Transdermal Delivery System of Testosterone Using Solid Lipid Particles

I
Transdermal Delivery System of

Testosterone Using Solid Lipid Particles

Presented By
Iman Mohammed Rashed Al-Fagih
(B. Pharm. Sci. 2000)

A Dissertation Submitted in
Partial Fulfillment of the Requirements for the Masters
Degree in Pharmaceutical Sciences
(Pharmaceutics)

Departments of Pharmaceutics
College of Pharmacy
King Saud University

1428 H-2007 G
II

SUPERVISORY COMMITTEE

Principle Supervisor
Dr. Amal H. El-Kamel

Associate Professor of Pharmaceutics,
College of Pharmacy

Co-Advisor
Dr. Ibrahim A. Alsarra

Associate Professor of Pharmaceutics,
College of Pharmacy

III

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IV
Acknowledgments

In the name of Allah the merciful, the compassionate. Peace be upon
prophet Mohammed and his followers.
First, I am deeply grateful to the almighty Allah for enabling me to
complete my thesis. The gracious has fortified me with patience and
endurance which were essential ingredients for the birth of this work.
I would like to thank the following people for their kind help during my
work:
To Dr. Amal H. El-Kamel: Associate Professor of Pharmaceutics, College
of Pharmacy, King Saud University, and my great Supervisor without
whom none of this could have happened. She was willing to guide,
explain and solve problems facing me during my work. She was
supportive from beginning to end and always encouraged me to follow
through. She consistently holds herself to a high standard, I feel blessed
by our relationship.
To Dr. Ibrahim A. Alsarra: Associate Professor of Pharmaceutics, College
of Pharmacy, King Saud University, and my Co-Advisor, for his kind
cooperation.
To Dr. Fars Al-Enzy: Chairman of Pharmaceutics Department, Associate
Professor of Pharmaceutics, College of Pharmacy, King Saud University,
for his kind help.
V
To Dr. Fouad Al-Dayel: Chairman of Department of Pathology and
Laboratory Medicine, King Faisal Hospital, Kingdom of Saudi Arabia,
for valuable assistance in interpretation of histological studies.
To Collage of High Educations: the Research Centre, King Saud
University, for the financial support.
To my parents: my wonderful mother, my faithful helper and moral
booster whose wisdom and unconditional love pulled me through the
bumpy road, and whose input I always hold valuable, I am eternally
grateful. To my father, who first approached me to pursue my education.
To my brothers and sisters: for their continuous assistance.
To my family: my dear husband and precious kids for the support they
gave me at home, infinite assistance and patience during my work.
Finally, I pray to almighty Allah so that I can pursue my education.

VI

Acknowledgment to King Abdullaziz City for
Science and Technology

I am grateful to King Abdulaziz City for Science and
Technology for their generous financial support.

I
Contents

List of abbreviations ................................................................................ IV
List of Tables ........................................................................................... IX
List of Figures.......................................................................................... XI
INTRODUCTION..........................................................................................1
1. Rational of transdermal drug delivery....................................................2
2. Anatomy and physiology of the skin......................................................3
3. Permeation pathways through the skin...................................................5
3.1. Transappendageal transport (shunt route transport) ........................5
3.2. Intracellular route (Transcellular) ....................................................6
3.3. Intercellular route .............................................................................6
4. Factors affecting transdermal drug delivery...........................................7
4.1. Physicochemical properties of permeant .........................................7
4.2. Physiological factors ........................................................................9
4.3. Pathological disorders ....................................................................12
5. Mathematical model for analysis of data..............................................13
6. Transdermal formulations.....................................................................14
7. Optimizing transdermal drug delivery..................................................15
7.1. Drug and vehicle interactions.........................................................16
7.2. Formulation approachs...................................................................19
7. 3. Stratum corneum modification......................................................21
7.4. Stratum corneum bypassed /removed ............................................25
7.5. Electrically assisted methods .........................................................27
8. Testosterone..........................................................................................38
9. An overview on the transdermal delivery systems of testosterone ......41
OBIECTIVE...............................................................................................44
METHODOLOGY.......................................................................................47
1. Materials ............................................................................................48
2. Apparatus ...........................................................................................49
3. Methods .............................................................................................51
3.1. High performance liquid chromatography (HPLC) assay of
testosterone............................................................................................51
3.2. HPLC calibration standards / quality control of testosterone.....51
3.3. Assay validation of testosterone .................................................52
3.4. Preparation of testosterone solid lipid microparticles ................52
3.5. Morphological examination of the prepared SLM.....................53
3.6. Particle size analysis of the prepared SLM.................................54
3.7. Rheological studies of the prepared SLM..................................55
3.8. Differential scanning calorimetry (DSC)....................................55
3.9. Powder X-ray diffractometry (PXRD) .......................................56
3.10. Drug entrapment efficiency of the prepared SLM (% EE) .....56
II
3.11. Occlusion test ..........................................................................57
3.12. Solubility study of testosterone...............................................58
3.13. In vitro release studies of testosterone through cellophane
membrane after application of different solid lipid microparticles
formulations ..........................................................................................58
3.14. In vitro permeation studies of testosterone through excised
abdomen rat skin after application of the selected testosterone SLM
formulations ..........................................................................................62
3.15. Effect of application of high frequency ultrasound (HUS)
and/or chemical enhancer on in vitro permeation of testosterone
through excised abdomen rat skin after application of the selected
testosterone SLM formulation...............................................................63
3.16. Effect of application of low frequency ultrasound (LUS)
and/or chemical enhancer on in vitro permeation of testosterone
through excised abdomen rat skin after application of the selected
testosterone SLM formulation...............................................................64
3.17. Stability studies .......................................................................65
3.18. Effect of freeze drying on the selected SLM formulation.......66
3.19. Skin irritation test ....................................................................67
3.20. Histological examination of excised rat skin ..........................67
3.21. Testosterone skin retention......................................................68
3.22. Statistics...................................................................................68
RESULTS AND DISCUSSION...................................................................69
1. High performance liquid chromatography (HPLC) assay of
testosterone ...............................................................................................70
2. Assay validation.................................................................................70
3. Morphological examination of the prepared SLM............................74
4. Particle size analysis of SLM............................................................74
5. Rheological studies............................................................................80
6. Differential scanning calorimetry (DSC) ..........................................86
7. Powder X-ray diffraction (PXRD).....................................................94
8. Drug entrapment efficiency of SLM (% EE).....................................97
9. Occlusion Study...............................................................................101
10. Permeability studies of testosterone.............................................101
10.1. Effect of type and concentration of the lipid.........................102
10.2. Effect of transporting membrane...........................................109
10.3. Effect of drug loading............................................................112
10.4. Effect of chemical enhancer ..................................................116
alone or in combination with chemical enhancers..............................120
10.6. Effect of application of low frequency ultrasound (LUS) alone
or in combination with chemical enhancers........................................123
11. Stability studies ............................................................................137
III
11.1. Stability of selected formulation ...........................................137
11.2. Stability of formulation containing 1% OA or 1% DA.........141
12. Effect of freeze-drying on the selected formulation ....................144
13. Skin irritation test .........................................................................149
GENERAL CONCLUSION.......................................................................151
SUMMARY..............................................................................................153
REFERENCES........................................................................................158
ARABIC SUMMARY..............................................................................181

IV
List of abbreviations

a Thermodynamic activity of drug
ANOVA Analysis of variance.
b Regression coefficient.
c Speed of sound in the medium.
C Initial concentration of drug in donor solution
cps Centipoise
CV % Percentage of coefficient of variation.
D Diffusion coefficient
D` Diffusion parameter
DA Dodecylamin
dC/dx Concentration gradient
dF Degree of freedom
DMSO Dimethylsulphoxide.
DSC Differential scanning calorimeter.
E Acoustic energy emitted.
EE Entrapment efficiency.
ER Enhancement ratio.
F Occlusion factor.
FDA Food and drug administration.
GB Glycerol dibehenate.
V
GD Glycerol distearate.
GM Glycerol monostearte
GRAS Generally recognized as safe.
h Thickness of the membrane
HIV Human immunodeficiency virus
HLB Hydrophilic lipophilic balance
HPLC High performance liquid chromatography.
HQC High concentration quality control
HUS High frequency ultrasound.
I Intensity
J Flux
K
o/w
Partition coefficient.
KDa Kilodalton
KHz Kilohertz
KSA Kingdom of Saudia Arabia
L Liter
Log K Log partition coefficient
LPP Lipid-protein partitioning.
LQC Low concentration quality control
LS Lipospheres.
L
t
Lag time
VI
LUS Low frequency ultrasound.
M Amount of material flowing through unit cross-section of a
barrier.
m Cumulative mass of diffusant
mA Milliampere
mg Milligram
MHz Megahertz
min Minute
ml Milliliter
mm Millimeter
MQC Medium concentration quality control
MS Mean squares.
MTS Microstructured transdermal systems.
nm Nanometer
OA Oleic acid
P Permeability coefficient
PBS Phosphate-buffered saline
PI Polydispersity index.
PTFE Polytetrafluoroethylene
PXRD Powder X ray diffractometry
QC Quality control.
R Mean radius
VII
R Coefficient of determination.
rpm Round per minute
S Unit cross-section of a barrier
s Standard deviation
SA Stearic acid
SC Stratum corneum
SD Standard deviation.
sec Second
SEM Scanning electron microscope.
SLM Solid lipid microparticles.
SLN Solid lipid nanoparticles.
SLS Sodium lauryl sulfate.
SPSS Statistical package for social studies.
SS Sum squares.
t Time
t
eff
Sum of the time when US is on.
TEWL Transepidermal water loss.
T
m
Transition temperature.
t
off
Time when US is off.
t
on
Length of the pulse when US is on.
TS Testosterone
VIII
t
us
Total exposure time.
UK United Kingdom
US Ultrasound.
USA United State of America
V Volt
l Microliter
m Micrometer
Effective activity coefficient in the skin barrier

IX
List of Tables
Table 1. HPLC calibration curve of TS. ...................................................71
Table 2. Intraday precision (CV %) and accuracy (% recovery) data for
HPLC quality control samples of TS. ......................................72
Table 3. Interday precision (CV %) and accuracy (% recovery) data for
HPLC quality control samples of TS. ......................................72
calibration standard concentrations of TS................................73
calibration standard concentrations of TS................................73
Table. 6. Reproducibility of peak area (run to run) in the analysis of TS
performed using five sets of quality control samples on five
different days............................................................................75
Table 7. Reproducibility of peak area (run to run) in the analysis of TS
performed using five sets of calibration standard concentrations
on five different days. ..............................................................75
Table 8. Mean particle size, polydispersity index (PI) and zeta potential
of different SLM formulations containing 2.5 mg TS/g of SLM
dispersion. ................................................................................78
Table 9. One-way analysis of variance showing the effect of lipid type on
the particle size of SLM. ..........................................................79
Table 10. One-way analysis of variance for viscosity of different SLM
formulations that contained 5 % lipid and 2.5 mg TS/g of SLM
dispersion at 0.5 rpm. ...............................................................85
Table 11. DSC results of bulk materials and SLM formulations. ............91
Table 12. Drug entrapment efficiency (% EE) of different SLM
formulations containing 2.5 mg TS/g of SLM dispersion. ......98
Table 13. One-way analysis of variance for drug entrapment efficiency
(% EE) of different SLM formulations. ...................................99
Table 14. Coefficient of determination (R) calculated after fitting the
permeation data of TS into Ficks and Higuchi equations.
X
Cellophane membrane was used as transporting membrane. All
formulations contained 2.5 mg TS/g of SLM dispersion.......105
Table 15. In vitro permeation parameters of TS after application of
different SLM formulations contained 2.5 mg TS/g of SLM
dispersion to cellophane membrane (n=3). ............................106
Table 16. Two-way analysis of variance for in vitro permeation of TS
after application of different SLM formulations to cellophane
membrane. All formulations contained 2.5 mg TS/g of SLM
dispersion. ..............................................................................107
Table 17. Permeation parameters of TS after the application of selected
SLM formulations to excised abdomen rat skin. (n=3) .........110
Table 18. Effect of application of chemical enhancers and/or HUS on
permeation parameters of TS through excised abdomen rat skin
after application of 10 % GB SLM containing 5 mg TS/ g of
SLM dispersion. .....................................................................118
Table 19. Effect of application of LUS at different duty cycles and
intensities and application of LUS and chemical enhancer on
SLM dispersion . ....................................................................127
Table 20. Scores for Draize test of skin irritation after application of the
examined formulations to the rabbit dorsal skin (n = 6)........150

XI
List of Figures
Figure 1. Skin structure
(9)
. .........................................................................4
Figure 2. Permeation routes through the stratum corneum: (1) via the lipid
matrix between the corneocytes (Intercellular route), and (2)
across the corneocytes and the intercellular lipid matrix
(Transcellular route)
(12)
. ............................................................7
Figure 3. Types of transdermal patches
(20)
. .............................................15
Figure 4. Scheme that summarizes techniques for circumventing the
stratum corneum barrier
(5)
. ......................................................16
Figure 5. Different types of microneedles array and patches
(59)
. ............26
Figure 6. Optical images of a 2.5 m-radius microbubble exposed to 5
cycles of 2.5 MHz ultrasound at 1.6 MPa pressure amplitude.
The left panel shows the bubble before exposure. The central
panel shows a streak photograph with the measured pressure
superimposed at the top of the panel. The right panel shows the
fragments produced by the collapse of the cavitating bubble
(65)
.
..................................................................................................30
Figure 7. Illustration of an asymmetric collapse of a bubble near a
surface, producing a jet of liquid toward the surface
(65)
. ........30
Figure 8. Schematic representation of various modes by which drug
delivery can be enhanced by ultrasound. A: therapeutic agent
(triangles); B: gas bubble undergoing stable cavitation; C:
microstreaming around cavitating bubble; D. collapse
cavitation emitting a shock wave; E: asymmetrical bubble
collapse producing a liquid jet that pierces the endothelial
lining; F: completely pierced and ruptured cell; G: non-
ruptured cells with increased membrane permeability due to
insonation; H: cell with damaged membrane from
microstreaming or shock wave; I: extravascular tissue; J: thin-
walled microbubble decorated with agent on surface; K. thick-
walled microbubble with agent in lipophilic phase; L: micelle
with agent in lipophilic phase; M: liposome with agent in
aqueous interior; N: vesicle decorated with targeting moieties
attached to a specific target
(65)
.................................................31
Figure 9. Second Generation SonoPrep
.
(77)
..........................................35
XII
Figure 10. Drug penetration pathway in low voltage iontophoresis and
high voltage electrophoresis .
(78)
..............................................38
Figure 11. Three stations vertical Franz diffusion system. ......................60
Figure 12. HPLC calibration curve of TS. (n=5) .....................................71
Figure 13. Scanning electron micrographs of SLM formulations
containing 2.5 mg TS/ g of: a) 5 % GB, b) 10 % GB, c) 5 %
GD, d) 10 % GD, e) 2.5 % GM, f) 5 % GM, g) 2.5 % SA and
h) 5 %.SA SLM dispersion. .....................................................76
Figure 14. Rheograms of different SLM formulations containing 2.5 mg
TS/g of SLM dispersion: a) 5 % GD, b) 10 % GD, c) 5 % GB
and d)10 % GB.........................................................................81
TS/g of SLM dispersion: a) 2.5 % GM, b) 5% GM, c) 2.5 %
SA, d) 5 % SA. .........................................................................82
Figure 16. Viscosity of different SLM formulations containing 2.5 mg TS
/ g of SLM dispersion at 0.5 rpm. ............................................84
Figure 17. DSC thermograms of TS, bulk GM, 2.5 % GM, and 5 % GM
SLM formulations containing 2.5 mg TS/g of SLM dispersion.
..................................................................................................87
Figure 18. DSC thermograms of TS, bulk GD, 5 % GD, and 10 % GD
..................................................................................................88
Figure 19. DSC thermograms of TS, bulk SA, 2.5 % SA, and 5 % SA
..................................................................................................89
Figure 20. DSC thermograms of TS, bulk GB, 5 % GB, and 10 % GB
formulations containing 2.5 mg TS/g of SLM dispersion. ......90
Figure 21. PXRD patterns of: a) TS, b) lyophilized SLM (10 % GB
containing 5 mg TS/g of SLM dispersion). .............................95
Figure 22. PXRD patterns of: a) bulk lipid (GB), b) lyophilized drug-free
SLM (10 % GB). ......................................................................96
Figure 23. Cumulative amount of TS released through cellophane
membrane after application of different SLM formulations
XIII
containing different types of lipid and 2.5 mg TS/g of SLM
dispersion. ..............................................................................103
containing different concentration of lipid and 2.5 mg TS/g of
SLM dispersion. .....................................................................104
Figure 25. Cumulative amount of TS permeated through excised
abdomen rat skin after application of 10% GB and 10% GD
SLM formulations containing 2.5 mg TS/ g of SLM dispersion.
................................................................................................111
abdomen rat skin after application of 10% GB SLM with
different drug loading concentrations. ...................................113
Figure 27. Correlation between the amounts of TS permeated through
excised abdomen rat skin and through cellophane membrane
over 24 h at the same time point. ...........................................115
abdomen rat skin using different chemical enhancers and/or
HUS. The examined formulation was 10% GB containing 5 mg
TS /g of SLM dispersion. .......................................................117
Figure 29. Histological changes of excised abdomen rat skin a) untreated
and after application of b) HUS, c) HUS +1% DA 30 min, d)
1% DA 30 min. ......................................................................124
Figure 30. Effect of total application time of LUS or duty cycle, over total
exposure time of 30 min with intensity of 2.5 W/cm on the
flux of TS through excised abdomen rat skin after application
of 10 % GB containing 5 mg TS/g of SLM dispersion.
2
........126
Figure 31. Histological characteristics of excised abdomen rat skin a)
untreated skin, and at different total application time of LUS or
duty cycle, b) 10:40 LUS, c) 10:15 LUS, d) 10:10 LUS over
total exposure time of 30 min with intensity of 2.5 W/cm .
2
..128
Figure 32. Flux of TS through excised abdomen rat skin after application
of 10 % GB containing 5 mg TS/g of SLM dispersion using
LUS at different intensities and total application time of 12 min
over total exposure time of 30 min. .......................................131
XIV
untreated, and after application of LUS at on/off 10:15 duty
cycle and at different intensity b) 2.5 W/cm , c) 3.5 w/ cm and
d) 5 w/cm , and e) LUS 2.5 W/cm after 24h.
2 2
2 2
.......................133
Figure 34. Effect of application of 1% DA for 30 min &/or LUS at
intensity of 2.5 W/cm at total application time t of 12 min
over total exposure time of 30 min on flux of TS through
excised abdomen rat skin after application of 10 % GB
containing 5 mg TS/g of SLM dispersion.
eff
.............................136
Figure 35. Cumulative amount of TS permeated after 24 h through
excised abdomen rat skin after application of the selected SLM
formulation (10 % GB containing 5 mg TS/ g SLM dispersion)
stored over16 week at 5 and 30 C. Bars with similar symbols
are statistically insignificant (A>B>C), (a>b>c>d). ..............139
Figure 36. Mean particle size of the selected SLM formulation (10 % GB
containing 5 mg TS/ g SLM dispersion) stored over16 week at
5 and 30 C. Bars with similar symbols are statistically
insignificant (A>B), (a>b). ....................................................140
Figure 37. Photographs showing the physical change in SLM
formulation: a) selected formulation, b) SLM containing 1 %
OA, and c) SLM containing 1 % DA after storage for 2 and 12
week at 5 C...........................................................................142
OA, and c) SLM containing 1 % DA after storage for 2 and 12
week at 30 C..........................................................................143
Figure 39. Effect of method of addition of trehalose before freeze drying
on cumulative amount of TS permeated through excised
abdomen rat skin. The applied formulation was 10 % GB
containing 5 mg TS/g of SLM dispersion..............................146
on the mean particle size. The examined formulation was 10 %
GB containing 5 mg TS/ g SLM dispersion. .........................148

1

INTRODUCTION

2
1. Rational of transdermal drug delivery
Transdermal delivery is the delivery of the drugs through the skin to
affect a pharmacological action at a location remote from the site of
application. Transdermal therapy can be regional or systemic
(1)
.
Transdermal route includes a large and varied surface as well as ease of
application
(1)
. Transdermal drug delivery offers several advantages over
the traditional methods. Firstly, compared to injections, it eliminates the
associated pain and the possibility of infection. Also, weighed against the
oral delivery, it avoids gastrointestinal drug metabolism (since it by
passes the hepatic first pass effect), reduces elimination by liver and
provides sustained release of drug. Transdermal drug delivery also offers
the relative ease of drug input termination in problematic cases as well as
maintaining stable plasma level and, moreover, transdermal systems do
not require high patient compliance
(1-3)
.
However, the negatives of transdermal drug delivery are skin irritation,
relatively high manufacturing costs and less-than-ideal cosmetic
appearance
(4)
.
Recently, the transdermal route has been vied with oral treatment as the
most successful innovative research area in drug delivery
(5)
. In the USA
(the most important clinical market), out of 129 drug delivery candidate
products under clinical evaluation, 51 are transdermal or dermal systems;
30 % of 77 candidate products in preclinical development represents such
3
drug delivery
(5)
. The worldwide transdermal patch market approaches
3 billion annually, yet is based on only few drugs scopolamine
(hyoscine), nitroglycerine, clonidine, estradiol (with and without
norethisterone or levonorgestrel), testosterone, fentanyl, nicotine,
lidocaine, and oxybutinin
(6)
. The fundamental reason for such few
transdermal drugs is that highly impermeable human skin limits daily
drug dosage, delivered from an acceptable sized patch, to about 20 mg
(7)
.
2. Anatomy and physiology of the skin
In order to design a successful transdermal drug delivery system, it is
important to understand the structure, physiology and function of the
skin. The skin of an average adult body covers around 2 m of surface
area and receives approximately one-third of all blood circulating through
the body. It is one of the most extensive and readily accessible organs on
the human body. With thickness of only fraction of millimeter, the skin
separates the underlying blood circulation network from the outside
environment and serves as a barrier against physical, chemical and
microbial attacks, act as a thermostat in maintaining body temperature,
and protect against the penetration of ultraviolet rays
(8, 9)
. It is covered
with an acid mantle that is believed to be responsible for the prevention
of the growth of microorganisms on the skin. The pH of the skin has been
reported to be between 4.8 and 6
(1)
.
The skin is a multilayer organ composed of many histological layers. It is
4
generally described in terms of three major layers: the epidermis, the
dermis and the hypodermis (Figure 1)
(8)
.
Figure 1. Skin structure
(9)
.
The epidermis is the outermost layer of the skin. It is also the thinnest
part of the skin, and its thickness varies depending on its location in the
body. It is considered as the rate limiting layer for transdermal
absorption of drugs. It is essentially a stratified epithelium, consisting of
four distinct layers: the stratum germinativum (basal layer); the stratum
spinosum (prickle cell layer); the stratum granulosum (granular layer);
and the stratum corneum (horny layer)
(8)
.
The outermost layer of the epidermis is the stratum corneum and
5
considered as the major barrier to permeation. It is 10 to 20 m thick and
consists of dead, anucleate, keratinized cells embedded in a lipid matrix.
These cells are termed as keratinocytes or corneocytes.
The dermis is the connective tissue layer that separates the epidermis
from the subcutaneous fat layer. There are two layers of the dermis, the
papillary dermis and the reticular dermis. The papillary dermis is a thin
superficial layer that lies adjacent to the epidermis. It contains capillary
venules, lymph vessels, and nerve tissue. The reticular dermis is the
thicker area of the dermis and form the bulk of the dermis. An extensive
network of dermal capillaries connects to the systemic circulation;
consequently, it acts as the systemic absorption site for drugs. Hair
follicles, sebaceous glands and sweat glands are found in the dermis and
subcutaneous fat layer
(8)
.
Hypodermis or subcutaneous fat layer, bridges between the overlying
dermis and the underlying body constituents providing mechanical
protection against physical shock
(8)
.
3. Permeation pathways through the skin
There are three pathways by which a molecule can traverse stratum
corneum:
3.1. Transappendageal transport (shunt route transport)
The appendages (hair follicles, sweat ducts) offer pores that bypass
6
the barrier of the stratum corneum. However, these openings onto the
skin surface occupy only around 0.1% of the total skin surface area
(10)
.
Sweat glands opening on the skin surface are very small and these ducts
are either evacuated or are actively secreting sweat that would be
expected to diminish inward diffusion of topically applied agents. The
opening of the follicular pore to the skin surface is considerably larger
than that of the sweat glands but again they also filled with fluid. The
shunt routes may be important for ions and large polar molecules that
struggle to cross intact stratum corneum
(11)
.
3.2. Intracellular route (Transcellular)
The pathway is directly across the stratum corneum
(12)
, as shown in
Figure 2, and the molecule crossing the intact stratum corneum faces
numerous repeating hurdles. First, there is partitioning into the
keratinocyte, followed by diffusion through the hydrated keratin. In order
to leave the cell, the molecule must partition into the bilayer lipids before
diffusing across the lipid bilayer to the next keratinocyte. For highly
hydrophilic molecules the transcellular route, Figure 2, may be
predominant
(13)
.
3.3. Intercellular route
In this rout the pathway is via lipid matrix between the keratinocytes
(12)
,
as shown in Figure 2. It is now accepted that this route provides the
7

Figure 2. Permeation routes through the stratum corneum: (1) via the
lipid matrix between the corneocytes (Intercellular route), and
(2) across the corneocytes and the intercellular lipid matrix
(Transcellular route)
(12)
.
principle pathway by which most small, uncharged molecules traverse stratum
corneum.
(13)
.
4. Factors affecting transdermal drug delivery
4.1. Physicochemical properties of permeant
4.1.1. Partition coefficient
For molecules with intermediate partition coefficient (log K 1 to 3) and
for highly lipophilic molecules (log K > 3), the intercellular route will be
almost the pathway used to traverse the stratum corneum. However, for
these molecules a further consideration is the ability to partition out of the
stratum corneum into the aqueous viable epidermal tissues. For more
8
hydrophilic molecules (log K < 1), the transcellular route probably
predominates
(13)
.
4.1.2. Molecular size
A second major factor in determining the flux of a material through
human skin is the size of the molecule. However, for simplicity, the
molecular weight is generally taken as an approximation of molecular
size. It has been suggested that an inverse relationship existed between
transdermal flux and molecular weight of the molecule. However, most
conventional therapeutic agents that are selected as candidates for
transdermal delivery tend to lie within narrow range of molecular weight
(100-500 Dalton)
(13)
.
4.1.3. Solubility/melting point
It is will known that most organic materials with high melting points have
relatively low aqueous solubility at normal temperature and pressure.
The lipophilic molecules tend to permeate through the skin faster than
more hydrophilic molecules. However, while lipophilicity is a desired
property of transdermal candidates, it is also necessary for the molecule
to exhibit some aqueous solubility since topical medicaments are
generally applied from an aqueous formulation
(13)
.
4.1.4. Ionization
According to pH-partition hypothesis, only the unionized form of the
drug can permeate through the lipid barrier in significant amounts
(13)
.
9
4.1.5. Other factors
Beyond the factors mentioned above, there are other molecular properties
that can affect drug delivery through the skin. Drug binding is a factor
that should be born in mind when selecting appropriate candidates.
Interactions between drug substances and the tissue can vary from
hydrogen bonding to weak Van der Waals forces, and the effect of drug
binding (if any) on flux across the tissue will vary depending on the
permeant. For example, with a poorly water-soluble drug in an aqueous
donor solution, significant binding to the stratum corneum may
completely retard drug flux. Consequently, there will be a delay between
applying a drug to the surface of the tissue and its appearance in a
receptor solution (in vitro) or the blood (in vivo)
(13)
.
Depending on the type of formulation selected, other factors may be
important in a transdermal delivery system. For example, if the drug is
suspended, then the particle size may become a key regulator of flux
(13)
.
4.2. Physiological factors
4.2.1. Skin barrier properties in the neonate and young infant
The skin of newborns is known to be relatively susceptible to irritants
(14)
.
Other variables related to stratum corneum function such as pH and
stratum corneum hydration may enhance the irritant potential to newborn
skin. Skin surface pH values in newborns are significantly higher in all
body sites than those in adult skin, but stabilize at values similar to adults
10
within the first month
(15)
. There are also significant changes in the
metabolic capacity of infants, whether full or preterm, and adult levels of
cutaneous enzyme activity are not observed until 2 months or even 612
months of age which may additionally account for the sensitivity of baby
skin to irritants
(14)
.
The skin surface of the newborn is slightly hydrophobic and relatively
dry and rough when compared to that of older infants. Stratum corneum
hydration stabilizes by the age of 3 months
(14)
.
4.2.2. Skin barrier properties in aged skin
There are changes in the physiology of aged skin (>65 years). The
corneocytes are shown to increase in surface area which may have
implications for stratum corneum function due to the resulting decreased
volume of intercorneocyte space per unit volume of stratum corneum.
The moisture content of human skin decreases with age. There is a
flattening of the dermoepidermal junction and, consequently, the area
available for diffusion into the dermis is diminished. Other age-related
changes include reductions in the absolute number of hair follicles, in the
diameter of the hair, and pilosebaceous units are also expected
(14)
.
4.2.3. Race
Racial differences between black and white skins have been shown in
some anatomical and physiological functions of the skin although data is
relatively sparse. In black skin, increased intracellular cohesion, higher
11
lipid content and higher electrical skin resistance levels compared to
whites have been demonstrated. Black skin appears to have a decreased
susceptibility to cutaneous irritants, but this difference is not detected in
stripped skin, suggesting the stratum corneum modulates the different
racial response to irritants. Black skin responds with a decrease in blood
flow and hence less erythematic than Hispanics or Caucasians.
By comparison, Chinese, Malay, Indian and Caucasian skin has shown no
difference in barrier integrity and function
(14)
.
4.2.4. Body site
It is readily apparent that skin structure varies to some degree over the
human body. However, the relative permeability of different skin sites is
not simply a function of stratum corneum thickness as different
permeants exhibit varied rank orders through different skin sites. It is
apparent that genital tissue usually provides the most permeable site for
transdermal drug delivery. The skin of the head and neck is also relatively
permeable compared to other sites of the body such as the arms and legs.
Intermediate permeability for most drugs is found on the trunk of the
body
(16)
.
4.2.5. Other factors
The level of hydration of the stratum corneum may have a dramatic effect
on drug permeation through the tissue, and increasing hydration is well
known to increase transdermal delivery of most drugs. Indeed, occlusive
12
dressings and patches are highly effective strategies to increase
transdermal drug delivery since they create elevated hydration of the
stratum corneum.
The human body maintains a temperature gradient across the skin from
around 37 C to around 32 C at the outer surface. Since diffusion
through the stratum corneum is a passive process, elevation of the skin
temperature can induce structural alterations within the stratum corneum,
and these modifications can also increase diffusion through the tissue
(16)
.
4.3. Pathological disorders
Numerous disorders result in an eruption of the skin surface. In such
cases, the barrier properties of the stratum corneum are compromised,
allowing the passage of drugs (and potentially toxic materials) into and
through the skin. Likewise, the erupted skin surface will allow increased
water loss from the body
(14)
.
Psoriasis is one of the most common skin diseases. It is associated with
reduced barrier skin function with transepidermal water loss (TEWL) up
to twenty times higher in active psoriasis. The reduced barrier function,
which is correlated with signs of scaling, enables increased percutaneous
absorption of topically applied compounds. The plaques are largely
devoid of intracellular lipid, reducing the convoluted lipid pathway to the
dermoepidermal junction, thus enhancing permeation
(14)
.

13
Eczema in the chronic stage is often characterized by lichenification, a
dry thickened leathery state, with increased cell markings caused by,
repeated scratching and rubbing. In such areas, where the skin is intact,
absorption may be retarded, since the absorptive path is increased
(16)
.
Non-eczematous skin of patients with a prior history of atopic eczema
shows abnormalities in lipid metabolism resulting in a decrease in stratum
corneum lipids. In addition to a reduced barrier function it is presumed
that these compositional differences are related to the decrease in water
binding capacity of eczematous skin. Total epidermal water loss is
elevated in patients with atopic eczema (up to 10 fold)
(16)
.
Infections cause damage to the skin barrier integrity varies depending on
the severity of the infection. This is usually favorable for topical
treatment of infection, but it must be remembered that the barrier is
dynamic and will be restored as the condition improves, therefore, drug
flux across the repairing tissue will be expected to be slow
(16)
.
5. Mathematical model for analysis of data
Commonly, Fick's first law is used to describe the transfer of a diffusing
substance through a particular material
(17)
. According to Fick's first law,
the amount, M, of material flowing through a unit cross-section, S, of a
barrier in unit time, t, is known as the flux, J
(18)
.
J=dM/(S.dt) [1]
The flux in turn is proportional to concentration gradient, dc/dx:
14
J=-D(dc/dx) [2]
Where, D is the diffusion coefficient (cm/sec). Equation 2 is known as
Fick's first law.
The permeability coefficient, P (cm/sec), is defined by the following
equation:
P=DK/h [3]
Where, K is the partition coefficient; D is the diffusion coefficient; and h
is the thickness of the barrier.
The lag time (L
t
) is given by:
L
t
= h/6P [4]
6. Transdermal formulations
Smith et al. (1999)
(19)
have classified topical dermatological vehicles as
liquids, semisolids and solids. Each vehicle has been sub classified as
monophase (solution, ointment and powder), diphase (suspension,
emulsion and some patches) and multiphase (multiple emulsions and
some patches).
Broadly speaking, most commercially available patches can be
categorized as reservoir systems, matrix systems without a rate-
controlling membrane or matrix systems with a rate-controlling
membrane
(20)
. Figure 3 illustrates types of transdermal patches.
15

Figure 3. Types of transdermal patches
(20)
.

7. Optimizing transdermal drug delivery
The fundamental reason for such few transdermal delivery systems is that
highly impermeable human skin limits daily drug dosage, delivered from
an acceptable sized patch, to about 20 mg
(7)
as mentioned before. How
to increase this low limit for topical systems in general provides a major
challenge to scientists. Human skin effectively inhibits drug permeation,
mainly because of the stratum corneum. Thus, to maximize drug flux,
formulators have to reduce the hindrance of this barrier. Figure 4 shows a
scheme that summarizes some applied techniques to circumvent the
(5)
.

16

OPTIMISING TRANSDERMAL
DRUG DELIVERY
Drug/Vehicle
Interactions
Formulation
Approach
Stratum
Corneum
Modified
Stratum Corneum
Bypassed /
Removed
Electrical
Approach
Drug / Prodrug
Chemical
potential
Ion Pair /
Coacervates
Eutectic
Systems
Liposomes &
vesicles
High Velocity
Particles
Hydration
Chemical
Enhancers
Microneedle
Array
Ablation
Follicular
Delivery
Ultrasound
Ionto-
phoresis
Electro-
poration
Magneto-
phoresis
Photo-
mechanical
Wave
Figure 4. Scheme that summarizes techniques for circumventing the
(5)
.

7.1. Drug and vehicle interactions
7.1.1. Selection of correct drug or prodrug
The simplest approach is to choose a drug from a pharmacological class
with the correct physicochemical properties to translocate across the
barrier at an acceptable rate. A useful way to consider factors affecting
drug permeation rate through stratum corneum is via equation 5
(21)
. If
the cumulative mass of diffusant, m, passing per unit area through the
membrane, was plotted versus time, the slop of linear portion of the graph
yields the steady state flux, dm/dt,
17
dm/dt=DC
SK/h [5]
Where C

is the constant concentration of drug in donor solution, K is the
partition coefficient of solute between membranes and bathing solution,
D is the diffusion coefficient and h is the thickness of membrane.
According to equation 5 the ideal properties of a molecule penetrating
stratum corneum are: low molecular mass, preferably less than 600 Da,
high diffusion coefficient D, adequate solubility in oil and water, high but
balanced (optimal) partition coefficient K, and low melting point.
The partition coefficient is crucially important in establishing a high
initial penetrante concentration in the first stratum corneum layer. If the
drug does not possess the correct physicochemical properties (usually K
is too low), a suitable prodrug may have an optimal partition coefficient
for skin entry. After permeation to viable tissues, enzymes activate the
prodrug
(5)
.
7.1.2. Chemical potential adjustment
An alternative form of equation 5 uses thermodynamic activity
(22)
:
dm/dt=aD/ h [6]
Where, a is the thermodynamic activity of drug in its vehicle and is the
effective activity coefficient in the skin barrier. For maximum penetration
rate, the drug should be at its highest thermodynamic activity
(5)
.
Supersaturated solutions (i.e. non-equilibrated systems of high
thermodynamics activity) may arise, either by design or via a cosolvent
18
evaporation on the skin. The theoretical maximum flux may then increase
many folds. Polymers may be incorporated to inhibit crystallization in
unstable supersaturated preparations. Megrab et al. (1995)
(23)
have
achieved an 18-fold increase in stratum corneum uptake and a 13-times
increase in flux of estradiol at 18-times saturations. However, Schwarb et
al. (1999)
(24)
were unable to show an effect of supersaturation in
increasing the delivery of fluocinonide to the skin, as assessed by the
vasoconstrictor assay.
7.1.3. Ion pairs
Charged molecules do not readily penetrate stratum corneum. One
technique forms a lipophilic ion pair, by adding an oppositely charged
species. The complex partitions into the stratum corneum lipids, as
charges temporarily neutralize. The ion pair diffuses to the aqueous
viable epidermis and dissociates into its charged species, which partition
into the epidermis and diffuse onward
(5)
.
7.1.4. Eutectic systems
The formulation advantages of a eutectic mixture of prilocaine and
lidocaine in EMLA
cream (Astra Zeneca, Australia) prompted study of

such systems for other drugs
(25)
. For example, Stott et al. (1998, 2001)
(26,
27)
have investigated eutectic systems of ibuprofen formed with seven
terpenes and propranolol with fatty acids, correlating their interactions
with increased transdermal permeation. Kang et al. (2000)
(28)
have shown
19
that the lidocaine /menthol system promoted permeation through snake
skin.
7.2. Formulation approachs
7.2.1. Liposomes and other vesicles
Liposomes are colloidal particles, typically consisting of phospholipids
and cholesterol, with other possible ingredients. These lipid molecules
form concentric bimolecular layers that may entrap and deliver drugs to
the skin
(29)
. Most reports have cited a localizing effect whereby vesicles
accumulate drugs in stratum corneum or other upper skin layers
(30-32)
.
Generally, liposomes are not expected to penetrate into viable skin,
although occasional transport processes have been reported
(33)
. Vesicles
transport drugs through the skin is debatable and represents an important
area for further study. This controversy grew with the introduction of
transfersomes, which incorporate surfactant molecules such as sodium
cholate
(34, 35)
. The inventors have claimed that such vesicles, being
ultradeformable squeeze through pores in stratum corneum which are less
than one-tenth the liposomes diameter. Traditional liposomes in this
situation are expected to confine themselves to surface or upper layers of
stratum corneum, where they dehydrate and fuse with skin lipids
(30, 36)
.
Ethosomes are liposomes high in ethanol content (up to 45%). They
penetrate skin and enhance compound delivery to deep skin strata or
systemically
(37, 38)
. Touitou et al. (2000)
(39)
have suggested that ethanol
20
fluidizes both ethosomal lipids and lipid bilayers of the skin. The soft,
malleable vesicles then penetrate through the disorganized lipid bilayers.
Niosomes use nonionic surfactants to form vesicles
(40, 41)
. Niosomes
have been much promoted by the cosmetic industry
(5)
.
Solid lipid nanoparticles (SLN) or solid lipid microparticles (SLM) that
are also called lipospheres (LS)
(42)
, have been proposed as a new type of
fat-based encapsulation system for drug delivery (especially lipophilic
compound)
(43)
. Solid lipid microparticles consist of solid microparticles
with a mean diameter usually comprised between 0.2 m and 500 m,
composed of a solid hydrophobic fat matrix, where the drug is dissolved
or dispersed
(43)
. Fat matrix (lipid) includes: glycerides (e.g. glycerol
dibehenate), fatty acids (e.g. stearic acid), waxes (e.g. carnauba wax),
steroids (e.g. cholesterol)
(44)
. A clear advantage of SLM is that the lipid
matrix is made from physiological lipids which decrease the danger of
acute and chronic toxicity
(44)
. SLM have some advantages over other
delivery systems, such as good physical stability, low cost of ingredients,
ease of preparation and scale-up and high entrapment yields for
hydrophobic drugs
(43)
. Also, the encapsulation in the lipid matrix enables
increased photostability and modified release
(45)
. Further favorable
properties of SLM include an occlusive effect due to film formation on
the skin surface which reduces transepidermal water loss. Occlusion can
enhance the penetration of drugs through the stratum corneum by
21
increased hydration
(46)
. LS have been used for the controlled delivery of
various types of drugs, including insulin
(47)
, antiplatelet drugs,
antibiotics, anti-inflammatory agents, vaccines, local anesthetic
(43 ,48)
and
sunscreen
(49)
.
7.2.2. High velocity particles
The PowderJect system fires solid particles (20100 m) through stratum
corneum into lower skin layers, using a supersonic shock wave of
compressed gas (usually helium)
(50)
. The claimed advantages of the
system include: pain-free deliveryparticles are too small to trigger pain
receptors in skin; improved efficacy and bioavailability; targeting to a
specific tissue, such as a vaccine delivered to epidermal cells; sustained
release, or fast release; accurate dosing; overcomes needle phobia. In
addition, the device avoids skin damage or infection from needles or
splash back of body fluids which is particularly important issue for HIV
and hepatitis B virus
(50)
. However, there have been problems with
bruising and particles bouncing off skin surfaces. This could damage the
skin structure and carry surface contaminants such as bacteria into viable
skin layers
(5)
.
7. 3. Stratum corneum modification
7. 3.1. Hydration
Hydration of stratum corneum increases the penetration rate of most
substances; water opens up the compact structure of horny layer
(51)
.
22
7. 3.2. Chemical penetration enhancers
Penetration enhancers are chemicals that interact with skin constituents to
promote drug flux
(52)
. The enhancers include: water, hydrocarbons,
sulphoxides (especially dimethylsulphoxide, DMSO) and their analogues,
pyrrolidones, fatty acids, esters and alcohols, azone and its derivatives,
surfactants (anionic, cationic and nonionic), amides (including urea and
its derivatives), polyols, essential oils, terpenes and derivatives,
oxazolidines, epidermal enzymes, polymers, lipid synthesis inhibitors,
biodegradable enhancers
(52)
.
An important theme in enhancer research is how to classify accelerant
action and explain the various mechanisms responsible for increased drug
permeation. One simple classification is via the lipidproteinpartitioning
concept (LPP)
(5)
. This hypothesis suggests that accelerants act by one or
more ways selected from three main possibilities. Studies by Aungst et
al. (1990)
(53)
have broadly supported this concept. This hypothesis can be
summarized as follows:
Lipid action
The enhancer disrupts stratum corneum lipid organization, making it
permeable. The essential action is to increase the drugs diffusion
coefficient. The accelerant molecules jump into the bilayer, rotate,
vibrate and translocate, forming microcavities and increasing the free
volume available for drug diffusion
(5)
. Many enhancers operate mainly in
23
this way (e.g. azone, terpenes, fatty acids, DMSO and alcohols). It was
assumed that such enhancers would penetrate into, and mix
homogeneously with the lipids
(5)
.
Some solvents (e.g. ethanol, DMSO) and micellar solutions may also
extract lipids, making the horny layer more permeable through forming
aqueous channels
(5)
.
Protein modification
The stratum corneum proteins are noted for their insolubility, which
results from extensive crosslinking of both cell envelope and intercellular
proteins. Thus, increased diffusion across the corneocytes (transcellular
transport) may result from swelling of the protein matrix or alterations in
its structure. A decrease in crosslinking density at the cell envelope
would allow access to the cells and, within the intracellular keratins and
matrix proteins; consequently, diffusivity through the cellular regions will
be enhanced. This would have a two-fold effect, namely: increase in the
surface area accessible for penetration and decrease in the penetration
pathway. Alteration in stratum corneum protein structures have been
frequently attributed to penetration enhancers, such as ethanol, and
DMSO
(54)
.
Partitioning promotion
Many solvents enter stratum corneum, change its solution properties by
altering the chemical environment, and thus increase partitioning of a
24
second molecule into the horny layer (i.e. raise K). This molecule may be
a drug, a coenhancer or a cosolvent (including water). For example,
ethanol increases the penetration of nitroglycerine and estradiol
(5)
.
Propylene glycol is also widely employed, particularly to provide
synergistic mixtures with molecules such as azone, oleic acid and the
terpenes i.e. to raise the horny layer concentration of these enhancers
(5)
.
In theory, nonsolvent enhancers that mainly act to raise drug diffusivity
by mechanisms discussed above (lipid action) should also increase the
partition coefficient for lipid drugs. That is, by disordering the lipid
interfacial domain they increase free volume and make a larger fraction
of the bilayer available for solute partitioning. The nonsolvent enhancer,
of course, also affects the chemical environment throughout the lipid
domain and thus, theoretically, modifies the solute partition coefficient.
Many chemical enhancers combine these three LPP mechanisms. Thus,
high concentrations of DMSO (above 60%) disturb intercellular
organization, extract lipids, interact with keratin and facilitate lipid drug
partitioning
(5)
.
Oleic acid is one of the most popular fatty acid chemical enhancer used to
improve transdermal drug delivery
(52)
. It is both GRAS listed and
included in the FDA Inactive Ingredients Guide
(55)
. Oleic acid has been
shown to be effective for many drugs, for example increasing flux of
salicylic acid 28-fold and 5-fluorouracil flux 56-fold through human skin
25
membrane in vitro
(52)
and of testosterone 3-fold through snake skin in
vitro
(56)
. It is clear from numerous literature reports that the oleic acid
interacts with and modifies the lipid domains of the stratum corneum, as
would be expected for a long chain fatty acid with a cis configuration.
Recently, electron microscopic studies have shown that a discreet lipid
domain is induced within stratum corneum bilayer lipids on exposure to
oleic acid. The formation of such pools would provide permeability
defects within the bilayer lipids thus facilitating permeation of
hydrophilic permeant through the membrane
(52)
.
Dodecylamine is a surfactant which is cationic at low pH and non ionic at
a high pH
(57)
. It is a typical saturated fatty amine of 12 carbon units,
which is known to increase the skin permeation rate of various drugs
(58)
.
It acts through the disruption of lipid bilayer
(58)
.
7.4. Stratum corneum bypassed /removed
7.4.1. Microneedle array
Microneedle patches (Figure 5) are microstructured transdermal systems
(MTS) that are developed by 3 M Microfabrication technology. ALZA
Corp, USA, has designed its microprojection patch, Macrofluxw
, with a
thin titanium screen with precisely manufactured microprojections. The
needles or projections on the surface of patch are sufficiently long to
penetrate through the SC, but short enough to not stimulate nerves and
hence pain receptors in the deeper tissues
(2)
.
26

Figure 5. Different types of microneedles array and patches
(59)
.

Microneedle patches are suited for delivery of vaccines, proteins or
peptide-based drugs
(59)
. Studies have achieved successful delivery of
water soluble, polar, ionic, and large molecules ( 19,500 Da) with
ALZA MTS system
(2)
. Microneedles used in transdermal delivery can be
classified into two categories: solid and hollow microneedles
(60)
.
7.4.2. Stratum corneum ablated
As the horny layer usually provides the permeation barrier, one could
consider simply removing it. Chemical peels may provide superficial or
light (epidermal), medium (epidermaldermal junction) or deep (deep
papillary or papillary reticular dermis) treatments. Microdermabrasion
uses a stream of aluminium oxide crystals and dermabrasion employs a
motor-driven abrasive fraise or cylinder
(61)
. Laser ablation applies high-
powered pulses to vaporize a section of the horny layer so as to produce
27
permeable skin regions
(62)
. The apparatus is costly and requires expert
operation to avoid damage such as burns.
Adhesive tape can remove stratum corneum prior to drug application;
tape-stripping is used to measure drug uptake into skin
(63)
. One other
method forms a blister by suction, an epidermatome removes the raised
tissue, after which a morphine solution delivered directly to the exposed
dermis produces fast pain relief
(64)
.
7.4.3. Follicular delivery
The pilosebaceous unit (hair follicle, hair shaft and sebaceous gland)
provides a route that bypasses intact stratum corneum. The rich blood
supply aids absorption, even though the shunt route cross-sectional area is
small
(11)
. Microparticulate systems, such as polystyrene, solid lipid
nanoparticles, and porous nylon were used in follicular drug delivery
researches
(11)
. In general, particles >10 m remain on the skin surface,
those 310 m concentrate in the follicle and when < 3 m, they
penetrate follicles and stratum corneum
(5)
.
7.5. Electrically assisted methods
7.5.1. Ultrasound (phonophoresis, sonophoresis)
Ultrasound therapies can broadly include lithotripsy, sonophoresis,
sonoporation, gene therapy and bone healing
(65, 66)
.
The effect of ultrasound on the movement of drugs through intact living
skin and into soft tissues is known as sonophoresis or phonophoresis
(67)
.
28
Ultrasound is a pressure wave having a frequency of more than 20 kHz
(68)
. Ultrasound is produced by a transducer composed of a piezoelectric
crystal which converts electric energy into mechanical energy in the form
of oscillations which generate acoustic waves.
(69)
.
Just as audio sound which is the transmission of pressure waves through a
medium such as air or water, ultrasound is the same type of transmission
of pressure waves, but at frequencies above human hearing, or above 20
kHz. As with light waves, these ultrasonic waves can be reflected,
refracted (bent), focused, and absorbed. On the other hand, unlike light
waves, ultrasonic waves cause actual movement of molecules as the
medium is compressed (at high pressure) and expanded (at low pressure),
and thus ultrasound can act physically upon biomolecules and cells
(65)
.
Most importantly, unlike visible light waves, ultrasonic waves are
absorbed relatively little by water, flesh and other tissues. Therefore,
ultrasound can see into the body (e.g., diagnostic ultrasound) and can
be used to transmit energy into the body at precise locations. This safe,
non-invasive and painless transmission of energy into the body is the key
to ultrasonic-activated drug delivery
(65)
.
7.5.1.1.. Mechanism of ultrasound
Although considerable attention has been given to the investigation of
sonophoresis in the past years, its mechanisms were not clearly
understood, reflecting the fact that several phenomena may occur in the
29
skin upon ultrasound exposure. These include: cavitation, thermal effects,
induction of convective transport and mechanical effects
(67, 71)
.
7.5.1.1.1. Cavitation
Cavitation is the formation and/or activity of gas-filled bubbles in a
medium exposed to ultrasound. As the pressure wave passes through the
media, gas bubbles of any size will expand at low pressure and contract at
high pressure. If the resulting oscillation in bubble size is fairly stable
(repeatable over many cycles), the cavitation is called stable or non-
inertial cavitation. Such oscillation creates a circulating fluid flow
(called microstreaming) around the bubble with velocities and shear rates
proportional to the amplitude of the oscillation
(65)
.
As the ultrasonic intensity increases, the amplitude of oscillation also
increases to a point in which the inward moving wall of fluid has
sufficient inertia that it cannot reverse direction when the acoustic
pressure reverses, but continues to compress the gas in the bubble to a
very small volume, creating extremely high pressures and temperatures.
This type of cavitation (called transient, inertial or collapse cavitation)
can be detrimental to cells because of the very high shear stresses in the
region of the collapse, the shock wave produced by the collapse, and the
free radicals produced by the high temperatures. The collapsed bubble
often fragments into smaller bubbles that serve as cavitation nuclei, grow
in size, and eventually collapse again (Figure 6)
(65)
.
30

Figure 6. Optical images of a 2.5 m-radius microbubble exposed to 5
cycles of 2.5 MHz ultrasound at 1.6 MPa pressure amplitude.
The left panel shows the bubble before exposure. The central
panel shows a streak photograph with the measured pressure
superimposed at the top of the panel. The right panel shows the
fragments produced by the collapse of the cavitating bubble
(65)
.

Furthermore, if the collapse is near a solid surface, an asymmetrical
collapse occurs which ejects a liquid jet at sonic speed toward the surface.
Figure 7 illustrates this type of collapse. If the rigid surface is skin then
the jet can pierce the surface
(65)
.

Figure 7. Illustration of an asymmetric collapse of a bubble near a
surface, producing a jet of liquid toward the surface
(65)
.
As mentioned previously, the sonic jet of fluid produced by collapse
cavitation near a solid surface also generates extreme shear stresses that
31
can shear open or perhaps pierce nearby vesicles. Some of these methods
of drug delivery are illustrated in Figure 8
(65)
.

Figure 8. Schematic representation of various modes by which drug
delivery can be enhanced by ultrasound. A: therapeutic agent
(triangles); B: gas bubble undergoing stable cavitation; C:
microstreaming around cavitating bubble; D. collapse
cavitation emitting a shock wave; E: asymmetrical bubble
collapse producing a liquid jet that pierces the endothelial
lining; F: completely pierced and ruptured cell; G: non-
ruptured cells with increased membrane permeability due to
insonation; H: cell with damaged membrane from
microstreaming or shock wave; I: extravascular tissue; J: thin-
walled microbubble decorated with agent on surface; K. thick-
walled microbubble with agent in lipophilic phase; L: micelle
with agent in lipophilic phase; M: liposome with agent in
aqueous interior; N: vesicle decorated with targeting moieties
attached to a specific target
(65)
.

32
7.5.1.1. 2. Thermal effect
The increase in the skin temperature resulting from the absorbance of
ultrasound energy may increase the skin permeability coefficient because
of an increase in the permeant diffusion coefficient
(67)
.
7.5.1.1. 3. Convective transport
Convective transport of permeant across the skin, especially through hair
follicles and sweet duct, occurs due to fluid velocities generated as a
result of interference of reflected and incident waves in the medium
(67)
.
7.5.1.1. 4. Mechanical effects
It occurs due to pressure variation induced by ultrasound. Since
ultrasound induces sinusoidal pressure variation in the skin there will be
generation of cyclic stresses. Lipid bilayer of the skin can easily disorder
by these stresses, which results in an increase in bilayer permeability
(67)
.
7.5.1.2. Physical characteristics of ultrasound
7.5.1.2.1. The frequency
The frequency of an emitted wave depends on the size of the crystal.
Attenuation of an acoustic wave is inversely proportional to its frequency,
and thus as the frequency increases, the ultrasound penetrates less deeply
into and under the skin
(69)
. There are two types of ultrasound waves; high
frequency range 1-3 MHz, and Low frequency range 20-100 kHz. It is
suggested that low frequency ultrasound (~ 20 kHz) induces a greater
perturbation of the skin barrier than conventional, therapeutic ultrasound
33
(~ 1 MHz) resulting in up to a 1000-fold difference in the level of
enhancement
(70)
.
7.5.1.2.2. Mode
Ultrasound waves can be emitted continuously (continuous mode) or in a
sequential mode (discontinuous or pulsed mode). The rise in temperature
is faster and more intense with the continuous mode
(69)
. Hikima et al.
(1998)
(73)
have shown an increase of transdermal diffusion of
prednisolone in vitro by 2-5 fold when increasing the exposure time from
10 to 60 min with 1 MHz ultrasound at intensity 4.3 W/cm in continuous
mode.
7.5.1.2.3. Intensity
The intensity I is directly dependent on the acoustic energy E emitted and
the speed of sound c in the medium:
I=cE [7]
Energy E is itself dependent on the density of the propagation medium r,
on the total pressure p (equal to the sum of the atmospheric pressure and
the pressure created by the ultrasound wave) and on the speed of sound c:
E=p
2
/rc
2
[8]
The employed intensities usually lie between 0.5 and 2 W/cm
2
. The
increase in pressure is approximately 0.2 bar with 1 W/cm
2
in water
(69)
.
7.5.1.3. Synergistic effect of ultrasound and chemical enhancers
A combination of ultrasound and chemical enhancers may result
34
in greater enhancement than each enhancement method alone
(74)
.
Mitragotri et al. (2000)
(75)
, have evaluated the synergistic effect of low-
frequency ultrasound with chemical enhancers and surfactants, including
sodium lauryl sulfate (SLS) and a model permeant, mannitol.
Application of ultrasound alone as well as SLS alone, both for 90 min,
increased skin permeability about 3 fold for SLS and 8 fold for
ultrasound. However, combined application of ultrasound and 1% SLS
solution induced an increase in skin permeability to mannitol in the order
of 200-fold. The cavitations produced by ultrasound may induce mixing
and facilitate the dispersion of an enhancer and subcutaneous lipids
(76)
.
7.5.1.4. Ultrasound commercially available device
In May 2006, Sontra, USA
(77)
, has introduced its 2
nd
generation
SonoPrep

skin permeation device for topical lidocaine delivery. The
SonoPrep

shown in Figure 9 consists of 8 components: control console,
battery charger, power adaptor/charger, hand piece, grip style and flat
style patient reference sensors, and decontamination stand. This system
permits the controlled increased permeability of human skin. The system
operates by transforming a low level of ultrasound energy for a short time
(less than 90 sec.) from the hand piece, causing stratum corneum to
become permeable. The size of the sonication site is 0.8 cm. By
applying a law voltage to the patient reference sensor, SonoPrep
35

n SonoPrep

(77)
. Figure 9. Second Generatio
measures the increase in skin conductance during the application of
ultrasound and stops the sonocation procedure when the desired level of
conductance is achieved
(77)
.
7.5.2. Iontophoresis
Iontophoresis simply defined as the application of an electrical potential
that maintains a constant electric current across the skin and enhances the
delivery of ionized as well as unionized moieties.
The iontophoretic technique is based on the general principle that like
charges repel each other. Thus during iontophoresis, if delivery of a
positively charged drug is desired, the charged drug is dissolved in the
electrolyte surrounding the electrode of similar polarity, i.e. the anode in
this example. On application of an electromotive force (small direct
36
current approximately 0.5 mA /cm
2
) the drug is repelled and moves
across the stratum corneum towards the cathode, which is placed
elsewhere on the body. Communication between the electrodes along the
surface of the skin has been shown to be negligible, i.e. movement of the
drug ions between the electrodes occurs through the skin and not on the
surface. When the cathode is placed in the donor compartment of a Franz
diffusion cell to enhance the flux of an anion, it is termed cathodal
iontophoresis and for anodal iontophoresis, the situation would be
reversed
(78)
.
An interesting development is reverse iontophoresis by which molecules
in the systemic circulation (such as glucose) can be extracted at the skin
surface using the electroosmotic effect. The GlucoWatch
G2

Biographer (Animas Technologies, LLC, USA) aims to monitor blood
glucose concentrations in diabetics using this procedure
(5)
. A problem
with iontophoresis is that, although the apparent current density per unit
area is low, most of the current penetrates via the low resistance route i.e.
the appendages, particularly hair follicles. Thus the actual current density
in the hair follicle may be high enough to damage growing hair. Pores,
whose identity has not been elucidated, may also contribute to
iontophoretic flux
(5)
.

37
7.5.3. Electroporation
Transdermal electroporation is the application of short (< 1 s), high
voltage (0.5500 V) pulses to the skin to cause disorganization of the
stratum corneum lipid structure and hence to enhance drug delivery
(79)
.
Drug is thought to utilize different penetration pathways as shown in
Figure 10
(78)
. Voltage, pulse length, number of pulses, and
physicochemical properties of drugs are among the factors affecting drug
permeation in electroporation. Fluxes increased 1010
4
fold for neutral
and highly charged molecules of up to 40 kDa
(50)
.
Clinical evaluation studies have been reported that the technique was
reasonably well tolerated by subjects. However, the subjects in one study
reported strong muscle contractions occurring with each electrical pulse
and around one quarter of them suffered mild muscle fatigue after
treatment. These side-effects, even if considered safe, will need to be
eliminated before the technique is likely to gain wide acceptance in the
transdermal drug delivery field
(50)
. Mitragotri et al. (2000)
(75)
have
published a review about synergistic interactions between chemical
enhancers and ultrasound, iontophoresis or electroporation.
7.5.4. Magnetophoresis
Limited work probed the ability of magnetic fields to move diamagnetic
materials through skin
(80)
. Langer (2000)
(81)
has discussed the
38
interesting idea of employing intelligent systems based on magnetism or
microchip technology to deliver drugs in controlled, pulsatile mode
(82)
.

low voltage iontophoresis and
high voltage electrophoresis
(78)
.

wave stresses the horny layer and enhances drug delivery
(83)
.
. Testosterone
Drug penetration pathway in Figure 10.
7.5.5. Photomechanical wave
A drug solution, placed on the skin and covered by a black polystyrene
target, is irradiated with a laser pulse. The resultant photomechanical
8
Chemical name: 17 -Hydroxyandrost-4-en-3-one

39
Molecular weight: 288.4.
Formula: C
19
H
28
O
2
Partition coefficient: K
o/w
is 2070 and log K is 3.3
(67)
.
Testosterone is a white or slightly creamy-white odorless or almost
odorless, crystals or crystalline powder. Practically insoluble in water,
freely soluble in alcohol, dioxin, and dichloromethane, soluble 1 in 6 of
anhydrous alcohol, 1 in 2 of chloroform, and 1 in 100 of ether, and
slightly soluble in ethyl oleate
(84)
.
Testosterone is an anabolic/androgenic hormone. Its anabolic properties
include the maintenance and growth of muscle and bone tissue. Its
androgenic properties are responsible for normal growth and development
of male sex organs and maintenance of secondary sexual characteristics.
Resting testosterone values for mature males range from 14 nmol/L to 28
nmol/ L
(84)
.
Testosterone is absorbed from gastrointestinal tract, skin and the oral
mucosa. However, it undergoes extensive first-pass hepatic metabolism
when administered by mouth and is therefore administered
intramuscularly, subcutaneously, or transdermally. Approximately 97 to
99 % of testosterone is transported in the blood bound to plasma proteins.
The remaining 1 to 3% is the biologically active, free testosterone.
Testosterone circulates in the blood approximately 15 to 30 minutes until
it is either bound to receptors or metabolized into active product
40
(dihydrotestosterone) by liver and subsequently excreted through the
urine. Testosterone can be converted to estradiol through aromatization
in adipose tissue, certain brain tissue, and other specific tissues. Its
plasma half-life is reported to range from 10 to 100 minutes
(84)
.
Testosterone deficiency is associated with symptoms that include
impotence, fatigue, mood depression, and regression of secondary sexual
characteristics. Testosterone deficiency may also adversely affect bone
mineralization, muscle strength, immune function and carbohydrate
metabolism
(84)
.
A variety of conditions have now been identified in which testosterone
production is diminished and replacement therapy may be beneficial. In
males, these include hypogonadism, HIV infection, non-androgenic
dependent cancer, chronic obstructive pulmonary disease, diabetes,
autoimmune diseases, and aging. In women, these encompass surgical
and natural menopause, HIV infection, cancers, autoimmune diseases,
premature ovarian failure, and premenstrual syndrome
(85)
.
Testosterone is considered to be a suitable candidate for transdermal
delivery. Its short half life (10-100 min) necessitate a sustained mode of
drug input , its extensive degradation by the gastrointestinal tract and
liver precludes efficient oral administration, and its physico-chemical
properties are suitable for formulation of transdermal delivery system
(85)
.
41
Other approaches for delivering testosterone are problematic. Intra-
muscular testosterone ester injections, a pro-drug concept from the 1950s,
are uncomfortable to administer and produce wide fluctuations in
testosterone levels
(86)
. Oral methyl-testosterone is potentially hepatotoxic
and increases cholesterol blood levels
(86)
.
9. An overview on the transdermal delivery systems of
testosterone
Currently, many testosterone transdermal systems are marketed
internationally. However, they are not available in the Saudi market: a
system applied to the scrotum that has no permeation enhancers
(Testoderm
, 6 mg, ALZA Corporation, USA) and two systems that

contain permeation enhancers for application to appendage or torso skin
(Androderm
, 2.5 mg and 5 mg, SmithKline Beecham Pharmaceuticals,

UK, and Testoderm
TTS, 5 mg ALZA Corporation, USA)

(84)
.
Recently, testosterone transdermal systems, namely, Androgel
gel 1 %
(Unimed pharmaceuticals, USA) and Testim

gel 1 % (Auxilium
pharmaceuticals, USA) have been approved by FDA in 2000 and 2002,
respectively
(87)
.
Scrotal patches produce high levels of circulating dihydrotestosterone due
to the high 5-alpha-reductase enzyme activity of scrotal skin. The mean
maximum serum concentration after application of transdermal patch
42
ranged from 635-939 ng/dL at 4-6 hours
(84)
.
Clinical studies of transdermal systems demonstrated their efficacy in
providing adequate testosterone replacement therapy.
However, skin irritation is usually associated with the use of transdermal
patches which are not well tolerated especially in a warm climate. A
severe reaction may be complicated by persistent post inflammatory
hyperpigmintation and scaring
(88)
. Bennett (1998)
(89)
has reported
localized skin reaction with reference to a burn-like lesion on the
shoulder of patients caused by testosterone transdermal patch delivery
systems. These reactions are usually localized to site of application of the
patch and sometimes present after many weeks or even months of
continuous exposure. Potential allergens include the adhesive, the
diffusion membrane and the vehicle
(89)
. However, the majority of
adverse reactions are due to direct contact of the active drug in high
concentration
(89)
. Wilson et al. (1998)
(90)
have suggested that application
of a topical corticosteroid cream prior to testosterone transdermal
application may be helpful in the management of skin irritation associated
with transdermal testosterone therapy. Consequently, it is important to
develop transdermal systems with minimal adverse effects on the skin.
Recent approaches have reported to deliver TS by transdermal route such
as gels
(91)
, spray
(92)
, ethosomes
(93)
, a reservoir -type transdermal delivery
system using ethanol/water as vehicle and dodecylamine as skin
43
permeation enhancer
(94)
and patches
(95)
. A matrix -type transdermal
delivery has been also developed using Span 80 and Tween 80 as
enhancer
(96)
. The effects of propylene glycol and octisalate as enhancer
in 95% ethanol solution w/v on the in vitro skin permeability of
testosterone have been investigated by Nicolazzo et al. ( 2005)
(97)
. The
effects of vehicles: phosphate-buffered saline (PBS), ethanol (50%
ethanol in PBS w/w), propylene glycol (50% propylene glycol in PBS
w/w) on in vitro transdermal penetration of testosterone have been
investigated in dogs by Mills et al. (2006)
(98)
. However, as far as the
latest scientific publications no study has been reported the development
of testosterone encapsulated in SLM for transdermal delivery.

44

OBIECTIVE

45
Objective
The main objective of the study was to formulate an improved
transdermal delivery system of testosterone in an attempt to:
1. Enhance the transdermal penetration of testosterone.
2. Minimize skin irritation (by drug encapsulation and by
avoiding the use of organic solvents or adhesives or
diffusion membrane).
The following three approaches were applied separately or in
combination to evaluate their ability to enhance the delivery of
testosterone systemically after its topical application: i) formulation
approach: by production and characterization of testosterone SLM, ii)
stratum corneum modification approach: by application of chemical
enhancer, and iii) electrically assisted approach: by application of
ultrasound waves (sonophoresis).
To reach the objective of the thesis a systematic study was designed
considering the following aspects:
1. Formulation of SLM dispersion using emulsion melts
homogenization technique.
2. Evaluation of the influence of formulation parameters on the
morphology, encapsulation efficiency, particle size, rheology,
thermal behavior, X-ray pattern and release characteristics of
testosterone from the prepared SLM formulations.
46
3. Evaluation of the influence of application of ultrasound waves
and/or chemical enhancers on the release characteristics of
testosterone from selected SLM formulations.
4. Assessment of stability of the selected testosterone SLM
formulation.
5. Evaluation of skin irritation after application of the selected
testosterone SLM formulation.

47

METHODOLOGY

48
1. Materials
Testosterone (TS), Fluka Chemie GmbH (Netherlands).
Poloxamer 188 (Pluronic F- 68), BASF (Ludwig-Shafen,
Germany).
Glycerol monostearate (GM), BDH laboratory supplies (Pooles,
England).
Stearic acid (SA), Winlab laboratory chemicals (Leicestershire,
UK).
Compritol 888 ATO (glycerol dibehenate, GB), Gattefosse (Lyon,
France).
Precirol ATO 5 (glycerol distearate, GD), Gattefosse (Lyon,
France).
Dodecylamin (DA), Merck-Schuchardt (Germany).
Oleic acid (OA), Riedel-de Haen (Germany).
Trehalose, Fluka Chemie GmbH (Netherlands).
Monopotassium phosphate, Winlab laboratory chemicals
(Leicestershire, UK).
Disodium phosphate, Winlab laboratory chemicals (Leicestershire,
UK).
Chloroform, BDH laboratory supplies (Pooles, England).
Sodium chloride 0.9% w/v, Pharmaceutical solution industry
(Jeddah, KSA).
49
Propylene glycol, Winlab laboratory chemicals (Leicestershire,
UK).
Acetonitrile HPLC grade, BDH laboratory supplies (Pooles,
England).
Ethanol HPLC grade, BDH laboratory supplies (Pooles, England).
Magnesium nitrate GPR grade, BDH laboratory supplies (Pooles,
England).
Formaldehyde 40 % stabilized with 10% of methanol, Farmitalia
carloerba S.P.A. (Milano).
2. Apparatus
Analytical balance (Mettler Toledo, Switzerland).
Ultra-Turrax
Ika
T18 basic (IKa Works, Inc. USA).

Ultrasonic bath transsonic 460/H (Elma, Germany).
Vertical jacketed Franz diffusion cell, 15 mm or 25 mm diameter,
12 ml or 20 ml volume, respectively, (Crown Glass Co. Inc.,
Somerville, NJ, USA).
3-Station vertical cell stirrer (Perma Gear, Advanced Engineering,
Inc., Milwaukee, Wisconsin, USA).
Circulating water bath (Julabo, Germany).
Cellophane membrane (molecular weight cut-off: 6000-8000)
(Spectra/ pro
membrane, Spectrum medical industries, Inc., USA)

50
High performance liquid chromatography (HPLC) system
(Shimadzu, Japan) consists of:
Intelligent Shimadzu pumping system LC-10.
Rheodyne injector with 20 l loop.
C
18
-Shimpack TM steel column 150 mm X 4.6 mm.
Intelligent UV detector SPD-10.
Shimadzu VP chromatography software, version: 6.12 SP5.
Copyright
1998-2003. Shimadzu Corporation.

Advanced digital laboratory microscope (Motic B series with
Moticam 2000 USB 2.0 M pixel camera and Motic Images
Advanced 3.2 software, Motic china group co. ltd., China)
Scanning electron microscope (SEM) (Joel, SEM model JSM-25
SII, Tokyo, Japan).
Freeze dryer (Christ alpha 1-2, Osterode a. H., Germany).
Centrifuge (Kubota Co., Tokyo, Japan).
Ultra low temperature freezer (Sanyo electric Co., Japan)
Oven: Karl Kolb (Scientific and technical supplies, Germany).
Brookfield viscometer DV-+PRO using spindle no. 61, 62, and
64 (Brookfield Engineering Laboratories, Inc., Middleboro, USA).
Differential scanning calorimeter (DSC) (Perkin Elmer, Shelton,
CT, USA).
Powder X-ray diffractometer, D-5000 (Siemens, Germany).
51
Submicron particle size analyzer 90 plus (Brookhaven Instrument
Co., Holtsville, NY, USA).
Shaking water Bath SS40-D (Hetro, Denmark).
Nemectroson 400 high frequency ultrasound probe, (Nemectron
GmbH, Daimlerstrasse.15, 76185 Karlsruhe)
Ultrasonic processor model VCX 500, 220 V (Sonic, USA)
Micrometer (Germany).
pH meter MP220 (Metter-Toledo GmbH, Switzerland).
3. Methods
3.1. High performance liquid chromatography (HPLC) assay of
testosterone
Testosterone was assayed by the method proposed by Morgan et al.
(1998)
(56)
. Testosterone was detected at 241 nm. The mobile phase was
55 % acetonitrile in water, filtered through 0.45 m membrane filter and
degassed. The flow-rate was 1 ml / min. The injection volume was 20
l. The retention time of the drug was 5.2 0.5 min. The column used
was reversed phase C
18
- Shimpack TM (150 mm X 4.6 mm) and
operated at ambient temperature.
3.2. HPLC calibration standards / quality control of testosterone
A stock solution of testosterone (1 mg/ml) in the ethanol was prepared.
One milliliter from the stock solution was diluted with ethanol to give
solution with concentration of 0.1 mg/10 ml which was further diluted
52
with the mobile phase to give the following standard concentrations:
0.005, 0.01, 0.02 and 0.03 mg/10 ml. Three concentrations, namely,
0.006 mg/10 ml (LQC), 0.015 mg/10 ml (MQC) and 0.025 mg/10 ml
(HQC) were used as quality control (QC) samples
(99)
.
3.3. Assay validation of testosterone
The assay data were validated in terms of linearity, precision, accuracy
and reproducibility
(100)
. Linearity was represented by the regression
analysis of the calibration data and quality control data. The mean and
standard deviation of the estimated concentration obtained by refitting
into the regression equation of each standard concentration analyzed on
different days (interday precision) and on the same day (intraday
precision) were calculated. The percent coefficient of variation was
obtained by dividing the standard deviation by the mean of each
corresponding standard concentration. Accuracy (% recovery) was
assessed by comparing the amount of the drug added in the standards to
the found value. Reproducibility of standard curve of TS was performed
by comparing sets of standard concentrations and QC samples developed
in five different days within one month and by evaluation of statistical
significance among various calibration curves.
3.4. Preparation of testosterone solid lipid microparticles
Solid lipid microparticles dispersion was prepared by hot homogenization
technique on a weight basis
(43)
. In hot homogenization technique, lipid
53
was melted at temperature ten degrees above its melting point.
Testosterone was added to the melted lipid. The dispersion was kept at
the same temperature until it appeared optically clear. Chemical enhancer
(1% w/w OA or DA based on the total weight of SLM dispersion) was
dissolved in the melted lipid when required. Poloxamer 188 (2.5 % w/w
of total weight of SLM dispersion as stabilizer) was dissolved in distilled
water and heated to the same temperature as lipid mixture. Hot poloxamer
solution was then added to the melted lipid-drug mixture and emulsified
by an Ultra-Turrax
Ika

T18 at 8000 rpm for 1 min. The formulation
was then removed from water bath and the dispersion of SLM was mixed
gently at room temperature until cooling.
The following SLM were prepared: 2.5% or 5% w/w GM; or 5% or 10%
w/w GD; or 5% or 10% w/w GB; or 2.5% or 5% w/w SA (based on the
total weight of SLM dispersion). The concentration of TS in SLM was
2.5% or 5% w/w calculated as a percentage of lipid matrix (e.g., for 100 g
of a 10% GB SLM dispersion loaded with 5% drug, the lipid phase
consisted of 9.5 g GB and 0.5 g drug, TS concentrations can be also
expressed as 2.5 or 5 mg/g of SLM dispersion).
3.5. Morphological examination of the prepared SLM
Solid lipid microparticles were coated uniformly with gold after
evaporation of aqueous phase under vacuum. All samples were examined
54
for morphology and surface properties using scanning electron
microscope (SEM).
3.6. Particle size analysis of the prepared SLM
Particle size of freshly prepared SLM formulations was measured by laser
particle size analyzer to detect the presence of nanoparticles. However,
further particle size measurements were performed by microscopic
method.
Concerning, the first technique, 200 l of SLM were diluted with 3 ml of
deionized water. The diluted samples were loaded into 4 ml cuvette and
the particle size measurement was conducted at ambient temperature.
Measurements were performed in triplicate.
Regarding microscopic method
(18)
an advanced biological microscope
with digital camera was used. According to this method, sample was
mounted on a slide and placed on a mechanical stage. The field was
projected onto a screen where the particle radius was measured along an
arbitrarily chosen three points. The data were recorded using Motic
images advanced software, version 3.1.
The counted number of particles was 300 particles in each specimen. For
each formulation, the particles in four different fields were counted. The
average diameter for each SLM formulation was calculated according to
the following equation
(101)
:
55
d
ave
=
n
n n
n n n
d n d n d n
.......
....
2 1
2 2 1 1
+ +
+ +
=
n
nd) (
[9]
Where d
ave
is the average diameter and n
1
, n
2
, and n
n
are the number of
particles having diameters d
1
, d
2
and d
n
, respectively. The polydispersity
index which stands for the width of particle size distribution
(46)
was also
calculated according to the following equation
(102)
:
PI = s / R [10]
Where, R and s are the mean radius and standard deviation. An ideal,
monodisperse formulation has a PI of zero
(102)
.
3.7. Rheological studies of the prepared SLM
Rheological properties of the prepared formulations were measured using
Brookfield viscometer. The rate of speed of the spindle was increased
from 0.5 to 50 rpm, and then in descending order from 50 to 0.5 rpm at
30 sec interval. The viscosity was read directly from the viscometer
display. The sample was equilibrated at 32 0.5 C prior to each
measurement. All measurements were made at least in duplicate.
3.8. Differential scanning calorimetry (DSC)
The transition temperature (T
m
) of lipid particles was measured by DSC.
Samples (about 5 mg) of TS, bulk lipid, lyophilized drug-containing SLM
after separation of aqueous phase were weighed and sealed in an
aluminum pan. They were heated from 25

C to 250

C at a heating rate
of 5

C per min, under a constant nitrogen stream. All measurements were
56
made in triplicate.
Percentage crystallinity of lipid in SLM was calculated taking the
enthalpy of lipid as being 100 % and considering the lipid content of
SLM using the following equation:
SLM dried in lipid of fraction lipid bulk of Enthalpy
phase aqueous of separation after SLM d lyophilize of Enthalpy ation Crystalliz
*
100 * % =
[11]

3.9. Powder X-ray diffractometry (PXRD)
Powder X-ray diffractometry studies were performed on the samples by
exposing them to Cu K radiation (40 kV/ 30 mA) and scanned from 5
to 70, and the scanning rate was 5 / min at a step size of 0.020 and a
step time of 1 sec. The examined samples were TS powder, bulk lipid
(GB) powder, lyophilized drug-free SLM after separation of aqueous
phase (10 % GB) and lyophilized drug-containing SLM after separation
of aqueous phase (10 % GB containing 5 mg TS/g of SLM dispersion).
3.10. Drug entrapment efficiency of the prepared SLM (% EE)
Testosterone content of the microparticles was determined by HPLC
assay after drug extraction from SLM. The detailed procedure was
dependent on the type of lipid. Solid lipid microparticles formulation was
centrifuged at 19,000 rpm and 5

C for 2 hr. Then supernatant was
removed and the sediment was washed with distilled water. The resultant
precipitant was frozen at -70

C overnight in a deep freezer. Then the
sample was dried by freeze dryer for 8 hours. Concerning GB SLM, ten
57
milligrams of the freeze-dried formulation was dissolved in 1 ml
dichloromethane and the volume was completed to 10 ml with ethanol.
On the other hand, in case of GM, SA and GD SLM, 10 mg of the freeze-
dried SLM was dissolved in 9 ml ethanol while heating at 60 C. After
cooling to room temperature the volume was then completed to 10 ml by
water. After filtration through PTFE filter (0.2 m) (Millipore
Corporation, USA) a suitable dilution was performed. An aliquot of 20
l was injected onto HPLC column and assayed for drug content. All
measurements were made at least in duplicate.
The entrapment efficiency was calculated as the percentage ratio between
the quantity of TS entrapped in SLM and the amount of TS added to the
melted lipid phase
(49)
.
3.11. Occlusion test
The in vitro occlusion test was performed according to Souto et al. (2004)
(103)
. Beakers (25 ml) were filled with 12.5 ml of water, covered with
filter paper (Whatman International Ltd., Maidstone, England, 55 mm;
cutoff size: 8 um and grade 2) and sealed. A weight of 550 mg of sample
was spread on the filter surface (9.07 cm). A visible film formation on
top of the filter paper was observed during the experiment. The samples
were stored at 32 C and relative humidity of 53 % using saturated
solution of magnesium nitrate. A beaker covered with filter paper but
without applied sample was served as reference. The samples were
58
weighed after 6, 24 h giving the water loss due to water evaporation at
each time. The occlusion factor F was calculated according to the
following equation:
F= (A-B / A) 100 [12]
Where A is the water loss without sample (reference) and B is the water
loss with sample. An occlusion factor of zero means no occlusive effect
compared to the reference and 100 is the maximum occlusion factor. All
measurements were made in triplicate.
3.12. Solubility study of testosterone
The saturated solubility of TS in 40 % (v/v) propylene glycol /normal
saline mixtures was determined. Excess amount of TS (100 mg) was
added into 10 ml glass bottle containing 2 ml of 40 % propylene
glycol/normal saline mixtures. Each bottle was vortexed for 1 min and
incubated in a shaking water bath at 32 0.5
o
C for 48 h. After
equilibrium, a sample of the supernatant was removed, filtered through
PTFE filter (0.45 m) and suitably diluted. An aliquot of 20 l was
injected onto HPLC column. All measurements were made in triplicate.
3.13. In vitro release studies of testosterone through cellophane
membrane after application of different solid lipid
microparticles formulations
In-vitro release across cellophane membrane was conducted using
jacketed vertical Franz diffusion cells (25 mm diameter orifice) as shown
59
in Figure 11. The cells have 20 ml receptor volume and spherical joint.
The area of diffusion was 4.91 cm. The cells were placed in a V-3
station vertical cell stirrer and connected to circulating water bath heated
to 32 0.5C.
Cellophane membrane (molecular weight cut-off: 6000-8000) previously
soaked in receptor medium was clamped by an O ring between the donor
and the receptor chamber of diffusion cell. A suitable aliquot of the
formulation (equivalent to 2.5 mg TS/g of SLM dispersion) was added to
the donor chamber of the diffusion cell which was occluded with a
parafilm. The receptor medium was normal saline solution containing
40% propylene glycol to maintain sink condition
(56)
. The receptor
medium was stirred by magnetic bar. Aliquots (200 l) were withdrawn
from the receptor compartment at the following time intervals: 2, 4, 6, 8,
18, 20, 22 and 24 h

and were replaced by equal volume of the fresh
receptor fluid. Samples were analyzed for TS content by HPLC after
suitable dilution. Each experiment was carried out at least in triplicate.
The concentration of TS was corrected for sampling effects according to
the equation described by Hayton and Chen (1982)
(104)
. It takes into
account the dilution of the receptor solution resulting from replacing the
sampling volume with equal amount of fresh receptor solution at each
sampling point:

60

Figure 11. Three stations vertical Franz diffusion system.

61
C
\
n
= C
n
(V
T
/V
T
-V
s
) (C
\

n-1
/C
n-1
) [13]
Where C
\
n
is the corrected concentration of the n
th
sample, C
n
the
measured concentration of TS in the n
th
sample, C
\

n-1
is the corrected
concentration of (n-1)
th
, C
n-1
is the measured concentration of TS in the
(n-1)
th
sample, V
T
is the total volume of the receiver compartment and V
s

is the volume of the sample drawn.
The cumulative amount of TS released into the receptor medium was
plotted versus time and steady state flux, J, (g/cm
2
/h), was determined
from the slope of the linear portion of the plot. Permeability coefficient,
P, (cm/sec) was calculated as
P = J/C
o
[14]
Where, C
o
is the initial donor drug concentration.
The diffusion coefficient, D, can be obtained from the following
equation:
D/h
2
= 1/6 *L
t
[15]
Where h is the thickness of the epidermis and L
t
is the lag time. Since it
is generally accepted that most molecules permeate through subcutaneous
layer mainly by a tortuous intercellular route, the thickness of the
epidermis is not equal to the diffusion path length. Since it is difficult to
determine the diffusion path length correctly, D/h
2
is replaced by D`, so
the equation becomes
(105)
:
62
D` =1/6 *L
t
[16]
Where, D` is the diffusion parameter. The results were reported as mean
SD.
The effect of each enhancement strategy was represented by an
enhancement ratio (ER) which was calculated using the following
equation
(97)
:
J with enhancer
ER =------------------------- [17]
J without enhancer
The values reported were mean ratios for a minimum of three replicates.
The examined formulations were: SLM contained 2.5 % and 5 % w/w
glycerol monostearte; or 5% and 10% w/w glycerol distearate; 5% and
10% w/w glycerol dibehenate; 2.5% and 5% w/w stearic acid. All
formulations contained 2.5 mg TS/ g of SLM dispersion.
3.14. In vitro permeation studies of testosterone through excised
abdomen rat skin after application of the selected testosterone
SLM formulations
The animals used for the preparation of skin were Albino male rats (150-
170 g). Rats were sacrificed with an overdose of chloroform inhalation.
Abdomen skin was excised after clipping of the hair and removal of
subcutaneous fat by means of blunt dissection. Skin was stored at - 5C
for maximum 24 h
(94)
.
63
The full thickness skin was clamped between the donor and the receptor
compartment of a jacketed Franz diffusion cell with stratum corneum in
contact with donor phase. The diffusion cell has 12 ml receptor volume
with spherical joint, 15 mm diameter orifice and diffusion area was 1.76
cm
2
.
The examined formulations were: 10 % GD SLM containing 2.5 mg
TS/g, 10 % GB SLM containing 2.5 or 5 mg TS/g; 10 % GB SLM
containing 5 mg TS/g after application of 1% w/w DA in ethanol to the
skin for 30 min then removed by gentle swabbing of the skin with cotton ;
freeze dried 10 % GB SLM containing 5 mg TS/g of SLM after
reconstitution, and 10 % GB SLM containing 5 mg TS/g and 1% OA or
1% DA. The in vitro experiments were operated as mentioned before in
section 3.13. Each experiment was carried out at least in triplicate.
and/or chemical enhancer on in vitro permeation of
testosterone through excised abdomen rat skin after
application of the selected testosterone SLM formulation
The ultrasound equipment with 1-3 MHz frequency and intensity of 1-3
W/cm was used. The diameter of the ultrasound probe was 2.5 cm. For
application, the ultrasound probe was fixed to a clamp that could be
easily raised or lowered. The ultrasound was applied to rat skin in a
continuous mode for 1 hour prior to skin permeation experiment at
64
frequency of 1 MHz and intensity of 0.5 W/cm of skin area. The
ultrasound transducer was located approximately 0.5 cm from stratum
corneum. Buffer of pH 7.4 was used as coupling medium
(106)
.
The examined formulations were: 10 % GB SLM containing 5 mg TS/g,
10 % GB SLM containing 5 mg TS/g after application of 1% DA to the
skin for 30 min then removed, and 10 % GB SLM containing 5 mg TS/g
and 1% OA. The permeation experiments were operated as mentioned
before in section 3.14. Each experiment was carried out at least in
triplicate.
3.16. Effect of application of low frequency ultrasound (LUS) and/or
chemical enhancer on in vitro permeation of testosterone
through excised abdomen rat skin after application of the
selected testosterone SLM formulation
The ultrasound equipment with 20 kHz frequency was used. The
diameter of the ultrasound probe was 13 mm. For application, the
ultrasound probe was fixed to a clamp that could be easily raised or
lowered. The ultrasound transducer was located approximately 0.5 cm
from stratum corneum. Buffer of pH 7.4 was used as coupling medium.
The ultrasound was applied to rat skin following three different protocols:
Effect of total application time (t
eff
) (6, 12, 15 min) when total
exposure time was 30 minutes at intensity of 2.5 W/cm and t
on

equals 10 sec.
65
Effect of three different intensities (2.5, 3.25 and 5 W/cm
2
) with
other parameters remaining constant (t
on
= 10 sec, t
eff
= 12 min) for
total exposure time 30 minutes.
Combined application of LUS and chemical enhancer: (t
eff
= 12
min) over total exposure time 30 minutes at intensity of 2.5 W/cm
followed by 1% DA in ethanol applied to the skin for 30 min then
removed prior to skin permeation experiment.
The examined formulation was 10 % GB SLM containing 5 mg TS/g of
SLM dispersion. Each experiment was carried out at least in triplicate.
The in vitro experiments were operated as mentioned before in section
3.13. Each experiment was carried out at least in triplicate. The
ultrasound parameters can be correlated by the following equation
(107)
;
t
eff
= t
us
(t
on
/(t
on
+t
off
)) [18]
where t
off
represents the time when US is off,
t
on
represents the length of the pulse when US is on,
t
eff
represents the sum of the time when US is on,
t
us
represents total exposure time.
3.17. Stability studies
Testosterone SLM (10% GB containing 5 mg TS/g and the same
formulation containing 1% OA or DA) was stored in glass container in
dark at temperature of 5 0.5
C and 30 0.5
C. The following stability

66
parameters were evaluated at time intervals of 2, 4, 6, 8, 12 and 16 weeks:
appearance, color changes, creaming, drug release and particle size
analysis.
3.18. Effect of freeze drying on the selected SLM formulation
The selected formulation (10% GB containing 5 mg TS/g of SLM
dispersion) was freeze dried in presence of cryoprotector (trehalose),
which was added to the selected formulation by one of the following
methods:
a) Incorporation of trehalose in the formulation
Trehalose was added to the aqueous phase of the selected formulation
during preparation in a ratio of 3 sugar: 1 lipid (w/w) before
homogenization
(108)
.
b) Dilution of the formulation with trehalose solution
The selected formulation was diluted in a ratio of 1:1 with 15% w/w
trehalose solution
(109)
.
The formulations (a, b) were frozen in a deep freezer at 70 C over
night and then lyophilized in freeze dryer for 24 hours. Reconstitution of
the lyophilized products by distilled water was performed using vortex
for 5 minutes (to give concentration equivalent to 5 mg TS/g SLM
dispersion)
(110)
. Particle size and release characteristics of TS from
reconstituted freeze dried SLM formulation were monitored.

67
3.19. Skin irritation test
The primary irritation to the skin was evaluated by the Draize test using
male New Zealand rabbits weighing 2.5-3 kg
(111)
. The examined
formulations were: pure drug solution (5 mg TS/g) in ethanol; drug free
10 % GB SLM; drug loaded 10 % GB SLM (5 mg TS/g); drug loaded
10 % GB SLM (containing 5 mg TS/g) and 1% OA; drug loaded 10 %
GB SLM (5 mg TS/g) after application of 1% DA for 30 min; 1% DA in
ethanol solution; 1% OA in ethanol solution, and ethanol. Petrolatum was
used as negative control and an aqueous solution of 5% sodium lauryl
sulfate as a positive control in each animal.
Six rabbits were used in each test group for each examined formulation.
The dorsal part of the rabbit was carefully shaved the day before the
experiment, and an aliquot (equivalent to 2.5 mg TS /g of SLM
dispersion) of the tested formulation was applied on the shaved skin for
24 hours. After gentle removal of the formulation, the conditions of the
dorsal skin was observed and classified into five grades (points 0-4);
point 0, without erythema or edema; point 1, very slight erythema or
edema; point 2, obvious erythema or edema; point 3, medium erythema
or edema; point 4, strong erythema or edema
(112)
.
3.20. Histological examination of excised rat skin
Histological changes in the excised abdomen rat skin were examined
immediately after treatment with US and/or chemical enhancer or after
68
performing the permeation study for 24 h. Each specimen was fixed in
40% formaldehyde stabilized with 10% of methanol for at least 48 h. The
specimen was cut vertically against skin surface. Each section was
dehydrated using ethanol and then embedded in paraffin wax, stained
with hematoxylin and eosin. Skin samples were examined under
advanced digital biological, microscope with digital camera
(113)
.
3.21. Testosterone skin retention
Drug retained in rat skin was determined. After performing permeation
experiment over 24 h, the skin was cleaned from the sample and washed
with water (skin surface area was 1.76 cm
2
). To extract the drug from the
skin, the skin was cut into small pieces and placed in ethanol then vortex-
mixed for 10 min and kept aside for 24 h
(56)
. The extract was then
filtered through Millipore 0.2 m filter unit. A suitable dilution was
performed. An aliquot of 20 l was injected onto HPLC column. All
measurements were made in triplicate.
3.22. Statistics
Statistical analyses of data were performed using statistical package for
social sciences (SPSS ver. 10.0). Analysis of variance (ANOVA) was
applied; p value of less than or equal to 0.05 was considered significant.
Multiple comparisons post hoc test was applied when necessary to
identify which of the individual formulations was significantly different.

69

RESULTS AND DISCUSSION

70
1. High performance liquid chromatography (HPLC) assay
of testosterone
The calibration curve of TS was linear over the concentration range of
0.005- 0.03 mg/10 ml with determination coefficient (R) of 0.9987. The
data of calibration curve as a mean of five runs were tabulated (Table 1)
and plotted in Figure 12. The value of regression coefficient (b) was
statistically significant (p 0.01) indicating linear relationship between
the concentration and the area of HPLC peak.
2. Assay validation
The intraday (Tables 2, 4) and interday (Tables 3, 5) coefficient of
variation were calculated and found to be less than 5.9 for both QC and
calibration samples.
The interday and intraday accuracy represented by the mean percentage
recoveries for QC samples ranged from 93.1 % to 100.8 % and for
calibration standards ranged from 95.7 to 108.4 %. The low values of SD
of mean percentage recoveries indicated high accuracy as shown in
Tables 2- 5. This result revealed that any small change in the drug
concentration in the solution can be accurately determined by this
method. The results were found to fulfill the requirement of drug analysis
in biological studies (CV % <15 %)
(99)
.
The reproducibility of QC and calibration samples performed in five

71

Table 1. HPLC calibration curve of TS.
Drug
concentration
(mg/10ml)
Peak area
SD (n=5)
CV%
0.005 355982153 6.1
0.01 672323306 4.9
0.02 1435237554 5.3
0.03 2231044817 2.2

igure 12. HPLC calibration curve of TS. (n=5)
y = 7E+06x - 2733.2
R
2
= 0.9987
0
50000
100000
150000
200000
250000
0 0.005 0.01 0.015 0.02 0.025 0.03 0.035
Concentrati on of TS (mg/10ml )
M
e
a
n

p
e
a
k

a
r
e
a

F

72

able 2. Intraday precision (CV %) and accuracy (% recovery) data for
HPLC quality control samples of TS.
TS added
(mg/10 ml) (mg/10 ml)
% Mean % recovery
SD (n=3)
T
Mean concentration found CV
SD (n=3)
0.006 (LQC) 01 1.8 0.00570.00 94.501.8
0.015 (MQC) 07 4.7 100.14.7 0.01500.00
0.025 (HQC) 0.02510.0001 0.4 100.50.5

HPLC quality control samples of TS.
TS added
(mg/10 ml) (mg/10 ml)
CV % Mean %
recovery
3)
Mean concentration found
SD (n=3) SD (n=
0.006 (LQC) 01 1.8 0.00560.00 93.101.7
0.015 (MQC) 03 2.0 0.01500.00 100.22.2
0.025 (HQC) 0.02520.0004 1.6 100.81.7

73

calibration standard concentrations of TS.
TS adde
(mg/10 ml) (mg/10 ml)
Mean %
recovery
3)
d Mean concentration found CV %
SD (n=3) SD (n=
0.005 0.00520.0002 3.8 104.603.4
0.01 04 4.2 0.00960.00 96.023.7
0.02 0.02050.0011 5.4 102.505.3
0.03 0.03250.0007 2.2 108.402.2

able 5. Interday precision (CV %) and accuracy (% recovery) data for
calibration standard concentrations of TS.
TS added
(mg/10 ml) (mg/10 ml)
% Mean %
recovery
3)

T
Mean concentration found CV
SD (n=3) SD (n=
0.005 0.00510.0003 5.9 101.85.3
0.01 04 4.2 0.00960.00 95.703.8
0.02 0.01980.0006 3.0 99.203.1
0.03 0.03200.0006 1.9 106.62.1

74
different days was shown in Tables 6, 7. Coefficient of variation (%) did
not exceed 6.1 %. Test of significance among several regression lines was
insignificant (p > 0.05) indicating good reproducibility of calibration
curve data. The minimum detected concentration was 0.07g/ml.
3. Morphological examination of the prepared SLM
It has been reported that different lipid types might influence the surface
morphology and the shape of the particles
(43)
. Scanning electron
micrographs of SLM containing 5% and 10% GB and 5% and 10% GD,
Figure 13: a, b, c and d respectively, revealed the formation of spherical
microparticles with irregular surface. However, GD microparticles
(Figure 13: c, d) showed large population of small particles. Glyceryl
monostearate microparticles (2.5 and 5%) Figure 13: e, f had relatively
smaller microparticles with rough and irregular surface. On the other
hand, most of SA microparticles (Figure 13: g, h) were somewhat
spherical although they had imperfection on their surfaces. It is worth
mentioning that during SEM sample preparation, both the water of the
bulk phase and the water present in the particles were completely
removed. In general, such drying apparently may cause the observed
shrinking of the surface of the particles.
4. Particle size analysis of SLM
The effect of lipid type and concentration on the size of SLM was
75

Table. 6. Reproducibility of peak area (run to run) in the analysis of TS
performed using five sets of quality control samples on five
different days.
Peak area found (n=3)
Conc. of
TS
(mg/10ml)
I II III IV V
Mean of
peak area
found
SD (n=5)
CV%
0.006 40122 39834 42223 42885 41470 413071315 3.2
0.015 106367 108669 108599 108712 106083 1076861338 1.2
0.025 178040 181244 188451 181990 175141 1809734990 2.8

Table 7. Reproducibility of peak area (run to run) in the analysis of TS
performed using five sets of calibration standard concentrations
on five different days.

Peak area found of TS (n=3)
Conc. of
TS
(mg/10ml)
I II III IV V
Mean of peak
area found
SD (n=5)
CV%
0.005 35819 35024 33480 34528 39139 355982153 6.1
0.01 62740 67949 65410 68610 71453 672323306 4.9
0.02 133713 148539 142421 139829 153115 1435237554 5.3
0.03 223524 222415 231027 219011 219544 2231044817 2.2
76

a) b)

d) c)
f) e)

g) h)
Figure 13. Scanning electron micrographs of SLM formulations
containing 2.5 mg TS/ g of: a) 5 % GB, b) 10 % GB, c) 5 %
GD, d) 10 % GD, e) 2.5 % GM, f) 5 % GM, g) 2.5 % SA
and h) 5 %.SA SLM dispersion.
77
investigated for SLM prepared using GM, GD, GB and SA. For the same
type of lipid matrix, statistical analysis followed by Duncan Multiple
post hoc test Table 8 showed no significant increase in particle size was
observed as the lipid concentration increased from 2.5% to 5% or from
5% to 10% (p < 0.05) Table 9 .
The minor increase in particle size with increasing lipid concentration can
be explained by the decrease in homogenization efficiency with
increasing content of dispersed phase (lipid phase)
(44)
. Similarly, Souto
et al. (2004)
(103)
have reported that the particle size of SLN increased
with the increase in lipid concentration from 9.5% to 19%. Generally, it
has been reported that increasing the lipid content over 5 -10 % in most
cases results in larger particles and broader size distribution
(114)
.
On the other hand, the type of lipid may affect the size of microparticles.
ANOVA (Table 9) followed by post hoc Dungcan test showed that 10%
GD particles were significantly larger than that formed using 2.5% or 5%
stearic acid. This was in agreement with the Cavalli et al. (1997)
(115)
who
reported that the choice of the lipid could affect SLM diameter. On the
other hand, no statistical significant differences in particle size were
observed between 10 % GD or SA SLM and the other SLM formulations.
Symbols presented in Table 8 show the results of post hoc Duncan test
for particle size. Contrary, Trotta et al. (2005)
(47)
reported that,
irrespective of lipid composition, the particle size was not changed.
78

Table 8. Mean particle size and polydispersity index (PI) of different

Formulation Mean particle size SD (m) PI
2.5% GM

18.5 2.3
ab
0.13
5% GM

25.5 3.9
ab
0.15
5% GD

26.2 4.3
ab
0.16
10% GD

30.6 7.2
a
0.23
5% GB

24.4 5.5
ab
0.22
10% GB

24.6 3.5
ab
0.14
2.5% SA

15.6 3.1
b
0.19
5% SA

16.3 6.9
b
0.42
10% GB*

24.1 2.9
ab
0.12
Means of same symbols are not statistically different. a>b>c
* N.B.: SLM contains 5mg TS/g of SLM dispersion.

79

Table 9. One-way analysis of variance showing the effect of lipid type on the
particle size of SLM.

Source of
variation
dF SS MS F
Particle
size
7 728.6 104.1 3.143
*
Error 23 761.6 33.1
Total 30 1490.1

* p 0.05

80
Polydispersity index values of SLM formulation were 0.3 (Table 8)
except for 5% SA SLM that have PI value of 0.42. The high PI value for
5% SA SLM could be due to high viscosity of SLM dispersion (as shown
in section 5, rheological studies) that could affect the homogenization
efficiency and resulted in a wide range of particle size distribution. It is
worth mentioning that a narrow size distribution is essential to prevent
particle growth due to Ostawld ripening being caused by different
saturation solubilities in the vicinity of differently sized particles
(116)
.
5. Rheological studies
Viscosity measurement is valuable tool for quality control of ingredients
and final products together with manufacturing process, such as mixing,
pumping, stirring, filling and sterilization
(117)
. The results of viscosity
measurement revealed that all the prepared formulations had plastic flow
characteristics, where the viscosity decreases with increasing shear rate
(Figure 14: a-d and Figure 15: a, c). The ascending and descending flow
curves overlapped and showed no time effects like, e.g. thixotropy. The
lipid particles in the dispersion tend to align with increasing shear stress
which is alleviating the flow. Unlike other formulations, the ascending
and descending curve of 5% GM (Figure 15, b) and 5% SA (Figure 15, d)
SLM formulations did not overlap, showing thixotropy. In other words,
increasing lipid content of SA and GM from 2.5% to 5% lead to different
flow characteristics. The same observation was documented for
81

0
1000
2000
3000
4000
5000
6000
7000
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
b)

0
100
200
300
400
500
600
700
800
900
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
a)

0
20
40
60
80
100
120
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
c)
0
100
200
300
400
500
600
700
800
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
d)

TS/g of SLM dispersion: a) 5 % GD, b) 10 % GD, c) 5 % GB
and d)10 % GB.

82

0
500
1000
1500
2000
2500
3000
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
0
2000
4000
6000
8000
10000
12000
14000
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
b) a)

0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
c)
0
5000
10000
15000
20000
25000
0 10 20 30 40 50 60
Speed (rpm)
V
i
s
c
o
s
i
t
y

(
c
p
s
)
d)

TS/g of SLM dispersion: a) 2.5 % GM, b) 5% GM, c) 2.5 %
SA, d) 5 % SA.

83
cetylpalmitate SLN which showed plastic flow with thixotropic
characteristics upon increasing the lipid concentration
(46, 117)
. Comparing
the viscosity of various formulations at 0.5 rpm (Figure 16) it was
observed that, for each type of lipid, as the concentration of lipid
increased the viscosity increased. In addition, the type of lipid affected
the viscosity of the final product. At 5% lipid concentration the rank
order of the viscosity of formulations according to the type of lipid was
GB< GD< GM< SA as revealed by analysis of variance (Table 10) that
followed by post hoc Duncan test.
The difference in viscoelastic behavior can be attributed to the presence
of different particle-particle interaction since there are different particle
sizes and particle size distributions
(46)
. Decreasing the particle size, as in
case of 5 % SA, is accompanied by a huge increase of surface. Therefore
the number of contact points increases and so particle- particle
interactions are more pronounced, leading to a three dimensional
network structure and hence increase the viscosity.
Another important factor is that viscosity is also a function of width of
particle size distribution
(118)
. It has been reported that by increasing the
polydispersity index, the viscosity can be reduced. This could be
explained as follows: the smaller particles may fit between the voids of
the larger particles thereby reducing the interactions between the latter
(118)
. This assumption was in disagreement with the obtained results. For
84

igure 16. Viscosity of different SLM formulations containing 2.5 mg

0
5000
10000
15000
20000
25000
5
%

G
B
1
0
%

G
B
2
.
5
%
G
M
5
%

G
M
5
%

G
D
1
0
%

G
D
2
.
5
%
S
A
5
%

S
A
Formulation
V
i
s
c
o
s
i
t
y

(
c
p
s
)

F
TS / g of SLM dispersion at 0.5 rpm.

85

Table 10. One-way analysis of variance for viscosity of different SLM
Source of MS F
formulations that contained 5 % lipid and 2.5 mg TS/g of SLM
dispersion at 0.5 rpm.
dF SS
variation
Formulations 7 76581884 10940269.086 4.478
*
Error 8 19543844 2442980.501
Total 15 96125728

* p 0.05

86
example, 5% SA SLM had the highest PI value (0.42), however, it had
order to assess the
ressed when
particle size effect as predicted by Thomson equation
(120)
. Similar
the highest viscosity relative to other formulations. This could be due to
the relative small mean size of 5% SA SLM (16.3 m).
6. Differential scanning calorimetry (DSC)
Differential scanning calorimetry was performed in
crystallinity of testosterone, the crystalline character (polymorphic
transition) of the lipid matrix as a function of lipid type and
concentration, and to detect the possible melting point change of lipid.
Therefore, the DSC thermograms of TS, bulk lipid and SLM formulations
were investigated. The thermograms of lyophilized SLM after separation
of aqueous phase did not show the characteristic melting peak for the TS
at 154.4 C (Figures 17-20). This suggested that TS may exist in SLM in
an amorphous state. Similar results has been reported by Venkateswarlu
and Manjunath (2004)
(119)
stating that rapid quenching of the formed
microemulsion prevents the drug to crystallize. In addition, the presence
of poloxamer as a surfactant may not allow TS to crystallize.
Melting points of GM in SLM formulations were slightly dep
compared to the melting points of corresponding bulk lipid, Table 11 and
Figure 17. This may suggest that GM existed mainly in a stable form in
SLM. The observed melting point depression could be due to small
87

Figure 17. DSC thermograms of TS, bulk GM, 2.5 % GM, and 5 % GM
SLM formulations containing 2.5 mg TS/g of SLM
dispersion.

88

Figure 18. DSC thermograms of TS, bulk GD, 5 % GD, and 10 % GD SLM
formulations containing 2.5 mg TS/g of SLM dispersion.

89

Figure 19. DSC thermograms of TS, bulk SA, 2.5 % SA, and 5 % SA
SLM formulations containing 2.5 mg TS/g of SLM
dispersion.

90

igure 20. DSC thermograms of TS, bulk GB, 5 % GB, and 10 % GB

F

91

Table 11. DSC results of bulk materials and SLM formulations.

point (C)
llinity Formulation Melting Enthalpy Crysta
(J /g) (%)
TS 154.4 96.1 --------------
GM 60.9 142.1 --------------
Main peak 59.4 75.8 59.2 2.5% GM
------- Shoulder 47 12.9 -------
Main peak 57.3 53.5 39.6 5 % GM
------- Shoulder 47 12.6 -------
Main peak 6 64.0 39.9 -------------- GD
Shoulder 58.3 8 --------------
5 % GD .7 61.3 98 52.2
10 % GD 60.5 108.9 56.1
SA 61.2 157.8 ---------------
Main peak 53.1 18.1 46.9 2.5 % SA
-------- Shoulder 47.3 48.5 -------
5 % SA 55.8 63.2 42.1
GB 71.5 107.8 -------- -------
5 % GB 72.2 87.9 85.8
10 % GB 71.8 83 78.9

92
behavior has been reported by Venkateswarlu and Manjunath (2004)
(119)

6 C
A microparticles, the melting point of 2.5% and 5% SA
and Mhlen et al. (1998)
(121)
for triglycerides and compritol SLN,
respectively. The second small peak appeared at ~ 47 C in the
thermogram of GM SLM may indicate the formation of a second
polymorph. This may indicate that a fraction of the particles possesses
the polymorph with transition temperature of 47 C, while the remaining
fractions of SLM still exhibited the crystal lattice of polymorph with
transition temperature of the main endotherm. The percentage
crystallinity of lipid in 2.5 and 5% GM SLM decreased to 59.2 % and
39.6 %, respectively, taking the enthalpy of the bulk lipid as 100 %.
Concerning GD, the melting endotherm of bulk lipid was at 64.0
with a shoulder at 58.3C as shown in Figure 18. For 5% GD SLM, the
shoulder changed to a plateau. For 10 % GD SLM, the shoulder
disappeared and gave a broad peak at 60.5 C. This may indicate the
transition of less stable polymorph to more stable one as the lipid
concentration increased from 5 to 10 % in SLM. The % crystallinity of
lipid in 5% and 10% GD SLM decreased to 52.2 % and 56.1%,
respectively.
Concerning S
SLM depressed by 8.1 and 5.4 C, respectively, compared to bulk lipid as
shown in Figure 19 and Table 11. In addition, there was another peak
93
appeared at about 47.3 C in the thermogram of 2.5% SA. Since the
amount of drug is fixed in the formulation, consequently, as the % of
lipid phase (drug and lipid) decreased the ratio of drug to lipid increased.
This indicated that in the presence of low concentration of lipid phase
(i.e. high concentration of drug) the polymorphic form of SA may be
affected. This may indicate the possibility of the presence of interaction
between TS and SA.
The % crystallinity of SA in 2.5 % and 5 % SA SLM was 46.9 and 42.1
of GB (Figure 20) shows the melting endotherm for GB
%, respectively.
The thermogram
as a bulk lipid and in SLM at ~71.5 1 C. This was in agreement with
Hamdani et al. (2003)
(122)
who have reported that SLN which are
composed of glycerides with heterogenous composition posses a less
pronounced melting point depression. The percentage crystallinity of GB
in 5% and 10% GB was 85.8 % and 78.9 %, respectively (Table 11). In
general, GB showed highest thermal stability among the examined lipids
and this was in agreement with the documented literature which reported
that the polymorphic transitions after crystallization of lipid in SLM are
slower for longer chain glyceride (GB, C
22
) than for shorter chain
glycerides (SA, C
18
)
(123)
. Additionally, Mhlen et al. (1998)
(121)
have
reported that SLN which are composed of glycerides with heterogeneous
composition, like compritol (64-72% mono-and diglycerides) possess a
94
less pronounced melting depression. Therefore, it was supposed that
stability problems can be avoided by using compritol as lipid matrix.
Generally, DSC examination suggested that the lipids in SLM were of
less ordered arrangement than the corresponding bulk lipid.
7. Powder X-ray diffraction (PXRD)
Powder X-ray diffraction patterns of TS, bulk lipid (GB), lyophilized
ngles 12.9,
ttered
agreement with the results established by DSC studies (section 6).
drug-free SLM (10 % GB) and lyophilized SLM (10 % GB containing 5
mg TS/g of SLM dispersion) are shown in Figures 21 and 22.
PXRD pattern of TS exhibits sharp peaks at 2 scattered a
15.2, 15.9, 18.1, 20.5 and 25.4 indicating crystalline nature. However,
there were no characteristic peaks for TS in lyophilized SLM (10 % GB
containing 5 mg TS/g of SLM dispersion). This may indicate that TS was
in amorphous form in SLM as shown before by DSC examination.
PXRD pattern of bulk lipid (GB) shows sharp peaks at 2 sca
angles 21.3 with a small additional peak of about 23.4. These peaks are
typical for the orthothrombic ` form of triglycerides
(122)
. These
characteristic peaks were also observed in lyophilized drug-free SLM and
lyophilized SLM (10 % GB containing 5 mg TS/g of SLM dispersion)
but with less intensity than the bulk lipid (GB) indicating that GB was in
less crystalline state. This could be due to the method of preparation that
could affect the crystalline state of lipid. These results were in a good
95

.

igure 21. PXRD patterns of: a) TS, b) lyophilized SLM (10 % GB
containing 5 mg TS/g of SLM dispersion).

a)
b)
F
96

igure 22. PXRD patterns of: a) bulk lipid (GB), b) lyophilized drug-free
SLM (10 % GB).

a)
b)

F
97
8. Drug entrapment efficiency of SLM (% EE)
Factors determining the % EE of drug in the lipid microparticles are:
solubility of the drug in melted lipid; chemical and physical structure of
solid matrix; and polymorphic state of lipid material .
Table 12 listed the % drug entrapment efficiency of TS in SLM. The drug
entrapment efficiency ranged from 80.7 %- 95.7 %. The remaining
percentage was the unentrapped drug that could be solubilized in water-
poloxamer phase. A reduction in drug entrapment by poloxamer has been
also reported by Venkateswarlu and Manjunath (2004) .
The chemical nature of the lipid is important because lipid which forms
highly crystalline particles with perfect lattice lead to drug expulsion
(125)
.
Complex lipids, e.g. GB, GD and GM being mixtures of mono-, di- and
triglycerides and also containing free fatty acids of different chain length
form less perfect crystals with many imperfections offering space to
accommodate the drug . This could explain the high entrapment
efficiency of TS in lipids. In addition, the presence of mono- and
diglycerides in the lipid used as matrix material promotes drug
solubilization in lipid .
The statistical analysis (Table 13) followed by Tukeys post hoc test
revealed the following order for drug entrapment efficiency in different
SLM formulations: 10% GB 5% GB 10% GB* 5% GD 10% GD
2.5% GM 5% GM 2.5% SA 5% SA.
(124)
(119)
(124)
(124)
98

Table 12. Drug entrapment efficiency (% EE) of different SLM

* SLM contains 5mg TS/g of SLM dispersion.

Formulation % EE SD
2.5% GM
88.57.1

5% GM

82.27.3

5

% GD
83.24.2

1

0% GD
83.33.4

5

% GB
91.71.8

1

0% GB
80.70.3

2

.5% SA
90.32.1

5

% SA
95.71.4

1

0% GB*
82.23.9

99

One-way analysis of variance for drug entrapment efficiency
(% EE) of different SLM formulations.
variation
Table 13.
Source of dF SS MS F
Formulations 8 615.767 76.971 3.940
*
Error 16 312.601 19.538
Total 24 928.368

1
*
p 0.0

100
It is obvious that the % EE of TS in 10% GB SLM was statistically lower
an that of 5% SA SLM. However, there was no statistically significant
.
stallinity in the
id concentration increased the
from 5% to
10% for 10% GB SLM the entrapment efficiency did not changed
th
difference in % EE of TS among the other formulations.
The highest entrapment efficiency was obtained for SA microparticles
This could be explained on the basis of % lipid cry
prepared formulations. Stearic acid crystallinity in SLM was 46.9% and
42.1%. Therefore, the structure of SA in SLM was of less ordered
arrangement compared with other lipids and this could be the reason for
the highest drug entrapment efficiency
(125)
. Contrary, Trotta et al. (2005)
(47)
have reported that the type of lipid did not affect the encapsulation
efficiency values achieved for insulin.
Concerning the effect of lipid concentration on the entrapment efficiency,
it has been reported that as the lip
entrapment efficiency increased
(103)
. This was in agreement with the
results obtained for SA SLM formulations and in disagreement with the
results obtained for GB SLM formulations. Since the % EE decreased
from 91.7 % to 80.7 % as the concentration of GB increased from 5 to 10
%. However, there was no significant change in % EE as the
concentration of lipid of other SLM formulations increased.
Regarding the effect of theoretical drug loading on % TS entrapment, the
results revealed that as the theoretical drug loading increased
101
significantly. This was in disagreement with Reithmeier et al. (2001)
(126)

who have reported that the efficiency of encapsulation decreased at
higher theoretical loading.
9. Occlusion Study
SLM was chosen as a carrier for TS to be applied transdermally due to its
tendency to adhere to cells and surfaces, and due to the film formation
t can be observed visually after application of SLM
rsion) were 100% and 35% after 6 h and 24 h, respectively.
n solubility of TS in the receptor medium. From the
he concentration of TS in
properties of SLM tha
to the skin. In addition, SLM has been reported to posses occlusive
properties that could favor drug penetration into the skin after application
of SLM
(127)
.
The calculated occlusion factors under the current experimental
conditions for the examined formulation (10% GB containing 5 mg TS/g
of SLM dispe
This could be due to solid state of lipid matrix in SLM that forms a thin
film on the surface which disable the evaporation of water from skin for
the first 6 h
(103)
.
10. Permeability studies of testosterone
During permeability study the sink conditions was assured by
determinatio of
results of the solubility study, it was found that t
receptor medium (assuming 100% drug permeation) was not exceed 20 %
of its saturation solubility (100.01 8 mg) in the receptor medium. Each
102
withdrawn sample volume was replaced by equal volume of fresh
medium.
The effect of the following factors on the permeation characteristics of
TS was examined:
10.1. Effect of type and concentration of the lipid
tion of SLM formulations with
formulations that
er values of coefficient of determination, (R
2
>
he permeability coefficient (P, cm/h) and
Figures 23 and 24 show the cumulative amounts of TS released across
cellophane membrane after applica
different type and concentration of lipid content SLM
contained 2.5 mg TS/g.
The release data were fitted into Ficks and Higuchi equations. Almost
all the SLM formulations followed Ficks law better than Higuchi model
as indicated by the high
0.9909) as shown in Table 14.
The in vitro release data were then treated in accordance to Ficks law to
calculate the flux (J) which is the amount of drug permeated per unit area
and per unit time (g/cm/h), t
diffusion parameter (D, h
-1
) as shown in Table 15. Statistical analysis of
release data using two way ANOVA without interaction followed by the
Duncan test (Table 16) revealed the following order for the release of TS
after application of different formulations: 10% GB > 10% GD > 2.5%
SA > 2.5% GM > 5% GB > 5% SA > 5% GD > 5% GM. It seemed that
the release of TS was affected not only by the concentration of lipid but
103

0
50
100
150
200
250
0 5 10 15 20 25 30
Time (h)
C
u
m
u
l
a
t
i
v
e

a
m
o
u
n
t

o
f

T
S
r
e
l
e
a
s
e
d

(
g
/
c
m
)
5% GM
5% GD
5% GB
5% SA

containing different types of lipid and 2.5 mg TS/g of SLM
dispersion.

104

containing different concentration of lipid and 2.5 mg TS/g
of SLM dispersion.

0
50
100
150
200
250
5 % GD 10% GD
Formulation
C
u
m
u
l
a
t
i
v
e

a
m
o
u
n
t

o
f

T
S

r
e
l
e
a
s
e
d

a
f
t
e
r

2
4

h

(
g
/
c
m
)
0
50
100
150
200
250
5 % GB 10% GB
Formulation
C
u
m
u
l
a
t
i
v
e

a
m
o
u
n
t

o
f

T
S

r
e
l
e
a
s
e
d

a
f
t
e
r

2
4

h

(
g
/
c
m
)
0
50
100
150
200
250
2.5% GM 5% GM
Formulation
C
u
m
u
l
a
t
i
v
e

a
m
o
u
n
t

o
f

T
S

r
e
l
e
a
s
e
d

a
f
t
e
r

2
4

h

(
g
/
c
m
)
0
50
100
150
200
250
2.5% SA 5% SA
Formulation
C
u
m
u
l
a
t
i
v
e

a
m
o
u
n
t

o
f

T
S

r
e
l
e
a
s
e
d

a
f
t
e
r

2
4

h

(
g
/
c
m
)
105

Table 14. Coefficient of determination (R) calculated after fitting the
release data of TS into Ficks and Higuchi equations.
Cellophane membrane was used as transporting membrane. All
Formulation R (Ficks) R (Higuchi)

2.5 % GM
0.9988 0.9854
5 % GM
0.9924 0.9944
5 % GD
0.9909 0.9733
10 % GD
0.9985 0.9888
5 % GB
0.9916 0.9969
10 % GB
0.9985 0.9832
2.5 % SA
0.9984 0.9826
5 % SA
0.9997 0.9880

106

Table 15. In vitro release parameters of TS after application of different
formulati ng 2.5 mg TS/g of SLM dispersion
cellophane m (n=3).

on J (g/cm
SD
P (cm/h)
x 10
2
SD
SLM ons containi
to embrane

Formulati /h) D` (h
-1
)
SD
2.5% GM 8.040.6 0.3200.0002 1.2610.500
5% GM 5.010.6 0.2000.0002 0.0640.016
5% GD 7.070.8 0.2820.0003 0.3300.200
10% GD 8.290.2 0.3310.0001

.035 0.2410
5% GB 7.590.3 0.3040.0001 0.4030.100
10% GB 8.780.1 0.3510.00002 0.0950.017
2.5% SA 8.420.4 0.3360.0001 0.1210.011
5% SA 7.100.3 0.2840.0001 0.4640.087
107

Table 16. Two-way analysis of variance for in vitro release of TS after
application of different SLM formulations to cellophane
membrane. All formulations containing 2.5 mg TS/g of SLM
dispersion.

variation
d SS Source of F MS F
Total 1 793274.512 91

***p 0.0001

Formulations 7 14269.731 2038.533 14.253***

Time 7 753689.353 107669.908 752.805
Error 177 25315.428 143.025

108
also by th
transdermal drug transport on the carrier medium is well documented in
e
(5)
differen ect of each ier related sp fically to
f the drug within the carrier
(128)
, only solubilized drug can
t matrix and ign release
ition, wit 24 h (the i the a id
ispersion lied to th e slowly turned into a semisolid
el format f SLM a o water evaporation could be
orrelated with polymorphic transition of lipid matrix
(130)
. Consequently,
e release of TS from various lipids microparticles could be correlated
ith polymorphic transition of lipid matrix. Since different polymorphic
rms differ in their ability to include host molecules in their lattice
(123)
,
rug expulsion as a consequence of this transition was likely to occur.
he mechanism of release of TS could be explained as follows: TS which
as in amorphous form (as shown in DSC study, section 6) dissolved in
pid, diffused to the surface, and partitioned between lipid and aqueous
hase. Soluble drug is partitioned into aqueous phase and from which it is
leased to the receptor medium.
This result indicated that the release process is consistent with skin-
e type of lipid used in the formulation. The dependence of
the literatur
solubility o
. The t eff carr eci
diffuse within he contribute s ificantly to rate
(129)
.
In add hin release exper ment period) pplied flu
SLM d app e membran
gel. G ion o s a function f
c
th
w
fo
d
T
w
li
p
re
The obtained results showed no relation between the viscosity of the
formulation and TS transport. For example, SA SLM showed
intermediate release behavior in spite that it had the highest viscosity.
109
controlled mechanism, since the viscosity of lipid formulation will play
an important role in controlling the release of the drug if the diffusion of
are water-filled pores or channels for
t matrix itself
drug through the matrix is the rate-determining step
(131)
.
10.2. Effect of transporting membrane
Based on the flux values and on the total amount permeated through the
cellophane membrane two formulations were chosen to be tested using
excised abdomen rat skin. These formulations were 10% GB and 10%
GD containing 2.5 mg TS /g of SLM dispersion.
The permeability of TS through cellophane membrane was significantly
higher than that from excised abdomen rat skin (p 0.0001) as indicated
by the flux and permeability coefficient values shown in Table 15 and 17.
The higher permeability can be due the less dense structure of cellophane
membrane relative to the rat skin. Considering the structure of
cellophane membrane, the molecular weight cut off of the membrane is ~
6000-8000 suggesting that there
drug molecules to diffuse freely. Accordingly, the penetration of drug
may depend on the speed of drug partitioning from the lipid matrix into
receptor phase
(131)
. On the other hand, the total cumulative amount of TS
released from 10% GB (19.4 g 3.1) was statistically higher (p
0.0001) than 10% GD SLM (9.74 g 1.2) using excised abdomen rat
skin as shown in Figure 25. The effect of matrix on drug penetration
cannot, however, be considered in isolation because the fa
110

Formulation J (ug/cm/h) SD P(cm/h)x10 SD

D`(h ) SD

Table 17. Permeation parameters of TS after the application of selected
SLM formulations to excised abdomen rat skin. (n=3)

4

-1
TS Concentration: 2.5 mg/g
10
-5
% GB 0.93490.135 3.745.4x10 0.0420.002
10 % GD 0.43160.068 1.732.7x10
-5
0.0470.003
TS Concentration: 5 mg/g
10 % GB 0.951560.109 3.814.4x10
-5
0.0510.004

111

0
10
25
0 5 10 15 20 25 30
Time (h)
S

p
e
r
m
5
m
u
l
a
15
t

o
f

T
c
m
)
20
e
a
t
e
d

C
u
t
i
v
e

a
m
o
u
n
(
g
/
0% GD
10%GB
1

abdomen rat skin after application of 10% GB and 10% GD
SLM formulations containing 2.5 mg TS/ g of SLM
dispersion.

112
may have enhancing effect on drug penetration or permeation
(132)
.
Based on these results, 10% GB SLM formulation containing 2.5 mg TS/
g of SLM dispersion was chosen to study the effect of increasing drug
loading on TS permeation.
10.3. Effect of drug loading
The permeation rate of drug can be altered by changing the drug
concentration in the lipid matrix. It has been reported that the increase of
drug concentration up to a certain level may enhance the permeability of
drug due to an increase in the drug thermodynamic activity
(133)
. In the
present study, statistical analysis revealed the cumulative amount of drug
sed abdomen rat skin was not significantly
changed (p> 0.05), Figure 26, as the theoretical drug loading of 10 % GB
SLM increas
TS, Table 17
that the conducting pathways of the skin have reached saturation
(134)
.
(133)
, have found that the permeation of
indomethacin increased as the initial drug concentration increased from 5
to 20%. However, Souto et al. (2004)
(103)
have reported that clotrimazole
as released more quickly when using lower drug concentration.
imillarly, Wissing and Mller (2002)
(135)
have reported that the release
ue to
steric hindrance effect of drug molecules at higher drug concentration.
permeated through exci
ed from 2.5 to 5 mg TS /g of SLM dispersion. The flux of
, did not also increase significantly (p> 0.05). This indicated
Contrary, Rao and Diwan (1998)
w
S
rate of oxybenzone decreased as the drug concentration increased d
113

abdomen rat skin after application of 10% GB SLM with
different drug loading concentrations.

0
5
0 5 10 15 20 25 30
Time (h)
C
m
u
l
a
t
i

a
m
o
u
n

o
f

T
S

p
m
e
a
t
e
d
g
/
c
m
5 mg TS/ g
10
15
20
25
u
v
e
t
e
r

(
)
2.5 mg TS/ g

114
Concerning further permeation studies 10 % GB SLM containing 5 mg
TS/ g of SLM dispersion formulation was chosen as selected formulation.
In an attempt to find a correlation between the amounts of TS released
through cellophane membrane and the amounts of TS permeated through
excised abdomen rat skin, the amount of TS permeated through the rat
skin was plotted against the amount released through cellophane
membrane after application of 10% GB SLM formulation containing 5
mg TS/g of SLM dispersion at the same time points over 24 h, as shown
in Figure 27. Regression analysis revealed high correlation between the
amounts of TS permeated through the excised abdomen rat skin and
embrane over 24 h, as indicated by the value of coefficient
ination (0.997).
y = -0.247 + [19]
here, y = amount of TS permeated through excised abdomen rat skin
nd x = amount of TS permeated through cellophane membrane. Pearson
orrelation was also calculated and revealed significant (p 0.0001)
ositive correlations (direct relationship) as indicated by its value 0.995.
onsequently, it could be concluded that under the current experimental
onditions the permeation of TS through excised abdomen rat skin could
be predicted using cellophane membrane by applying equation [19].
cellophane m
of determ
This relation was best fitted into the following cubic equation:
0.048x + 0.00036x
2
- 3.257 x
3

W
a
c
p
C
c
115

0
0 50 100 150 200 250
Amount of TS permeated through cellophan membrane (g/cm
2
)
A
o
u
n
t

T
S
r
m
e
e
d

t
h
u
g
h

e
x
c
i
s
e
d
d
o
m
e

r
a
t
i
n

(
c
m
2
)
5
10
15
20
25
m

o
f

p
e
a
t
r
o

a
b
n

s
k
g
/
Figure 27. Correlation between the amounts of TS permeated through
excised abdomen rat skin and through cellophane membrane
over 24 h at the same time point.

116
10.4. Effect of chemical enhancer
igure 28 shows the effect of addition of 1% OA and 1% DA to SLM
formulation or pretreatment of excised abdomen rat skin with 1% DA in
ethanol for 30 min before application of the selected formulation (10%
GB containing 5 mg TS/g of SLM dispersion) on the cumulative amount
of TS permeated. The permeation parameters and the enhancement ratios
are summarized in Table 18. The highest flux and enhancement ratio
(2.59 ug/cm
2
/h and 2.73, respectively) were obtained when 1% DA was
applied to the skin for 30 min then removed before application of the
selected formulation. On the other hand, the addition of 1 % OA or 1 %
DA to the selected formulation produced an enhancement ratio of 1.15
nd 2.42, respectively. The higher enhancement obtained for DA was in
greement with Tanojo et al. (1997)
(136)
who have reported that the chain
saturated f , brings about an optimal
alance between partition coefficient of solubility parameter and affinity
skin.
tatistically, there was no significant difference (p>0.05) in the
permeation of TS when SLM containing 1% DA was applied and when
the skin was pretreated with 1% DA for 30 min then removed before
ulation.
t were also significantly increased
F
a
a
length of saturated fatty acid of about 12 carbon (DA is a typical
atty amine of 12 carbon units)
(137)
b
to
S
application of the selected form
The values of permeability coefficien
117

0
10
20
30
40
50
60
70
0 5 10 15 20 25 30
Time (h)
C
u
m
u
t
i
v
e

a
m
o
u
n
t

o
f
S

p
e
r
e
a
t
e
d

(
g
/
c
m
)
HUS
1% OA
1 % DA for 30 min
1 % DA for 30 min only & HUS
1 % DA

fferent
mg TS /g of SLM dispersion.

l
a

T
m
No enhancement
1 % OA & HUS
abdomen rat skin using di chemical enhancers and/or
HUS. The examined formulation was 10% GB containing 5

118

Effect of application of chemical enhancers and/or HUS on
SLM dispersion.
Enhancement
method
J
(g/cm
2
/h)
SD
P 10
4
(cm/h)
SD
D`
(hr
-1
)
SD

ER Skin
retention
(g/cm
2
)
SD

Table 18.

Selected formulation
(10% GB containing
5 mg TS/g)

0.950.11

3.810.00004

0.05070.004

-----

46.13.4

SLM containing 1%
DA

2.300.32

9.210.0001

0.07630.016

2.42

80.94.3

SLM containing 1%
OA

1.090.08

4.370.00003

0.05930.014

1.15

63.30.5

30 min 1% DA

2.590.39

10.360.0001

0.06100.018

2.73

56.94.3

HUS

1.590.35

6.380.0001

0.04880.006

1.68

34.52.8

HUS& SLM
containing 1% OA
0.05140.004 1.64

55.912.3 1.560.23

6.260.00009

HUS&30 min 1%
DA

1.780.42

7.140.0001

0.06440.027

1.88

62.210.5
119
(p 0.001) in the presence of investigated enhancer in comparison with
elected formulation (10 % GB contained 5 mg TS/g). DA significantly
creased the permeation rate of TS by increasing the permeability
9.21x10
-4

the skin w
application of the selected formulation). These results were in agreement
n et . (2003)
(58)
creased the ate isic acid at a concentration o
Enhanced permeation rate of TS has been also documented when 1% DA
3 as u ina %
n .
Concerning OA, it affects the fluidity of the lipids in intercellular layers
of the stratum corneum because of their resemblance in structure to the
8)
.
Fatty acids an in g bee h
atio
39)
. T att ru o
at a he ex ace m eu
It is worth mentioning that, the results of stability studies (Data are in
stability studies, section 11) indicated that SLM containing 1% DA was
physically unstable. Consequently, it was excluded from further studies.
There was a significant statistical difference (p 0.05) in skin residuals
s
in
coefficient (3.81x 10
-4
cm/h for selected formulation compared with
cm/h for SLM containing 1% DA, and 10.36 x 10
-4
cm/h when
as pretreated with 1% DA for 30 min then removed before
with Bia
in
al who have reported that DA significantly
permeation r of gent f 1 %.
(94)
or when % DA w sed in comb tion with 10 Span 80 as
permeation e hancer
(95)
lipid
(13
d amines, eneral, have n reported to ave a potent
skin perme n effect
(1
heir effect is ributed to dis ption f lipid
bilayers th re filling t tracellular sp s of the stratu corn m.
120
of TS determined after application of enhancers compared with the
ted SLM formulation increased the flux of TS
merous. Ultrasound causes mechanical disturbance in the
th
application of selected SLM formulation alone, Table 18. The highest
skin residuals were found after application of SLM containing 1% DA
followed by SLM containing 1% OA and when the skin was pretreated
with 1% DA for 30 min then removed before application of the selected
formulation.
10.5. Effect of application of high frequency ultrasound (HUS) alone
or in combination with chemical enhancers
Figure 28 shows the cumulative amounts of TS permeated across excised
abdomen rat skin after application of HUS alone or in combination with
chemical enhancer. The permeation parameters and the enhancement
ratios are reported in Table 18. Pretreatment of skin with HUS for 1 h
before application of selec
by 1.68 fold compared with the selected formulation. The possible
mechanisms for the observed enhancement using continuous mode HUS
can be nu
absorbing medium. As ultrasound propagates through a medium, some of
its energy is absorbed and converted into heat. This will increase the
temperature leading to enhancement of drug transport. It is wor
mentioning that under the current experimental conditions the change in
temperature after application of HUS for one hour did not exceed 2C
which cannot explain the increase in transdermal absorption of TS.
121
This was in agreement with what has been reported by Merino et al.
e reversible
(141)
. Furthermore, ultrasound may affect the
corneu , which in turn may
lower the flux of TS through rat skin (ER = 1.88). Similar results were
(2003)
(71)
.
Another possible explanation for the increased transport during
phonophoresis may be due to cavitation, which involves creation and
subsequent collapse of microbubbles from dissolved gas
(140)
. It is a very
energetic phenomenon that cause cellular damage to cells, this damage is
thought to b
stratum corneum itself by disordering the lipids within the stratum
m, as suggested by Boucaud et al. (2001)
(142)
increase the diffusion of certain compounds. Consequently, transdermal
transport in presence of HUS is expected to be higher than passive
transport.
In an attempt to further improve the transdermal permeation of TS over
what was obtained by application of HUS alone, another approach which
was the combination of HUS and chemical enhancers was examined. The
results showed that there was no statistically significant difference (p>
0.05) in the permeation of TS after application of HUS alone (6.38 x 10
-4

cm/h) and application of either 1% DA for 30 min (7.14 x 10
-4
cm/h) or
SLM containing 1% OA (6.26 x 10
-4
cm/h) after pretreatment with HUS
for 1 h. On the other hand, compared with the ER obtained after
application of 1% DA for 30 min (ER = 2.73), pretreatment with HUS
122
documented by Lui et al. (2006)
(143)
who have reported that the
cumulative amount of cyclosporine A diffused to receptor medium
with application of DMSO alone. Contrary, by comparing the
the enhancement ratios applying US remained relatively low
decreased after application of US in combination with DMSO in
comparison
enhancement effect produced by 1% OA, there was a synergistic effect
for pretreatment of skin with HUS before application of SLM containing
1 % OA (ER = 1.64, 1.15 for HUS + SLM containing 1% OA and SLM
containing 1 % OA, respectively). This was in agreement with Tiwari et
al. (2004)
(106)
who have reported that when HUS was combined with d-
limonene/ethanol, diffusion resistance of stratum corneum barrier is
further expected to drop because HUS may enhance the penetration of
enhancer inside the stratum corneum and further loosing of stratum
corneum cells.
All in all,
either in presence or in absence of permeation enhancers. This was in
agreement with what have been reported by Fang et al. (1999)
(144)
.
Taken together statistical analysis revealed the following order for the
permeation of TS through the excised abdomen rat skin: 1% DA for 30
min > HUS +1% DA for 30 min HUS HUS +SLM containing 1% OA
> SLM containing 1% OA selected formulation (10 % GB contained 5
mg TS/g of SLM dispersion). This order was confirmed by the
histological changes in the rat skin observed after each treatment. The
123
most severe skin disorder was obtained after application of 1% DA for 30
min as shown in Figure 29, d. A thin epidermis layer was observed for all
treated rat skin compared with untreated skin (Figure 29, a). Vascular
initial hyperplasia was noticed for skin exposed to HUS (Figure 29, b),
while dermal fibrosis was observed for skin exposed to HUS and 1% DA
for 30 min (Figure 29, c). On the other hand, severe dermal edema was
noticed for skin treated with 1% DA for 30 min (Figure 29, d). However,
macroscopic alteration of the skin can not be seen after in vitro treatment
with HUS. This was in disagreement with Machet et al. (1996)
(141)
who
have reported macroscopical changes of human skin treated with HUS (1-
3 MHz) at intensities ranging from 2-3 W/cm
2
. On the other hand,
histological studies performed on hairless rat skin exposed to therapeutic
US have reported that application of US (1 MHz, 2 W/cm
2
) induced no
skin damage
(72)
.
There was no statistical difference in skin residuals of TS determined
alone
after application of HUS or HUS + enhancers compared with the
application of selected SLM formulation alone, Table 18.
10.6. Effect of application of low frequency ultrasound (LUS)
or in combination with chemical enhancers
10.6.1. Effect of LUS alone
10.6.1.1. Effect of total application time t
eff
(or duty cycle)
The effect of three t
eff
(6, 12, and 15 min) corresponding to 10:40, 10:15
124

and after application of b) HUS, c) HUS +1% DA 30 min, d)
a) b)
c) d)

Figure 29. Histological changes of excised abdomen rat skin a) untreated

1% DA 30 min.

125
and 10:10 sec (on/off) duty cycle, respectively, over total exposure time

S was studied. Statistical analysis showed the following rank order for
e permeation of TS: 15 min 12 min > 6 min selected formulation
o application of LUS). As shown in Figure 30, at t
eff
15 and 12 min the
ux of TS increased by 4.77 and 4.63 fold, respectively, compared with
the selected formulation. While at t
ef
n, the increased in TS flux was
.86 fold only as shown in Table 19. The 10 and 15 sec off durations may
ot be enough to allow the skin to recover from abnormal status which
ay explain the higher TS permeability in these two protocols. Indeed,
e histological changes presented in Figure 31 demonstrated that the
horter off periods produced greater epidermal and dermal necrosis
igure 31: c, d) while long off period produced epidermal thinning
igure 31, b). These observations confirmed the enhancement effect in
after exposure to smaller LUS on/ off
ratios. These results were in ag
have reported that the application of smaller on/off ratio US on skin
duced greater histological changes. The results were also consistent
with a cavitationbased mechanism since cavitation activity increased
with increasing application time
(107)
. Cavitation which is the generation,
oscillation and subsequent violent collapse of gaseous micro-bubbles
within the coupling medium and/or within the skin, is probably the
of 30 min with intensity of 2.5 W/cm
2
on the transdermal permeation of
T
th
(n
fl
f
6 mi
1
n
m
th
s
(F
(F
transdermal permeation o TS f
reement with Fang et al. (1999)
(144)
who
in
126

F
exposure time of 30 min with intensity of 2.5 W/cm
2
on the

igure 30. Effect of total application time of LUS or duty cycle, over total
flux of TS through excised abdomen rat skin after application
of 10 % GB containing 5 mg TS/g of SLM dispersion.

0
1
5
Total application time

(
m
2
3
4
6
F
l
u
x
g
/
c
2
/
h
)
No LUS 6 min. 12 min. 15 min.
127

Table 19. Effect of application of LUS at different duty cycle

s and
intensities and application of LUS and chemical enhancer on
SLM dispersion .
in
)
Enhancement
method
J
(g/cm
2
/h)
SD
P 10
4
(cm/h)
SD
D`
(h
-1
)
SD

ER TS Sk
retention
(g/cm
2
SD
tion (10
% GB contained
5 mg TS/ G)

0.950.1

3.810.00004

0.05070.004
-------
46.13.4
LUS (10:10) at
2.5 W/cm

4.530.50

18.140.0001

0.05220.004

4.77

119.316.5
LUS (10:15) at
2.5 W/cm

4.411.21

17.630.0004

0.0950.014

4.63

110.712.1
LUS (10:40) at

1.770.18

7.060.00007

0.04550.001

1.86

33.19.2
Selected
formula

2.5 W/cm

LUS (10:15) at
3.25 W/cm

2.680.22

10.700.0001

0.06540.006

2.81

60.77.3

LU
W/cm
S (10:15) at 5

2.320.04

9.290.00001

0.05140.007

2.44

48.211.3

4.580.58

18.300.0002

0.07280.014

4.81

108.119.
LUS (10:15) at
2.5 W/cm& 30
min DA
5

128

Histological characteristics of excised abdo en rat in a)
untreated skin, and at different t ion of
duty cycle, b) 10: 0 LUS, c) 10:15 LUS, d) 10:10 LUS over
tota tim i f /cm

Figure 31.

m sk
otal applicat time LUS or
4
l exposure e of 30 min w th intensity o 2.5 W
2
.

a) b)
c) d)

129
main mechanism that could explain the enhancement of TS permeability.
he cavitation causes disorder of the stratum corneum lipids, resulting in
ignificant water penetration into the disordered lipid region. This might
ause the formation of aqueous channels through the intracellular lipids
f stratum corneum through which permeants could move
(145)
.
has been reported that cavitation may occur more readily with LUS
an with HUS
(146)
. This could be due to the fact that at higher
equencies it becomes increasingly difficult to generate cavitation
ecause the fact that the time between the positive and negative acoustic
ressures becomes too short, diminishing the ability of dissolved gas
ithin the medium to diffuse into the cavitation nuclei
(67)
. In addition,
e number and size of cavitation bubbles is inversely correlated with
application frequency
(147)
. The resonance radius of gas bubbles R
r
(m) is
roughly related to frequency (F in kHz) by the following equation
(69)
:
F x R
r
=3000 [20]
m at 20 kHz and 3 m with 1
MHz .
skin and t
Moreover ling
during application of LUS due to presence of dissolved gas.
Furthermore, raising the temperature during exposure to LUS (about 10-
T
s
c
o
It
th
fr
b
p
w
th
Thus the micro-bubble size was about 150
(69)
This suggested that the cavitation may affect the structure of
hus increase drug permeability.
, bubbles due to cavitation could be easily seen in the coup
medium
130
15C under the current experimental conditions) may lead to
nhancement of drug transport by increasing the fluidity of skin lipids
(69)
.
he literature supported the observation that increasing temperature lead
eri s of LUS
re lts
results showed that the effect
e
T
to an increment in skin permeability
(148)
. The skin residuals of TS
showed a trend of 10:40 (33.1 9.2 g/ cm
2
) < 10:15 (110.7
12.1g/cm
2
) 10:10 s (119.8 16.5 g /cm
2
), which is consistent with
the order of TS flux and ER, Table 19. The permeation and partition (skin
retention) results both suggested that the shorter the off p od
the higher the amount of TS existed in the skin and therefore the higher
amount of TS permeated. This result was in accordance with previously
reported studies using pulsed ultrasound to improve permeability of
clobetasol 17-propionate through skin
(144)
.
10.6.1.2. Effect of intensity
Of the protocols presented above, the t
eff
that produced the greatest
enhancement of TS transdermal permeation was at t
eff
15, 12 min for 30
min at 2.5 W/cm. Therefore, the later t
eff
(12 min) was selected to study
the effect of increasing LUS intensity to 3.25 and 5 W/cm. The su
demonstrated that the LUS was effective in enhancing the permeability of
TS for all the intensities studied as shown in Figure 32.
It has been reported that the cavitation induced by LUS is directly
correlated with intensity
(71)
. However, the
131

Figure 32. Flux of TS through excised abdomen rat skin after application
LUS at different intensities and total application time of 12
min over total exposure time of
of 10 % GB containing 5 mg TS/g of SLM dispersion using
30 min.

0
5

(
m
1
2
3
4
6
No LUS 2.5 W/cm 3.45 W/cm 5 W/cm
Intensity
F
l
u
x
g
/
c
2
/
h
)
132
of enhancement was not directly proportional to the magnitude of
ultrasound intensity. The flux and ER (Table 19) of TS decreased as the

tensity of US showed more skin damage as well as lower permeability
f drugs through skin tissues
(144)
. The decrease in ER beyond application
of 2.5 W/cm may be due to acoustic decoupling, a process which
tensity due to acoustic decoupling.
o clarify the obtained results, the histological characteristics of the skin
sever epide
other hand, s was observed in the
in when LUS having intensity of 3.5 W/cm was applied (Figure 33, c)
nd only epidermal thinning and hyalinized dermis were observed in the
intensity increased. These results were consistent with previously
reported findings which have documented that the application of higher
in
o
decreases the intensity seen by the skin due to the presence of the
cavitations cloud
(147)
. Tezel et al. (2001)
(149)
have reported that there
exists an intensity below which no detectable enhancement is observed.
This intensity is referred to as the threshold intensity. Once the intensity
exceeds this threshold, the enhancement increases strongly with the
intensity until another threshold intensity, referred to as decoupling
intensity is reached. Beyond this intensity, (in this study it equals to 2.5
W/cm), the enhancement does not increase with further increase in the
in
T
were examined. Application of LUS at intensity 2.5 W/cm resulted in
rmal and dermal necrosis as shown in Figure 33, b. On the
moderate epidermal and dermal necrosi
sk
a
133

a)
b) c)

, c) 3.5 w/ cm

d) e)
2 2
and d) 5 w/cm
2
, and e) LUS 2.5 W/cm
2
after 24h.

untreated, and after application of LUS at on/off 10:15 duty
cycle and at different intensity b) 2.5 W/cm

134
skin when LUS having intensity of 5 W/cm was applied (Figure 33, d).
This could explain the obtained results, since among the three applied
ultrasound protocols, the intensity of 2.5 W/cm
2
LUS at t
eff
12 min
showed the highest TS flux (4.41 g/cm
2
/h). This could be due to severe
skin damage or disorganization of the lipid bilayers of stratum corneum
occurred at intensity 2.5 W/cm
2
. Fortunately, the damaged skin (exposed
LUS 2.5 W/cm
2
at t
eff
12 min for total exposure time of 30 min)
eemed to resume its original structure after 24 h as shown in Figure 33,
. Consequently, it could be concluded that the action of LUS was
versible. This was in agreement with Tyle and Agrawala (1989)
(150)
.
ortunately, Machet and Boucaud (2002)
(69)
have reported that the
reshold tolerance of human skin to US is high in vitro and probably also
vivo compared to animal skin.
he skin residuals of TS showed a trend of 2.5 W/cm
2
(110.7 12.1
g/cm
2
) >3.25 W/cm (60.7 7.3 g/cm
2
) 5 W/cm
2
(48.2 4.3 g/cm
2
),
hich is consistent with e order of TS flux and ER, Table 19.
ll things considered, the results showed that LUS significantly enhanced
the permeability of TS across excised abdomen rat skin. Delivering the
me amount of ultrasonic energy in different modes of application and
ifferent intensities markedly influenced the flux and skin residuals of
S.
to
s
e
re
F
th
in
T
w th
A
sa
d
T
135
10.6.2. Effect of LUS and chemical enhancer
tocols presented above, the LUS protocol that produced the
ancement of TS transdermal delivery was t
Of the pro
greatest enh n and total
posure time of 30 min at 2.5 W/cm. Consequently, it was selected as
US protocol to examine the effect of pretreatment of excised abdomen
t skin with LUS before application of 1 % DA in ethanol for 30 min on
S permeation. The results showed that no significant (p > 0.05)
he
in.

eff
12 mi
ex
L
ra
T
synergistic or additive effect on flux of TS was obtained when 1% DA
applied for 30 min after exposure to LUS (30 min, 2.5 W/cm
2
, t
eff
12 min)
as shown in Figure 34 and as indicated by ER values (4.81 and 4.63 for
LUS + 30 min 1 % DA and LUS alone, respectively) as shown in Table
19. This result was in accordance with Liu et al., 2006
(143)
who have
found that the combination of LUS and chemical enhancers had no
synergistic effect on the transdermal delivery of cyclosporine A.
Contrary; Mitragotri et al. (2000)
(75)
have reported a synergistic effect of
LUS with SLS.
On the other hand Fang et al. (1999)
(144)
have reported that although t
enhancement ratio obtained by combination of LUS and chemical
enhancer was statistically significant it remained relatively low in some in
vitro studies performed on mice sk
136

ure 34. Effect of application of 1% DA for 30 min &/or LUS at intensity
of 2.5 W/cm at total application time t
Fig
exposure time of 30 min on flux of TS through excised abdomen

eff
of 12 min over total
rat skin after application of 10 % GB containing 5 mg TS/g of
SLM dispersion.
0
1
2
5
No enhancement 1 % DA for 30 LUS LUS & DA
x

(
c
)
3
4
6
F
l
u
g
/
m
2
/
h
min
Method of enhancement
137
Statistical analysis followed by Duncan post hoc test revealed that the
skin residuals of TS showed the following rank order LUS 1% DA for
in had no advantage over
pplication of either LUS or HUS alone. In general, application of LUS
sulted in higher TS permeation than HUS.
ased on histological examination, the effect of US on skin is derived
irectly from the application parameters, which include application
duration, frequency and intensity.
he cumulative amount of TS permeated through excised rat skin over 24
after application of the selected formulation was significantly decreased
30 min + LUS > DA for 30 min which is consistent with the order of TS
flux and ER, Table 19.
To sum up, the pretreatment of excised abdomen rat skin with HUS or
LUS before application of 1% DA for 30 m
a
re
B
d
11. Stability studies
11.1. Stability of selected formulation
In order to evaluate the physical stability of the selected formulation (10
% GB containing 5 mg TS /g of SLM dispersion), both release behavior
and particle size measurement were evaluated after storage for a period of
16 weeks at 5C and 30 C in the dark.
During the period of storage the selected examined formulation showed
no change in color and no creaming or phase separation.
T
h
138
after16 and 12 weeks of storage at 5 and 30 C, respectively, as shown in
35.
tempt to correlate the change in permeation of TS with the particle
Figure
In an at
size of SLM, particle size measurement was performed by microscopic
method over 16 week storage at 5C and 30C (Figure 36).
Statistically, the particle size increased significantly after storage for 12
and 16 weeks at 30C. It was assumed that the high temperature (25 C)
increased the kinetic energy of system, which could accelerate the
collision of particles
(151)
and, consequently, increased the possibility of
Contrary, there was no significant increase in particle size of SLM stored
similar to data reported by Hu
(152)
onoglycerides and 70%
(153)
hus prevent particles aggregation and
m stability
(154)
.
aggregation of particles. Therefore, the exposed surface area was
expected to decrease and could affect the drug release negatively.
at 5C after 16 weeks. These results were
et al. (2006) who have found that the particle size of monostearin
SLN increased significantly after 30 day storage at 25 C, however, at 4
C no significant change in particle size was observed. It was proposed
that the partial glycerides like GB (15% m
diglycerides) posses improved surfactant properties (HLB 2-5) .
These surface active partial glycerides facilitate emulsification and form
more rigid surfactant films and t
therefore improve long ter
139

Figure 35. Cumulative amount of TS permeated after 24 h through excised
elected SLM
are statistically insignificant (A>B>C), (a>b>c>d).

abdomen rat skin after application of the s
formulation (10 % GB containing 5 mg TS/ g SLM dispersion)
stored over16 week at 5 and 30 C. Bars with similar symbols

0
Fresh 2 4 6 8 12 16
m
i

o
f

p
e

a

2
5
10
15
C
u
u
l
a
t
v
e

a
m
e
r
m
a
t
e
d
f
t
e
r
20
25
o
u
n
t

T
S
4

h

(
g
/
c
m
30
Time (week)
)
at 5 C
at 30 C
B
B B
B
A
B
C
ab
cd bc
a ab
d d
140

15
25
35
45
n

p
t
i
c
l
e
i
z
e
m
)
at 5 C
C
AB
B
B
A A
b
ab
ab
ab
ab
a
a

Figure 36. Mean particle size of the selected SLM formulation (10 % GB
containing 5 mg TS/ g SLM dispersion) stored over16 week at
5 and 30 C. Bars with similar symbols are statistically
insignificant (A>B), (a>b).

0
5
10
20
30
40
50
Fresh 2 4 6 8 12 16
Time (week)
M
e
a
a
r

s

(
at 30
AB
AB

141
In addition, the poloxamer film around the microparticles steric stabilized
the particles and prevents aggregation
(155)
. Borgia et al. (2005)
(156)
who
have documented that SLN composed of 10% GB did not show change in
particle size or particle size distribution after storage for almost 12 week
at 8C reported similar results. Consequently, other factors were expected
to affect the release of drug from SLM after storage for 16 weeks at 5C.
During storage, rearrangement of the lipid crystal lattice might occur in
favor of thermodynamically stable configurations and this is often
connected with expulsion of the drug molecules
(44)
. This could be
resulted i
opposed b
storage at
low-viscosity system into gel . Gelling of SLM may lead to increase
microviscosity and retard drug diffusion and consequently its release.
he final consequences of the last mentioned two effects may be the
ecrease in overall amount of drug release.
1.2. Stability of formulation containing 1% OA or 1% DA
igures 37 and 38 show the photographs of the stored SLM formulations
0 % GB containing 5 mg TS/ g and containing 1 % OA or 1 % DA)
fter 12 weeks at 5 and 30 C in comparison to SLM formulation
ontaining no chemical enhancer. No change in appearance was
observed of the stored SLM formulation containing 1% OA stored at 5 C
n enhanced drug release after storage of SLM. This effect is
y slight gelling of SLM that could be observed after 16

week
30 C. Gelling phenomenon described the transformation of a
(151)
in
T
d
1
F
(1
a
c
142

OA, and c) SLM containing 1 % DA after storage for 2 and
12 week at 5 C.

b)
a)
c)
143

igure 38. Photographs showing the physical change in SLM
OA, and c) SLM containing 1 % DA after storage for 2 and
12 week at 30 C.

F
a)
b)
c)
144
and ulation
ontaining 1 % DA suffered from change in color to yellow and phase
paration and creaming after 2 weeks storage at 5 C and 30 C.
Consequently, SLM formulation containing 1 % DA was excluded from
further studies.
12. Effect of freeze-drying on the selected formulation
pt to improve the long term stability of SLM formulation the
eeze-dried formulations were prepared.
lthough, lyophilization has been widely used to improve the chemical
nd physical stability of SLM over an extended period of time, it may
amage the surfactant film around the microparticles due to freezing out
ffect and may also cause particle aggregation during the redispersion
process
(44)
. Various cryoprotectors have been used to prevent the
problems associated with lyophilization. Sucrose and trehalose were
ined in preliminary experiments to select the cryoprotector of
ighest potential. The use of 5 % sucrose as cryoprotector by dilution
method and by addition to aqueous phase at ratio of (1:3) sugar: lipid
resulted in
to handle. On the other hand, addition of trehalose as cryoprotector gave
dried free flowing powder. Consequently, trehalose was used for further
studies. This result was in a good agreement with what have been
reported by Mehnert and Mder (2001)
(44)
who have documented that
30 C after 12 weeks. On the other hand, SLM form
c
se
In an attem
fr
A
a
d
e
exam
h
the formation of sticky glassy dried mass that is very difficult
145
trehalose was more efficient as cryoprotector than sucrose. In general,
ehalose proved to be most effective cryoprotector in preventing particle
rowth during the freeze drying process
(110, 157)
.
he method of addition of trehalose influenced the quality of the final
rmulation. The results showed that the formulation to which trehalose
as added to the aqueous phase before homogenization, in a ratio of 3:1
/w (sugar: lipid), the freeze-dried powder was rapidly reconstituted after
ddition of water and the lipid phase was homogenously dispersed in the
queous phase. On the other hand, when 15% trehalose solution was used
dilute the final formulation in a ratio of 1:1 before freeze-drying, the
constituted formulation needs ogenously
dispersed and large aggregates were macroscopically observed in SLM
uspension. Similarly, it has been also reported that the best results were
btained when cryoprotector was added to the sample prior to
omogenization
(110)
.
igure 39 shows the cumulative a unt of TS released from selected
trehalose was added to aqueous pha
dilution of ion by 15% trehalose in a ratio of 1:1 in
tr
g
T
fo
w
w
a
a
to
re longer time to be hom
re
s
o
h
F mo
formulation (10% GB containing 5 mg TS/ml SLM) after 24 h to which
se before homogenization or by
the final formulat
comparison with the freshly prepared formulation. Statistical analysis
revealed the following order of TS release: fresh SLM SLM diluted
with 15% trehalose > SLM-containing trehalose before homogenization.
146


on cumulative amount of TS permeated through excised
abdomen rat skin. The applied formulation was 10 % GB
containing 5 mg TS/g of SLM dispersion.

0
5
10
15
20
(3:1)
u
m
u
t
i
v
e

a
m
o
u
n

o
f

T
p
e
a
t

a
f
t
4

h
g
/
25

Fresh Dilut ed wit h 15 % t rehalose Cont aining t rehalose at
Method of addition of trehalose
C
l
a
t
S
e
r
m
e
d
e
r

2

(
c
m
)
147
Figure 40 shows the mean particle size of the reconstituted formulations
in presence and in absence of trehalose in comparison with freshly
prepared formulation and freeze dried SLM without addition of trehalose.
Statistical analysis revealed the following rank order for the mean particle
size: freeze dried SLM without addition of trehalose SLM diluted with
15% trehalose (1:1) > freshly prepared SLM > SLM containing trehalose
in aqueous phase 3:1 w/w (sugar: lipid) before homogenization.
It could be concluded that dilution of SLM formulation with trehalose has
no stabilizing effect on the mean particle size of the reconstituted
formulation and resulted in SLM suspension of larger particle compared
to the freshly prepared SLM. Similarly, Cavalli et al. (1997)
(115)
have
observed increase in particle sizes after lyophilization. However,
e surfactant and serves as a kind of pseudo hydration shell which may
elp in a good dispersion and formation of smaller particles
(158)
.
addition of trehalose in the aqueous phase before homogenization
resulted in SLM suspension of smaller mean particle size compared with
the freshly prepared SLM.
The cryoprotector effect of trehalose has been reported to arise from the
formation of a protective capping layer around SLM. It can be considered
as place holders which prevent the contact between discrete lipid
particles. Furthermore, trehalose interacts with the polar head group of
th
h

148

Figure 40.
formulation was 10
% GB containing 5 mg TS/ g SLM dispersion.

Effect of method of addition of trehalose before freeze drying
on the mean particle size. The examined

0
5
10
15
20
25
30
35
40
45
Fresh No t rehalose Dilut ed wit h 15%
t rehalose solut ion
Cont aining t rehalose
(3:1)
Method of addition of trehalose
M
e
a
n

p
a
r
t
i
c
l
e

s
i
z
e

(
m
)
149
The lower cumulative amount of TS released from SLM that contained
trehalose in aqueous medium in spite of smaller mean particle size could
be due to possible H-bond formation between TS and hydroxyl functions
of trehalose.

ned six rabbits. However, application of drug
1% OA,
ure ethanol and 1% OA in ethanol to rabbit dorsal skin, Draize irritation
cores were zero (no skin reaction).

13. Skin irritation test
The results of evaluation of skin irritation test (Table 20) indicated that
only one out of six rabbits gave Draize score 1 (very slight erythema)
after application of TS ethanolic solution. However, no erythema was
observed for the examined six rabbits after application of TS loaded SLM
(10% GB containing 5 mg TS/g SLM dispersion). On the other hand,
application of 1% DA in ethanol gave Draize score 2 (obvious
erythematic) for the exami
loaded SLM after skin treatment with 1% DA for 30 min gave Draize
score 1 (very slight erythematic) for five rabbits while only one rabbit
gave score 2 (obvious erythematic). Consequently, it could be concluded
that application of drug loaded SLM offered skin protection against the
irritation effect produced by TS and 1% DA. It is worth mentioning that
after application of drug free SLM, drug loaded SLM containing
p
s
150

Table 20. Scores for Draize test of skin irritation after application of the
examined formulations to the rabbit dorsal skin (n = 6).
Formulation Draize score Number of rabbit
Pure ethanol 0 6
TS ethanolic solution 1 6
1% OA in ethanol 0 6
1% DA in ethanol 2 6
TS free SLM 0 6
TS loaded SLM 0 6
TS loaded SLM
containing 1% OA,
0 6
TS loaded SLM after
skin treatment with
1% DA for 30 min
1
2
5
1

151

GENERAL CONCLUSION

152
All things considered, the choice of type and lipid concentration can
SLM formulation. The selected SLM
mg TS rsion) has po on as
transdermal delivery system for TS by virtue of its high encapsulation
efficie re ase characteristics, and stability during
storag
As a chemical enhancer, 1% DA applied for 30 min showed higher
enhancement in TS permeation across rat abdomen skin than 1% OA.
Application of 1% DA for 30 min after exposure of skin to HUS or LUS
had no enhancement effect over application of US alone as revealed by
ER. Consequently, the application of LUS without the use of chemical
enhancer would be recommended to enhance the permeation of TS
formulated as SLM through excised abdomen rat skin under the adapted
experi r, safe application of LUS should be
practiced by careful selection of exposure parameters.
pplication of drug loaded GB SLM offered skin protection against the
irritation effect produced by the drug and the chemical enhancer.
In General, the development of SLM for the controlled transdermal
delivery of TS is feasible, and further clinical studies should be
performed to confirm the efficacy of the prepared system in vivo.

affect the final physicochemical and release characteristics of TS from
formulation (10% GB containing 5
/g SLM dispe shown high tential for applicati
ncy, thermal behavior, le
e.
mental conditions. Howeve
A

153

SUMMARY

154
The main objective of the present study was to formulate an
improved transdermal delivery system of testosterone (TS) in an attempt
to enhance the transdermal penetration of testosterone and minimize skin
irritation.
The following three approaches were applied separately or in
combination to evaluate their ability to deliver the drug systemically after
its topical application: i) formulation approach: by production and
char
The formulations
ological
h. The
permeation experiments were performed using Franz diffusion cells and
acterization of testosterone solid lipid microparticles (SLM), ii)
stratum corneum modification approach: by application of chemical
enhancer, and iii) electrically assisted approach: by application of low
frequency ultrasound waves (LUS) and high frequency ultrasound (HUS)
on TS transdermal permeation after application of testosterone SLM.
Testosterone SLMs were formulated using emulsion melt
homogenization technique. Various types and concentrations of lipids,
namely, glyceryl monostearate (GM), glyceryl distearate (GD), stearic
acid (SA), and glyceryl behanate (GB) were used.
containing 2.5 or 5 mg TS/g of SLM dispersion.
Morphology, particle size, entrapment efficiency (EE), rhe
properties, thermal behavior and X-ray differaction pattern of the
prepared SLM were examined. In vitro permeation characteristics of TS
from various prepared SLM were also evaluated over 24

155
either cellophane membrane or exci d abdomen rat skin. A mixture of
propylene glycol: normal saline (40:60 v/v) was used as receptor solution.
The examined permeation enhancers were 1% oleic acid (OA) or 1
% dodecylamine (DA). HUS (1 MHz) was applied in a continuous mode
for 1h at intensity 0.5 W/cm
2
. Different intensities (2.5, 3.25, and 5
W/cm
2
) and application time (6, 12, and 15 min.) of pulsed LUS (20 kHz)
were also examined. Additionally, the effect of application of US and
OA or DA was investigated. Skin irritation and histological changes
were also evaluated. In addition, effect of storage and freeze-drying on
particle size and release pattern of TS from the selected formulation (10%
GB SLM containing 5 mg TS/g) was evaluated.
The results of morphological examination indicated that the type of
lipid affected the morphology and particle size of SLM.
A relative high drug % EE ranged from 80.7-95.7% was also
obtained.
Rheological studies showed plastic flow characteristics of the
prepared formulations. Unlike other formulations, 5% GM and 5% SA
SLM formulations showed thixotropic properties. For each type of lipid,
as the concentration of lipid increase the viscosity increased. In addition,
the type of lipid affected the viscosity of the final product.
DSC examination revealed that TS existed in amorphous form in
the prepared SLM. The results revealed that GB as lipid matrix showed
se

d

156
high th
rmed by X-ray diffraction studies.
t of
TS
r the permeation of TS:
1 %
ermal stability compared with other types of lipid. Generally, DSC
examination suggested that the lipids in SLM were in less ordered
crystalline arrangement than the corresponding bulk lipid. These results
were confo
Release studies revealed the following rank order of TS permeation
through cellophane membrane after application of various SLM
formulations: 5% GM< 5% GD<5% SA <5% GB <2.5% GM<2.5%
SA<10% GD <10% GB. It seemed that the permeation of TS was
affected by not only the concentration of lipid but also by the type of lipid
used in the formulation. The drug permeation through excised abdomen
rat skin after application of 10% GB-2.5 mg TS/g SLM was lower than
that permeated through cellophane membrane. In addition, the amoun
permeated through excised abdomen rat skin was not statistically
changed as the theoretical drug loading of 10 % GB SLM increased from
2.5 to 5 mg TS/g.
Concerning the effect of HUS and or chemical enhancers,
statistical analysis revealed the following order fo
DA for 30 min >HUS + 1 % DA for 30 min HUS HUS + SLM
containing 1 % OA > SLM containing 1% OA selected formulation
without enhancement.
Regarding the effect of LUS, at total application time of LUS 6, 12,
and 15 min the flux of TS increased by 1.86, 4.63, and 4.77 fold,

157
respectively. The enhancement effect of different intensities of LUS was
not directly proportional to the magnitude of its intensity. Skin exposure
to H
as TS transdermal delivery system. The pretreatment of
excise
US or LUS before application of 1% DA for 30 min had no superior
enhancement effect over application of either LUS or HUS alone.
Stability studies indicated that SLM containing 10% GB-5 mg
TS/g stored at 5C have good physical stability as indicated by release
study and particle size analysis.
Trehalose showed high potential as cryoprotector during freeze
drying of the selected SLM formulation.
Application of drug loaded SLM offered skin protection against the
irritation effect produced by TS and 1% DA.
Histological characteristics of the rat skin were affected to various
extents by application of enhancers or ultrasound.
In general, the developed TS SLM delivery system seemed to be
promising
d abdomen rat skin with HUS or LUS before application of 1% DA
for 30 min had no advantage over application of either LUS or HUS
alone. Application of LUS gave higher TS permeation than HUS.
However, safe application of LUS should be practiced by careful
selection of exposure parameters.

158

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181

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Transdermal Delivery System of Testosterone Using Solid Lipid Particles

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I

Transdermal Delivery System of

cream (Astra Zeneca, Australia) prompted study of

, 6 mg, ALZA Corporation, USA) and two systems that

, 2.5 mg and 5 mg, SmithKline Beecham Pharmaceuticals,

TTS, 5 mg ALZA Corporation, USA)

T18 basic (IKa Works, Inc. USA).

membrane, Spectrum medical industries, Inc., USA)

1998-2003. Shimadzu Corporation.

C. The following stability

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