formulation and evaluation of colchicine...
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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
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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