FORMULATION AND EVALUATION OF

COLCHICINE LOADED ETHOSOMAL GEL FOR

TRANSDERMAL DELIVERY

IBRAHIM M. ABDULBAQI ALMALLA YOUSIF

UNIVERSITI SAINS MALAYSIA

2017

FORMULATION AND EVALUATION OF

COLCHICINE LOADED ETHOSOMAL GEL FOR

TRANSDERMAL DELIVERY

by

IBRAHIM M. ABDULBAQI ALMALLA YOUSIF

Thesis submitted in fulfilment of the requirements

for the degree of

Master of Science

March 2017

ii

ACKNOWLEDGMENT

Praise is to our almighty gracious Allah for enabling me to finish and present this work.

I would like to express my greatest gratitude to my supervisor Associate Professor Dr.

Yusrida Darwis for providing unconditional support and high standard of scientific

guidance as well as continuous encouragement. Her insightful suggestions and

generosity to assure that all the needed working conditions are available to me, played

a central role in shaping this research project and its publications until successfully

completed. One simply could not wish for a better or friendlier supervisor than she is.

I am also extremely indebted to my co-supervisor Associate Professor Dr. Nurzalina

Abdul Karim Khan for sharing with me her wealth of knowledge and experiences. I

attribute the level of my work to both of them for their encouragement and effort.

Without them this thesis could not see the light of day.

I would also thank Professor Dr. Munavvar Zubaid Abdul Sattar, Dean of the school

of Pharmaceutical sciences for giving me opportunity to carry out this research in the

school of Pharmaceutical sciences, Universiti Sains Malaysia.

My deepest appreciation to the staff members of the school especially the staff of

pharmaceutical technology department, Mr. Samsudin Bakar, Mr. Ibrahim Zainal

Abidin, Mr. Mohd Hafiz Abdul Rahim, and Mr. Rosli Hassan who helped me

throughout my research work.

I am also thankful to the Institute of Postgraduate Studies, and the school of pharmacy

for granting me the graduate assistantship, and much thanks to the Universiti Sains

Malaysia for giving me this opportunity and providing me the necessary facilities.

iii

Special thanks to my colleagues especially Ms. Reem, Mr. Arshad, Mr. Jahanzeb, Ms.

Safia, Mr. Gabriel, Ms. Emily, Mr. Nasir, Ms. Nida, Ms. Ang Lee Fung, and Ms. Nor

Azah, for their co-operation and timely help in this work.

I would like to thank all my family members especially my parents, for their care to

nourish me to what I am today. I also like to thank my sister Janan for being a reference

in my life, my sister Iman for being the big heart, and my brothers Abdullah and

Ammar for being there for me, when I needed. I also like to thank my family in law

for their emotional support and advices. Your advocacy to my passion had taught me

a lot and helped me to grow. Last but not least, to my wife Reem for being an amazing

lab mate and wife at the same time and always being there but never asking why it is

taking so long.

Ibrahim M. Abdulbaqi Almalla Yousif

iv

TABLE OF CONTENTS

ACKNOWLEDGMENT ii

TABLE OF CONTENTS iv

LIST OF TABLES xi

LIST OF FIGURES xiii

LIST OF PLATES xv

LIST OF ABBREVIATIONS AND SYMBOLS xvi

LIST OF APPENDICES xviii

ABSTRAK xix

ABSTRACT xxi

CHAPTER 1

INTRODUCTION

1.1 Gout 1

1.2 Pseudo gout 2

1.3 Familial Mediterranean fever (FMF) 3

1.4 Colchicine 3

1.4.1 Physicochemical properties 3

1.4.2 Pharmacodynamics 4

1.4.3 Pharmacokinetics 5

1.5 Skin and the transdermal drug delivery 6

1.5.1 Skin structure 6

1.5.2 Transdermal drug delivery 6

1.6 Ethosomal nanocarriers 7

1.6.1 Ethosomal systems classifications 8

v

1.6.1(a) Classical ethosomes 8

1.6.1(b) Binary ethosomes 9

1.6.1(c) Transethosomes 9

1.6.2 Mechanisms of ethosomal systems skin permeation 11

1.6.3 Preparation Methods 13

1.6.3(a) The classical cold method 13

1.6.3(b) The ethanol injection–sonication method 13

1.6.3(c) The hot method 14

1.6.3(d) The thin-film hydration method 14

1.6.3(e) The reverse-phase evaporation method 14

1.6.3(f) Transmembrane pH-gradient method 15

1.7 Literature review 15

1.8 Problem statement 17

1.9 Study Objectives 18

vi

CHAPTER 2

DEVELOPMENT AND CHARACTERIZATION OF COLCHICINE

ETHOSOMAL SYSTEMS

2.1 Introduction 19

2.2 Materials 20

2.3 Methods 20

2.3.1 Preparation of the ethosomal systems 20

2.3.2 Effects of formulation variables on the ethosomal systems properties 21

2.3.3 Characterization of the ethosomal systems by measuring the

vesicular size, PdI and ZP

24

2.3.4 Incorporation of colchicine in ethosomal systems 24

2.3.5 Separation of colchicine-ethosomes from the free colchicine 25

2.3.6 Quantification of colchicine using Ultraviolet-Visible

spectrophotometer

26

2.3.7 Statistical analysis 26

2.4 Results and discussion 27

2.4.1 Preparation and optimization of the ethosomal systems 27

2.4.2 Analysis of the full factorial designs 29

2.4.3 Effects of formulation variables on the ethosomal systems properties 45

2.4.3(a) Effects of the formulation variables on the vesicular size

and PdI

45

2.4.3(b) Effects of the formulation variables on the ZP 48

2.4.4 Quantification of colchicine using Ultraviolet-Visible

spectrophotometer

49

vii

2.4.5 Determination and optimization of the entrapment efficiency (EE%) 50

2.5 Conclusion 54

CHAPTER 3

DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR THE

QUANTIFICATION OF COLCHICINE IN ETHOSOMAL GEL AND THE

DRUG RELEASE MEDIA

3.1 Introduction 55

3.2 Materials and methods 56

3.2.1 Materials 56

3.2.2 Instrumentation 57

3.2.3 Chromatographic condition 57

3.2.4 Preparation of stock solution, calibration standards and quality

control samples

57

3.2.5 System suitability 58

3.2.6 Specificity 58

3.2.7 Stress degradation studies 58

3.2.7(a) Acid and alkali degradation studies 59

3.2.7(b) Oxidative hydrogen peroxide (H2O2) degradation 59

3.2.7(c) Heat degradation study 60

3.2.7(d) UV light degradation 60

3.2.8 Method validation 61

3.2.8(a) Linearity 61

3.2.8(b) Precision and accuracy 61

viii

3.2.8(c) Limit of Detection (LOD) and Limit of Quantification

(LOQ)

62

3.2.8(d) Robustness 62

3.2.8(e) Stock solution stability 62

3.2.9 Assay of colchicine in the ethosomal gel 63

3.2.10 Recovery of colchicine 63

3.2.11 Statistics 63

3.3 Results and discussion 64

3.3.1 System suitability studies 64

3.3.2 Specificity 65

3.3.3 Stress degradation studies 67

3.3.4 Linearity 73

3.3.5 Precision and accuracy 75

3.3.6 Limit of Detection (LOD) and Limit of Quantification (LOQ) 75

3.3.7 Robustness 76

3.3.8 Stock solution stability 77

3.3.9 Assay of colchicine in the formulation 77

3.3.10 Recovery studies of colchicine in the ethosomal gel 77

3.4 Conclusion 78

ix

CHAPTER 4:

PREPARATION AND CHARACTERIZATION OF ETHOSOMAL GEL

FORMULATIONS, EX-VIVO SKIN PERMEATION AND STABILITY

STUDIES

4.1 Introduction 79

4.2 Materials 80

4.3 Methods 80

4.3.1 Preparation of the ethosomal gel formulation 80

4.3.2 Characterization of the colchicine-loaded ethosomal gel

formulations

81

4.3.2(a) Determination of pH 81

4.3.2(b) Vesicular size, polydispersity index (PdI) and Zeta

potential (ZP)

81

4.3.2(c) Rheological behavior and viscosity measurements 82

4.3.3 Ex-vivo skin permeation studies 83

4.3.3(a) Preparation of the skin 83

4.3.3(b) Skin permeation studies 84

4.3.3(c) Data analysis of the skin permeation studies 85

4.3.4 Drug content determination of the optimized ethosomal gel

formulation

86

4.3.5 Transmission electron microscopy 86

4.3.6 Stability studies 87

4.3.7 Statistical analysis 87

4.4 Results and discussion 88

x

4.4.1 Preparation of the ethosomal gel formulation 88

4.4.2 Characterization of the colchicine-loaded ethosomal gel

formulations

89

4.4.3 Ex-vivo skin permeation studies 92

4.4.4 Transmission electron microscopy 94

4.4.5 Stability studies 95

4.4.5(a) Physical stability 95

4.4.5(b) Vesicular size, PdI and ZP 95

4.4.5(c) pH, drug content, viscosity and yield 96

4.5 Conclusion 102

CHAPTER 5 103

SUMMARY AND GENERAL CONCLUSION

CHAPTER 6 106

SUGGESTIONS FOR FUTURE RESEARCH

REFERENCES

108

APPENDICES

124

LIST OF PUBLICATIONS

xi

LIST OF TABLES

Page

1.1 Comparison of Classical Ethosomes, Binary Ethosomes and

Transethosomes.

10

2.1 Factorial designs for the preparation of the three types of ethosomal

systems.

23

2.2 Experimental design and value of each factor and response for

Classical ethosomes (CE) design; A: Ethanol concentration (%w/w),

B: PL90G concentration (%w/w), C: Number of extrusions. Mean ±

SD, n = 3.

30

2.3 Experimental design and value of each factor and response for binary

ethosomes (BE) design; A: Ethanol concentration (%w/w), B: PL90G

concentration (%w/w), C: PG concentration (%w/w), D: Number of

extrusions. Mean ± SD, n = 3.

30

2.4 Experimental design and value of each factor and response for

transethosomes with Tween 20® (TET) design; A: Ethanol

concentration (%w/w), B: PL90G concentration (%w/w), C: Tween

20® concentration (%w/w), D: Number of extrusions. Mean ± SD, n

= 3.

31

2.5 Experimental design and value of each factor and response for

transethosomes with sodium taurocholate (TENa) design; A: Ethanol

concentration (%w/w), B: PL90G concentration (%w/w), C: Sodium

taurocholate concentration (%w/w), D: Number of extrusions. Mean

± SD, n = 3.

32

2.6 Experimental design and value of each factor and response for

transethosomes with Labrafil® (TEL) design; A: Ethanol

concentration (%w/w), B: PL90G concentration (%w/w), C:

Labrafil® concentration (%w/w), D: Number of extrusions. Mean ±

SD, n = 3.

33

2.7 Results of regression analysis for the responses size, PdI and ZP for

all the designs.

34

2.8 Coded equations of the size, PdI and ZP for all the designs. 35

xii

2.9 The effect (increase/reduce) of individual factor on the vesicular size,

PdI and ZP in each design.

45

2.10 Characteristics of ethosomal systems loaded with (0.2 %w/w) of

colchicine. Mean ± SD, n = 3.

51

2.11 Characteristics of ethosomal systems loaded with (0.3 %w/w) of

colchicine. Mean ± SD, n = 3.

52

2.12 Characteristics of ethosomal systems loaded with (0.5 %w/w) of

colchicine Mean ± SD, n = 3.

52

3.1 Results of system suitability studies of quality control samples of

colchicine. Mean ± SD, n =6.

64

3.2 Results of the stress degradation studies. Mean ± SD, n =3. 68

3.3 Weighted least squares regression analysis. Mean ± SD, n=3. 74

3.4 Results of intra-day and inter-day precision and accuracy. Mean, n=6. 75

3.5 Analysis of method robustness using colchicine at concentration of

4000 ng/ml. Mean ± SD, n=3.

76

4.1 Composition of the gel base formulation. 81

4.2 Characterization of various colchicine-loaded ethosomal gels. Mean

± SD, n = 3.

90

4.3 The flux at steady state (Jss), permeability coefficient (Kp) of

colchicine from various gel formulations through rat skin. Mean ±

SD, n = 3.

93

4.4 Stability study results of the TET 2 gel formulation stored at 4 ± 2

°C. Mean ± SD, n = 3.

97

4.5 Stability study results of the TET 2 gel formulation stored at 25 ± 2

°C / 60 ± 5 % RH. Mean ± SD, n = 3.

98

xiii

LIST OF FIGURES

Page

1.1 The chemical structure of colchicine. 4

1.2 Schematic representations of the different types of ethosomal

systems.

8

1.3 Proposed mechanism for permeation of molecules from ethosomal

system through the skin.

12

2.1 Preparation of the ethosomal systems using the cold method. 21

2.2 Response surface plots showing influences of the independent factors

on the response parameters in CE design.

36

2.3 Response surface plots showing influences of the independent factors

on the response parameters in BE design.

37

2.4 Response surface plots showing influences of the independent factors

on the response parameters in TET design.

39

2.5 Response surface plots showing influences of the independent factors

on the response parameters in TENa design.

41

2.6 Response surface plots showing influences of the independent factors

on the response parameters in TEL design.

43

2.7 The ultraviolet-visible spectra of colchicine from 190 to 450 nm. 49

2.8 Calibration curve for colchicine in methanol at 353 nm (n=3). 50

3.1 Chromatograms of A: Mobile phase, B: Standard colchicine solution

(6000 ng/ml), C: Blank ethosomal gel, D: Colchicine ethosomal gel

(6000 ng/ml), E: Phosphate buffer saline solution.

66

3.2 Chromatograms of stressed standard colchicine solution under A:

Acid (0.5 M HCl) (24 hr), B: Base (0.5 M NaOH) (24 hr), C:

Oxidation (3 % H2O2) (24 hr), D: UV light (365 nm) (24 hr), E: Heat

(80 ºC) (2 hr).

69

3.3 Chromatograms of the stressed colchicine ethosomal gel samples

under A: Acid (0.5 M HCl) (24 hr), B: Base (0.5 M NaOH) (24 hr),

C: Oxidation (3 % H2O2) (24 hr), D: UV light (365 nm) (24 hr), E:

Heat (80 ºC) (2 hr).

70

xiv

3.4 Chromatograms of the stressed mobile phase under A: Acid (0.5 M

HCl) (24 hr), B: Base (0.5 M NaOH) (24 hr), C: Oxidation (3 % H2O2)

(24 hr), D: UV light: (365 nm) (24 hr), E: Heat (80 ºC) (2 hr).

71

3.5 Chromatograms of the stressed blank ethosomal gel samples under

A: Acid (0.5 M HCl) (24 hr), B: Base (0.5 M NaOH) (24 hr), C:

Oxidation (3 % H2O2) (24 hr), D: UV light (365 nm) (24 hr), E: Heat

(80 ºC) (2 hr).

72

4.1 Representative rheograms of the prepared ethosomal gels. 91

4.2 Ex-vivo skin permeation of colchicine from (TET 2, TENa 4 and

Non-ethosomal gel) gel formulations. Mean ± S.D., n=3.

92

4.3 Representative rheogram of a freshly prepared TET 2 gel

formulation.

98

4.4 Representative rheograms of the TET 2 gel formulation stored at the

refrigerated temperature (4 ± 2 °C) for specified time intervals.

99

4.5 Representative rheograms of the TET 2 gel formulation stored at the

room temperature (25 ± 2 °C / 60 ± 5 % RH) for specified time

intervals.

100

4.6 Estimated shelf life based on the drug content percent results of the

TET 2 gel at 4 ± 2 °C.

101

4.7 Estimated shelf life based on the drug content percent results of the

TET 2 gel at 25 ± 2 °C / 60 ± 5 % RH.

102

xv

LIST OF PLATES

Page

4.1 TEM images of TET 2 formulation in its ethosomal suspension and

ethosomal gel dosage forms.

95

xvi

LIST OF ABBREVIATIONS AND SYMBOLS

% Percent

°C Celsius

µl Microliter

ANOVA Analysis of Variance

BE Binary ethosomes

CE Classical ethosomes

cm Centimetre

cm2 Squared centimetre

FDA Food and Drug Administration

HCL Hydrochloric acid

HETP Height equivalent of a theoretical plate

HLB Hydrophilic Lipophilic Balance

HPLC High Performance Liquid Chromatography

hr Hour

LOD Limit of Detection

LOQ Limit of Quantification

M Molar

m2 Square metre

mg Milligram

mg/ml Milligram per millilitre

min Minute

ml Millilitre

ml/min Millilitre per minute

mM Millimole

mV Millivolt

NaOH Sodium hydroxide

NE gel Non-ethosomal gel

ng Nanogram

ng/ml Nanogram per millilitre

nm Nanometer

xvii

Pa Pascal

Pa.s Pascal second

PdI Polydispersity index

PTFE Polytetrafluoroethylene

RH Relative Humidity

rpm Revolutions per minute

RSD Relative Standard Deviation

SD Standard deviation

Sec Second

TEL Transethosomes containing Labrafil®

TENa Transethosomes containing Sodium taurocholate

TEM Transmission Electron Microscopy

TET Transethosomes containing Tween 20®

UK United Kingdom

USA United States of America

USP United State Pharmacopoeia

UV Ultraviolet

w/v Weight per volume

w/w Weight per weight

η Viscosity

ZP Zeta potential

xviii

LIST OF APPENDICES

Page

Appendix A1 Animal Ethics Approval. 125

Appendix A2 Cumulative amount of colchicine permeated per unit area

(µg/cm2) from different formulations.

127

xix

FORMULASI DAN PENILAIAN GEL ETOSOM BERMUATAN

COLCHICIN UNTUK PENGHANTARAN TRANSDERMAL

ABSTRAK

Colchicin digunakan untuk rawatan gout, pseudo-gout dan demam

Mediterranean. Ia diketahui mempunyai terapetik indeks rendah dan biokeperolehan

oral rendah yang disebabkan oleh kesan laluan pertama yang teruk, dan ia juga

dikaitkan dengan kesan sampingan gastrousus teruk berikutan pemberian oral. Untuk

mengatasi kelemahan pengambilan oral, gel etosom bermuatan colchicin disediakan

dalam usaha untuk penghantaran drug secara transdermal. Tiga jenis etosom pembawa

nano telah disediakan dengan menggunakan kaedah sejuk. Lima set reka bentuk

faktorial eksperimen telah dijalankan, satu factorial 23 untuk etosom klasik, satu

factorial 24 untuk etosom binari, dan tiga factorial 24 untuk transetosom. Sistem etosom

dengan purata saiz vesikel ≤ 150 nm, polydispersity indeks (PdI) ≤ 0.3, dan zeta

berpotensial (ZP) ≥ -20 mV dikaji lebih lanjut untuk penggabungan drug dalam

etosom. Sistem etosom optimum dengan kecekapan pemerangkapan > 75 %,

kemudian digabungkan ke dalam gel asas (Carbopol 940®). Gel etosom itu dicirikan

mengikut purata saiz vesikel, PdI, ZP, pH, kelikatan dan nilai alah. Semua formulasi

gel etosom menujukkan aliran plastik bukan Newtonian tanpa thixotropy. Dua gel

etosom optimum dipilih iaitu TET 2 dan TENa 4 gel. Gel TET 2 memiliki purata saiz

vesikel, PdI, ZP, pH, kelikatan dan nilai alah, masing-masing 111.4 ± 1.6 nm, 0.157

± 0.01, -40.56 ± 5.32 mV, 6.74 ± 0.05, 66.25 ± 2.75 Pa.s, 131.91 ± 1.94 Pa. Manakala

TENa 4 gel memiliki purata saiz vesikel, PdI, ZP, pH, kelikatan dan nilai alah masing-

masing 110.5 ± 1.1 nm, 0.162 ± 0.014, -43.54 ± 2.56 mV, 6.81 ± 0.04, 55.68 ± 0.76

xx

Pa.s, 122.71 ± 0.99 Pa. Kaedah HPLC dibangunkan dan divalidasi bagi kuantifikasi

colchicin dalam gel etosom dan dalam cecair media. Kajian ex-vivo ketelapan kulit

dijalankan untuk menilai dan membandingkan parameter ketelapan kulit antara

formulasi gel etosom optimum dan gel bukan etosom. Formulasi gel TET 2

mempunyai penelapan kulit lebih tinggi (188.90 kali ganda) berbanding gel colchicin

bukan ethosom. Gel TET 2 dipilih sebagai formulasi akhir gel etosom optimum.

Kajian kestabilan gel TET 2 menunjukkan bahawa formulasi stabil pada keadaan

penyimpanan 4 ± 2 °C. Anggaran jangka hayat formulasi gel TET 2 adalah 14.44

bulan. Kesimpulannya, formulasi gel colchicin etosom untuk penghantaran

transdermal telah berjaya disediakan, dan ia mungkin berguna sebagai laluan alternatif

kepada pemberian colchicin secara oral.

xxi

FORMULATION AND EVALUATION OF COLCHICINE LOADED

ETHOSOMAL GEL FOR TRANSDERMAL DELIVERY

ABSTRACT

Colchicine is used for the treatment of gout, pseudo-gout and familial

Mediterranean fever. It is known to have low therapeutic index and low oral

bioavailability due to extensive first pass effect, and also associated with sever

gastrointestinal side effects following oral administration. To overcome its oral intake

drawbacks, colchicine loaded ethosomal gels were prepared in an attempt to deliver

the drug transdermally. The three types of ethosomal nanocarriers were prepared using

the cold method. Five sets of factorial designs experiments were conducted, one of 23

for classical ethosomes, one of 24 for binary ethosomes, and three of 24 for

transethosomes. Ethosomal systems with vesicular size ≤ 150 nm, polydispersity

index (PdI) ≤ 0.3, and zeta potential (ZP) ≥ -20 mV were further investigated for drug

incorporation studies. The optimized ethosomal systems with entrapment efficiency >

75 % were then incorporated into gel base (Carbopol 940®). The prepared ethosomal

gels were characterized for vesicular size, PdI, ZP, pH, viscosity and yield values. All

the formulated ethosomal gels exhibited non-Newtonian plastic flow with no

thixotropy. Two optimized ethosomal gels were selected TET 2 and TENa 4 gels. TET

2 gel possessed mean vesicular size, PdI, ZP, pH, viscosity and yield values of 111.4

± 1.6 nm, 0.157 ± 0.01, -40.56 ± 5.32 mV, 6.74 ± 0.05, 66.25 ± 2.75 Pa.s, and 131.91

± 1.94 Pa, respectively. While TENa 4 gel possessed mean vesicular size, PdI, ZP, PH,

viscosity and yield value of 110.5 ± 1.1 nm, 0.162 ± 0.014, -43.54 ± 2.56 mV, 6.81 ±

0.04, 55.68 ± 0.76 Pa.s, 122.71 ± 0.99 Pa, respectively. HPLC method was developed

xxii

and validated for the quantification of colchicine in ethosomal gel and dissolution

media. Ex-vivo skin permeation studies were conducted to evaluate and compare the

skin permeation parameters between the optimized ethosomal formulations and the

non-ethosomal gel. The TET 2 gel formulation had higher skin permeation (188.90

folds) compared to non-ethosomal colchicine gel. The TET 2 gel was selected as the

final optimized ethosomal gel formulation. The stability studies of TET 2 gel

formulation revealed that the formula was stable at the storage condition of (4 ± 2 °C).

The estimated shelf life for the TET 2 gel was estimated and was found to be 14.44

months. In conclusion, colchicine ethosomal gel formulation for transdermal delivery

was successfully prepared, and could be useful alternative route to oral administration

of colchicine.

1

CHAPTER 1

INTRODUCTION

1.1 Gout

Gout is a form of inflammatory arthritis where swelling and severe pain develop in

joints, and negatively interfering with patient’s basic daily activities. It was first

recognized in 2640 BC by the Egyptians (Omole et al., 2009). The worldwide reported

prevalence of gout ranges from 0.1 % to approximately 10 %, while its incidence vary

from 0.3 to 6 cases per 1,000 person-years. Over the past 50 years, the global burden

of gout management was substantial, and seems to be increasing in many parts of the

world (Kuo et al., 2015). Gout can manifest as acute or chronic arthritis.

Acute gout is an intense acute inflammatory reaction that occurs due to articular

deposits of monosodium urate (MSU) crystals. It is one of the most painful conditions

experienced by humans (Martillo et al., 2014). Acute gout typical symptoms include

sever red, hot, and swollen joint with an excruciating pain. The acute form of the

disease may advance to chronic gout if not properly treated. Acute gout treatment relies

on various therapeutic approaches such as anti-inflammatory agents including

nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, uricosuric agents and

corticosteroids (Choi et al., 2005), as well as systemic and intraarticular

glucocorticoids, and biological agents that inhibit the action of interleukin (IL)-1 beta

(Terkeltaub, 2010). In 2009, the United States food and drug administration (FDA)

approved colchicine for treatment of acute gout. A scheduled oral dose for the first 24

hours of the attack was set with an initial dose of 1.2 mg, one hour later another 0.6

2

mg is administrated, making a sum of 1.8 mg as a total dose on the first day of therapy

(Burns & Wortmann, 2012)

Chronic gout is a progressive disorder of urate metabolism characterized by

symptomatic hyperuricemia, and monosodium urate crystals deposition in joints as

well as soft tissues (Novak et al., 2007), which leads to a series of repeated pain

episodes, as well as inflammation and degenerative ramification, in addition to joint

damage and motion loss. The treatment of chronic gout goal is to completely dissolve

MSU crystals and prevent new crystals formation. To achieve such status, the serum

urate, and uric acid levels within the tissues and joints should decrease below MSU

saturation point (Perez, 2009), by using uricosuric agents such as allopurinol (> 300

mg), and prophylactic colchicine doses (0.5 – 1 mg daily), and/or a non-steroidal anti-

inflammatory drug (NSAID) (Roddy et al., 2007).

1.2 Pseudo-gout

It is known as acute attacks of synovitis due to calcium pyrophosphate dihydrate

(CPPD) crystals. Clinically, the disease looks like gouty arthritis. CPPD crystals

deposition can lead to symptoms like septic arthritis, as well as poly-articular

inflammatory arthritis which usually are misdiagnosed as rheumatoid arthritis or

degenerative osteoarthritis (McCarthy, 2008). The diagnosis of pseudo-gout is

conducted by the demonstration of CPPD crystals in synovial fluid. Both gout and

pseudo-gout can also coexist in a single inflammatory effusion, as 20 % of patients

with CPPD will have hyperuricemia, and later, a quarter of these patients will develop

gout (Rosenthal., 2005). The current available treatments of pseudo-gout are limited

to fixing the ongoing metabolic abnormalities, and treating their associated symptoms,

3

these treatments include NSAIDs, colchicine, corticosteroids and adrenocorticotropic

hormone (Choy, 2005).

1.3 Familial Mediterranean fever (FMF)

Familial Mediterranean fever (FMF) is a hereditary inflammatory disease

characterized by recurrent febrile episodes accompanied by peritonitis, pericarditis,

pleuritis and/or arthritis, it is sometimes associated with an erysipelas-like skin rash.

Serious complications can happen if the disease is not treated such as end-stage renal

disease and malabsorption secondary to amyloid deposition in the kidneys and

digestive tract, male and female infertility, and growth retardation in children (Zadeh

et al., 2011). FMF treatment relies mainly on preventing the development of painful

attacks and amyloidosis. Colchicine is the only available drug for treating FMF and is

considered as the gold standard in FMF therapy. It is thought to inhibit the increased

chemotactic activity of neutrophils during FMF attacks (Ben-Chetrit & Levy, 1998).

Colchicine can also improve height development and weight parameters in children

with FMF (Ozcakar et al., 2010). A daily lifelong administration of colchicine is

required to prevent both the fever/pain attacks, and the silent amyloid deposition.

NSAIDs are used to mask the FMF symptoms of fever and pain. (Zadeh et al., 2011).

1.4 Colchicine

1.4.1 Physicochemical properties

Colchicine (C22H25NO6) is a yellowish-white amorphous or crystalline powder with a

structure described in figure 1.1. It is a potent alkaloid obtained from the dried corns

and seeds of plants of the genus colchicum, which belongs to Liliaceae family. Among

4

this family, the commonly used plants are colchicum autumnale “meadow saffron”

and colchicum autumn crocus. The drug has a neutral chemical nature, with a partition

coefficient (log P) and pKa values of 1.3 and 1.85 respectively. It has a molecular

weight of 399.437 Da, water solubility of 45 mg/ml and a melting point of 156 °C

(Singh et al., 2015; Bombuwala et al., 2006).

Fig. 1.1: The chemical structure of colchicine.

1.4.2 Pharmacodynamics

Colchicine has been used for the treatment of acute gout since more than 2000 years

ago. It was also used to treat pseudo-gout and familial Mediterranean fever (FMF) for

several decades. It is highly effective in the treatment of acute gout especially when

given in the first 12 – 36 hr of the gouty attack (Terkeltaub, 2009). Colchicine relieves

pain and inflammation of gouty arthritis without altering the metabolism or excretion

of urates and without other analgesic effects. It produces its anti-inflammatory effects

by binding to the intracellular protein tubulin, thereby preventing its polymerization

into microtubules, leading to the inhibition of leukocyte migration and phagocytosis.

It also inhibits the formation of leukotriene B4 (Katzung et al., 2009).

5

Colchicine oral administration linked to several mild to severe gastrointestinal side

effects including nausea, vomiting, diarrhea, stomach cramps or pain. (Ahern et al.,

1987). Even at therapeutic doses, around 80 percent of patients will develop these side

effects (Harris et al., 1999; Putterman et al., 1991) While the intravenous

administration is associated with potential serious adverse effects including tissue

necrosis cytopenias, disseminated intravascular coagulation, and death (Evans et al.,

1996), accordingly the intravenous route of administration was banned by the FDA

(Burns & Wortmann, 2012).

1.4.3 Pharmacokinetics

Colchicine has a low therapeutic index (Terkeltaub, 2009). After oral administration,

colchicine is absorbed in the jejunum and ileum with a single zero-order rate process,

showing a low bioavailability of 25 % to 50 % due to the extensive first pass effect

(Kesselheim & Solomon 2010). Within the first 24 hours of colchicine intake, 15 %

to 30 % of it excreted in the urine, and its excretion still can be measured for day 10

of the intake. Up to two thirds of the drug eliminated via feces. After around one hour

of oral intake the maximum serum concentration is reached, and it varies between 2.2

and 6.7 ng/ml (Rochdi et al.,1994; Girre et al.,1989). Serum half-life time of colchicine

is speculated to be ~10 to 20 hours (Girre et al., 1989; Poffenbarger & Brinkley, 1974).

In FMF patients with renal amyloidosis and are on hemodialysis, it was found that the

drug elimination half-life was increased significantly (400 %), and total clearance was

reduced compared to healthy patients (Ben-Chetrit et al.,1994).

6

1.5 Skin and the transdermal drug delivery

1.5.1 Skin structure

The skin covers a total body surface area of approximately 1.8 m2, it is providing the

contact between the human body and the outer environment. It is the largest and most

readily accessible organ of the body, serving as a potential route of drug administration

for systemic effects. Skin has three main layers namely: the epidermis which is the

outermost layer of skin, beneath the epidermis layer there is the dermis layer which

contains tough connective tissue, hair follicles, and sweat glands, the third deeper layer

is the subcutaneous tissue (hypodermis) layer which is made of fat and connective

tissue. The epidermis is itself divided into at least four separate parts, a fifth part is

present in some areas of our body. The order from the most superficial layer of the

epidermis to the deepest, these layers (strata) are: the stratum corneum, stratum

lucidum, stratum granulosum, stratum spinosum and stratum basale. One of the main

challenges for percutaneous drug delivery is the nearly insuperable nature of stratum

corneum layer of the epidermis (Barry, 2001), its permeability is approximately 1000

times less than other biological membranes and this is due to the (brick and mortar)

arrangement of corneocytes, flattened mononucleated keratinocytes, with interspersed

lipids and proteins (Hadgraft, 2001a; Hadgraft, 2001b).

1.5.2 Transdermal drug delivery

Transdermal drug delivery is currently one of the most appealing alternatives to oral

drug delivery as well as hypodermic injection route. Transdermal drug delivery

systems offer many advantages, such as avoidance of first-pass metabolism by the

liver, controlled delivery of drugs, reduced dosing frequency, and improved patient

7

compliance, as they are noninvasive and can be self-administered (Prausnitz & Langer,

2008; Schoellhammer et al., 2014).

The first transdermal patch containing scopolamine for the treatment of motion

sickness was approved in the US in 1979. A decade later, nicotine patches became the

first transdermal blockbuster. Till 2008 a variety of 19 transdermal delivery systems

for single or multiple drug delivery were available in the market such as (fentanyl HCl,

methylphenidate, selegiline and rotigotine) with an estimation of more than one billion

transdermal patches that are manufactured each year (Prausnitz & Langer, 2008).

1.6 Ethosomal nanocarriers

A new era of research in the field of transdermal drug delivery was opened with the

use of liposomes for topical delivery of triamcinolone (Mezei & Gulasekharam, 1980),

and since then a wide range of novel lipid-based vesicular systems were developed.

The deformable or elastic liposomes which are currently known as transfersomes®

were introduced by Cevc and Blume in 1992 (Cevc & Blume, 1992), and followed by

the innovative work of Elka Touitou and her team (Touitou et al., 1996), that led to the

discovery of a novel lipid vesicular system called ethosomes. Ethosomal systems differ

from liposomes because they contain relatively high concentrations of ethanol in

addition to phospholipids and water (Touitou et al., 2000). New generations of

ethosomal systems have been introduced since then by adding other compounds to the

basic ethosomal formula in an attempt to enhance the vesicular characteristics and the

skin permeation.

8

1.6.1 Ethosomal systems classification

Ethosomal systems are classified into three classes on the basis of their compositions

namely: classical ethosomes, binary ethosomes, and transethosomes (Figure 1.2)

(Abdulbaqi et al., 2016).

Fig. 1.2: Schematic representations of the different types of ethosomal systems.

1.6.1(a)Classical ethosomes

Classical ethosomes are a modification of classical liposomes and are composed of

phospholipids, high concentration of ethanol up to 45 %w/w and water. The classical

ethosomes were reported to be superior over the classical liposomes for transdermal

drug delivery because they have smaller size, negative zeta potential, higher

entrapment efficiency. Moreover, classical ethosomes showed better skin permeation

and stability profile compared to classical liposomes (Touitou et al., 2000; Sarwa et

al., 2014; Jain et al.,2014). The molecular weights of drugs entrapped in classical

9

ethosomes were ranging from 130.077 Daltons to 24 K Daltons (Zhang et al., 2012;

Mishra et al., 2010).

1.6.1(b) Binary Ethosomes

Binary ethosomes were first introduced by Zhou et al in 2010 (Zhou et al., 2010).

Basically, they were developed by adding another type of alcohol to the classical

ethosomes. The most commonly used alcohols in binary ethosomes are propylene

glycol (PG) and isopropyl alcohol (IPA) (Li et al., 2012; Zhang et al., 2012; Akhtar &

Pathak, 2012; Dave et al., 2010; Shen et al., 2014).

1.6.1(c) Transethosomes

Transethosomes are the new generation of ethosomal systems which were first

reported by Song et al in 2012 (Song et al., 2012). This ethosomal system contains the

basic components of classical ethosomes and additional compound such as a

penetration enhancer or an edge activator (surfactant) in their formula. These novel

vesicles were developed in an attempt to combine the advantages of classical

ethosomes and deformable liposomes (Transfersomes®) in one formula to produce

transethosomes. Many researchers have reported superior properties of transethosomes

over classical ethosomes (Ainbinder & Touitou, 2011; Bragagni et al., 2012; Meng et

al., 2013; Chen et al., 2014; Chenet al., 2014; Fang et al., 2008; Ascenso et al., 2015).

Different types of edge activators and penetration enhancers have been investigated to

produce ethosomal systems with better characteristics. Transethosomes were reported

to entrap drugs with molecular weights ranging from 130.077 Daltons to 200–325 K

Daltons (Ainbinder & Touitou, 2011; Chen et al., 2014). Table 1.1 shows the

10

comparison of classical ethosomes, binary ethosomes and transethosomes properties

in their initial suspension form.

Table 1.1: Comparison of Classical Ethosomes, Binary Ethosomes and

Transethosomes

Parameter Classical

ethosomes

Binary ethosomes Transethosomes

Composition 1. Phospholipids

2. Ethanol

3. Stabilizer

5. Charge inducer

6. Water

7. Drug/agent

1. Phospholipids

2. Ethanol

3. Propylene glycol

(PG) or other alcohol

4. Charge inducer

5. Water

6. Drug/Agent

1. Phospholipids

2. Ethanol

3. Edge activator

(surfactant) or

penetration

enhancer

4. Charge inducer

5. Water

6. Drug/agent

Morphology Spherical Spherical Regular or

irregular spherical

shapes

Size Smaller than the

classical

liposomes

Equal to or smaller

than classical

ethosomes

Size based on

type and

concentration of

penetration

enhancer or edge

activator used

Zeta Potential Negatively

charged

Negatively charged Positively or

negatively

charged

Entrapment

efficiency

Higher than

classical

liposomes

Typically higher than

classical ethosomes

Typically higher

than classical

ethosomes

Skin permeation Typically higher

than classical

liposomes

Typically equal to or

higher than classical

ethosomes

Typically higher

than classical

ethosomes

Stability More stable than

classical

liposomes

More stable than

classical ethosomes

No particular

trend determined

11

1.6.2 Mechanisms of ethosomal system skin permeation

Ethanol and phospholipids are reported to act synergistically to enhance the skin

permeation of drugs in ethosomal formulations. Ethanol fluidizes the lipid bilayers of

the ethosomal vesicles and the stratum corneum simultaneously, changing the

arrangement and decreasing the density of skin lipids. Therefore, the highly malleable

and soft vesicles of an ethosomal system will penetrate the altered structure of the

stratum corneum and create a pathway through the skin. The release of the therapeutic

agent occurs by the fusion of these vesicles into cell membranes in the deeper layers

of the skin (Touitou et al., 2000; Ainbinder et al., 2010; Singh et al., 2014). It is

suggested that transethosomes have superior skin-permeation properties over classical

ethosomes. This is because transethosomes contain both ethanol and the edge activator

or the penetration enhancer, which both act together to increase vesicular malleability

and skin-lipid perturbation (Bragagni et al., 2012; Ascenso et al., 2015). A proposed

mechanism is illustrated in figure 1.3.

12

Fig 1.3: Proposed mechanism for permeation of molecules from ethosomal system

through the skin. Notes: (A) Normal skin; (B) Skin-lipid perturbation by ethanol

effects; (C) Penetration of the soft malleable ethosomal system vesicles.

13

1.6.3 Preparation methods

1.6.3(a) The classical cold method

This is the simplest and most widely used method for the preparation of ethosomal

systems. The organic phase is obtained by dissolving the phospholipids, (in addition

to the surfactant or penetration enhancer for the preparation of transethosomes) in

ethanol, or in mixture of solvents (ethanol/PG for binary ethosomes) (Zhang et al.,

2012), at room temperature (Cortesi et al., 2010), or at 30 °C (Touitou et al., 2000).

The used aqueous phase is either water (Dubey et al., 2007; De la Presa et al., 2009;

Chourasia et al.,2011), buffer solution (Dubey et al., 2010; Cortesi et al., 2010) or

normal saline solution (Raza et al., 2013). The aqueous phase is added to the organic

phase in a fine stream (Touitou et al., 2000) dropwise (Zhou et al., 2010) or using a

syringe pump at a constant rate of 175 or 200 µl/min (Paolino et al., 2005). The mixture

is stirred at a speed of 700 – 2,000 rpm (Touitou et al., 2000; Chourasia et al., 2011;

Kaur & Saraf, 2011; Chen et al., 2010) using an overhead (Touitou et al., 2001) or

magnetic (Elsayed et al., 2006) stirrer. The intended drug for incorporation within the

ethosomal system will be dissolved in either the aqueous or the organic phase,

depending on its physicochemical properties.

1.6.3(b) The ethanol injection–sonication method

In this method, the organic phase containing the dissolved phospholipid in ethanol is

injected to the aqueous phase, using a syringe system (Zhaowu et al., 2009) at a flow

rate of 200 µl/min, then homogenized with an ultrasonic probe for 5 min (Liu et al.,

2011).

14

1.6.3(c) The hot method

In one vessel, phospholipid is dispersed in water and placed in a water bath at 40 °C

until a colloidal suspension is obtained. In another vessel, ethanol is heated to 40 °C

and then added dropwise to the phospholipid dispersion, under continuous mixing

using a mechanical or magnetic stirrer (Ashoniya & Meenakshi, 2011). The drug is

dissolved in either the organic or the aqueous phase, based on its

hydrophilic/hydrophobic properties.

1.6.3(d) The thin-film hydration method

In this method the phospholipid is first dissolved in chloroform only (Park et al., 2014)

or a chloroform–methanol mixture at ratios of 3:1 or 2:1 in a clean, dry, round-bottom

flask, later, organic solvents are removed by a rotary vacuum evaporator at a

temperature above the lipid-phase transition temperature. The traces of the solvents

are removed from the deposited lipid film under vacuum overnight, then the lipid film

is hydrated with a water–ethanol solution (Lopez-Pinto et al.,2005) or phosphate

buffered saline–ethanol solution (Mishra et al., 2008).

1.6.3(e) The reverse-phase evaporation method

This is the least used method and specially designed to produce large unilamellar

vesicles. The organic phase is prepared by dissolving the phospholipid in diethyl ether

and then mixing it with the aqueous phase at a ratio of 3:1 v/v in an ultrasonic bath at

0 °C for 5 min to form a water-in-oil emulsion. The organic solvent is removed under

15

reduced pressure to produce a gel, which turns into a colloidal dispersion upon

vigorous mechanical agitation (Maestrelli et al., 2009).

1.6.3(f) Transmembrane pH-gradient method

In this method, the drug is loaded “actively”, based on the pH-gradient difference

between the acidic interior of the internal phase and the basic exterior of the external

phase of the ethosomal system. At first, the empty ethosomal suspension is prepared

using any of the aforementioned methods, but the aqueous phase or the hydration

process uses an acidic buffer (usually citrate buffer, pH 3). Secondly, the active drug

is loaded into the empty ethosomal suspension, followed by continuous stirring. In

order to make the external phase more alkaline and to establish the pH gradient

between the acidic internal (pH 3) and basic external phases of the ethosomal system,

an alkali, usually a sodium hydroxide solution of 0.5 M is added to make the external

pH 7.4. In the third stage, the ethosomal system is incubated at a specified time and

temperature (30 – 60 °C) to give the opportunity for the unionized drug to actively

pass the bilayer of the ethosomal vesicles and get entrapped (Zhou et al., 2010).

1.7 Literature review

The ability of ethosomal systems to penetrate intact skin, delivering molecules with

diverse physicochemical properties in therapeutic amounts to the blood circulation,

has been reported by many researchers (Abdulbaqi et al., 2016). For instance, the

pharmacokinetics and pharmacodynamics effects of a valsartan-loaded ethosomal

system were studied in albino Wistar rats. The bioavailability of transdermal

16

ethosomal valsartan was significantly higher (3.03 times) than the oral valsartan

suspension; the area under the concentration–time profile curve from time 0 to infinity

for the transdermal ethosomal formulation was 177,298.82±665.01 ng/ml/hr., while

for the oral suspension it was 55,554.54±774.01 ng/ml/hr. The dose of valsartan used

in both routes was 3.6 mg/kg. The reported maximum drug concentration (Cmax) and

the time taken to reach maximum concentration (Tmax) values for the oral

administration were 13,100±101.12 ng/ml and 1±0.01 hr, respectively. The Cmax and

Tmax values for the transdermal route were 7,944±134.32 ng/ml and 5.0±0.83 hr.,

respectively (Ahad et al., 2013). Ethosomal valsartan was shown to effectively reduce

blood pressure by 34.11 % for 48 hr in hypertensive rats (Ahad et al., 2015).

The incorporation of ethosomal dispersions in suitable vehicles like gels, patches, and

creams represents an important step to get better skin-permeation and therapeutic

results. Several researchers have studied the skin permeation and disposition properties

of various drugs from ethosomal gels, and reported the high efficiency and superiority

of these formulations in dermal or transdermal drug delivery over the traditional or

marketed gels or creams. (Puri & Jain, 2012) compared the in vitro skin permeation

properties of 5-fluorouracil from ethosomal gel and marketed cream using Franz

diffusion cell and albino rat skin, and found that the transdermal flux of 5-fluorouracil

from ethosomal gels was 4.9 folds higher than the marketed cream. Moreover, the skin

disposition of the drug from the ethosomal gel was 9.4-fold higher than the marketed

cream (Puri & Jain, 2012). Other researchers found that in vitro transdermal flux of

aceclofenac from ethosomal gel was higher (226.1 μg/cm2/hr) than Zynac gel (131.1

μg/cm2/hr) (Dave et al., 2010). The development of colchicine loaded ethosomal gel

for transdermal delivery may offer a new route for administration of this venerable

17

drug to solve the several dose dependent side effects associated with its oral

administration and enhance its bioavailability.

1.8 Problem statement

Colchicine is a highly effective drug in the treatment of gout, pseudo-gout, FMF and

many other illnesses, but it is known to have a low therapeutic index and oral

bioavailability of 25 % to 50 % due to the extensive first-pass metabolism, its oral

administration is associated with mild to severe gastrointestinal side effects including:

abdominal cramps, pain, nausea, vomiting and diarrhea that will develop in around 80

percent of patients. Colchicine was used to be administered intravenously until it was

banned by the United States food and drug administration (FDA) in 2008 due to its

potential and serious adverse effects such as tissue necrosis cytopenias, disseminated

intravascular coagulation, and even death.

Therefore, colchicine transdermal administration by ethosomal gels may offer an

alternative route of administration of this drug to overcome its adverse reactions, and

low oral bioavailability problems associated with the oral route.

18

1.9 Study Objectives

The objectives of the proposed study are:

I. To optimize colchicine ethosomal systems using 23 and 24 factorial designs.

II. To develop and validate an HPLC method for the quantification of colchicine

in ethosomal gel and in the release media.

III. To formulate and optimize colchicine loaded ethosomal gel.

IV. To evaluate the stability of the optimized gel formulation.

V. To study the skin permeation behavior of the drug from the optimized gel

formulations.

19

CHAPTER 2

DEVELOPMENT AND CHARACTERIZATION OF COLCHICINE

ETHOSOMAL SYSTEMS

2.1 Introduction

Colchicine is used for the treatment of acute gout, familial Mediterranean fever (FMF)

and pseudo-gout (Martinon et al., 2006). However, colchicine is known to have low

oral bioavailability (25 – 50 %) due to the extensive first-pass metabolism (Ben-Chetrit

& Levy, 1998) and low therapeutic index (Terkeltaub, 2009).

Oral administration of colchicine is causing severe gastrointestinal side effects,

including abdominal cramps and pain, nausea, vomiting and diarrhea (Niel &

Scherrmann, 2006).

To overcome these drawbacks, and to improve colchicine bioavailability, some

researchers studied and prepared microemulsions (Ganju et al., 2012) nanoemulsions

(Shen et al., 2011), transdermal patches (Kumar et al., 2012), iontophoresis (Kulkarni

et al., 1996) and elastic liposomes (Singh et al., 2009).

Ethosomal systems are novel nanocarriers especially designed for the delivery of drugs

dermally or transdermally, and may represent useful tools for the delivery of

colchicine. Therefore, the aim of present study was to prepare colchicine ethosomes

for transdermal delivery. The three types of ethosomal systems were prepared namely:

classical ethosomes (CE), binary ethosomes (BE) and transethosomes (TE). The 23 and

24 factorial designs were used in the optimization of ethosomal formulations.

20

2.2 Materials

Colchicine (purity > 97 %) was purchased from (Acros organics, New Jersey, USA).

Phospholipon 90G® (PL90G) (contains > 90 % Phosphatidylcholine from soybean)

generously donated by (Lipoid LLC, New Jersey, USA). Absolute ethanol (purity ~

99.7 %) was bought from (Fine Chemicals Inc., USA). Tween 20® (purity ≥ 99 %) and

sodium stearate were purchased from (R & M Chemicals, Essex, UK). Sodium

taurocholate, cholesterol and Sephadex® G-25 were bought from (Sigma-Aldrich, St.

Louis, MO, USA). Propylene glycol (PG) acid was obtained from (QRëC, Selangor,

Malaysia). Labrafil® M 1944 CS (Oleoyl macrogol-6 glycerides EP) was purchased

from (Gattefossé, Lyon, France). Ultrapure water was prepared by Elga PURELAB

Classic UVF water purification system, France.

2.3 Methods

2.3.1 Preparation of the ethosomal systems

The three types of ethosomal systems namely classical ethosomes (CE), binary

ethosomes (BE) and transethosomes (TE) were prepared using the cold method

(Abdulbaqi et al., 2016; Touitou et al., 2000). In this method the organic phase was

obtained by dissolving the PL90G at 30 °C in ethanol for preparation of CE or mixture

of solvents (ethanol/polyethylene glycol) for the preparation of BE. Surfactants (either

Tween 20®, Sodium taurocholate or Labrafil®) were added into organic phase for the

preparation of TE. The aqueous phase (Ultrapure water) was heated to 30ºC as well

and then added to organic phase dropwise with continuous stirring at 1200 rpm, using

magnetic stirrer. The mixing continued for 45 min to get the ethosomal dispersions

which are further subjected to size-reduction process by multiple extrusions through