. aceclofenac
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
Figure 4.74: 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 Rabbit
AR 12
Figure 4.75: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg (microemulsion based gel) in Rabbit 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
Figure 4.76: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 1
Figure 4.77: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
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
Table 4.51: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 2
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.78: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 2
Figure 4.79: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 2
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
Table 4.52: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 3
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.80: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 3
Figure 4.81: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 3
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
Table 4.53: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 4
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.82: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 4
Figure 4.83: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 4
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
Table 4.54: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 5
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.84: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 5
Figure 4.85: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 5
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
Table 4.55: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in Rabbit
R 6
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.86: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 6
Figure 4.87: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 6
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
Table 4.56: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 7
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.88: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 7
Figure 4.89: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 7
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
Table 4.57: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 8
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.90: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 8
Figure 4.91: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 8
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
Table 4.58: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 9
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.92: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 9
Figure 4.93: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 9
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
Table 4.59: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 10
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.94: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 10
Figure 4.95: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 10
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
Table 4.60: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 11
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.96: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 11
Figure 4.97: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 11
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
Table 4.61: Plasma concentration (µg/ml) of aceclofenac marketed gel
administered as a topical dose of 2 mg aceclofenac in
Rabbit R 12
Sr. No. Time
(hour)
Plasma conc. aceclofenac
(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
Figure 4.98: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 2 mg in Rabbit R 12
Figure 4.99: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 2 mg in Rabbit R 12
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
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 1
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
Figure 4.100: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 20 mg marketed gel in volunteer 1
Figure 4.101: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
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
Table 4.63: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 2
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
Figure 4.102: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 20 mg marketed gel in volunteer 2
Figure 4.103: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg marketed gel in volunteer 2
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
Table 4.64: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 3
Sample No. Time
(hour) Peak Height Ratio
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
plotted on rectangular co-ordinate graph, administered as
a topical dose of 20 mg marketed gel in volunteer 3
Figure 4.105: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg marketed gel in volunteer 3
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
Table 4.65: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 4
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.106: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 20 mg marketed gel in volunteer 4
Figure 4.107: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg marketed gel in volunteer 4
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
Table 4.66: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 5
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
Figure 4.108 : Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 5
Figure 4.109: Plasma concentration verses time profile of aceclofenac
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
Table 4.67: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 6
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.110: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 6
Figure 4.111: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 6
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
Table 4.68: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 7
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.112: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 7
Figure 4.113: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 7
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
Table 4.69: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 8
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.114: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 8
Figure 4.115: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 8
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
Table 4.70: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 9
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.116: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 9
Figure 4.117: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 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 6 7
Pla
sma
co
nc.
µg
/ml
Time (Hour)
213
Table 4.71: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration of
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
Figure 4.118: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 10
Figure 4.119: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 10
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
Table 4.72: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 11
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.120: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 20 mg marketed gel in volunteer 11
Figure 4.121: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg marketed gel in volunteer 11
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
Table 4.73: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 12
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.122 : Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 12
Figure 4.123: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 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 7 8
Pla
sma
co
nc.
µg
/ml
Time (Hour)
219
Table 4.74: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 13
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.124: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 13
Figure 4.125: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 13
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
Table 4.75: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 14
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.126: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered as
a topical dose of 20 mg marketed gel in volunteer 14
Figure 4.127: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg marketed gel in volunteer 14
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
Table 4.76: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 15
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.128: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 15
Figure 4.129: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 15
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
Table 4.77: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 16
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.130: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 16
Figure 4.131: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 16
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
Table 4.78: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 17
Sample No. Time
(hour) Peak Height Ratio
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
Figure 4.132: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 17
Figure 4.133: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg marketed gel in volunteer 17
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
Table 4.79: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of marketed aceclofenac gel in volunteer 18
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.134: Plasma concentration verses time profile of aceclofenac
plotted on rectangular co-ordinate graph, administered
as a topical dose of 20 mg marketed gel in volunteer 18
Figure 4.135: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
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.
Table 4.80: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 1
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.136: 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 1
Figure 4.137: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
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
Table 4.81: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 2
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.138: 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 2
Figure 4.139: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg microemulsion based gel in 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
Table 4.82: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 3
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.140: 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 3
Figure 4.141: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
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
Table 4.83: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 4
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.142: 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 4
Figure 4.143: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.84: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 5
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.144: 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 5
Figure 4.145: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.85: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after
administration of microemulsion based aceclofenac gel in
volunteer 6
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.146: 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 6
Figure 4.147: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.86: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 7
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.148: 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 7
Figure 4.149: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg microemulsion based gel in 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
Table 4.87: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 8
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.150: 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 8
Figure 4.151: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg microemulsion based gel in 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
Table 4.88: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 9
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.152: 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 9
Figure 4.153: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical
dose of 20mg microemulsion based gel in 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
Table 4.89: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 10
Sample No. Time (hour) Peak Height Ratio Plasma conc.
aceclofenac µg/ml
1 0 0 0
2 0.5 0.651 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
Table 4.90: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 11
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.156: 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 11
Figure 4.157: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.91: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 12
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.158: 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 12
Figure 4.159: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.92: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 13
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.160: 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 13
Figure 4.161: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.93: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 14
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.162: 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 14
Figure 4.163: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.94: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 15
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.164: 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 15
Figure 4.165: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.95: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 16
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.166: 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 16
Figure 4.167: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.96: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 17
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.168: 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 17
Figure 4.169: Plasma concentration verses time profile of aceclofenac
plotted on semi log graph, administered as a topical dose
of 20mg microemulsion based gel in 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
Table 4.97: Concentration of ACF in human plasma calculated from
chromatograms by forecasting method after administration
of microemulsion based aceclofenac gel in volunteer 18
Sample No. Time (hour) Peak Height Ratio Plasma conc.
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
Figure 4.170: 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 18
Figure 4.171: Plasma concentration verses time profile of aceclofenac
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