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 Master’s

Degree in Pharmaceutical Sciences (Pharmaceutics)

Departments of Pharmaceutics College of Pharmacy King Saud University

1428 H-2007 G

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SUPERVISORY COMMITTEE

Principle Supervisor Dr. Amal H. El-Kamel

Associate Professor of Pharmaceutics,

College of Pharmacy King Saud University

Co-Advisor

Dr. Ibrahim A. Alsarra

Associate Professor of Pharmaceutics, College of Pharmacy

King Saud University

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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.

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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.

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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

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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 10.5. Effect of application of high frequency ultrasound (HUS) 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

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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.

Cº 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.

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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.

Lt Lag time

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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

R² 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

teff Sum of the time when US is on.

TEWL Transepidermal water loss.

Tm Transition temperature.

toff Time when US is off.

ton Length of the pulse when US is on.

TS Testosterone

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tus Total exposure time.

UK United Kingdom

US Ultrasound.

USA United State of America

V Volt

µl Microliter

µm Micrometer

γ 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

Table 4. Intraday precision (CV %) and accuracy (% recovery) data for calibration standard concentrations of TS................................73

Table 5. Interday precision (CV %) and accuracy (% recovery) data for 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 (R²) calculated after fitting the permeation data of TS into Fick’s 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 permeation parameters of TS through excised abdomen rat skin after application of 10 % GB SLM containing 5 mg TS/ g of 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

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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 µm-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

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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

Figure 15. Rheograms of different SLM formulations containing 2.5 mg 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 SLM formulations containing 2.5 mg TS/g of SLM dispersion...................................................................................................88

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...................................................................................................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

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containing different types of lipid and 2.5 mg TS/g of SLM dispersion. ..............................................................................103

Figure 24. Cumulative amount of TS released through cellophane membrane after application of different SLM formulations 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

Figure 26. Cumulative amount of TS permeated through excised 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

Figure 28. Cumulative amount of TS permeated through excised 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

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Figure 33. Histological characteristics of excised abdomen rat skin a) 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/cm² 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 ºC. 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 ºC. 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 ºC...........................................................................142

Figure 38. 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 30 ºC..........................................................................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

Figure 40. Effect of method of addition of trehalose before freeze drying on the mean particle size. The examined formulation was 10 % GB containing 5 mg TS/ g SLM dispersion. .........................148

1

INTRODUCTION

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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 m² 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 µm 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 6–12

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 ºC to around 32 ºC 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 (cm²/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 (Lt) is given by:

Lt = 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

stratum corneum barrier (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

ChemicalEnhancers

Microneedle Array

Ablation

Follicular Delivery

Ultrasound

Ionto-phoresis

Electro- poration

Magneto-phoresis

Photo-mechanical

Wave

Figure 4. Scheme that summarizes techniques for circumventing the

stratum corneum barrier (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=DCº SK/h [5]

Where Cº 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/ γh [6]

Where, a is the thermodynamic activity of drug in its vehicle and γ 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 liposome’s 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 µm and 500 µm,

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 (20–100 µm) 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 delivery—particles 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 lipid–protein–partitioning

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 drug’s 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 (epidermal–dermal 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 µm remain on the skin surface,

those ≈ 3–10 µm concentrate in the follicle and when < 3 µm, 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 µm-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/cm² 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=p2/rc2 [8]

The employed intensities usually lie between 0.5 and 2 W/cm2. The

increase in pressure is approximately 0.2 bar with 1 W/cm2 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 2nd 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 cm². 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 /cm2) 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 (0.5–500 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 10–104 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 inFigure 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 ß-Hydroxyandrost-4-en-3-one

39

Molecular weight: 288.4.

Formula: C19 H28O2

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 µl loop.

C18 µ-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-ІІ+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 (Siemen’s, 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 µm membrane filter and

degassed. The flow-rate was 1 ml / min. The injection volume was 20

µl. The retention time of the drug was 5.2 ± 0.5 min. The column used

was reversed phase C18 µ- 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 µl 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

dave = n

nn

nnndndndn

...........

21

2211

++++ =

∑∑

nnd )( [9]

Where dave is the average diameter and n1, n2, and nn are the number of

particles having diameters d1, d2 and dn, 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 (Tm) 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 ºC to 250 ºC at a heating rate

of 5 ºC 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:

SLMdriedinlipidoffractionlipidbulkofEnthalpy

phaseaqueousofseparationafterSLMdlyophilizeofEnthalpyationCrystalliz

*

100*% = [11]

3.9. Powder X-ray diffractometry (PXRD)

Powder X-ray diffractometry studies were performed on the samples by

exposing them to Cu Kα radiation (40 kV/ 30 mA) and scanned from 5º

to 70º, and the scanning rate was 5 º/ min at a step size of 0.020º 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 ºC for 2 hr. Then supernatant was

removed and the sediment was washed with distilled water. The resultant

precipitant was frozen at -70 ºC 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 ºC. After

cooling to room temperature the volume was then completed to 10 ml by

water. After filtration through PTFE filter (0.2 µm) (Millipore

Corporation, USA) a suitable dilution was performed. An aliquot of 20

µl 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 cm²). A visible film formation on

top of the filter paper was observed during the experiment. The samples

were stored at 32 ºC 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) Х 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 ± 0.5oC for 48 h. After

equilibrium, a sample of the supernatant was removed, filtered through

PTFE filter (0.45 µm) and suitably diluted. An aliquot of 20 µl 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 cm². The cells were placed in a V-3

station vertical cell stirrer and connected to circulating water bath heated

to 32 ± 0.5°C.

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 µl) 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 = Cn (VT/VT-Vs) (C\ n-1/Cn-1) [13]

Where C\n is the corrected concentration of the nth sample, Cn the

measured concentration of TS in the nth sample, C\ n-1 is the corrected

concentration of (n-1) th, Cn-1 is the measured concentration of TS in the

(n-1) th sample, VT is the total volume of the receiver compartment and Vs

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, (µg/cm2/h), was determined

from the slope of the linear portion of the plot. Permeability coefficient,

P, (cm/sec) was calculated as

P = J/Co [14]

Where, Co is the initial donor drug concentration.

The diffusion coefficient, D, can be obtained from the following

equation:

D/h2= 1/6 *Lt [15]

Where h is the thickness of the epidermis and Lt 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/h2 is replaced by D`, so

the equation becomes (105):

62

D` =1/6 *Lt [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 - 5°C

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

cm2.

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.

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

The ultrasound equipment with 1-3 MHz frequency and intensity of 1-3

W/cm² 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/cm² 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 (teff) (6, 12, 15 min) when total

exposure time was 30 minutes at intensity of 2.5 W/cm² and ton

equals 10 sec.

65

• Effect of three different intensities (2.5, 3.25 and 5 W/cm2) with

other parameters remaining constant (ton= 10 sec, teff = 12 min) for

total exposure time 30 minutes.

• Combined application of LUS and chemical enhancer: (teff = 12

min) over total exposure time 30 minutes at intensity of 2.5 W/cm²

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);

teff= tus (ton/(ton +toff)) [18]

where toff represents the time when US is off,

ton represents the length of the pulse when US is on,

teff represents the sum of the time when US is on,

tus 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 ºC 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 cm2). 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 µm filter unit. A suitable dilution was

performed. An aliquot of 20 µl 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 (R²) 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 35598±2153 6.1 0.01 67232±3306 4.9 0.02 143523±7554 5.3 0.03 223104±4817 2.2

igure 12. HPLC calibration curve of TS. (n=5)

y = 7E+06x - 2733.2R2 = 0.9987

0

50000

100000

150000

200000

250000

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035

Concentration of TS (mg/10ml)

Mea

n pe

ak a

rea

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 0.0057±0.00 94.50±1.8 0.015 (MQC) 07 4.7 100.1±4.7 0.0150±0.000.025 (HQC) 0.0251±0.0001 0.4 100.5±0.5

Table 3. Interday precision (CV %) and accuracy (% recovery) data for

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 0.0056±0.00 93.10±1.70.015 (MQC) 03 2.0 0.0150±0.00 100.2±2.2 0.025 (HQC) 0.0252±0.0004 1.6 100.8±1.7

73

Table 4. Intraday precision (CV %) and accuracy (% recovery) data for

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 0.0052±0.0002 3.8 104.60±3.40.01 04 4.2 0.0096±0.00 96.02±3.7 0.02 0.0205±0.0011 5.4 102.50±5.3 0.03 0.0325±0.0007 2.2 108.40±2.2

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 0.0051±0.0003 5.9 101.8±5.3 0.01 04 4.2 0.0096±0.00 95.70±3.8 0.02 0.0198±0.0006 3.0 99.20±3.1 0.03 0.0320±0.0006 1.9 106.6±2.1

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 0.07µg/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 41307±1315 3.2 0.015 106367 108669 108599 108712 106083 107686±1338 1.2 0.025 178040 181244 188451 181990 175141 180973±4990 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 35598±2153 6.1 0.01 62740 67949 65410 68610 71453 67232±3306 4.9 0.02 133713 148539 142421 139829 153115 143523±7554 5.3 0.03 223524 222415 231027 219011 219544 223104±4817 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

SLM formulations containing 2.5 mg TS/g of SLM dispersion.

Formulation Mean particle size ± SD (µm) 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 ± 5.5ab 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)

Vis

cosi

ty (c

ps)

b)

0

100

200

300

400

500

600

700

800

900

0 10 20 30 40 50 60

Speed (rpm)

Visc

osity

(cps

)a)

0

20

40

60

80

100

120

0 10 20 30 40 50 60

Speed (rpm)

Vis

cosi

ty (c

ps)

c)

0

100

200

300

400

500

600

700

800

0 10 20 30 40 50 60

Speed (rpm)

Vis

cosi

ty (c

ps)

d)

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.

82

0

500

1000

1500

2000

2500

3000

0 10 20 30 40 50 60

Speed (rpm)

Vis

cosi

ty (c

ps)

0

2000

4000

6000

8000

10000

12000

14000

0 10 20 30 40 50 60

Speed (rpm)

Visc

osity

(cps

)

b) a)

0

20004000

60008000

1000012000

14000

1600018000

0 10 20 30 40 50 60

Speed (rpm)

Vis

cosi

ty (c

ps)

c)

0

5000

10000

15000

20000

25000

0 10 20 30 40 50 60

Speed (rpm)

Vis

cosi

ty (c

ps)

d)

Figure 15. Rheograms of different SLM formulations containing 2.5 mg

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

10%

GB

2.5%GM

5% G

M5%

GD

10%

GD

2.5%

SA5%

SA

Formulation

Vis

cosi

ty (c

ps)

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 µm).

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 ºC (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

formulations containing 2.5 mg TS/g of SLM dispersion.

91

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

point (ºC)

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 Mühlen 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 ºC, 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 58.3°C 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 ºC, 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 ºC 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, C22) than for shorter chain

glycerides (SA, C18) (123). Additionally, Mühlen 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 θ 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 θ sca

angles 21.3 with a small additional peak of about 23.4. These peaks are

typical for the orthothrombic β` 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 Tukey’s 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

formulations containing 2.5 mg TS/g of SLM dispersion.

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

Formulation % EE ± SD 2.5% GM 88.5±7.1 5% GM

82.2±7.3

5 % GD 83.2±4.2

1 0% GD 83.3±3.4

5 % GB 91.7±1.8

1

0% GB 80.7±0.3

2

.5% SA 90.3±2.1

5

% SA 95.7±1.4

1

0% GB* 82.2±3.9

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, (R2>

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 Fick’s and Higuchi equations. Almost

all the SLM formulations followed Fick’s 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 Fick’s law to

calculate the flux (J) which is the amount of drug permeated per unit area

and per unit time (µg/cm²/h), t

diffusion parameter (D̀, 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)

Cum

ulat

ive

amou

nt o

f TS

rele

ased

(µg/

cm²)

5% GM5% GD5% GB5% SA

Figure 23. Cumulative amount of TS released through cellophane

membrane after application of different SLM formulations

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

dispersion.

104

Figure 24. Cumulative amount of TS released through cellophane

membrane after application of different SLM formulations

containing different concentration of lipid and 2.5 mg TS/g

of SLM dispersion.

0

50

100

150

200

250

5 % GD 10% GDFormulation

Cum

ulat

ive

amou

nt o

f TS

rele

ased

afte

r 24

h (µ

g/cm

²)

0

50

100

150

200

250

5 % GB 10% GBFormulation

Cum

ulat

ive

amou

nt o

f TS

rele

ased

afte

r 24

h (µ

g/cm

²)0

50

100

150

200

250

2.5% GM 5% GMFormulation

Cum

ulat

ive

amou

nt o

f TS

rele

ased

afte

r 24

h (µ

g/cm

²)

0

50

100

150

200

250

2.5% SA 5% SAFormulation

Cum

ulat

ive

amou

nt o

f TS

rele

ased

afte

r 24

h (µ

g/cm

²)

105

Table 14. Coefficient of determination (R²) calculated after fitting the

release data of TS into Fick’s and Higuchi equations.

Cellophane membrane was used as transporting membrane. All

formulations containing 2.5 mg TS/g of SLM dispersion.

Formulation R² (Fick’s) R² (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 (µg/cm ± SD

P (cm/h) x 102 ± SD

SLM ons containi

to embrane

Formulati ²/h) D` (h-1) ± SD

2.5% GM 8.04±0.6 0.320±0.0002 1.261±0.500

5% GM 5.01±0.6 0.200±0.0002 0.064±0.016

5% GD 7.07±0.8 0.282±0.0003 0.330±0.200

10% GD 8.29±0.2 0.331±0.0001 .035 0.241±0

5% GB 7.59±0.3 0.304±0.0001 0.403±0.100

10% GB 8.78±0.1 0.351±0.00002 0.095±0.017

2.5% SA 8.42±0.4 0.336±0.0001 0.121±0.011

5% SA 7.10±0.3 0.284±0.0001 0.464±0.087

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 µg ± 3.1) was statistically higher (p ≤

0.0001) than 10% GD SLM (9.74 µg ± 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/cm²/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 0.9349±0.135 3.74±5.4x10 0.042±0.002 10 % GD 0.4316±0.068 1.73±2.7x10 -5 0.047±0.003 TS Concentration: 5 mg/g 10 % GB 0.95156±0.109 3.81±4.4x10 -5 0.051±0.004

111

0

10

25

0 5 10 15 20 25 30

Time (h)

S pe

rm

5

mul

a

15

t of T

cm²)

20eate

d C

utiv

e am

oun

(µg/

0% GD10%GB1

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.

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 Müller (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

Figure 26. Cumulative amount of TS permeated through excised

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)

Cm

ulat

i a

mou

n o

f TS

pm

eate

dg/

cm²

5 mg TS/ g

10

15

20

25

uve

ter

(µ)

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 + 0.00036x2 - 3.257 x3

W

a

c

p

C

c

115

00 50 100 150 200 250

Amount of TS permeated through cellophan membrane (µg/cm2)

Aou

nt T

Srm

eed

thug

h ex

cise

ddo

me

rat

in (µ

cm2 )

5

10

15

20

25

m o

f p

eat

ro a

bn

skg/

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/cm2/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 30Time (h)

Cum

utiv

e am

ount

of

S pe

rea

ted

(g/

cm²)

HUS

1% OA

1 % DA for 30 min

1 % DA for 30 min only & HUS

1 % DA

Figure 28. Cumulative amount of TS permeated through excised

fferent

mg TS /g of SLM dispersion.

la T

mµ

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

permeation parameters of TS through excised abdomen rat skin

after application of 10 % GB SLM containing 5 mg TS/ g of

SLM dispersion.

Enhancement method

J (µg/cm2/h) ± SD

P Χ 104

(cm/h) ± SD

D` (hr-1) ± SD

ER Skin retention (µg/cm2) ± SD

Table 18.

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

0.95±0.11

3.81±0.00004

0.0507±0.004

-----

46.1±3.4

SLM containing 1% DA

2.30±0.32

9.21±0.0001

0.0763±0.016

2.42

80.9±4.3

SLM containing 1% OA

1.09±0.08

4.37±0.00003

0.0593±0.014

1.15

63.3±0.5

30 min 1% DA

2.59±0.39

10.36±0.0001

0.0610±0.018

2.73

56.9±4.3

HUS

1.59±0.35

6.38±0.0001

0.0488±0.006

1.68

34.5±2.8

HUS& SLM containing 1% OA

0.0514±0.004 1.64

55.9±12.3 1.56±0.23

6.26±0.00009

HUS&30 min 1% DA

1.78±0.42

7.14±0.0001

0.0644±0.027

1.88

62.2±10.5

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 2°C

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/cm2. 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/cm2) 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 teff (or duty cycle)

The effect of three teff (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 teff 15 and 12 min the

ux of TS increased by 4.77 and 4.63 fold, respectively, compared with

the selected formulation. While at tef 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 cavitation–based 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/cm2 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/cm2 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

Flux

µg/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

permeation parameters of TS through excised abdomen rat skin

after application of 10 % GB SLM containing 5 mg TS/ g of

SLM dispersion .

in

)

Enhancement method

J (µg/cm2/h) ± SD

P Χ 104

(cm/h) ± SD

D` (h-1) ± SD

ER TS Skretention (µg/cm2

± SD

tion (10 % GB contained 5 mg TS/ G)

0.95±0.1

3.81±0.00004

0.0507±0.004

------- 46.1±3.4

LUS (10:10) at 2.5 W/cm²

4.53±0.50

18.14±0.0001

0.0522±0.004

4.77

119.3±16.5

LUS (10:15) at 2.5 W/cm²

4.41±1.21

17.63±0.0004

0.095±0.014

4.63

110.7±12.1

LUS (10:40) at 1.77±0.18

7.06±0.00007

0.0455±0.001

1.86

33.1±9.2

Selected formula

2.5 W/cm² LUS (10:15) at 3.25 W/cm²

2.68±0.22

10.70±0.0001

0.0654±0.006

2.81

60.7±7.3

LUW/cm²

S (10:15) at 5 2.32±0.04

9.29±0.00001

0.0514±0.007

2.44

48.2±11.3

4.58±0.58

18.30±0.0002

0.0728±0.014

4.81

108.1±19.

LUS (10:15) at 2.5 W/cm²& 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 Rr (µm) is

roughly related to frequency (F in kHz) by the following equation (69):

F x Rr=3000 [20]

µm at 20 kHz and 3 µm 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

15°C 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 µg/ cm2) < 10:15 (110.7 ±

12.1µg/cm2) ≈ 10:10 s (119.8 ± 16.5 µg /cm2), 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 teff that produced the greatest

enhancement of TS transdermal permeation was at teff 15, 12 min for 30

min at 2.5 W/cm². Therefore, the later teff (12 min) was selected to study

the effect of increasing LUS intensity to 3.25 and 5 W/cm². 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/cm² 3.45 W/cm² 5 W/cm²

Intensity

Flux

µg/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/cm² 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/cm² 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/cm²), the enhancement does not increase with further increase in the

in

T

were examined. Application of LUS at intensity 2.5 W/cm² 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)

Figure 33. Histological characteristics of excised abdomen rat skin a)

2 2

and d) 5 w/cm2, and e) LUS 2.5 W/cm2 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/cm² was applied (Figure 33, d).

This could explain the obtained results, since among the three applied

ultrasound protocols, the intensity of 2.5 W/cm2 LUS at teff 12 min

showed the highest TS flux (4.41 µg/cm2/h). This could be due to severe

skin damage or disorganization of the lipid bilayers of stratum corneum

occurred at intensity 2.5 W/cm2. Fortunately, the damaged skin (exposed

LUS 2.5 W/cm2 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/cm2 (110.7± 12.1

g/cm2) >3.25 W/cm² (60.7± 7.3 µg/cm2) ≈ 5 W/cm2 (48.2± 4.3 µg/cm2),

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

µ

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/cm². 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/cm2, teff 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/cm² 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

Flu

µg/

m2 /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 5°C and 30 ºC 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 ºC, 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 5°C and 30°C (Figure 36).

Statistically, the particle size increased significantly after storage for 12

and 16 weeks at 30°C. It was assumed that the high temperature (25 ºC)

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 5°C 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 ºC, however, at 4

ºC 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 ºC. Bars with similar symbols

0Fresh 2 4 6 8 12 16

mi

of

p

e a

2²

5

10

15

Cu

ulat

ve a

mer

mat

edfte

r

20

25

ount

TS

4 h

(µg/

cm

30

Time (week)

) at 5 °Cat 30 °C

B B BB

A

B

C

ab

cd bca ab

d d

140

15

25

35

45

n p

ticle

ize

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 ºC. Bars with similar symbols are statistically

insignificant (A>B), (a>b).

0

510

20

30

40

50

Fresh 2 4 6 8 12 16

Time (week)

Mea

ar s

(µ

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 8°C reported similar results. Consequently, other factors were expected

to affect the release of drug from SLM after storage for 16 weeks at 5°C.

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 ºC 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 ºC

n enhanced drug release after storage of SLM. This effect is

y slight gelling of SLM that could be observed after 16 week

30 ºC. 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 ºC.

Figure 37. Photographs showing the physical change in SLM

formulation: a) selected formulation, b) SLM containing 1 %

b)

a)

c)

143

igure 38. 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 30 ºC.

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 ºC and 30 ºC.

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 Mäder (2001) (44) who have documented that

30 ºC 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

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.

0

5

10

15

20

(3:1)

umu

tive

amou

n o

f Tp

eat

aft

4 h

µg/

25 ²

Fresh Diluted with 15 % trehalose Containing trehalose at

Method of addition of trehalose

Cla

tS

erm

eder

2 (

cm)

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 trehalose Diluted with 15%trehalose solution

Containing trehalose(3:1)

Method of addition of trehalose

Mea

n pa

rtic

le si

ze (µ

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/cm2. Different intensities (2.5, 3.25, and 5

W/cm2) 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 5°C 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

ARABIC SUMMARY

182

ر ا توصيل هرمون ف الرئيسي للدراسة هو صياغة نظام لتحسين إن الهد ستوستيرون عب د الت لجل

ة في د المتمثل ى الجل ق تح التهاب الج انبية الموضعية عل ك عن طري د وذل ل ل اآلثار الجتقليللو مي

ة

ة تقنيو صلبة بطريق ة ال م ا قد تمت صياغة الجسيمات الدهني ت

( ل

ق عل

ص

رون

د وق

من

روبي . منطقة البطن ول : لينوقد استخدم مزيج من جليكول الب ل

. مين

ا

ضة

، 6 5 ، 2.5 ( مختلف التردد ذات نبض

ة 15 ، و12 تخدام إض. ) دقيق أثير اس م فحص ت د ت ك فق ى ذل ع افة إل صوتية م وق ال ات ف الموج

.تهابات الجلد والتغيرات النسيجيةآما تم تقييم ال. أو مع دوديسيالمينحمض األوليك

ة الدواء في جسيمات ل العضوية أو الم دهنية صلبة دقيق واد الالصق أو وتجنب استخدام المحالي

. غشية النفاذةاأل

سة للمستحلب ة المتجان آم .ة اإلذاب

سيريل استياريك رآيز المواد الدهنية ، وهي أحادي من تام آميات مختلفةاستخد ، ) GM( الجلي

ائي تياريكوثن سيريل اس تياريك ) GD( الجلي ض اس اته، وب ) SA ( ، وحم سيريان الجلي

GB . ( ى وقد احتو ستوستيرون في آل جرام من م ملجم 5 أو 2.5ت هذه الجسيمات عل ت

.لدهنية الصلبة االجسيمات

ي الجسيمات، وخصائ دواء ف ل ال ى تحمي درة عل م الجسيمات، والق م فحص شكل وحج د ت وق

يم . اللزوجة، والسلوك الحراري للجسيمات الدهنية الصلبة ستوستيآما تم تقي اذ الت خواص نف

ري من ة محضرة من المخب واع مختلف ى مدى أن صلبة عل ة ال . ساعة 24الجسيمات الدهني

سيلوفا أجريت تجارب النفاذ باستخدام خالي د ا فرانز النفاذة مع غشاء ال أر الن أو جل أخوذالف م

ح طبيعي آمح ) v/v 60: 40( مل

.مستقِبل

اذ وقد استخدمت ز معززات النف ة بترآي سيال% 1 من حمض األوليك أو %1 الكيميائي دودي

ردد الموجات آما استخدمت ة الت صوتية عالي وق ال دة س) اهيرتز ميج 1( ف ستمر لم شكل م عة ب

ة لموجات .سم مربع / واط 0.5بشدة تبلغ وق صوتية منخف وآذلك تم فحص شدات مختلف ف

ع / واط 5 ، .3 ة ) سم مرب ة مختلف رات زمني (ضمن فت

183

زين أثير التخ يم ت م تقي ك ت ى ذل الوة عل الق ع سيمات وانط م الج ى حج د عل ف بالتجمي والتجفي

ستوستيرون صيالت ن ال ارة ا م سيريل هب% 10(غة المخت ات الجلي ى ) GB( ان ة عل 5و المحتوي

).جرام / وستيرون تستملجم

د ائج وق وع اشارت النت ى أن ن ى شكل وإل أثير عل ه ت ان ل ستخدمة آ دهون الم سيما ال م الج حج

. %95.7 - 80.7 ما بين الدهنية الصلبة و آانت نسبة تحميل الدواء تتراوح

ة التي غاصيال األخرى فقد أظهرت اتغاوبخالف الصي آما أوضحت دراسات قياس اللزوجة أنه

تياريك أحادي %5تحتوي على سيريل و اس ة % 5الجلي تياريك في الجسيمات الدهني حمض االس

ز الصلبة خصائص تم اد ترآي د مع ازدي زي

.ن يؤثر في لزوجة المنتج النهائيوعالوة على ذلك فإن نوع الدهن آا. الدهن

ان موجود ) DSC( فحص ت نتائج وقد أظهر ة أن التستوستيرون آ ر بلوري ا في صورة غي

ا له )GB( انات الجليسيريل ه ب أن وأظهرت النتائج . الجسيمات الدهنية الصلبة ا حراري ثبات

دهون في ) DSC( فحص وبشكل عام أوضح . ع الدهون األخرى مقارنة بأنوا بأن ال

.ظيما من الدهون النقية التابعة لها آانت عبارة عن مجموعة من البلورات األقل تنالدهنية الصلبة

).X-ray(ة السينية و قد أآدت هذه النتائج دراسة األشع

ة ل لنتائجوقد أظهرت ا الي في المرتب سلوفا نظام التدرج الت ستوستيرون خالل غشاء ال ة الت نفاذي

د تطبيق صي ة ات متنوعة من جسيم غابع صلبة الدقيق ة ال ستوستيرون الدهني : ات الت

GD<5% SA <5% GB <2.5% GM<2.5% SA<10% GD <10% GB . دو ويب

ةنفاذ التستوستيرون قد تأثر ليس فقط بترآيز الدهن وإنما أيضا بنوع الدهن المستخدم في الترآيب

زوع د استخدام وقد آان نفاذ الدواء خالل جلد بطن الفأر المن ات الج من ب % 10 بع سيريل هان ( لي

GB ( ل جرام من الجسيمات الدهني / ون تستوستير ملجم 2.5التي تحتوي على صلبة أق ة ال

.نخالل غشاء السيلوفا

ت

وفي آل نوع من الدهون آانت اللزوجة ت. يعية هالمية

ي ف

ا عالي

الجسيمات

ن

5%< 5%

أن

.

ه من

184

ردد و ة الت صوتية عالي وق ال أثير الموجات ف د أظهر / وفيما يخص ت ة ، فق أو المعززات الكيميائي

: الترتيب التالينفاذ التستوستيرونالتحليل اإلحصائي لنتائج

دة % 1 سيالمين لم ة 30دودي دة % 1> دقيق سيالمين لم ة 30دودي ≈ HUS + HUS ≈ دقيق

+HUS 10 %انات الجليسيريل هب )GB ( ى ست ملجم 5و المحتوية عل 1جرام و / وستيرون ت

4.77 و 4.63 ، 1.86 معدل انطالق الدواء بمقدار إلى زيادة في

د أظهرت ثب 5مخزنة عند درجة حرارة الجرام / تستوستيرون ملجم 5.-ل ة ق ا مئوي ات

و\ززات أو

. الموجات فوق الصوتية

ست ملجم 5و المحتوية على ) GB( انات الجليسيريل هب% 10 >حمض األوليك % / وستيرون ت

رام و ك % 1ج ض األولي ارة ≈ حم صيغة المخت سيريل هب% 10(ال ات الجلي و ) GB( ان

).جرام/ وستيرون تست ملجم5المحتوية على

وفيما يخص تأثير الموجات فوق الصوتية منخفضة التردد ، فإن إجمالي وقت تطبيقها الذي هو

دقيقة أدى15 ، و12 ، 6

ة منخفضة ولم يكن تأثير التعزيز للشدات المختلفة لألمواج فوق الصوتي . ضعفا على التوال

وإن تعريض الجلد للموجات فوق الصوتية عالية . التردد متناسبا بشكل مباشر مع مقدار شدته

دقيقة لم يكن ذا تأثير تعزيزي 30دوديسيالمين لمدة % 1التردد أو منخفضة التردد قبل تطبيق

. التردد أو منخفضة التردد بمفردهاأقوى من تطبيق الموجات فوق الصوتية عالية

ى وأ ة عل صلبة المحتوي ة ال ى أن الجسيمات الدهني ات % 10شارت دراسات التخزين إل من بهان

الجليسيري

.جيدا

ر صلبة وأظه ة ال سيمات الدهني أثير واٍق للج ه ت كرالتريهالوز ل ائج أن س ف ت النت اء التجفي أثن

. المختارةغةاتجميد المطبق على الصيبال

اتج عن االلتهاب وقد وفر استخدام الجسيمات الدهنية الصلبة المحملة بالدواء حماية جلدية ضد الن

. دوديسيالمين %1التستوستيرون أو

سيجية ل صائص الن أثرت الخ ا ت دآم أرجل ق المع الف ل تطبي ة بفع درجات متفاوت ب

185

ستوستيرون من ح لنقل التستوستيرون يبدو نظاما واعدا لتوصيل رلنظام المقتن اوبشكل عام فا الت

زوع آما أن العال . خالل الجلد أر المن د بطن الف صوتية ج المسبق لجل وق ال باستخدام الموجات ف

ردد ة الت رعالي ضة الت ق أو منخف ل تطبي سيالمين %1دد قب دة دودي ن ذا30 لم م يك ة ل أثير دقيق ت

ن ستوستيرون م للت

آما اوضحت . ية التردد أو منخفضة التردد بمفردها أفضل من تطبيق الموجات فوق الصوتية عال

ائج أن ى النت اذا أعل ر نف ردد وف ضة الت صوتية منخف وق ال ات ف ق الموج تطبي

صوتية منخفضة . الموجات الصوتية عالية التردد وق ال على أي حال ، يجب استخدام الموجات ف

. التردد بشكل آمن يتمثل في االختيار الحذر لمستويات التعرض لها

186

رسالة مقدمة من

)صيدالنيات(

كلية الصيدلة -النيات

2007-1428

نظام توصيل التستوستيرون عبر الجلد باستخدام

الجسيمات الدهنية الصلبة

إميان حممد راشد الفقيه )2000(بكالوريوس العلوم الصيدلية

بات احلصول على درجة املاجستري قدمت هذه الرسالة استكماال ملتطل يف العلوم الصيدلية

قسم الصيد

جامعة امللك سعود