FORMULATION AND EVALUATION OF CERTAIN TOPICALLY ...

FORMULATION AND EVALUATION OF
CERTAIN TOPICALLY APPLIED DRUGS
A Thesis Submitted for the
Degree of Master
In
Pharmaceutical Sciences (Pharmaceutics)
By
Rasha Ali AL-Hussiny
Under the Supervision of
Prof. Dr. Fakhr El-Din S. Ghazy
Professor of Pharmaceutics
Faulty of Pharmacy
Zagazig University
Dr. Mohamed A. Hammad Dr. Nagia A. El-Megrab
Assistant Professor of Assistant Professor of
Pharmaceutics Pharmaceutics
Faulty of Pharmacy Faulty of Pharmacy
Zagazig University Zagazig University
Department of Pharmaceutics
And Industrial Pharmacy
Faculty of Pharmacy
Zagazig University
2010
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ACKNOWLEDGEMENTS
I am deeply thankful to GOD, by the grace of whom the progress and
success of this work was possible.
I would like to express my heartfelt gratitude and profound indebtedness
to my guide Prof. Dr. F. S. Ghazy, Professor of Pharmaceutices, Faculty
of Pharmacy, Zagazig University; the greatest supporting person for this
work. Under his guidance I have worked. His constant enlightening
support, timely advice all throughout my work and encouragement have
been instrumental in the completion of this study.
Also, I have to thank Dr. M.A. Hammad, Assistant Professor of
Pharmaceutices, Faculty of Pharmacy, Zagazig University; for
supervising the work, for his encouragement and for his great efforts to
make this work possible.
Also, I thank Dr. N. A. EL-Megrab, Assistant Professor of
Pharmaceutices, Faculty of Pharmacy, Zagazig University; appreciating
her continous encouragement and help supporting me with much scientific
materials and with valuable instructions.
I also extend my sincere thanks to all my colleagues and members of the
department of Pharmaceutics, Faculty of Pharmacy, Zagazig University
for their help.
And finally I would like to thank my family, for their support during this
study …Thank You.
Rasha
2010
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ABBREVIATIONS
ABBREVIATION THE WORD
Glz Gliclazide
Glib Glibenclamide
PEG Polyethylene glycol
UR Urea
glu Glucose
O/W Oil in water
W/O Water in oil
HPMC Hydroxypropylmethyl cellulose
WSB Water soluble base
IPP Isopropyl palmitate
IPM Isopropyl myristate
OA Oleic acid
LOA Linoleic acid
Lab Labrafil
Tc Transcutol
SLS Sodium lauryl sulphate
Tw 80 Tween 80 ( Polyoxyethylene Sorbitan
Monooleate)
PG Propylene glycol
Span 80 Sorbitan mono-oleate
i.p intraperitoneal
NIDDM Non insulin dependant diabetes mellitus
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List of Tables
List of Figures
Contents
Abstract ………………………………………………………………. i
General Introduction …………………………………………………. 1
Scope of work ………………………………………………………... 35
Part One
Formulation and Evaluation of Topically Applied Gliclazide
- Introduction ………………………………………………………… 37
Chapter (I)
Formulation and Characterization of Gliclazide Solid Dispersions
-Introduction …………………………………………………………. 40
-Experimental and methodology …………………………………….. 67
-Results and discussion ……………………………………………… 74
-Conclusion ………………………………………………………….. 117
Chapter (II)
In Vitro and In Vivo Studies on Topical Applications of Gliclazide
Solid Dispersions
-Introduction ………………………………………………………. 118
-Experimental and methodology ………………………………….. 119
-Results and discussion …………………………………………… 134
-Conclusion ……………………………………………………….. 157
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Part Two
Formulation and Evaluation of Topically Applied Glibenclamide
-Introduction …………………………………………………… 158
-Experimental and methodology ………………………………… 176
-Results and discussion …………………………………………… 186
-Conclusion ……………………………………………………….. 235
General Conclusion …………………………………………………. 237
References …………………………………………………………… 238
Arabic Summary ……………………………………………………..
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Figure
List of Figures
Number Description
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Page
Number
1 Diagrammatic representation of the skin structure. 3
2 Diagrammatic representation of the stratum
corneum and the intercellular and transcellular
routes of penetration
3 Schematic representation of types of external
medicines.
4 Structure of gliclazide
5 Diagrammatic representation of process of
solubilization
6 Phase diagram for eutectic system 55
7 Phase diagram for Discontinuous solid solutions 56
8 Substitutional crystalline solid solutions
9 Interstitial crystalline solid solutions.
10 Amorphous crystalline solid solution 58
11 UV spectra of gliclazide in methanol. 74
12 Calibration curve of gliclazide in methanol at max
227 nm.
13 Calibration curve of gliclazide in phosphate buffer
(7.4)at max 227 nm .
14 Phase solubility diagram of gliclazide in water at
25°C in presence of PEG 4000 and PEG 6000.
15 Phase solubility diagram of gliclazide in water
at 25°C in presence of glucose and urea.
16 Dissolution profile of gliclazide-PEG 6000 systems. 82
17 Dissolution profile of gliclazide-PEG 4000 systems. 84
10
20
37
41
57
58
75
75
78
78

18 Dissolution profile of gliclazide-glucose systems. 87
19 Dissolution profile of gliclazide-urea systems 89
20 Ratio between % of gliclazide dissolved from (A)
drug in different solid dispersions and (B) drug
alone at t = 60 min.
21 FTIR spectra of gliclazide –PEG 6000 systems.
22 FTIR spectra of gliclazide –PEG 4000 systems. 100
23 FTIR spectra of gliclazide –glucose systems.
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92
99
101
24 FTIR spectra of gliclazide –urea systems. 102
25 DSC spectra of gliclazide –PEG 6000 systems. 106
26 DSC spectra of gliclazide –PEG 4000 systems. 107
27 DSC spectra of gliclazide –glucose systems.
108
28 DSC spectra of gliclazide –urea systems. 109
29 X-ray spectra of gliclazide –PEG 6000 systems. 113
30 X-ray spectra of gliclazide –PEG 4000 systems. 114
31 X-ray spectra of gliclazide –glucose systems. 115
32 X-ray spectra of gliclazide –urea systems. 116
33 Diagrammatic representation of the drug diffusion
apparatus.
34 In vitro release profile of gliclazide from different
topical preparations.
35 In vitro release profile of gliclazide and (8:92)
gliclazide –PEG 6000 solid dispersion from
different topical bases.
36 In vitro release profile of gliclazide and (1:10)
gliclazide –glucose solid dispersion from different
topical bases.
37 In vitro release profile of gliclazide and (8:92)
gliclazide –PEG 4000 solid dispersion from
different topical bases.
125
136
141
143
145

38 In vitro release profile of gliclazide and (1:10)
gliclazide –urea solid dispersion from different
topical bases.
39 Release of gliclazide from different bases with
different solid dispersions.
40 Percent reduction in blood glucose levels after oral
and topical administration of gliclazide in normal
rats.
41 Percent reduction in blood glucose levels after oral
and topical administration of gliclazide in diabetic
rats.
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147
148
153
156
42 Glibenclamide structure. 158
43 Techniques to optimize drug permeation across the
skin.
44 UV absorption spectra for glibenclamide in
methanol.
45 Calibration curve of glibenclamide in phosphate
buffer (7.4) at max 227 nm.
46 Release profile of glibenclamide from different
topical bases.
47 Percentage drug released from different topical
bases.
48 Release profile of glibenclamide from water soluble
base containing different concentrations of
cetrimide.
49 Release profile of glibenclamide from water soluble
base containing different concentrations of SLS.
163
186
188
192
194
199
201

50 Release profile of glibenclamide from water soluble
base containing different concentrations of Tween
80.
51 Release profile of glibenclamide from water
soluble base containing different concentrations of
labrafil.
52 Percentage drug released from water soluble base
containing different concentrations of different
surfactants
53 Release profile of glibenclamide from water soluble
base containing different concentrations of oleic
acid.
54 Release profile of glibenclamide from water soluble
base containing different concentrations of linoleic
acid.
55 Percentage drug released from water soluble base
containing different concentrations of fatty acids.
56 Release profile of glibenclamide from water soluble
base containing different concentrations of
isopropyl myristate.
57 Release profile of glibenclamide from water soluble
base containing different concentrations of
isopropyl palmitate .
58 Release profile of glibenclamide from water soluble
base containing different concentrations of
Transcutol.
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203
205
206
209
211
212
215
217
220

59 . Percentage drug released from water soluble base
containing different concentrations of fatty acid
esters and Transcutol.
60 Percentage drug released from water soluble base
containing the best concentrations of different
penetration enhancers used.
61 Percent reduction in blood glucose levels after oral
and topical administration of glibenclamide in
normal rats.
62 Percent reduction in blood glucose levels after oral
and topical administration of glibenclamide in
diabetic rats.
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221
222
231
234

Table
Number
List of Tables
Description Page
1 Methods for the characterization of solid
dispersion.
2 Types of carriers and their ratios in gliclazide solid
dispersions and physical mixtures.
3 Solubility enhancement data of gliclazide in various
carrier solutions at 25°C.
4 Effect of change in pH on the solubility of
gliclazide.
5 Dissolution parameters (±SD) of gliclazide in
distilled water from different gliclazide - PEG 6000
systems.
6 Dissolution parameters (±SD) of gliclazide in
distilled water from different gliclazide - PEG 4000
systems.
7 Dissolution parameters (±SD) of gliclazide in
distilled water from different gliclazide – glucose
systems.
8 Dissolution parameters (±SD) of gliclazide in
distilled water from different gliclazide –urea
systems.
9 Collective data for dissolution of gliclazide
obtained from different carriers used.
10 FTIR spectra of gliclazide solid dispersions and
physical mixtures compared with individual
components.
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Number
64
69
77
79
81
83
86
88
91
95

11 Fusion temperatures (Tc) and heat of fusion (
of gliclazide solid dispersions and physical mixtures
compared with individual components.
12 º)
for some gliclazide solid dispersions and physical
mixtures compared with individual components.
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105
111
13 Composition of different topical bases 124
14 Amounts of sample and standard used 131
15 In vitro release data of gliclazide from
different topical bases
135
16 Viscosity of different topical bases. 138
17 In vitro release of gliclazide and (8:92) gliclazide-
PEG 6000 solid dispersion from different topical
bases
18 In vitro release of gliclazide and (1:10) gliclazide-
glucose solid dispersion from different topical
bases.
19 In vitro release of gliclazide and (8:92) gliclazide-
PEG 4000 solid dispersion from different topical
bases.
20 In vitro release of gliclazide and (1:10) gliclazide-
urea solid dispersion from different topical bases.
21 Kinetic data of the release of gliclazide and its solid
dispersions from different topical bases.
22 Reduction in blood glucose level after oral and
topical application of gliclazide and 10:90
gliclazide- PEG 6000 solid dispersion in normal
rats. All values are expressed as mean ± sd.
140
142
144
146
149
152

23 Reduction in blood glucose level after oral and
topical application of gliclazide and 10:90
gliclazide- PEG 6000 solid dispersion in diabetic
rats. All values are expressed as mean ± sd.
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155
24 Composition of different topical formulations. 180
25 Types of penetration enhancers and percentages
used.
26 In vitro release of glibenclamide from different
topical bases.
27 In vitro release of glibenclamide from water soluble
base containing different concentrations of
cetrimide
28 In vitro release of glibenclamide from water soluble
base containing different concentrations of Sodium
lauryl sulphate (SLS).
29 In vitro release of glibenclamide from water soluble
base containing different concentrations of Tween
80.
30 In vitro release of glibenclamide from water soluble
base containing different concentrations of labrafil.
31 In vitro release of glibenclamide from water soluble
base containing different concentrations of oleic
acid.
32 In vitro release of glibenclamide from water soluble
base containing different concentrations of linoleic
acid.
33 In vitro release of glibenclamide from water soluble
base containing different concentrations of
Isopropylmyristate (IPM).
182
198
200
202
204
208
210
214

34 In vitro release of glibenclamide from water soluble
base containing different concentrations of
Isopropylpalmitate (IPP).
35 In vitro release of glibenclamide from water soluble
base containing different concentrations of
Transcutol.
36 Kinetic data of the release of Glib from different
topical bases
37 Reduction in blood glucose level after oral and
topical application of glibenclamide and
glibenclamide with 1% oleic acid in normal rats.
38 Reduction in blood glucose level after oral and
topical application of glibenclamide and
glibenclamide with 1% cetrimide in normal rats
39 Reduction in blood glucose level after oral and
topical application of glibenclamide and
glibenclamide with 1% isopropyl myristate (IPM) in
normal rats..
40 Reduction in blood glucose level after oral and
topical application of glibenclamide and
glibenclamide with 5 % Labrafil in normal rats.
41 Reduction in blood glucose level after oral and
topical application of glibenclamide and
glibenclamide with 1% cetrimide in diabetic rats.
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216
219
224
227
228
229
230
233

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Abstract
Part One
Formulation and Evaluation of Topically Applied Gliclazide.
Chapter One
Formulation and Characterization of Gliclazide Solid Dispersions.
The purpose of this study was to improve the dissolution of Gliclazide
(Glz) for enhancing its bioavailability and therapeutic efficacy.
Physical mixtures (PMs) and solid dispersions (SDs) of Glz with each of
polyethylene glycol 4000 (PEG 4000) and polyethylene glycol 6000 (PEG
6000) in ratios 10: 90, 8: 92, 5: 95 and 1: 99 (drug-to-carrier w/w) were
prepared. Glucose (glu) and urea (UR) in ratios 1:1, 1:2, 1: 3, 1: 5 and 1: 10
(drug-to-carrier w/w) were also prepared. All SDs were prepared by solvent
evaporation method. The equilibrium solubility of Glz in presence of
different concentrations of the above mentioned carriers was determined at
25°C and the influence of different pH on the solubility of Glz was also
examined. The dissolution of all prepared samples (PMs and SDs) was
carried out in media of pure distilled water pH 6.5. All SDs and PMs as well
as individual components were subjected to inspection by FTIR
spectroscopy, DSC and X-ray powder diffraction.
The results revealed that, the aqueous solubility of Glz was favoured
by the presence of PEG 4000 and PEG 6000 while the aqueous solubility
was slightly improved when glu or UR was used as a carrier. The solubility
of Glz increased with increasing pH (higher in alkaline medium rather than
acidic one). The type of carrier and drug to carrier ratio had great influence
on the rate and extent of dissolution of Glz from its SDs. All the investigated
carriers improved the dissolution rate of Glz. The highest rates were obtained
from PEG 6000 followed by PEG 4000, glu and finally UR SDs at mixing
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atios of (1:99), (1:99), (1:10) and (1:10) respectively. Physical
characterization of all systems prepared revealed structural changes in the
prepared SDs from the plain drug, which may account for increased
dissolution rates.
It was concluded that SDs showed increased dissolution rate as compared
to the pure drug.
Chapter Two
In Vitro and In Vivo Studies on Topical Application of
Gliclazide Solid Dispersions
The aim of this study to enhance the release of Glz from topical
preparations by incorporating it in the form of solid dispersion with water
soluble carriers. Another aim was to determine whether a Glz would be
absorbed through the skin and consequently lower blood glucose levels.
Glz was formulated in different topical formulations. For this
purpose, a set of traditional formulations such as ointment bases, cream
bases and gel bases were utilized. The traditional classes of ointment
bases studied were water soluble base (WSB), emulsion bases and
absorption base. The gel base studied was hydroxylpropyl
methylcellulose gel (HPMC gel). The emulsion bases chosen were oil in
water (O/W) and water in oil (W/O) emulsions. Investigation of the
release studies from topical formulation bases were carried in vitro over a
period of six hours at a thermostatically controlled water bath operating at
37°C and 100 rpm using the rate limiting membrane technique , at
concentration of 1 % w/w Glz for all topical preparations. The receptor
media employed throughout this investigation was sörensen’phosphate
buffer of pH 7.4. The release studies of drug from (8:92) PEG 6000,
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(8:92) PEG 4000, (1:10) glu and (1:10) UR w/w drug to carrier ratio SDs
from WSB, HPMC gel and O/W emulsion were investigated. In vitro skin
permeation of Glz and its SDs from different topical formulations was
studied. The blood glucose reducing hypoglycemic activity of Glz
systems was studied in both normal and diabetic rats.
The results revealed that, the percentage amount of drug released
from WSB, gel base are greater than that released from other bases. The
rate of drug release can be arranged in the following descending order:
WSB (64.15 %) > HPMC gel (43.38 %) > O/W emulsion base (8.43 %).
There is no drug is released fromabsorption base and W/O emulsion
base. The amount of drug released from topical bases incorporating SDs
can be arranged in the following descending order: Topical preparations
containing drug: PEG 6000 (8:92) SD > (1:10) drug: glu (1/10) SD >
drug: PEG 4000 (8:92) SD > drug: UR (1:10) SD > pure drug.Isolated
skin permeation studies indicated that, the amount of Glz permeated
across hairless rabbit skin was too small to be measured
spectrophotometrically. The present study showed that Glz was absorbed
through the skin and lowered the blood glucose levels. Topical
preparations of Glz or its SDs exhibited better control of blood glucose
level than oral Glz administration in rats as topical route effectively
maintained normoglycemic level in contrast to the oral group which
produced remarkable hypoglycemia. The blood glucose reducing activity
of ointment contained (10:90) Glz –PEG 6000 solid dispersions was
significantly more when compared to ointment contained Glz alone.
The results suggest the possibility of transdermal administration of
Glz for the treatment of NIDDM.
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General introduction
Skin anatomy and physiology
Skin is the largest organ of the body and, in addition to its primary
function as a barrier for protection of the internal biological milieu from
the external environment, has a variety of roles in the maintenance of
physiological homeostasis (Monteiro-Riviere, 2001a) .
1. The main funcnion of the skin:
There are many different structures within the skin. Together these
structures impart many protective properties to the skin that help to avoid
damage to the body from outside influences. In this way, the skin serves
many purposes:
Protects the body from water loss and from injury due to bumps,
chemicals, sunlight or microorganisms, and some glands (sebaceous)
may have weak anti-infective properties.
Helps to control body temperature through sweat glands.
Is the sensor to inform the brain of changes in immediate environment.
Produces vitamin D in the epidermal layer, when it is exposed to the
sun's rays.
Uses specialized pigment cells to protect us from penetration of
ultraviolet rays of the sun.
Act as channel for communication to the outside world.
Plays an important role in regulation of body blood pressure (Chine,
1982).
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2. Skin anatomy:
As shown in (Figure 1), anatomically, skin is comprised of two principal
components: a stratified, a vascular epidermis and the underlying dermis.
The epidermis is further classified into layers called the stratum corneum,
stratum lucidum, stratum granulosum, stratum spinosum, and the stratum
basale. Together, these cell layers function to anchor the epidermis to the
underlying dermis, to replenish cells that are naturally sloughed off from
the surface epidermis, and to form a permeability barrier that protects the
internal biological environment from the external milieu. The dermis
consists of a dense irregular network of collagen, elastic, and reticular
fibers that provides mechanical support for the tissue. An extensive
network of capillaries, nerves, and lymphatics also located in the dermis
facilitate the exchange of metabolites between blood and tissues, fat
storage, protection against infections, and tissue repair. Below the dermis
is the hypodermis, which anchors skin to underlying muscle or bone by
loose connective tissue of collagen and elastic fibers (Monteiro-Riviere,
2001a, 2004, 2006; Taylor et al., 2006).
2.1. The epidermis:
The epidermis is derived from ectoderm and consists of stratified
squamous keratinized epithelium. The thickness and number of stratified
layers varies among mammalian species and anatomical location. In
general, porcine skin in the thoracolumbar area is an acceptable model for
percutaneous absorption studies and has an epidermal thickness of about
52 (Monteiro-
Riviere, 2004). The vascular epidermis continuously undergoes an
orderly process of proliferation, differentiation, and keratinization to
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eplenish the epidermis as stratum corneum cells are naturally sloughed
from the skin’s surface (Monteiro-Riviere, 2006).
Figure 1: Diagrammatic representation of the skin structure.
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Keratinocytes are the predominate cell type of the epidermis, accounting
for approximately 80% of the cell population (Monteiro-Riviere, 2004).
These cells originate in the stratum basale and, upon mitosis, undergo a
continual differentiation process, known as keratinization. During this
process, the epidermal cells migrate upward, increase in size, and produce
differentiation products such as tonofilaments, keratohyalin granules, and
lamellated bodies. Epidermal layers are easily identified by distinct
differences in cell morphology and differentiation products that result due
to keratinization. The remaining group of epidermal cells, known as
nonkeratinocytes, consists of melanocytes, Langerhans cells, and Merkel
cells and do not participate in the process of keratinization (Smack, et al.,
1994).
Stratum basale:
The stratum basale is the layer of skin located closest to the dermis
and is comprised of a single layer of columnar or cuboidal cells that are
attached to the overlying stratum spinosum cells and to adjacent basale
cells by desmosomes and to the underlying basement membrane by
hemidesmosomes. Desmosomes are small, localized adhesion sites that
mediate direct cell-to-cell contact by providing anchoring sites for
intermediate filaments of the cellular cytoskeletons. Hemidesmosomes,
on the other hand, function to provide strong attachment sites between the
intermediate filaments of cells and the extracellular matrix of the
underlying basal lamina (Taylor et al., 2006). In addition to their role in
synthesizing the basement membrane, basale cells also function as stem
cells to continuously produce keratinocytes that subsequently undergo
keratinization. Immature keratinocytes of the stratum basale are capable
of engaging in the synthesis of keratin, which are later assembled into
keratin filaments called tonofilaments. Other nonkeratinocytes cells are
also present in the stratum basale. Merkel cells are closely associated with
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nerve fibers and function as mechanoreceptors capable of relaying
sensory information to the brain. Additionally, melanocytes, which
produce and secrete melanin and provide protection from ultraviolet
irradiation, reside near the basement membrane and are responsible for
transferring melanin to surrounding keratinocytes.
Stratum spinosum:
The stratum spinosum or “prickle cell layer” is located above the
stratum basale and consists of several layers of irregularly shaped
polyhedral cells. Tight junctions and desmosomes connect adjacent cells
and the underlying stratum basale. Additionally, Langerhans cells,
important for the skin’s immune response, are found in this epidermal
layer. This layer is morphologically distinguished from other epidermal
layers by the presence of tonofilaments. As keratinocytes mature and
move upward through this layer, the cells increase in size and become
flattened in a plane parallel to the surface of the skin. Keratinocytes
within the upper part of the stratum spinosum begin to produce
keratohyalin granules and lamellar bodies, which are distinctive features
of the cells in the stratum granulosum.
Stratum granulosum:
The next epidermal layer, the stratum granulosum, contains several
layers of flattened cells positioned parallel to skin’s surface. The
numerous granules those are present in the cells of this layer contain
precursors for the protein filaggrin, which is responsible for the
aggregation of keratin filaments present within the cornified cells of the
stratum corneum. These granules fuse with the cell membrane and secrete
their contents via exocytosis into the intercellular spaces between the
stratum granulosum and stratum corneum layers. The lipid contents of the
- 26 -

granules then form the intercellular lipid component of the stratum
corneum barrier.
Stratum lucidum:
Present only in areas of thick skin, such as the palms of the hands
and soles of the feet, is a subdivision of the stratum corneum called the
stratum lucidum. This epidermal layer is a thin, translucent layer of cells
devoid of nuclei and cytoplasmic organelles. These cells are keratinized
and contain a viscous fluid, eleidin, which is analogous to keratin.
tratum corneum:
The stratum corneum is the outermost layer of the epidermis and its
composition and organization significantly contribute to the skin’s
permeability barrier. The stratum corneum consists of terminally
differentiated cells arranged in multicellular stacks perpendicular to the
surface of the skin. The cells are devoid of nuclei and cytoplasmic
organelles and are almost completely filled with keratin filaments. The
interlocking columns of cells are embedded in a structured lamellar
matrix that consists of specialized lipids secreted from the granules of the
stratum granulosum cells. This barrier functions to restrict the penetration
of hydrophilic substances and large entities through the skin and to
prevent excess loss of body fluids (Mackenzie, 1975; Menton, 1976;
Monteiro-Riviere, 1991, 2001a, 2001b, 2006; Smack et al., 1994;
Taylor et al., 2006)
2.2. The dermis
Collagen, elastic, and reticular fibers embedded in an amorphous
ground substance of proteoglycans create a network of dense connective
tissue that makes up the dermis. Fibroblasts, mast cells, and macrophages
are the predominate cell types found in the dermis; however, plasma cells,
- 27 -

fat cells, chromatophores, and extravasated leukocytes are often also
present. The more superficial layer of the dermis, the papillary layer, lies
immediately beneath the basement membrane and contains a less dense,
irregular framework of type I and type III collagen molecules and elastic
fibers. This region also contains blood and lymphatic vessels that serve
but do not enter the epidermis and nerve processes that either terminate in
the dermis or penetrate into the epidermis. Fingerlike protrusions of the
dermal connective tissue into the underside of the epidermis are called
dermal papillae. Likewise, epidermal ridges are similar protrusions of the
epidermis into the dermis. Increased mechanical stress on the skin
increases the depth of the epidermal ridges and length of the dermal
papillae, thus, creating a more extensive interface between the dermis and
epidermis. The reticular layer of the dermis lies beneath the papillary
layer. This layer is substantially thicker than its superficial layer and is
characterized by thick bundles of mostly type I collagen, coarser elastic
fibers and fewer cells (Monteiro-Riviere, 1991, 2001a, 2001b, 2006).
2.3. The hypodermis:
The hypodermis is superficial fascia that lies below the skin and helps to
anchor the dermis to underlying muscle and bone. It is comprised of
connective tissue containing a loose arrangement of collagen and elastic
fibers that allows for flexibility and free movement of the skin over the
underlying structures (Monteiro-Riviere, 2006).
2.4. Skin appendages:
Hair follicles, associated sebaceous glands, arrector pili muscles,
and sweat glands are appendageal structures commonly found in skin.
Hairs are produced by hair follicles and are keratinized structures derived
from epidermal invaginations that traverse the dermis and may extend
- 28 -

into the hypodermis. Although skin penetration through a hair follicle still
requires a compound to traverse the stratum corneum, follicles represent
regions of greater surface areas and can, therefore, contribute to increased
transdermal absorption (Monteiro-Riviere, 2004). Connective tissue at
the base of the hair follicle provides an attachment site for the arrector pili
muscle, which upon contraction not only erects the hair but also assists in
emptying the sebaceous glands. Sebaceous glands release their secretory
product, sebum, into ducts that empty into the canal of the hair follicle.
Sebum is an oily secretion that acts as an antibacterial agent. Apocrine
and eccrine sweat glands are also located in skin and function to produce
secretions involved in communication and thermoregulation, respectively
(Monteiro-Riviere and Stinsons, 1993).
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Percutaneous absorption
The primary barrier against the passage of foreign hydrophilic
substances into the skin is the stratum corneum. The stratum corneum
consists of 10-15 layers of nonviable, protein rich cells surrounded by an
extracellular lipid matrix. The intercellular lipid lamellae, composed
mainly of ceramides, cholesterol, and fatty acids, are primarily
responsible for restricting the passage of aqueous entities through the skin
(Wertz, 2004). The importance of the lipid moieties in barrier function
has been demonstrated by the removal of lipids from the stratum corneum,
which subsequently results in an increased penetration of compounds
(Hadgraft, 2001; Monteiro-Riviere et al., 2001). The stratum corneum
serves as the rate-limiting barrier to percutaneous absorption because the
underlying epidermal layers are much more aqueous in nature and, thus,
allow the passage of substances to occur more easily. Once penetration
through the epidermis occurs, there is little resistance to diffusion, and
substances have access to systemic circulation via absorption into the
blood and lymphatic vessels located in the dermis. Additionally,
keratinocytes possess metabolizing enzymes that interact with the
diffused compound and produce metabolites that can easily be absorbed
by cutaneous vasculature (Monteiro-Riviere, 2001a; Riviere, 1990;
Bronaugh et al., 1989).
1. Pathways for transdermal drug delivery:
Drugs can be diffused through the following pathways:
1.1. Transappendagel:
Diffusion occurs through hair follicle, sebaceous glands and
eccrine glands
- 30 -

1.2. Transepidermal:
It is the most important pathway of drug permeation. As shown in
(Figure 2) it is divided into:
1.2.1. Intercellular bathway:
It is the main route for permeation of the most drugs through
intercellular spaces between the cells of stratum corneum, which is filled
with a lipid, based lamellar crystalline structure (Moghimi et al., 1996,
1997, and 1998).
1.2.2. Transcellular pathway:
Transport via corneocytes e.g. through protein-filled cell cytoplasm
and protein-lipid cellular envelope (Moghimi et al., 1999).
Figure 2: Diagrammatic representation of the stratum
corneum and the intercellular and transcellular routes of penetration
(Barry, 2001)
- 31 -

2. Factors affecting percutaneous absorption:
2.1. Physicochemical properties of the penterant molecules:
2.1.1. Partition coefficient:
The majority of topically applied drugs are covalent compounds in
nature. Regardless of the types of vehicle used, at some point during the
process of transdermal penetration the drug molecules have to dissolve
and diffuse within the endogenous hydrated tissues of the stratum
corneum. Drugs possessing both water and lipid solubility are favorably
absorbed through the skin. Transdermal permeability coefficient a linear
dependency on partition coefficient .A lipid/ water partition coefficient of
one or greater is generally required for optimal tarnsdermal permeability.
The drug substances should have a greater physicochemical attraction to
the skin than to the vehicle in which it is presented (Chine, 1982).
Molecules showing intermediate partition coefficients (log P
octanol/water of 1-3) have adequate solubility within the lipid domains of
the stratum corneum to permit diffusion through this domain whilst still
having sufficient hydrophilic nature to allow partitioning into the viable
tissues of the epidermis (Heather, 2005).
2.1.2. pH conditions:
The pH condition of the skin surface and in the drug delivery
systems affect the extent of dissociation of ionogenic drug molecules and
their transdermal permeability. The pH dependence of the transdermal
permeability was related to the effect of the solution pH on the
concentration of lipophilic, nonionized species of the drugs.
- 32 -

2.1.3. Penetrant concentration :
Transdermal permeability across mammalian skin is passive
diffusion process and thus, depends on the concentration of penetrant
molecules on the surface layers of the skin
2.1.4. Penetrant solubility:
According to Meyer-Overton theory of absorption , lipid soluble
drugs pass through cell membrane owing to its lipid content while water
soluble substances pass after hydration of protein particles in the cell
wall which leaves the cell permeable to water soluble substances.
2.1.5. Penetrant molecular weight:
Rate of drug penetration is inversely proportional to its molecular
weight, low molecular weight drugs penetrate faster than high molecular
weight drugs.
2.2. Physiological and pathological conditions of the skin:-
2.2.1. Skin hydration:
The moisture balance in the stratum corneum has been attributed to
the presence of a combination of water soluble substances, known as
natural moisturizing factor in the superfacial barrier layers .This factor is
produced in the skin and is responsible for the hydration of the skin.
Hydration of stratum corneum can enhance the transdermal permeability.
Skin hydration can be achieved simply by covering or occluding the skin
with pasting sheeting, leading to accumulation of sweat and condensed
transpired water vapor .Increased hydration of stratum corneum appears
to open up its dense, closely packed cells and increase its porosity
resulting into increased permeation of drug molecules (Scheuplein and
Ross, 1974).
- 33 -

2.2.2. Skin temperature:
A rise in skin temperature has been shown to have a definite effect
on the percutaneous absorption of the drugs .This temperature-depentant
increase in transdermal permeability was rationalized as due to the
thermal energy required diffusivity and solubility of the drug in the skin
tissues. Rises in skin temperature may also increase vasodilatation of the
skin vessels leading to an increase in percutaneous absorption.
2.2.3. Regional variation
The permeation of water varies in different regions of the skin due
to difference in the nature and thickness of the barrier layer (Wester and
Maibach, 1999).
2.2.4. Traumatic and pathologic injury to the skin:
Injuries to the skin that disrupt the continuity of the stratum
corneum are reported to increase skin permeability .The observed
increase in the permeability may be due to the noticeable vasodilatation
caused by the removal of barrier layer (Scott, 1991).
2.2.5. Lipid film:
The lipid film on the skin surface which contains emulsifying
agents may provide a protective film to prevent the removal of natural
moisturizing factor from the skin and play some limited role in
maintaining the barrier function of the stratum corneum .
2.3. Physicochemical properties of drug delivery system:
2.3.1. Release characteristics:
The affinity of the vehicle for the drug molecules can influence the
release of the drug molecules from the vehicle. Solubility in the vehicle
- 34 -

will determine the release rate of the drug. Generally, the more easily the
drug is released from the drug delivery system, the higher the rate of
tranedermal permeability. The mechanisms of the drug release depend on
whether the drug molecules are dissolved or suspended in the delivery
system and on the interfacial partition coefficient of the drug from the
delivery system to the skin tissue.
2.3.2. Composition of drug delivery system :
The composition of drug delivery systems has a great influence on
the percutaneous absorption of drug species. It may affect not only the
rate of drug release, but also the permeability of the stratum corneum by
means of hydration, mixing with lipids, or other sorption-promoting
effects (Howes et al., 1999).
2.3.3. Enhancement of transdermal permeation:
Transdermal permeation of drugs can be improved by the addition
of sorption or permeations promoters into the drug delivery systems.
Sorption and permeation promoters are agents that have no therapeutic
properties of their own but can promote the absorption of the drugs from
the drug delivery systems onto the skin. Examples of permeation
promoters are organic solvents and surface active agents
3. Methods for studying percutaneous absorption:
3.1. In vitro methods:
The general advantage of in vitro method is to control the
laboratory environment and so elucidate the individual factors, which
modify drug penetration. Those methods are valuable for deducing
physicochemical parameters such as fluxes, partition coefficient, and
diffusion coefficient.
- 35 -

3.1.1. Release method without a rate-limiting membrane :
These procedures record the kinetics from a formulation to a
simple immiscible phase, which is supposed to corresponding properties
with human skin .Such techniques measure drug-vehicle interactions and
the release characteristics of the formulation.
3.1.2. Diffusion methods with a rate controlling membrane :
3.1.2.1. Simulated skin membrane:
Because human skin may be difficult to obtain and varies in its
permeability, many workers use other materials to simulate it such as
cellulose acetate membrane (Gary-Bobo et al., 1969; Diplo et al., 1970),
silicone rubber (Flynn and Roseman, 1971; Bottari et al., 1977; Di colo
et al., 1980), collagen (Nakano et al., 1976), and egg shell membrane.
3.1.2.2. Natural skin membrane:
Excised skin from a variety of animal including rats, mice, rabbits,
guinea pigs has been used. Skin may be used immediately or stored at -24
°C for a long time, and it may be subjected to greater extreme of heat,
humidity, pH, and various fluids other biological tissues without
irreversibly changing its barrier properties. Storage up to 6 months at -
20°C leaves human skin permeability unaffected (Astley and Levine,
1976). Elias et al., (1981) claims that a temperature as low as -70°C does
not affect barrier properties.
3.2. In-vivo methods:
These include:-
3.2.1. Animal models:
3.2.2. Techniques:
3.2.2.1. Observation of physiological or pharmacological response :
- 36 -

If the penetrant stimulates a biological reaction when it reaches the
viable tissue, then this response may provide the basis for determining the
penetrant kinetics. The most productive technique in terms of
biopharmaceutical application is the vasoconstrictor or balancing
response to topical steroids.
3.2.2.2. Physical properties of the skin:
There are several methods used for measuring physical properties
of the skin such as determination of transepidermal water loss, in addition
to thermal determinations, mechanical analysis, and spectral analysis.
3.2.2.3. Analysis of body tissues or fluids :
Urinary analysis is often used to study percutaneous absorption
(Wurster and Kramer, 1961; Butler, 1966; Fledmann and Maibach ,
1965, 1966, 1967, 1968, 1969, 1970 ). Combination of blood, urine, and
faeces analysis was used with rats, monkeys, and human volunteers to
examine the percutaneous absorption and excretion of tritium-labeled
diflorasone diacetate (Wickrema sinha et al., 1978).
3.2.2.4. Surface loss :
Measurements of the rate of loss of penetrant from an applied
vehicle should lead to a determination of the flux of the material into the
skin. The main use of a loss technique has been to monitor the decrease in
radioactivity at skin surface (Malkinson, 1956, 1958, 1964; Ainsworth,
1960; Wahlberg, 1965).
3.2.2.5. Histology
Histological techniques have elucidated absorption profiles and
penetration routes for these few compounds which produce colored end
- 37 -

products after chemical reaction for example, certain drugs change
epidermal sulfhydryl groups in an easily detectable way (Bradshaw,
1961; Chayen et al., 1970) few compounds fluoresce, and their behavior
in skin may revealed by microscopy such as vitamin A, tetracycline, and
benzpyrene.
4. Theoretical advantages of transdermal routes for systemic therapy:
Transdermal administration of drugs possesses several advantages
in therapy compared with oral or parenteral adminsteration (Barry, 1991).
These include:
The avoidance of hepatic (first pass) metabolism by which the liver
enzymes may reduce the amount of medicament passing into the
system circulation.
Transdermal input of a drug would avoid several variables which make
gastrointestinal absorption a problem like dramatic change in pH,
stomach emptying, intestinal motility, and the action of human and
bacterial enzymes and the effect of food on drug absorption
The percutaneous delivery may control the administration of highly
potent drug, produce a relatively constant plasma level of drugs,
concurrent decrease in side effects and improves patient compliance.
The percutaneous administration can be valuable for drugs with low
therapeutic indices and for which significant variation in plasma
concentration are dangerous.
Substitution for oral or potential administration in certain clinical
situations (pediatrics, geriatrics and nausea).
Ease of self administration.
- 38 -

Types of skin preparations
There are a large number of different types of external medicines,
ranging from dry powders through semi-solid to liquids. (Figure 3)
illustrates the formulation of the main types of preparation used on the
skin.
1. Solids:
Dusting powders are applied to the skin for a surface effect such as
drying or lubricating, or an antibacterial action. They are made of fine
particle size powders together with any medicament (Winfield, 1998).
2. Liquids:
Soaks have an active ingredient dissolved in aqueous solvent and are
often used as astringents, for cooling or to leave a film of solid on the
skin. Oily vehicles can be used in bath additives to leave an emollient
film on the skin surface.
Liniments are alcoholic or oily solutions or emulsions designed to be
rubbed into the skin. The medicament is usually a rubefacient.
Lotions are aqueous solutions, suspensions or emulsions that cooled
inflamed skin and deposit a protective layer of solid.
Paints and tinctures are concentrated aqueous or alcoholic
antimicrobial solutions.
Collodions are organic solvents containing a polymer and keratolytic
agent for treating corns and calluses.
Emulsion is a dispersion in which the dispersed phase is composed of
small globules of a liquid distributed through a vehicle in which it is
immiscible by the aid of surfactant but it is thermodynamically unstable.
pomades and foot washes (Winfield, 1998).
- 39 -

- 40 -

3. Semi-solids:
3.1. Ointments:
Ointments are usually oily vehicles that may contain a surfactant to
allow them to be washed off easily (barrier creams). They are used as
emollients, or for drug delivery either to the surface or for deeper
penetration (Winfield, 1998).
Ointment bases are classified into:
3.1.1. Hydrocarbon bases (oleaginous bases):
These bases are immiscible with water and are not absorbed by the
skin. They are almost inert and absorb very little water from a
formulation or from skin exudates. However, they inhibit water loss from
the skin by forming a waterproof film and by improving hydration, may
encourage absorption of the medicaments through the skin.
The constituents of hydrocarbon bases includes
* Soft paraffin: There are two varieties, one is yellow and the other
(bleached) form is white.
* Hard paraffin which is used to stiffen ointment bases.
* Liquid paraffin: It is used to soften ointment bases and to reduce the
viscosity of creams.
Hydrocarbon bases may contain ingredients additional to
petrolatum, for example, paraffin ointment B.P. is a blend of white
beeswax, hard paraffin, cetostearyl alcohol and soft paraffin (Collett,
1991).
3.1.2. Absorption bases:
Absorption bases are less occlusive than the hydrocarbon bases and are
easier to spread. They are good emollients. These bases absorb water and
aqueous solutions to produce water-in-oil (W/O) emulsions. They consist
- 41 -

of a mixture of sterol- type emulgent with one or more paraffins (Collett,
1991).
3.1.2.1. Non-emulsified:
These constituents include:
* Wool fat (anhydrous lanolin): It can absorb about 50% of its weight
water.
* Wool alcohols: This is the emulsifying fraction of wool fat.
* Beeswax and cholesterol : They are included in some ointment bases to
increase water-absorbing power.
3.1.2.2. Water-in-oil emulsions:
These are similar in properties to the previous group and are
capable of absorbing more water. The constituents of emulsified
absorption base include Hydrous Wool Fat BP (Lanolin) and Oily cream
BP.
3.1.3. Water-miscible bases (Emulsifying bases):
Despite their hydrophilic nature, absorption base are difficult to
wash from the skin. Although they can emulsify a large quantity of water
they are immiscible with an excess. Ointments made from water-miscible
bases are easily removed after use. The three emulsifying ointments from
water-miscible bases, i.e. Emulsifying Ointment BP (anionic), Cetrimide
Emulsifying Ointment BP (cationic) and Cetomacrogol Emulsifying
Ointment BP (non-ionic). These contains paraffins and O/W emulgent
and have the general formula:
Anionic, cationic or non-ionic emulsifying wax 30%
White Soft Paraffin 50%
Liquid Paraffin 20%
They are used for preparing O/W creams and as ointment bases
when easy removal from the skin is advantageous. Other advantages of
- 42 -

this type of base include, miscibility with exudates, good contact with the
skin, high cosmetic acceptability and easy removal from the hair (Collett,
1991).
3.1.4. Water -soluble bases:
Completely water-soluble bases have been developed the
macrogols (polyethylene glycols). The macrogols vary in consistency
from viscous liquids to waxy solids. They are non-toxic and non-irritating
to the skin unless it is badly inflamed. Products with ointment-like
consistency can be obtained by mixing liquid and waxy forms in suitable
proportions. The water-soluble bases have the advantages of being non-
occlusive, miscible with exudates, non-staining and easily removed by
washing.
The macrogol bases, being water-soluble, have the disadvantage of
having a very limited capacity to take up water without a physical change,
They are less bland than the paraffins and reduce the activity of a number
of antimicrobial substances. They may also react with plastic closures
(Collett, 1991).
3.2. Pastes:
Pastes are vehicles (aqueous or oily) with a high concentration of
added solid. This makes them thick so they don not spread and so
localizes drug delivery. They can also be used for sun-blocks (Winfield,
1998).
.
3.3. Gels:
Gels are transparent or translucent, non greasy, aqueous
preparations (Collett, 1991). They are usually used for lubrication or
applying a drug to the skin. Oily gels are also available where occlusion
is required (Winfield, 1998).
- 43 -

Bases for gels formulations:
3.3.1. Tragacanth:
Tagacanth gels are susceptible to microbial degradation and to
changes in PH outside the range pH 4.5-7. Concentrations of tragacanth
from 2% to 5 % produce gels of increasing viscosity.
3.3.2. Sodium alginate:
The viscosity of alginate gels is more standardized than that of
tragacanth. A concentration of 1.5 % produces fluid gels and 5-10 % gels
are suitable as dermatological vehicles.
3.3.3. Pectin:
Pectin gels are suitable for acid products. They are prone to
microbial contamination and to water loss by evaporation and may
require the inclusion of humectants.
3.3.4. Starch gels:
Starch gels are little used dermatological bases. Mucilages
prepared with water alone lose by evaporation and are prone to microbial
contamination. Glycerol concentrations of 50% or greater combine
humectant and preservative functions.
3.3.5. Gelatin:
Gelatin forms gels at concentrations of 2-15 %. Gelatin gels are
rarely used alone as a dermatological base but may be combined with
other ingredients such as pectin.
3.3.6. Polyvinyl alcohols:
Polyvinyl alcohols (PVAs) have been used to prepare gels that dry
very quickly. The residual film is strong and plastic, giving good contact
between the skin and the medicament. The required concentration is
usually between 10 %and 20 % depending on the grade of PVA and the
desired viscosity.
3.3.7. Clays:
- 44 -

Gels containing 7-20 % of bentonite are used as dermatological
bases. They are opalescent and lack the attractive clear appearance of
many other types of gels.
3.3.8. Carbomers :
Neutralized carbomer gels are also used as bases for lubricants
(0.3-1 %) and in dermatological preparations (0.5-5%). These gels are
clear provided that an excessive amount of air is not incorporated during
preparation.
3.3.9. Cellulose derivatives:
These are widely used because they produce neutral gels of stable
viscosity, good resistance to microbial attack, high clarity and good film
strength when dried on the skin. Methylcellulose 450 at a concentration
of 3-5% produces satisfactory gels. Carmellose sodium (sodium
carboxymethylcellulose) is easier to dissolve and the medium viscosity
grade produces lubricant gels at a concentration of 1.5-5 % and
dermatological gels at greater concentrations. Hypromellose
(hydroxypropyl methylcellulose) form exceptionally clear gels which are
used in ophthalamic products.
Hypromellose, short for hydroxypropyl methylcellulose (HPMC),
is a semisynthetic, inert, viscoelastic polymer used as an ophthalmic
lubricant, as well as an excepient and controlled-delivery component in
oral medicaments, found in a variety of commercial products (Collett,
1991).
3.4. Emulgels:
Emulgel is a system consists of hydrophilic surfactant(s), oil, water,
and gelling agent. Emulgel bases offer many advantages over other
preparations:
- 45 -

(i) They permit incorporation of aqueous and oleaginous ingredients, and
their rheological properties can be controlled easily.
(ii) They are easy to remove from a container in the desired quantity
without waste.
(iii) Upon application these preparations exhibit good spreadability; they
can easily be applied to the desired part of the body without running or
dripping and they are not tacky.
The selection of oil phase, emulsifier and gelling agent is one of the most
important factors in the preparation of emulgel bases.
* Choice of oil phase:
Many emulsions for external use contain oil which is present solely as a
carrier for the active agent. It must be realized, however, that the type of
oil used may also have an effect on the transport of the drug into the skin.
One of the most widely used oil for this type of preparation is liquid
paraffin. A variety of fixed oils of vegetable origin are also available, the
most widely used being arachis, sesame, cotton seed and maize oils.
* Choice of emulsifying agent:
The inclusion of an emulsifying agent or agents is necessary to facilitate
actual emulsification during manufacture and also to ensure emulsion
stability during the shelf life of the product.
* Choice of gelling agent:
They are different gelling agents as mentioned before. They differ
in their characteristics that affect the consistency of the emulgel (Balata,
1999).
- 46 -

4. Others:
4.1. Submicron emulsions (SME):
SME is liquid dispersion system formed by processing a medium-
chain triglycerides emulsion with high-pressure homogenizer. SME has
microparticles with diameter ranging from 3 to 10
layers of stratum corneum , increase its fluidity, disrupt barrier continuity,
this result in slow, continuous, and controlled systemic delivery of the
drug (Gupta and Garg, 2002).
4.2. Microemulsion:
Microemulsion is a liquid dispersion of water and oil with
surfactant and co-surfactant. It is transparent, homogeneous, and
thermodynamically stable, which provides sustained release effect after
application on the skin over 24 hours (El-Nokaly and Cornell, 1990).
4.3. Microsponges :
Microsponges are polymeric delivery system consisting of porous
microspheres that can entrap several active substances such as anti-fungal,
anti-infective, and anti-inflammatory. Those systems can be incorporated
into creams, lotions, powders, soaps from which the entrapped substances
are released to the skin in controlled- release manner (Report, 1992).
4.4. Transfersomes:
Transfersomes are vesicles made from phosphatidylcholine and
contained at least one component that controllably destabilizes lipid
bilayers and makes the vesicles very deformable. Such additives are bile
salts, polysorbate, glycolipids, and alkyl or acyl-polyethoxylenes.
Transfersomes are applied to the skin to achieve sustained drug release,
- 47 -

and in this way skin surface acting as reservoir for drug as well as carrier
(Cevc, 2003).
4.5. Niosomes:
Niosomes are unilameller or multilamellar vesicles where in
aqueous solutions are enclosed in highly ordered bilayers made up of non
ionic surfactants with or without cholesterol and diacetyl phosphate
(Namdeo and Jain, 1996). Niosomes are supposed to give desirable
interaction with human skin when applied in topical preparations by
reducing transepidermal water loss and by increasing smoothness via
replenishing lost skin lipids.
4.6. Liposomes :
Liposomes are concentric bilayered structures made of amphipathic
phospholipids and depending on the number of bilayers; liposomes are
classified as multilamaller vesicles (MLVs), small unilamaller vesicles
(SUVs), or large unilamaller vesicles (LUVs). They range in size
from .025 –
regulated by the method of preparation and composition (Kshirsagr,
2000).
4.7. Solid lipid nanoparticles (SLNs):
SLNs consist of physiological and biocompatible lipids, prepared
by several techniques such as hot and cold dispersion of lipids and high
pressure homogenization of melted lipids (Schwartz et al., 1992; Domb,
1995; Westesen et al., 1998; Yang et al., 1999). SLNs posses the
advantages of better drug penetration because their small particle size
ensure close contact to stratum corneum and increase the amount of
encapsulated drug penetrating skin. SLNs provide both burst and
- 48 -

sustained drug release and they can be incorporated into aqueous gel or
creams in which stability is maintained (Gupta and Garg, 2002).
4.8. Microneedles:
Microneedle concept employs an array of micron-scale needles that
inserted into skin sufficiently far that it can deliver drug into the body, but
not so far that it hits nerves there by avoids causing pain (Prausnitz et al.,
2003).
4.9. Metred- dose transdemal spray (MDTS):
MDTS is a topical solution made up of a volatile:non-volatile
vehicle containing drug dissolved as a single phase solution. Upon
application to the skin, evaporation of the volatile component of the
vehicle occurs leaving the remaining non-volatile penetration enhancer
and drug to partition into the stratum corneum during the first minute
after application, resulting in stratum corneum reservoir of drug and
enhancer which releases the drug in sustained pattern (Morgan et al.,
1998).
4.10. Macroflux tehnology:
Macroflux system incorporates a titanium microprojection array
that creates superficial pathways through the skin barrier layers to allow
transportation of therapeutic proteins and vaccines that currently require
parentral administration (Cormier and Daddona, 2003).
4.11. Transdemal drug delivery devices (TDDS) :
1987):
TDDS are broadly classified into the following types (Chien,
- 49 -

4.11.1. Reservoir systems:
In these systems, the drug reservoir is embedded between an
impervious backing layer and a rate controlling membrane. The drug
release only through the rate controlling membrane, can be microporous
or non-porous. In the drug reservoir compartment, the drug can be in the
form of solution, suspension, gel, or embedded in a solid polymer matrix.
On the outer surface of the polymeric membrane a thin layer of drug-
compatible, hypoallergenic adhesive polymer can be applied.
4.11.2. Matrix systems. Drug in adhesive system:
The drug reservoir is formed by dispersing the drug in an adhesive
polymer and then spreading the medicated polymer adhesive by solvent
casting or by melting the adhesive onto an impervious backing layer. On
top of the reservoir, layers of unmedicated adhesive polymer are applied.
4.11.3. Matrix dispersion system:
The drug is dispersed homogeneously in a hydrophilic or lipophilic
polymer matrix. This drug containing polymer disc then is fixed onto on
occlusive base plate in a compartment fabricated from a drug-
impermeable backing layer. Instead of applying the adhesive on the face
of the drug reservoir, it is spread along the circumference to form a strip
of adhesive rim.
4.11.4. Microreservoir system:
This drug delivery system is a combination of reservoir and matrix
dispersion system. The drug reservoir is formed by first suspending the
drug in an aqueous solution of water-soluble polymer and then dispersing
- 50 -

the solution homogeneously in a lipophilic polymer to form thousands of
unleachable, microscopic spheres of drug reservoirs. The
thermodynamically unstable dispersion is stabilized quickly by
immediately cross-linking the polymer insitu.
- 51 -

Diabetes mellitus
Diabetes mellitus is a group of disorders of carbohydrate
metabolism in which the action of insulin is diminished or absent through
altered secretion, decreased insulin activity, or a combination of both
factors. It is characterised by hyperglycaemia. As the disease progresses
tissue or vascular damage ensues leading to severe complications such as
retinopathy, nephropathy, neuropathy, cardiovascular disease, and foot
ulceration.
Diabetes mellitus may be categorised into several types but the two
major types are type 1 (insulin-dependent diabetes mellitus; IDDM) and
type 2 (non-insulin-dependent diabetes mellitus; NIDDM). The term
juvenile-onset diabetes has sometimes been used for type 1 and maturity-
onset diabetes for type 2 (Martindale, 1996) .
Oral Antidiabetics
If patients with type 2 diabetes have not achieved suitable control
after about 3 months of dietary modification and increased physical
activity, then oral antidiabetics (oral hypoglycaemics) may be tried. The
two major classes are the sulfonylureas and the biguanides. Sulfonylureas
act mainly by increasing endogenous insulin secretion, while biguanides
act chiefly by decreasing hepatic gluconeogenesis and increasing
peripheral utilisation of glucose. Both types function only in the presence
of some endogenous insulin production. More recently developed classes
of oral antidiabetics include the alpha-glucosidase inhibitors, the
meglitinides, and the thiazolidinediones. Alpha-glucosidase inhibitors act
by delaying the absorption of glucose from the gastrointestinal tract;
meglitinides increase endogenous insulin secretion; and
- 52 -

thiazolidinediones appear to increase insulin sensitivity (Martindale,
1996).
1.Mode of action:
Sulphonylurea
Sulfonylureas appear to have several modes of action, apparently
mediated by inhibition of ATP-sensitive potassium channels. Initially,
secretion of insulin by functioning islet beta cells is increased. However,
insulin secretion subsequently falls again but the hypoglycaemic effect
persists and may be due to inhibition of hepatic glucose production and
increased sensitivity to any available insulin; this may explain the
observed clinical improvement in glycaemic control (Martindale, 1996).
2.
Uses and Administration:
The sulfonylurea antidiabetics are a class of oral antidiabetic drugs
used in the treatment of type 2 diabetes mellitus. They are given to
supplement treatment by diet modification when such modification has
not proved effective on its own, although metformin is preferred in
patients who are obese (Martindale, 1996) .
3. Adverse effects:
Gastrointestinal disturbances such as nausea, vomiting, heartburn,
anorexia, diarrhoea, and a metallic taste may occur with sulfonylureas
and are usually mild and dose-dependent; increased appetite and weight
gain may occur. Skin rashes and pruritus may occur and photosensitivity
has been reported. Rashes are usually hypersensitivity reactions and may
progress to more serious disorders . Facial flushing may develop in
patients receiving sulfonylureas, particularly chlorpropamide, when
alcohol is consumed .
Mild hypoglycaemia may occur; severe hypoglycaemia is usually
an indication of overdosage and is relatively uncommon. Hypoglycaemia
- 53 -

is more likely with long-acting sulfonylureas such as chlorpropamide and
glibenclamide, which have been associated with severe, prolonged, and
sometimes fatal hypoglycaemia.
Other severe effects may be manifestations of a hypersensitivity
reaction. They include altered liver enzyme values, hepatitis and
cholestatic jaundice, leucopenia, thrombocytopenia, aplastic anaemia,
agranulocytosis, haemolytic anaemia, erythema multiforme or the
Stevens-Johnson syndrome, exfoliative dermatitis, and erythema
nodosum.
The sulfonylureas, particularly chlorpropamide, occasionally
induce a syndrome of inappropriate secretion of antidiuretic hormone
(SIADH) characterised by water retention, hyponatraemia, and CNS
effects. However, some sulfonylureas, such as glibenclamide, glipizide,
and tolazamide are also stated to have mild diuretic actions (Martindale,
1996) .
4. Precautions:
Sulfonylureas should not be used in type 1 diabetes mellitus. Use in
type 2 diabetes mellitus is contra-indicated in patients with ketoacidosis
and in those with severe infection, trauma, or other severe conditions
where the sulfonylurea is unlikely to control the hyperglycaemia; insulin
should be used in such situations.
Insulin is also preferred for therapy during pregnancy.
Sulfonylureas with a long half-life such as chlorpropamide or
glibenclamide are associated with an increased risk of hypoglycaemia.
They should therefore be avoided in patients with impairment of renal or
hepatic function, and a similar precaution would tend to apply in other
groups with an increased susceptibility to this effect, such as the elderly,
debilitated or malnourished patients, and those with adrenal or pituitary
- 54 -

insufficiency. Irregular mealtimes, missed meals, changes in diet, or
prolonged exercise may also provoke hypoglycaemia. Where a
sulfonylurea needs to be used in patients at increased risk of
hypoglycaemia, a short-acting drug such as tolbutamide, gliquidone, or
gliclazide may be preferred; these three sulfonylureas, being principally
inactivated in the liver, are perhaps particularly suitable in renal
impairment, although careful monitoring of blood-glucose concentration
is essential (Martindale, 1996) .
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- 56 -

Scope of Work
Sulfonylureas are widely used as oral hypoglycemic drugs in the
treatment of non insulin dependent diabetes mellitus (NIDDM). Since
sulfonylureas are usually taken for a long period, the compliance of the
patients is very important. Therefore, for the improvement of the
compliance of the patients, the development of a transdermal dosage form
of sulfonylureas was attempted in this study (Takahashi et al., 1997). In
addition, Sulfonylureas have associated with severe and sometimes fetal
hypoglycemia and gastric disturbances like nausea, vomiting, heartburn,
anorexia and increased appetite after oral therapy. The feasibility of
application of transdermal delivery for some sulfonylureas was also
previously reported (Srinivas and Nayanabhirama, 2005).
The present work is concerned with the pharmaceutical
formulation of certain sulfonylureas namely, gliclazide and glibenclamide
in different bases for topical application. The bases include water soluble,
emulsion, oleaginous, absorption, gel and emulgel bases. The in vitro
release of these drugs from the above mentioned bases was also studied
Gliclazide is practically insoluble in water; therefore, the
improvement of its dissolution is an important issue for enhancing its
bioavailability and therapeutic efficacy. Accordingly, solid dispersions of
gliclazide in different carrier systems (PEG 4000, PEG 6000, glucose and
urea) were prepared. The solid phases obtained were characterized by
Fourier transform infrared spectroscopy, differential scanning calorimetry
and X-ray powder diffraction. Solubility diagrams and dissolution studies
were also carried out.
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The role of improvement of gliclazide dissolution on the release
rate of gliclazide from different topical preparations mentioned above was
also studied.
Studies have been carried out to find suitable enhancers to
promote the percutaneous absorption of glibenclamide; therefore, the
effect of certain penetration enhancers with different concentrations on
the release of the glibenclamide from water soluble base was
demonstrated.
In vivo experiments were carried out in order to demonstrate
the blood glucose reducing hypoglycemic activity of gliclazide and
glibenclamide systems in both normal and diabetic rats. Drugs were
applied topically and compared to an orally administrated doses.
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- 59 -

1. Description:
Introduction
Gliclazide
1.1.Name, formula, molecular weight:
1-(3-Azabicyclo [3.3.0] oct-3-yl)-3-tosyl urea
Figure 10: Structure of Gliclazide.
Molecular weight: 323.4 C15 H21 N3 O3 S
1.2. Appearance, odour and colour:
without taste.
Glz is a white, crystalline, odourless powder and practically
2. Physical properties:
2.1. Melting point:
181°C.
2.2. Solubility:
Practically insoluble in water; slightly soluble in alcohol;
sparingly soluble in acetone; freely soluble in dichloromethane.
3. Pharmacokinetics:
Glz is readily absorbed from the gastrointestinal tract. It is
extensively bound to plasma proteins. The half-life is about 10 to 12
- 60 -

hours. Glz is extensively metabolized in the liver to metabolites that have
no significant hypoglycaemic activity. Metabolites and a small amount of
unchanged drug are excreted in the urine (Martindale, 1996).
4. Mode of action:
5. Dosage and adminstration:
As mentioned before under sulfonylureas.
It is given by mouth in the treatment of type 2 diabetes mellitus and
has duration of action of 12 to 24 hours. Because its effects are less
prolonged than those of chlorpropamide or glibenclamide it may be more
suitable for elderly patients, who are prone to hypoglycaemia with longer-
acting sulfonylURs. The usual initial dose is 40 to 80 mg daily, gradually
increased, if necessary, up to 320 mg daily. Doses of more than 160 mg
daily are given in 2 divided doses. A modified-release tablet is also
available: the usual initial dose is 30 mg once daily, increased if
necessary up to a maximum of 120 mg daily (Martindale, 1996).
.
6. Precautions:
7. Adverse Effects:
8. Interactions:
As mentioned before under sulfonylureas.
As mentioned before under sulfonylureas.
An increased hypoglycaemic effect has occurred or might be
expected with ACE inhibitors, alcohol, allopurinol, some analgesics
(notably azapropazone, phenylbutazone, and the salicylates), azole
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antifungals (fluconazole, ketoconazole, and miconazole),
chloramphenicol, cimetidine, clofibrate and related compounds, coumarin
anticoagulants, fluoroquinolones, heparin, MAOIs, octreotide (although
this may also produce hyperglycaemia), ranitidine, sulfinpyrazone,
sulfonamides (including co-trimoxazole), tetracyclines, and tricyclic
antidepressants.
Beta blockers have been reported both to increase hypoglycaemia
and to mask the typical sympathetic warning signs. There are sporadic
and conflicting reports of a possible interaction with calcium-channel
blockers, but overall any effect seems to be of little clinical significance.
In addition to producing hypoglycaemia alcohol can interact with
chlorpropamide to produce an unpleasant flushing reaction. Such an
effect is rare with other sulfonylureas and alcohol (Martindale, 1996).
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- 63 -

Introduction
Therapeutic effectiveness of a drug depends upon the bioavailability
and ultimately upon the solubility of drug molecules. Solubility is one of
the important parameter to achieve desired concentration of drug in
systemic circulation for pharmacological response to be shown. Currently
only 8% of new drug candidates have both high solubility and
permeability .
The solubility of a solute is the maximum quantity of solute that can
dissolve in a certain quantity of solvent or quantity of solution at a
specified temperature .
In the other words the solubility can also define as the ability of one
substance to form a solution with another substance.
The substance to be dissolved is called as solute and the dissolving
fluid in which the solute dissolve is called as solvent, which together
form a solution (Anil et al., 2007).
1. Process of solubilisation:
As shown in (Figure 4), the process of solubilisation involves the
breaking of inter-ionic or intermolecular bonds in the solute, the
separation of the molecules of the solvent to provide space in the solvent
for the solute, interaction between the solvent and the solute molecule or
ion (Anil et al., 2007).
.
Step 1: Holes opens in the solvent.
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Step2: Molecules of the solid breaks away from the bulk.
Step 3: The freed solid molecule is intergrated into the hole in the
solvent.
Figure 4: Diagramatic representation of process of solubilization.
2. Factors affecting solubility:
The solubility depends on the physical form of the solid, the nature
and composition of solvent medium as well as temperature and pressure
of system (Anil et al., 2007).
2.1. Particle Size:
The size of the solid particle influences the solubility because as a
particle becomes smaller, the surface area to volume ratio increases. The
larger surface area allows a greater interaction with the solvent.
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2.2. Temperature:
Temperature will affect solubility. If the solution process absorbs
energy then the solubility will be increased as the temperature is
increased. If the solution process releases energy then the solubility will
decrease with increasing temperature. Generally, an increase in the
temperature of the solution increases the solubility of a solid solute. A
few solid solutes are less soluble in warm solutions. For all gases,
solubility decreases as the temperature of the solution increases (Anil et
al., 2007).
2.3. Pressure:
For gaseous solutes, an increase in pressure increases solubility and
a decrease in pressure decrease the solubility. For solids and liquid
solutes, changes in pressure have practically no effect on solubility.
2.4. Nature of the solute and solvent:
While only 1 gram of lead (II) chloride can be dissolved in 100
grams of water at room temperature, 200 grams of zinc chloride can be
dissolved. The great difference in the solubilities of these two substances
is the result of differences in their natures.
2.5. Molecular size:
Molecular size will affect the solubility. The larger the molecule or
the higher its molecular weight the less soluble the substance. Larger
molecules are more difficult to surround with solvent molecules in order
to solvate the substance. In the case of organic compounds the amount of
carbon branching will increase the solubility since more branching will
reduce the size (or volume) of the molecule and make it easier to solvate
the molecules with solvent.
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2.6. Polarity:
Polarity of the solute and solvent molecules will affect the solubility.
Generally non-polar solute molecules will dissolve in non-polar solvents
and polar solute molecules will dissolve in polar solvents .
2.7. Polymorphs:
Polymorphs can vary in melting point. Since the melting point of the
solid is related to solubility, so polymorphs will have different solubilities
(Anil et al., 2007). Generally the range of solubility differences between
different polymorphs is only 2-3 folds due to relatively small differences
in free energy (Singhal and Curatolo, 2004)
3. Techniques of solubility enhancement:
There are various techniques available to improve the solubility of
poorly soluble drugs. Some of the approaches to improve the solubility
are (Pinnamaneni et al., 2002):
3.1.
Particle size reduction:
Particle size reduction can be achieved by micronisation and
nanosuspension. Each technique utilizes different equipments for
reduction of the particle size.
3.1.1 Micronization:
Micronisation increases the dissolution rate of drugs through increased
surface area, it does not increase equilibrium solubility (Chaumeil, 1998).
Micronization of drugs is done by milling techniques using jet mill, rotor
stator colloid mills etc. Micronization is not suitable for drugs having a
high dose number because it does not change the saturation solubility of
the drug.
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3.1.2. Nanosuspension:
Nanosuspensions are sub-micron colloidal dispersion of pure
particles of drug, which are stabilised by surfactants ( Anil et al., 2007).
The advantages offered by nanosuspension to increase dissolution rate is
due to larger surface area exposed, while absence of Ostwald ripening is
due to the uniform and narrow particle size range obtained, which
eliminates the concentration gradient factor.
3.2. Modification of the crystal habit:
Polymorphism is the ability of an element or compound to
crystallize in more than one crystalline form. Different polymorphs of
drugs are chemically identical, but they exhibit different physicochemical
properties including solubility, melting point, density, texture, stability
etc.
Some drugs can exist in amorphous form (i.e. having no internal
crystal structure). Such drugs represent the highest energy state and can
be considered as super cooled liquids. They have greater aqueous
solubility than the crystalline forms because they require less energy to
transfer a molecule into solvent.
3.3. Complexation:
Complexation is the association between two or more molecules to
form a nonbonded entity with a well defined stichiometry. Complexation
relies on relatively weak forces such as London forces, hydrogen bonding
and hydrophobic interactions.
3.4. Solubilization by surfactants:
Surfactants are molecules with distinct polar and nonpolar regions.
Most surfactants consist of a hydrocarbon segment connected to a polar
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group. The polar group can be anionic, cationic, zwitterionic or nonionic
(Swarbrick and Boylan, 2002). When small apolar molecules are added
they can accumulate in the hydrophobic core of the micelles. This process
of solubilization is very important in industrial and biological processes.
The presence of surfactants may lower the surface tension and increase
the solubility of the drug within an organic solvent (Anil et al., 2007).
3.5. Cosolvency:
The solubilisation of drugs in co-solvents is an another technique for
improving the solubility of poorly soluble drug (Amin et al., 2004). It is
well-known that the addition of an organic cosolvent to water can
dramatically change the solubility of drugs (Yalkowsky and Roseman,
1981).
Weak electrolytes and nonpolar molecules have poor water
solubility and it can be improved by altering polarity of the solvent. This
can be achieved by
addition of another solvent. This process is known as cosolvency. Solvent
used to increase solubility known as cosolvent. Cosolvent system works
by reducing the interfacial tension between the aqueous solution and
hydrophobic solute. It is also commonly referred to as solvent blending
(Joseph, 2002).
3.6. Chemical Modifications:
For organic solutes that are ionizable, changing the pH of the system
may be simplest and most effective means of increasing aqueous
solubility. Under the proper conditions, the solubility of an ionizable drug
can increase exponentially by adjusting the pH of the solution. A drug
that can be efficiently solubilized by pH control should be either weak
acid with a low pKa or a weak base with a high pKa (Anil et al., 2007) .
- 69 -

The use of salt forms is a well known technique to enhanced
dissolution profiles (Agharkar et al., 1976). Salt formation is the most
common and effective method of increasing solubility and dissolution
rates of acidic and basic drugs (Serajuddin, 2007). An alkaloid base is,
generally, slightly soluble in water, but if the pH of medium is reduced by
addition of acid, the solubility of the base is increased as the pH continues
to be reduced. The reason for this increase in solubility is that the base is
converted to a salt, which is relatively soluble in water (e.g. Tribasic
calcium phosphate)
3.7. Solid dispersions:
A solid dispersion may be defined as a dispersion of one or more
active ingredients in an inert carrier or matrix in the solid state prepared
by the melting, solvent, or melting-solvent method (Chiou and
Riegelman, 1971).
3.7.1. Advantages of solid dispersions over other strategies to
improve bioavailability of poorly water soluble drugs:
Improving drug bioavailability by changing their water solubility
has been possible by chemical or formulation approaches (Majerik et al.,
2007; Yoshihashi et al., 2006; Cutler et al., 2006).
Chemical approaches to improving bioavailability without changing
the active target can be achieved by salt formation or by incorporating
polar or ionizable groups in the main drug structure, resulting in the
formation of a pro-drug. Solid dispersions appear to be a better approach
to improve drug solubility than these techniques, because they are easier
to produce and more applicable. For instance, salt formation can only be
used for weakly acidic or basic drugs and not for neutral. Furthermore, it
- 70 -

is common that salt formation does not achieve better bioavailability
because of its in vivo conversion into acidic or basic forms (Serajuddin,
1999; Karavas et al., 2006).
Formulation approaches include solubilization and particle size
reduction techniques, and solid dispersions, among others. Solid
dispersions are more acceptable to patients than solubilization products,
since they give rise to solid oral dosage forms instead of liquid as
solubilization products usually do (Serajuddin, 1999; Karavas et al.,
2006). Milling or micronization for particle size reduction are commonly
performed as approaches to improve solubility, on the basis of the
increase in surface area ( Pouton, 2006; Craig, 2002). Solid dispersions
are more efficient than these particle size reduction techniques, since the
latter have a particle size reduction limit around 2–5
is not enough to improve considerably the drug solubility or drug release
in the small intestine (Pouton, 2006; Karavas et al, 2006; Muhrer et al.,
2006) and, consequently, to improve the bioavailability (Serajuddin,
1999; Karavas et al., 2006; Rasenack and Muller, 2004). Moreover,
solid powders with such a low particle size have poor mechanical
properties, such as low flow and high adhesion, and are extremely
difficult to handle (Pouton, 2006; Karavas et al, 2006; Muhrer et al.,
2006) .
3.7.2. Solid dispersions disadvantages:
Despite extensive expertise with solid dispersions, they are not
broadly used in commercial products, mainly because there is the
possibility that during processing (mechanical stress) or storage
(temperature and humidity stress) the amorphous state may undergo
crystallization (Pokharkar et al., 2006; Van den Mooter et al., 2006;
Chauhan et al., 2005; Vasanthavada et al., 2004). The effect of
- 71 -

moisture on the storage stability of amorphous pharmaceuticals is also a
significant concern, because it may increase drug mobility and promote
drug crystallization (Vasanthavada et al., 2004; Johari et al., 2005).
Moreover, most of the polymers used in solid dispersions can absorb
moisture, which may result in phase separation, crystal growth or
conversion from the amorphous to the crystalline state or from a
metastable crystalline form to a more stable structure during storage. This
may result in decreased solubility and dissolution rate (Van den Mooter
et al., 2006; Wang et al., 2005). Therefore, exploitation of the full
potential of amorphous solids requires their stabilization in solid state, as
well as during in vivo performance (Pokharkar et al., 2006).
3.7.3. The advantageous properties of solid dispersions:
Management of the drug release profile using solid dispersions is
achieved by manipulation of the carrier and solid dispersion particles
properties. Parameters, such as carrier molecular weight and composition,
drug crystallinity and particle porosity and wettability, when successfully
controlled, can produce improvements in bioavailability (Ghaderi et al.,
1999).
3.7.3.1. Particles with reduced particle size:
Molecular dispersions, as solid dispersions, represent the last state
on particle size reduction, and after carrier dissolution the drug is
molecularly dispersed in the dissolution medium. Solid dispersions apply
this principle to drug release by creating a mixture of a poorly water
soluble drug and highly soluble carriers (Leuner and Dressman, 2000).
A high surface area is formed, resulting in an increased dissolution rate
and, consequently, improved bioavailability (Leuner and Dressman,
2000; Bikiaris et al., 2005).
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3.7.3.2. Particles with improved wettability:
A strong contribution to the enhancement of drug solubility is
related to the drug wettability improvement verified in solid dispersions
(Karavas et al., 2006). It was observed that even carriers without any
surface activity, such as urea (Sekiguchi and Obi, 1964) improved drug
wettability. Carriers with surface activity, such as cholic acid and bile
salts, when used, can significantly increase the wettability properties of
drugs. Moreover, carriers can influence the drug dissolution profile by
direct dissolution or co-solvent effects (Pouton, 2006; Leuner and
Dressman, 2000; Kang et al., 2004). Recently, the inclusion of
surfactants (Van den Mooter et al., 2006; Ghebremeskel et al., 2007) in
the third generation solid dispersions reinforced the importance of this
property.
3.7.3.3. Particles with higher porosity:
Particles in solid dispersions have been found to have a higher
degree of porosity (Vasconcelos, and Costa, 2007). The increase in
porosity also depends on the carrier properties, for instance, solid
dispersions containing linear polymers produce larger and more porous
particles than those containing reticular polymers and, therefore, result in
a higher dissolution rate. The increased porosity of solid dispersion
particles also hastens the drug release profile (Ghaderi et al.,1999;
Vasconcelos, and Costa, 2007).
3.7.3.4. Drugs in amorphous state:
Poorly water soluble crystalline drugs, when in the amorphous state
tend to have higher solubility (Pokharkar et al., 2006; Lloyd et al.,
1999). The enhancement of drug release can usually be achieved using
the drug in its amorphous state, because no energy is required to break up
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the crystal lattice during the dissolution process (Taylor and Zografi,
1997) . In solid dispersions, drugs are presented as supersaturated
solutions after system dissolution, and it is speculated that, if drugs
precipitate, it is as a metastable polymorphic form with higher solubility
than the most stable crystal form ( Leuner and Dressman, 2000;
Van den Mooter et al., 2006; Karavas et al., 2006).
For drugs with low crystal energy (low melting temperature or heat
of fusion), the amorphous composition is primarily dictated by the
difference in melting temperature between drug and carrier. For drugs
with high crystal energy, higher amorphous compositions can be obtained
by choosing carriers, which exhibit specific interactions with them
(Vippagunta et al., 2006).
3.7.4.
Method for preparation of solid dispersions:
3.7.4.1.
Melting method:
The physical mixture of a drug in a water soluble carrier is heated
directly until it melts. The melted mixture is then cooled and solidified
rapidly while vigorously stirred. The final solid mass is crushed,
pulverized and sieved. A disadvantage is that many substances either
drugs or carriers may decompose or evaporate during the fusion process
at high temperatures. However, this evaporation problem may be avoided
if the physical mixture is heated in a sealed container. Melting under a
vacuum or blanket of an inert gas such as nitrogen may be employed to
prevent oxidation of the drug or carrier. Another disadvantage is the
drug/carrier immiscibility and the consequent irregular crystallization
may lead to only moderate increases in dissolution rate and difficulties in
formulation (Wrenn and Simeon, 1998). Currently, the melting method
is known as “hot melt technology” and provides pharmaceutical
technologists with new possibilities.
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3.7.4.1.1. Direct melt filling:
In 1978, Francois and Jones, further developed the solid dispersion
method by directly filling hard gelatin capsules with semisolid materials
as a melt, which solidified at room temperature. Catham 1987 reported
the possibility of preparing PEG-based solid dispersions by filling drug-
PEG melts into hard gelatin capsules.
3.7.4.1.2
Melt extrusion:
Melt extrusion is a new method for producing solid dispersions.
Special equipment is needed to develop the dosage form solid dispersions,
which limits the use of the extrusion method. Forster at al., 2002,
reported the use of melt extrusion to prepare glass solutions of poorly
water-soluble drugs with hydrophilic excipients. It is claimed that the
method is an improvement to existing formulation methods such as spray-
drying and co-melting because it uses smaller quantities of drug reduces
particle size and speeds up the formulation process (Breitenbach 2002).
3.7.4.1.3.
Hot spin melting
method:
A further alternative for processing thermolabile substances is by hot
spin melting. Here, the drug and carrier are melted together over an
extremely short time in a high-speed mixer and, in the same apparatus,
dispersed in air or an inert gas in a cooling tower. Some drugs have been
processed into solid dispersions using hot-spin-meIting include
progesterone (Fricke et al., 1995) and dienogest (Kaufmann et al.,
1995).
3.7.4.2.
Solvent method:
Prepared by dissolving a physical mixture of two solid components
in a common solvent, followed by evaporation of the solvent. The choice
of solvent and its removal rate are critical to the quality of the dispersion.
- 75 -

The main advantage of the solvent method is that thermal decomposition
of drugs or carriers may be prevented because of the low temperature
required for the evaporation of organic solvents. However , some
disadvantages associated with this method are the high cost of preparation,
the difficulty in completely removing liquid solvent , the possible adverse
effect of its supposedly negligible amount of the solvent on the chemical
stability of the drug (Wrenn and Simeon , 1998).
Mallick et al., 2003, prepared albendazole solid dispersions by
solvent evaporation technique using water soluble carriers such as
polyethylene glycol and polyvinyl pyrrolidone.
3.7.4.3.
Melting-solvent
method:
Prepared by first dissolving a drug in a suitable liquid solvent and
then incorporating the solution directly into a melt of carrier. The fluid is
then cooled to room temperature. Such a unique method possesses the
advantages of both the melting and solvent methods (Craig 1990).
3.7.4.4. Other methods:
Other methods for preparation of solid dispersions including co-
grinding ( Babu et al ., 2002 ) , kneading ( Singh and Udupa 1997 ) ,
spray drying ( Palmieri et al ., 2002 ) , freeze-drying (Emara et al .,
2002 ) and supercritical fluid technique e.g.supercritical C02 ( Moneghini
et al ., 2001).
Sethia and Squillante 2004, compared the physicochemical and
dissolution properties of carbamazepine solid dispersions prepared by
either a conventional solvent evaporation versus supercritical fluid
process. They found that, the supercritical based process produced solid
dispersions with intrinsic dissolution rate better than conventional solid
dispersions.
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3.7.5. Proposed Structures of solid dispersions:
The physicochemical structures of solid dispersions play an
important role in controlling their drug release. Four representative
structures have been outlined as representative of interactions between
carrier and drug.
3.7.5.1.
Simple eutectic mixtures:
No review of solid dispersions would be complete without a brief
description of eutectic mixtures, which are the cornerstone of this
approach to improving bioavailability of poorly soluble compounds. A
simple eutectic mixture consists of two compounds that are completely
miscible in the liquid state but only to a very limited extent in the solid
state. Solid eutectic mixtures are usually prepared by rapid cooling of a
co-melt of the two compounds in order to obtain a physical mixture of
very fine crystals of the two components. As shown in (Figure 5), when a
mixture with composition E, consisting of a slightly soluble drug and an
inert, highly water soluble carrier, is dissolved in an aqueous medium, the
carrier will dissolve rapidly, releasing very fine crystals of the drug
(Sekiguchi and Obi, 1961; Goldberg et al., 1966). The large surface
area of the resulting suspension should result in an enhanced dissolution
rate and thereby improved bioavailability.
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Figure 5: Phase diagram for eutectic system
(reproduced from Castellan, 1983).
3.7.5.2.
Solid solutions:
Solid solutions are comparable to liquid solutions, consisting of just
one phase irrespective of the number of components. Solid solutions of a
poorly water-soluble drug dissolved in a carrier with relatively good
aqueous solubility are of particular interest as a means of improving oral
bioavailability (Schachter et al., 2004). In the case of solid solutions, the
drug’s particle size has been reduced to its absolute minimum viz. the
molecular dimensions (Goldberg et al., 1965). Furthermore, the
dissolution rate is determined by the dissolution rate of the carrier. By
judicious selection of a carrier, the dissolution rate of the drug can he
increased by up to several orders of magnitude. Solid solutions can be
classified according to two methods. First, they can be classified
according to their miscibility (continuous versus discontinuous solid
solutions) or second, according to the way in which the solvate molecules
are distributed in the Solvendum (substitutional, interstitial or
amorphous).
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3.7.5.2.1. Continuous solid solutions
The components are miscible in all proportions. Theoretically, this
means that the bonding strength between the two components is stronger
than the bonding strength between the molecules of each of the individual
components. Leuner and Dressman (2000) stated that solid solutions of
this type have not been reported in most literatures.
3.7.5.2.2.
Discontinuous solid solutions:
The solubility of each of the components in the other is limited. A
typical phase diagram is shown in (Figure 6),
regions of true solid solutions. In these regions, one of the solid
components is completely dissolved in the other solid component. Note
that below a certain temperature, the mutual solubilities of the two
components start to decrease.
Figure 6: Phase diagram for Discontinuous solid solutions
(reproduced from Castellan, 1983).
3.7.5.2.3.
Substitutional crystalline solid solutions:
Classical solid solutions have a crystalline structure, in which the
solute molecules can either substitute for solvent molecules in the crystal
- 79 -

lattice or fit into the interstices between the solvent molecules. A
substitutional crystalline solid dispersion is depicted in (Figure 7).
Substitution is only possible when the size of the solute molecules differs
by less than 15% or so from that of the solvent molecules (Leuner and
Dressman, 2000).
Figure 7: Substitutional crystalline solid solutions.
(reproduced from Chiou and Riegelman, 1971)
3.7.5.2.4.
Interstitial crystalline solid solutions:
In interstitial solid solutions, the dissolved molecules occupy the
interstitial spaces between the solvent molecules in the crystal lattice
(Figure 8) The relative molecular size is a crucial criterion for classifying
the solid solution type. In the case of interstitial crystalline solid solutions,
the solute molecules should have a molecular diameter that is no greater
than 0.59 of the solvent molecule’s molecular diameter (Leuner and
Dressman, 2000). Furthermore, the volume of the solute molecules
should be less than 20% of the solvent.
Figure 8: Interstitial crystalline solid solutions.
- 80 -

(reproduced from Chiou and Riegelman, 1971)
3.7.5.2.5. Amorphous crystalline solid solutions:
In an amorphous solid solution, the solute molecules are dispersed
molecularly but irregularly within the amorphous solvent (Figure
9) .Using griseofulvin in citric acid, Chiou and Riegelman (1969) were
the First to report the formation of an amorphous solid solution to
improve a drug’s dissolution properties. Polymer carriers are particularly
likely to form amorphous solid solutions, as the polymer itself is often
present in the form of an amorphous polymer chain network. In addition,
the solute molecules may serve to plasticize the polymer, leading to a
reduction in its glass transition temperature.
Figure 9: Amorphous crystalline solid solution
(reproduced from Kreuter, 1999)
3.7.5.3.
Glass solution and glass suspensions:
A glass solution is a homogenous glassy system in which a solute
dissolves in a glassy solvents e.g. sugars, citric acid. It is often
characterized by transparency and brittleness below the glass transition
temperature. The lattice energy in glass solution is less than in solid
solutions because of its similarity with liquid solutions. Consequently,
faster dissolution rates of drugs from their glass solutions are expected
compared to those from solid solutions (Mummaneni and Vasavada,
1990).
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3.7.5.4. Combination of systems:
The possibility exists that some or indeed all systems may show
characteristics of more than one of the above structures. For example, the
formation of eutectic mixtures must involve solid-state complexation to
some extent (Juppo et al., 2003).
3.7.6.
Carriers for solid dispersions:
The carrier used for solid dispersion formulation has been a water-
soluble or water miscible polymer such as polyethylene glycol (PEG) or
polyvinylpyrrolidone (PVP) or low molecular weight materials such as
sugars. However, the proliferation of publications in the area since the
first solid dispersions were described (Sekiguchi and Obi, 1961) has led
to a broadening of these definitions to include water insoluble matrices
such as Gelucires and Eudragits that may yield either slow or rapid
release. Consequently, the properties of the carrier have a great influence
on the dissolution characteristics of the dispersed drug. A carrier, as
suggested by Kerc et al. (1998), should be: i) freely water soluble with
intrinsic rapid dissolution properties; ii) non-toxic and pharmacologically
inert; iii) chemically compatible with the drug and in the solid-state
should not form strongly bonded complexes that could reduce the
dissolution rates; iv) preferably, can increase the aqueous solubility of the
drug; v) soluble in a variety of organic solvents (for carriers intended for
solvent processes); and vi) chemically, physically and thermally stable
with a low melting point to avoid the use of excessive heat during
dispersion preparation(for carriers intended for fusion processes). With
references to these criteria there now follows brief review of the carriers
described in the literature with particular emphasis on their potentials and
limitations.
- 82 -

3.7.6.1.
Polyethylene glycol:
Polyethylene glycols are polymers of ethylene oxide, with a
molecular weight usually falling in the range 200: 300000. For the
manufacture of solid dispersions and solutions, PEGs with molecular
weights of 1500:35000 are usually employed. As the molecular weight
increases, so does the viscosity of the PEG. At molecular weight of up to
600, PEGs are fluid, in the range 800: 1500 they have a consistency that
is best described as vaseline like, from 2000 to 6000 they are waxy and
those of 20 000 and above form hard, brittle crystals at room temperature.
Their solubility in water is generally good. Furthermore a particular
advantage of PEGs for the formation of solid dispersions is that they also
have good solubility in many organic solvents. The melting point of the
PEGs of interest lies under 65°C in every case (e.g. the m.p. of PEG 1000
is 30: 40°C, the m.p. of PEG 4000 is 50: 58°C and the m.p. of PEG 20000
is 60:63°C) (Price, 1994). These relatively low melting points are
advantageous for the manufacture of solid dispersions by the melting
method. Additional attractive features of the PEGs include their ability to
solubilize some compounds (Fini et al., 2005) as well as to improve
compound wettability (Ambike et al., 2004). Even the dissolution rate of
a relatively soluble drug like aspirin can be improved by formulating it as
a solid dispersion in PEG 6000 (Corrigan et al., 1979).
3.7.6.2.
Polyvinylpyrrolidone:
Polymerization of vinylpyrrolidone leads to polyvinylpyrrolidone
(PVP) of molecular weights ranging from 2500 to 3000000 (Walking,
1994).
Similarly to the PEGs, PVPs have good water solubility and can
improve the wettability of the dispersed compound in many cases
(Mendyk and Jachowicz, 2005). Improved wetting and thereby an
- 83 -

improved dissolution rate from a solid dispersion in PVP has been
demonstrated for tolbutamide. The chain length of the PVP has a very
significant influence on the dissolution rate of the dispersed drug from the
solid dispersion. The aqueous solubility of the PVPs becomes poorer with
increasing chain length and a further disadvantage of the high MW PVPs
is their much higher viscosity at a given concentration (Takeuchi et al.,
2004).
3.7.6.3.
Urea:
Urea is the final product of human protein metabolism. Its solubility
in water is greater than 1 in 1 and it exhibits good solubility in many
common organic solvents. It has a relatively low melting point of 131 °C.
Consequently, both solvent and fusion processes could be used to prepare
urea dispersions. In one of the first bioavailability studies of solid
dispersions, it was shown that sulphathiazole was better absorbed in
rabbits when given as eutectic mixture with urea (Sekiguchi and Obi,
1961). Although urea is not often used as a carrier these days, it has been
shown that the dissolution rate of the poorly soluble compound ofloxacin
can be improved by more than three fold by incorporating it in a
coevaporate with urea (Okonogi et al., 1997). Similarly, urea was used in
combination with PEG to increase the dissolution rate of piroxicam (Pan
et al., 2000).
3.7.6.4.
Sugars:
Although sugars and related compounds are highly water-soluble
and have few, if any, toxicity issues, they are less suitable than other
carriers for the manufacture of solid dispersions. The melting point of
most sugars is high, making preparation by the hot melt method
- 84 -

problematic, and their solubility in most organic solvents is poor, making
it difficult to prepare co-evaporates.
3.7.6.5.
Chitosan:
Chitosan, a derivative of the polysaccharide chitin that is formed by
deacetylation at the N position, has also been used as a carrier in solid
dispersions. It exhibits good biocompatibility and safety after oral and
parenteral administration. Low molecular weight chitosan is a good
candidate as a carrier for enhancing the dissolution and bioavailability of
a number of poorly water soluble drugs (Asada et al., 2004; Takahashi
et al., 2005).
3.7.6.6.
Emulsifiers:
The release behaviour of many drugs can also be improved by the
use of emulsifying agents. Two mechanisms are possible here:
improvement of wetting characteristics and solubilisation of the drug.
Owing to their potential toxicity problems, such as damage to mucosal
surfaces, they are usually used in combination with another carrier. For
example, the release of naproxen from solid dispersions in PEG 4000,
6000 and 20000 could be further enhanced when either sodium lauryl
sulphate or Tween® 80 was added to the system (Mura et al., 1999).
Inclusion of alkali dodecylsulphate surfactants in carrier systems can lead
to conversion of a solid dispersion to a solid solution. Melts of
griseofulvin and PEG 6000 normally contain crystalline areas but in the
presence of sodium lauryl sulphate, a solid solution is formed (Wulff et
al., 1996).
3.7.6.7.
Other carriers:
- 85 -

Many other substances have been tested as carriers for solid
dispersions. A hydrolysis product of collagen, Gelita® Collagel, was
reported to improve the release rate of oxazepam by a factor of six when
prepared as a solid dispersion by spray drying (Jachowicz et al., 1993).
Even after tabletting, the solid dispersion displayed better release
characteristics than the physical mixture or the drug powder alone
(Jachowicz and Nurnberg, I 997). Other materials tested include
phospholipids (Sammour et al., 2001), inulin (Visser et al., 2004), silica
(Watanabe et al., 2003) ......etc.
3.7.7. Characterization of solid dispersions
The methods that have been used to characterize solid dispersions
are summarized in (Table1). In addition to characterizing the solid
dispersion, these methods can be used to differentiate between solid
solutions (molecularly dispersed drug), solid dispersions and physical
mixtures of drug and carrier. It is usually assumed that dispersions in
which no crystallinity can be detected are molecularly dispersed. The
absence of crystallinity is used as a criterion to differentiate between solid
solutions and solid dispersions (Leuner and Dressman, 2000).
Table 1: Methods for the characterization of solid dispersion.
1- Dissolution testing
2- Thermoanalytical methods: * Differential thermoanalysis (DTA).
* Differential scanning caloimetry (DSC).
* Hot stage microscopy.
3- X-Ray diffraction (XRD).
4- Spectroscopic methods, e.g. FTIR spectroscopy.
5- Microscopic methods: * Polarization microscopy.
* Scanning electron microscopy (SEM).
- 86 -

3.7.7.1. Dissolution testing:
When the goal of preparing a solid dispersion is to improve the
dissolution characteristics of the drug in question, the results of the
release rate experiments are obviously of prime importance in assessing
the success of the approach. A well-designed release experiment will
show whether the solubility of the drug and its dissolution rate has been
enhanced, as well as whether the resulting supersaturated solution is
stable or tends to precipitate quickly. Comparison of results with those for
pure drug powder and physical mixtures of the drug and carrier can help
to indicate the mechanism by which the carrier improves dissolution
(Leuner and Dressman, 2000).
3.7.7.2.
Thermoanalytical methods:
It includes all methods that examine a characteristic of the system as
a function of temperature. Differential scanning calorimetry (DSC) is the
most highly regarded method. DSC enables the quantitative detection of
all processes in which energy is required or produced (i.e. endothermic
and exothermic phase transformations). The usual method of
measurement is to heat the reference and test samples in such a way that
the temperature of the two is kept identical. The additional heat required
is recorded and used to quantitate the energy of the phase transition.
Exothermic transitions, such as conversion of one polymorph to a more
stable polymorph, can also be detected. Lack of a melting peak in the
DSC of a solid dispersion indicates that the drug is present in an
amorphous rather than a crystalline form. Since the method is quantitative
in nature, the degree of crystallinity can also be calculated for systems in
which the drug is partly amorphous and partly crystalline. However,
crystallinities of fewer than 2% cannot generally be detected with DSC
(Kreuter, 1999).
- 87 -

3.7.7.3.
X-ray
diffraction ( XRD):
The principle behind X-ray diffraction is that when an X-ray beam is
applied to the sample, interference bands can he detected. The angle at
which the interference bands can be detected depends on the wavelength
applied and the geometry of the sample with respect to periodicities in the
structure. Crystallinity in the sample is reflected by a characteristic
fingerprint region in the diffraction pattern. Owing to the specificity of
the fingerprint, crystallinity in the drug can be separately identified from
crystallinity in the carrier. Therefore, it is possible with X-ray diffraction
to differentiate between solid solutions, in which the drug is amorphous,
and solid dispersions, in which it is at least partly present in the
crystalline form, regardless of whether the carrier is amorphous or
crystalline. However, crystallinities of less than 5- 10% cannot generally
be detected with X- ray diffraction (Villiers et al., 1998).
3.7.7.4.
Infrared spectroscopy:
Structural changes and lack of a crystal structure can lead to changes
in bonding between functional groups that can be detected by infrared
spectroscopy. Since not all peaks in the IR spectrum are sensitive to
crystalline changes, it is possible to differentiate between those that are
sensitive to changes in crystallinity and those that are not (Taylor and
Zografi, 1997).
- 88 -

1- Materials and supplies:
Experiment and methodology
* Gliclazide was kindly supplied by Egyptian International
Pharmaceutical
Industries Company (EIPICO)
* Chloroform, glucose, methanol and urea (El-Gomhouria Co.,
Egypt).
* Polyethylene glycol 4000, 6000 (Hoechest Chemikalien, Werk Gendort,
Germany).
2- Equipment:
* UV/VIS spectrophotometer (Schimadzu U.V.-1201, Cat NO.
206-62409, Schimadzu Corporation, Japan).
* Thermostatic shaker water bath (Julpo SW 20C, Japan).
* Vacuum oven (Lab-line instruments, Inc., USA).
* Dissolution tester, rotating paddle (Erweka RT6- Frankfurt,
Germany).
* Perkin-Elmer FTIR spectrophotometer (1600 series, Perkin-Elmer
Corporation, Norwalk, USA).
* Differential scanning calorimeter (model 50, Schimadzu,
Japan).
* D-5000 x- ray diffractometer (Kristallofex D-5000 Powder
Diffractometer, Siemens, Germany).
Set of sieves (Mettler, Germany).
Electronic Digital Balance (Mettrt-Toledo, Ag,CH 8606,
Greifensee,Switzerland).
* Buchi rotavapor R-3000, (Switzerland).
- 89 -

3- Software:
Microsoft Office XP, Microsoft Corporation, USA.
4. Methods:
4.1. UV scanning of Glz:
Ten mg of Glz were dissolved in 100 ml methanol to obtain a
solution; 1 ml is diluted to 10 ml with methanol to produce a solution
containing 10 μg /ml of Glz in methanol. The obtained solution was
scanned spectrophotometerically from 200 to 400 nm using methanol as
blank.
4.2. Construction of calibration curve of Glz in methanol:
0.1 gram of Glz was dissolved in 100 ml methanol to obtain a
solution, 2.5 ml is diluted to 25 ml with methanol to produce a solution
containing 100 μg /ml of Glz. Aliquots of 1, 1.5, 2, 2.5, and 3 ml were
further diluted to 10 ml with methanol. After dilution, the solution
contained 10, 15, 20, 25, and 30 μg/ml of Glz respectively.
The calibration equation was constructed by regressing the relative
absorbances against the corresponding Glz solutionsconcentrations at
227 nm using methanol as blank.
4.3. Construction of calibration curve of Glz in S
buffer of pH 7.4:
0.1 gram of Glz was dissolved in 5 ml methanol, then completed to
100 ml with sörensen’ buffer. 2.5 ml is diluted to 25 ml with methanol to
produce a solution containing 100 μg /ml of Glz . Aliquots of 0.5, 0.75, 1,
1.5, 2, and 2.5 ml were furtherly diluted to 10 ml with sörensen’ buffer.
After dilution, the solution contained 5, 7.5, 10, 12.5, 15, 20, and 25
μg/ml of Glz respectively. The calibration equation was constructed by
- 90 -

egressing the relative absorbances against the corresponding Glz
solutionsconcentrations at 227 nm using sörensen’phosphate buffer as
blank.
4.4. Preparation of solid dispersions:
Gines’ et al., 1996, stated that, the technology employed to prepare
the solid dispersion, the proportion and properties of the carrier used
present an important influence on the properties of the resulting (SD). So,
in this study different types and proportions of carriers were examined
(Table 2).
Table 2: Types of carriers and their ratios in Glz solid dispersions
and physical mixtures.
Carrier Drug: Carrier weight ratio Solvent used
PEG 6000 10:90 8:92 5:95 1:99 Chloroform
PEG 4000 10:90 8:92 5:95 1:99 Chloroform
Glucose 1:1 1:2 1:3 1:5 1:10 Methanol
Urea 1:1 1:2 1:3 1:5 1:10 Methanol
Types of solvent used in solvent evaporation method.
4.4.1. Preparation of urea solid dispersions:
The calculated amounts of Glz with UR were dissolved in methanol
with continuous stirring in a dish followed by evaporation of the solution
under vacuum at 40°C. Dispersions were dried in a vacuum oven at room
temperature for 24 hr. The dry products were removed from the
containers and ground in laboratory mortar (Etman, 2000).
- 91 -

4.4.2. Preparation of glucose solid dispersions:
The co-precipitates were prepared using solvent evaporation
method. The calculated amounts of Glz and glucose were dispersed
homogeneously in the least amount of methanol at 40°C, and the solvent
was evaporated at 40°C under vacuum. The obtained co-precipitates were
dried in a vacuum oven at room temperature for 24 hr, and then the dried
mass was pulverized (Greenhalgh et al., 1999)
4.4.3. Preparation of PEG 4000 and PEG 6000 solid dispersions:
The required amounts of Glz and PEG 4000 or PEG 6000 were
accurately weighted and dissolved in chloroform. Mixtures were
evaporated using a rotary evaporator at 45°C and further drying was
performed using a vacuum dessicator for 48 hours at room temperature
.Subsequently, the dispersions were pulverized in a mortar (Law et al.,
1992).
4.5. Preparation of physical mixtures:
(PMs) were prepared simply by triturating appropriate quantities of
Glz and carriers using a porcelain mortar and a pestle, then transferring to
a vacuum dessicator until ready for use.
*** All samples were sieved. Powdered samples below 420 um (40
mesh) were stored in closed containers away from the light and humidity
until use.
4.6. Solubility measurements:
4.6.1. Effect of different carriers on the solubility of Glz:
- 92 -

Solubility studies were carried out according to the method of
Higuchi and Connors, (1965). An excess of the Glz (10 mg) was placed
into 25-ml glass vial containing various concentrations of each carrier,
ranging from 1 to 7 %, in 10 ml distilled water. All glass vials were
closed with stopper and cover-sealed with cellophane membrane to avoid
solvent loss .The content of the suspension was equilibrated by shaking in
a thermostatically controlled water bath at 25°C for 72 hr.
After attainment of equilibrium, the content of each vial was then
filtered through a double filter paper (Whatman 42). The filtrate was
suitably diluted and assayed spectrophotometrically at 227 nm to measure
the amount of dissolved drug. There was no interference from all the used
carriers at this wavelength except urea interfered with analysis of Glz,
thus the solutions containing urea were measured against a blank of urea.
The average of triplicate measurements was reported. The solubility of
Glz alone in water at the same temperature was also determined following
the same procedure mentioned above.
4.6.2. Effect of pH change on the solubility of Glz:
The solubility of Glz in Sorensen' phosphate buffer with different
pH ranging from 5 to 7.4 at the same temperature was also determined
following the same procedure mentioned above.
4.7. Dissolution studies:
The dissolution of Glz from the prepared (SDs), and (PMs) was
carried out according to the USP-25, NF 20 (2002), rotating paddle
method. Dissolution medium consisting of 500 ml distilled water. The
stirring rate was 100 rpm and the temperature was maintained at 37 ±
0.5°C. A sample of 40 mg of Glz or its equivalent of the (SDs), or the
- 93 -

(PMs) was placed on the surface of the dissolution medium. At a
appropriate time intervals (5, 10, 20, 30, 45, 60, 90, and 120 min), 5 ml
sample were withdrawn and replaced with an equivalent amount of the
fresh dissolution medium kept at 37°C. The samples were filtered rapidly
through a double layered filter paper (Whatman 42). The filtrates were
suitably diluted and assayed spectrophotometrically at 227 nm without
interference from the carriers. In case of (SDs) containing UR, the
solutions measured against a blank of UR.
The amount of Glz dissolved at different time intervals was calculated
using a standard calibration curve .Each experiment was carried out in
triplicates. The cumulative amount of the drug released was calculated as
follows (AL-Suwayeh, 2003):
Receptor compartment volume = VR
Sample volume withdrawn =5 ml
Sample #1(5min),# 2 (10 min), # 3 (20 min), # 4 (30 min), # 5 (45 min), #
6 (60 min), # 7 (90min), # 8 (120min).
Concentration C1 (5 min), C2 (10 min), C3 (20 min), C4 (30 min), C5
(45 min), C6 (60 min), C7 (90 min), C8 (120 min).
Cumulative amount at sample # 1 (5 min) = VR X C1
Cumulative amount at sample # 2 (10 min) = VR X C2 +5 ml X (C1)
Cumulative amount at sample # 3 (20 min) = VR X C3 + 5 ml X (C1+
C2)
Cumulative amount at sample # 4 (30min) = VR X C4 + 5 ml X
(C1+C2+C3). And so on.
- 94 -

4.8. Fourier transform infrared (FTIR) spectroscopy:
FTIR spectra were obtained on a Prekin-Elmer 1600 FTIR
spectrophotometer using KBr disc method. The scanning range was 400-
4000 cm -1 .
4.9. Differnntial scanning calorimetry (DSC):
The DSC thermograms were recorded on a Schimadzu-DSC 50.
Samples (1.3 mg) were heated in hermetically sealed aluminum pans over
the temperature range 50-200°C at a constant rate of 10°C/min under a
nitrogen purge 30 ml/min.
4.10. X-ray diffraction:
X-ray diffraction patterns were obtained using a Siemens
Kristallofex D-
of 0-80°.
- 95 -

1. UV scanning of Glz:
Results and discussion
UV scanning of Glz in methanol was carried out (Figure 11). Two
absorption maxima were observed at 227 nm and 273 nm The ratio of the
maxmax 273 nm is
0.825 to 0.034. So, measurements were done at 227 nm (Tadeusz et al.,
2005).
Wavelength
Figure 11: UV spectra of Glz in methanol.
2. Calibration curves of Glz in methanol and sörensen’s phosphate
buffer pH 7.4:
(Figures 12, 13) show a linear relationship between the absorbance
and the concentration of Glz in either methanol or in sörensen’s
max in the
concentration range used.
- 96 -

Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
y = 0.0329x + 0.0233
R 2 = 0.9973
R= 0.9986
0 5 10 15 20 25 30 35
Concentration (ug/ml)
Figure 12: Calibration curve of Glz in methanol at max 227 nm.
Absorbance
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.0361x + 0.0296
R 2 = 0.9887
R= 0.9943
0 5 10 15
concentration (ug/ml)
20 25 30
Figure 13 : Calibration curve of Glz in phosphate buffer (7.4) at max
227 nm.
- 97 -

3. Solubility measurements:
3.1. Effect of different carriers on the solubility of Glz :
In the present study, the solubility of Glz in distilled water at 25°C
was found to be 42.13 μg /ml.
(Figure 14, 15) depict the effect of different carriers on Glz
solubility in distilled water at 25°C. In case of PEG 4000, 6000, the
solubility of Glz linearly increased as the carrier concentration increased,
showing the feature of an AL-type solubility phase diagram (Higuchi and
Corner, 1965) .This result illustrates that the complex formed was
soluble and did not form a precipitate over the range of carrier
concentration.
As shown in (Table 3), the solubilizing power of PEGS slightly decreased
with increasing PEG molecular weight. As the solubility factors were 1.9
and 1.6 for PEG 4000 and PEG 6000, respectively. This is in accordance
with Mura et al.,1999, who found that, the solubility of naproxen is
affected by PEG molecular weight as the solubilizing power of PEG 4000
> PEG 6000 > PEG 20,000.
On other hand the solubility plot of glucose, and urea showed a Bs-type
curve (Higuchi and Corner, 1965). The initial rising portion was
followed by plateau region and finally a decrease in total concentration of
Glz. As shown in (Table 3), the solubility factor were 1.28 and 1.4 for
glucose and urea, respectively. Consequently, these carriers can be ranked
according to its effect on increasing the solubility of Glz as PEG 4000
PEG 6000 glucose urea. The increased solubility of Glz in carrier's
solution may be attributed to both complex formation and reduction in
interfacial tension of water and hence intermolecular forces and polarity
caused by the presence of these carriers (Al-Angary et al., 1996).
- 98 -

Table 3: Solubility enhancement data of Glz in various carrier
solutions at 25°C.
Item Glz PEG-
Phase solubility
diagram type
Optimum
carrier
concentration
%(w/v)
Solubility
(ug/ml)
Solubility factor
a
4000
- 99 -
PEG-
6000
Glu UR
----- AL AL BS BS
------ 7% 7% 3% 5%
42.13 80.23 67.63 54.3 48.19
------- 1.9 1.6 1.28 1.14
Solubility factor a = Total solubility / intrinsic solubility.

Solubility (ug/ml)
Solubility (ug/ml)
90
80
70
60
50
40
30
20
10
0
0 1 2 3 4
Carrier % (w/v)
5 6 7 8
- 100 -
PEG 4000
PEG 6000
Figure 14: Phase solubility diagram of Glz in water
at 25°C in presence of PEG 4000 and PEG 6000.
100
90
80
70
60
50
40
30
20
10
0
Urea Glucose
0 1 2 3 4 5 6 7 8
Carrier % (w/v)
Figure 15: Phase solubility diagram of Glz in water
at 25°C in presence of glucose and urea.

3.2. Effect of pH change on the solubility of Glz:
Table 4 demonstrate the solubility of Glz in different pH's. Glz
contains an -hydroxyl secondary amine, with a pKa of 7.8. It exhibits
pH dependent solubility. It can be noted that the solubility increased with
increasing pH (higher in alkaline rather than acidic one). This can be
attributed to the effect of pH on the degree of the ionization and hence the
solubility of the drug.
Table 4: Effect of change in pH on the solubility of Glz.
4. Dissolution studies:
pH Solubility (g/ml)
5 40.11 ± 0.26
5.6 52.34 ± 0.22
6 156.77 ± 2.07
6.4 227.4 ± 5.02
7.4 745.5 ± 13.45
The dissolution profiles of pure Glz, its physical mixtures and solid
dispersions with different carriers are shown in (Figures 16-19) Data are
average of three measurements. The shapes of the dissolution profiles
were examined using the following parameters:
I) The initial dissolution rate (IDR) calculated as percent dissolved of the
- 101 -

drug over the first twenty minutes per minutes.
II) The percentage of the drug dissolved after 20 and 60 minutes (PD20
and
PD60).
III) The dissolution efficiency (DE %) parameter after sixty minutes
(Arias
et al., 1995).
The dissolution efficiency can be defined as the area under the curve
up to a certain time. It is measured using the trapezoidal method and is
expressed as a percentage of the area of the rectangle described by 100%
dissolution in the same time (Torrado et al., 1996).
The calculated dissolution parameters revealed that, pure Glz
yielded the slowest dissolution rate with only about 28.4 % of the drug is
dissolved in 120 min. The hydrophobic property of Glz prevented its
contact with the dissolution medium which led to a slow dissolution rate
(Tantishaiyakul et., al 1996).
As shown in Tables 5-8, all (PMs) released the Glz at the faster rate than
the drug alone as reflected by higher (IDR) and greater extent of
dissolution after 120 min. These results can be explained on the basis that
the dry mixing brings the drug in close contact with the hydrophilic
polymer (Van den Mooter et al., 1998). Also may be due to; a possible
solubilization effect by the carrier operating the microenvironment
(diffusion layer) immediately surrounds the drug particle in the early
stage of solubilization (Arias et al., 1996). Indeed, during dissolution
experiments, it was noticed that (PMs) immediately sink to the bottom of
the dissolution vessels as (SDs) do.
- 102 -

Table 5: Dissolution parameters (±SD) of gliclazide in distilled water from different gliclazide - PEG 6000
systems.
DE*100
(%)
PD60
(%)
PD20
(%)
Composition (w/w) IDR
(%dissolved/min)
Gliclazide powder 0 ± 0 0 ± 0 13.05 ± 1.2 3.83 ± 0.06
Gliclazide-to-PEG 6000
PM 10:90
1.12 ± 0.12 22.58 ± 0.4 38.08 ± 0.19 24.44 ± 0.64
SD 10:90
3.27 ± 0.07 65.53 ± 1.4 72.89 ± 1.5
63.54 ± 1.1
PM 8:92
1.12 ± 0.09 22.59 ± 1.9 36.89 ± 0.18 25.30 ± 0.96
SD 8:92
3.2 ± 0.04 64.04 ± 0.87 69.29 ± 1.76 60.83 ± 0.91
PM 5:95
1.08 ± 0.08 21.63 ± 1.6 38.1 ± 1.2 24.82 ± 1.1
SD 5:95
3.66 ± 0.04 73.25 ± 0.9 93.23 ± 0.42 76.45 ± 0.30
34.76 ± 0.65
89.78 ± 3.48
41.19 ± 0.49
100.49 ± 1.00
35.79 ± 0.48
92.36 ± 1.5
1.79 ± 0.02
4.6 ± 0.07
PM 1:99
SD 1:99
IDR = Initial dissolution rate.
PD20 = Extent of dissolution after 20 min.
PD60 = Extent of dissolution after 60 min.
DE% = Dissolution efficiency after 60 min.
103

plain drug 10:90 PM 10:90 SD 8:92 PM 8:92 SD 5:95 PM
5:95 SD 1:99 PM 1:99 SD
120
100
80
60
40
% Drug dissolved
20
0
0 20 40 60 80 100 120
Time (min)
Figure 16: Dissolution profile of gliclazide-PEG 6000 systems.
104

Table 6: Dissolution parameters (±SD) of gliclazide in distilled water from different gliclazide - PEG 4000
systems.
Composition (w/w) IDR
PD20
PD60
DE*100
(%dissolved/min) (%)
(%)
(%)
Gliclazide powder 0 ± 0 0 ± 0 13.05 ± 1.2 3.83 ± 0.06
Gliclazide-to-PEG 4000
PM 10:90 1.08 ± 0.032 21.69 ± 0.65 38.39 ± 0.19 25.38 ± 1.09
SD 10:90 1.62 ± .047 32.53 ± 0.45 42.32 ± 1.5
34.58 ± 0.85
PM 8:92
1.2 ± 0.013 24.13 ± 1.69 36.49 ± 0.18 26.99 ± 1.7
SD 8:92
2.07 ± 0.028 64.04 ± 0.57 50.85 ± 1.76 42.23 ± 0.28
PM 5:95
1.02 ± 0.11 20.75 ± 1.3 33.67 ± 1.2 23.26 ± 1.6
SD 5:95
3.17 ± 0.04 63.49 ± 0.89 64.29 ± 0.42 61.81 ± 0.44
PM 1:99
1.77 ± 0.004 35.41 ± 0.08 43.98 ± 0.49 36.07 ± 1.9
SD 1:99 3.34 ± 0.075 66.94 ± 1.5 68.58 ± 1.56 62.8 ± 1.9
IDR = Initial dissolution rate.
PD20 = Extent of dissolution after 20 min.
PD60 = Extent of dissolution after 60 min.
DE% = Dissolution efficiency after 60 min.
105

plain drug 10:90 PM 10:90 SD 8:92 PM 8:92 SD 5:95 PM
5:95 SD 1:99 PM 1:99 SD
100
90
80
70
60
50
40
% Drug dissolved
30
20
10
0
0 20 40 60 80 100 120
Time (min)
Figure 17: Dissolution profile of gliclazide-PEG 4000 systems.
106

It is also apparent that, the rate and the extent of dissolution of Glz from
(SDs) exceeded those of pure Glz or the corresponding (PMs). The DE%
of (8:92) Glz-PEG 6000 co-precipitate (Table 5), for example, was
60.83%.While, the DE% of the corresponding physical mixture was only
25.3%.
The observed higher dissolution of the prepared (SDs) could
possibly due to the solubilizing effect of the carriers that may be operate
in the diffusion layer immediately surrounding the drug particles. Also,
each single crystallite of the drug was very intimately encircled by the
soluble carrier particles which can readily dissolve and cause the aqueous
medium to contact and wet the drug particles easily (Etman, 2000).
Moreover, it can be generally assumed that the increased dissolution via
(SDs) could be explained on the basis of alterations in the solid-state
structures of the carriers and the drug particles. These structural changes
include the formation of solid solution, eutectic mixtures or soluble
complex between the drug and the carriers and formation of amorphous
drug particles or loss of crystallinity of the drug. For most (SDs), more
than one of these factors may probably be responsible for the dissolution
enhancement (Trapani et al., 1999; Mura et al., 1999). Therefore, the
IR spectra, differential scanning calorimetry and x-ray diffraction patterns
of the pure drug, carriers and their (PMs) and (SDs) were performed.
4.1. Effect of different carriers on the dissolution of Glz from
(SDs):
PEG 6000 had the most influential effect on the rate and the extent
of dissolution of Glz, followed by PEG-4000, glucose and finally urea.
107

Table 7: Dissolution parameters (±SD) of gliclazide in distilled water from different gliclazide – glucose
systems.
Composition (w/w) IDR
PD20
PD60
DE*100
(%dissolved/min) (%)
(%)
(%)
Gliclazide powder 0 ± 0 0 ± 0 13.05 ± 1.2 3.83 ± 0.06
Gliclazide-to-Glucose
PM 1:1
1.12 ± 0.038 22.51 ± 0.65 32.13 ± 1.03 22.84 ± 0.75
SD 1:1
1.16 ± .045 23.23 ± 0.45 40.1 ± 1.18
25.52 ± 0.05
PM 1:2
1.03 ± 0.067 20.66 ± 1.69 34.18 ± 01.15 24.08 ± 1.4
SD 1:2
1.36 ± 0.028 27.27 ± 0.57 43.51 ± 1.30 29.51 ± 0.51
PM 1:3
1.06 ± 0.025 21.35 ± 1.3 38.53 ± 0.4 25.74 ± 0.54
SD 1:3
1.37 ± 0.027 27.58 ± 0.89 42.16 ± 0.8 32.45 ± 0.25
PM 1:5
1.42 ± 0.022 28.42 ± 0.08 42.76 ± 0.81 30.05 ± 0.43
SD 1:5
1.61 ± 0.075 32.38 ± 1.5 46.58 ± 1.00 35.02 ± 0.79
PM 1:10
1.3 ± 0.006 26.07 ± 0.13 41.68 ± 1.51 31.56 ± 1.29
SD 1:10
1.8 ± 0.004 36.04 ± 0.09 45.56 ± 1.06 37.05 ±0.87
IDR = Initial dissolution rate
PD20 = Extent of dissolution after 20 min.
PD60 = Extent of dissolution after 60 min.
DE% = Dissolution efficiency after 60 min.
108

Plain drug 1:1PM 1:1 SD 1:2PM 1:2 SD 1:3PM
1:3 SD 1:5PM 1:5 SD 1:10PM 1:10 SD
100
90
80
70
60
50
40
% Drug Released
30
20
10
0
0 20 40 60 80 100 120
Time (min)
Figure 18: Dissolution profile of gliclazide-glucose systems.
109

Table 8: Dissolution parameters (±SD) of gliclazide in distilled water from different gliclazide –urea
systems.
Composition (w/w) IDR
PD20
PD60
DE*100
(% dissolved/min) (%)
(%)
(%)
Gliclazide powder 0 ± 0 0 ± 0 13.05 ± 1.2 3.83 ± 0.06
Gliclazide-to-Glucose
PM 1:1 0.28 ± 0.08 5.6 ± 0.60 24.53 ± 1.05 11.85 ± 1.3
SD 1:1 0.68 ± 0.05 13.56 ± 1.005 24.81 ± 1.15
16.49
± 1.03
PM 1:2 0.67 ± 0.016 13.65 ± 0.32 25.8 ± 0.69 16.25 ± 0.02
SD 1:2
0.95 ± 0.13 19.18 ± 1.6 34.31 ± 1.9 22.48 ± 1.6
PM 1:3
0.74 ± 0.033 14.88 ± 0.67 29.01 ± 0.45 17.93 ± 0.64
SD 1:3
1.29 ± 0.07 25.99 ± 1.44 36.45 ± 1.4 27.11 ± 1.003
PM 1:5
0.78 ± 0.01 15.78 ± 0.38 29.3 ± 0.29 18.66 ± 0.09
SD 1:5
1.28 ± 0.07 25.70 ± 1.5 35.06 ± 1.00 26.81 ± 1.79
PM 1:10 1.09 ± 0.05 21.96 ± 1.055 34.9 ± 0.35 25.15 ± 0.52
SD 1:10 1.42 ± 0.03 28.51 ± 0.74 39.18 ± 0.24 31.22 ±1.47
IDR = Initial dissolution rate.
PD20 = Extent of dissolution after 20 min.
PD60 = Extent of dissolution after 60 min.
DE% = Dissolution efficiency after 60 min.
110

Plain drug 1:1PM 1:1 SD 1:2PM 1:2 SD 1:3PM
1:3 SD 1:5PM 1:5 SD 1:10PM 1:10 SD
100
90
80
70
60
50
40
% Drug Released
30
20
10
0
0 20 40 60 80 100 120
Time (min)
Figure 19: Dissolution profile of gliclazide-urea systems.
111

The DE% after 60 minutes was found to be 89.78%, 62.8%, 37.05%
and 31.22% from (1:99) PEG 6000, (1:99) PEG 4000, (1:10) glucose, and
(1:10) urea solid dispersions respectively.
This is in agreement with the results of the phase solubility diagram,
as it was observed that, the solubility of Glz in PEGs solutions was more
than that of glucose and urea. Although the solubility factor of PEG 4000
was more than PEG 6000, it was found that PEG 6000 is a better carrier
than PEG 4000. This is in an agreement with (Mura et al., 1999), who
found that the dissolution capacity of PEG 20000
4000 although the solubilizing power of PEG 4000
20000. This may be due to the higher viscosity of dissolution medium
provided by the PEG 6000 than PEG 4000 retards aggregation and
agglomeration of drug particles (Doshi, 1997).
4.2. Effect of carrier concentration on the dissolution of Glz from
(SDs):
The dissolution data of Glz from its different systems suggested that,
drug-to-carrier ratio had a great influence on the drug dissolution
enhancement. For example, the dissolution profile of (SDs) containing
PEG 4000 (Figure 17) show different dissolution rates for dispersions
containing 90%, 92%, 95%, and 99% of PEG 4000. Dispersions
containing 99% of PEG 4000 appeared to be the best preparation showing
a DP60 value of 68.58% which is about 5.25-fold increase compared with
Glz alone.
In case of all carriers (Figure 16-19), the dissolution of Glz was
enhanced as the proportion of the polymer increased. This is consistent
with that reported by Gul and Zhu 1998, who stated that, the
dissolution rate of ibuprofen increased with increasing PEG 10000
loading, and this may be attributed to the finer subdivision of the drug
particles in dispersions containing higher carrier loading. On the other
112

hand, Moneghini et al., 1998 and Chutimaworapan et al., 2000a stated
that, when the proportion of PEG increased, the dissolution was
suppressed. This result could be ascribed to the formation of a viscous
hydrophilic layer around the particles of the drug that slowed the drug
release into the dissolution medium.
It was important to find the optimal drug – carrier ratio in order to
achieve the optimal dissolution profile. When the weight ratio of carrier
decreased below its critical concentration, the concentration being too
small was probably insufficient to enhance dissolution to the maximum
extent hence, as the proportion of carrier increased, the dissolution rate
also increased. Above this critical concentration, as the proportion of
carrier increased, the longer time required for diffusion of the drug from
the matrix probably resulted in a decreased dissolution rate
(Tantishaiyakul et al., 1996).
All data are summarized in Table 9 and Figure 20
Table 9: Collective data for dissolution of Glz obtained from
different carriers used.
System a
% Released b
113
% Increase c
Glz 13.05 -
Drug : carrier
(1:99) PEG 6000 SD 100.49 670.038
(1:99) PEG 4000 SD 68.58 425.51
(1:10) Glu SD 45.56 249.11
(1:10) UR SD 39.18 200.22
a 40 mg of the drug or its equivalent were used.
b After 60 min.
c In relation to drug alone.

10
1:99 PEG 6000
9
8
1:99 PEG 4000
7
6
1:10 glucose
5
1:10 urea
A / B
4
3
2
1
0
Figure 20: Ratio between % of gliclazide dissolved from (A) drug in different solid dispersions and (B) drug
114

In order to shed light on the mechanism of dissolution enhancement
from solid dispersions, further studies were performed on the investigated
solid dispersions, physical mixtures and individual components. In case
of urea and glucose solid dispersions and their respective physical
mixtures the studies were performed at drug to carrier ratios (1:5), while
in case of PEG 4000 and PEG 6000, the studies were performed at (1:9)
drug to carrier ratio, as higher drug content is more suitable for practical
use (Okonogi et al., 1997).
5. Fourier-transform infrared spectroscopy:
FTIR spectra were performed to investigate the possible type of
interaction between Glz and different carriers (Figures 21-24).
(Table 10) showed that the characteristic shoulders of Glz were
traced at 3274.2 cm -1 (N – H stretching), 3192.9, 3113.2 cm -1 (C – H
aromatic), 2950-2836 cm -1 (C-H aliphatic), 1350, 1164.3 cm -1 (S=O
asymmetrical and symmetrical band) and 1596 (N-H deformation). The
major peak of C=O was at 1709 cm -1 .
In case of PEG 4000 and PEG 6000 systems, the carbonyl stretching
band of Glz that appeared at 1710.3 cm -1 decreased in the intensity with
the disappearance of the aromatic C-H stretching band and N-H
stretching band and predominance of O-H band corresponding to PEGs.
It was concluded from the chemical structures that an interaction of a
significant magnitude could be present between the aromatic hydrogens
of the drug and the hydroxyl groups of PEG, Mukne and Nagarsenker,
2004 attributed the complete disappearance of the aromatic stretching
vibrations of the phenyl group of triametrene by its complexation with ß-
cyclodextrin to be due to the significant interaction between the phenyl
group of triametrene and the cyclodextrin. On contrary, Glz – glucose
systems showed peaks at 3410, 3280, 2943, 2872 and 1709 cm -1 which
were the superimposed peaks of the two components. In spectra of
1

Glz systems with UR, no differences in the positions of the absorption
bands was observed, hence providing evidence for the absence of any
chemical interactions in the solid state between Glz and these carriers. In
the physical mixture and solid dispersion spectra, C=O and N-H peaks of
UR were overlapped with C=O and N-H of Glz, which formed a two
broad bands around 3300 cm -1 . If the drug and these carriers interact, then
the functional groups in the FTIR spectra will show bands changes and
broadening compared to the spectra of the plain carriers (Silverstein et
al., 1991).
2

Table 10: FTIR spectra of Glz solid dispersions and physical
mixtures compared with individual components.
System Assignment max (cm -1 )
Glz
PEG 6000
Glz – PEG 6000
(PM)(10:90)
Glz – PEG 6000
(SD)(10:90)
N – H (stretching)
C – H (aromatic)
C – H (aliphatic)
C=O
N-H (deformation band)
S=O (asymmetrical and
symmetrical band)
-O-H (stretching)
C-H (stretching)
C-O (ether)
O-H (bending)
-O-H (stretching)
C-H (stretching)
C=O
C-O (ether)
O-H (bending)
-OH (stretching)
C-H (stretching)
C=O
C-O (ether)
O-H (bending)
3
3274.2
3192.9 - 3113.2
2950 - 2867 - 2836
3447
2886.8
1710.2
1112.3
1345.7
1709
1596
1350 -1164
3445.8
2887
1110.7
1344
3446
2888.2
1710.3
1110.9
1345.5

Cont. Table 10: FTIR spectra of Glz solid dispersions and physical
mixtures compared with individual components.
System Assignment max (cm -1 )
Glz
PEG 4000
Glz – PEG 4000
(PM)(10:90)
Glz – PEG 4000
(SD)(10:90)
N – H (stretching)
C – H (aromatic)
C – H (aliphatic)
C=O
N-H (deformation band)
S=O (asymmetrical and
symmetrical band)
-OH (stretching)
C-H (stretching)
C-O (ether)
O-H (bending)
-OH (stretching)
C-H (stretching)
C=O
C-O (ether)
O-H (bending)
-O-H (stretching)
C-H (stretching)
C=O
C-O (ether)
O-H (bending)
4
3274.2
3192.9 - 3113.2
2950 - 2867 - 2836
1709
1596
1350 -1164
3414.3
2887.6
1110.4
1344.7
3447.2
2887.23
1710.3
1110.5
1345.2
3422.6
2888.0
1710.3
1112.1
1346.2

Cont. Table 10: FTIR spectra of Glz solid dispersions and physical
mixtures compared with individual components.
System Assignment max (cm -1 )
Glz
Glucose
Glz – glu
(PM)(1:10)
Glz – glu
(SD)(1:10)
N – H (stretching)
C – H (aromatic)
C – H (aliphatic)
C=O
N-H (deformation band)
S=O (asymmetrical and
symmetrical band)
-O-H (stretching)
5
(broad)
C-H (stretching)
O-H (bending)
-OH (stretching)
-NH (stretching)
C-H (stretching)
C=O
O-H (bending)
-O-H (stretching)
-NH (stretching)
C-H (stretching)
C=O
O-H (bending))
3274.2
3192.9 - 3113.2
2950 - 2867 - 2836
1709
1596
1350 -1164
3411.6 -3316.0
2944.1
1342.0
3410.4
3280.7
2943.4
1709.9
1346.7
3408.8
3276.3
2941.5
1709.9
1348.0

Cont. Table 10: FTIR spectra of Glz solid dispersions and physical
mixtures compared with individual components.
System Assignment max (cm -1 )
Glz
UR
Glz – UR
(PM)(1:10)
Glz – UR
(SD)(1:10)
N – H (stretching)
C – H (aromatic)
C – H (aliphatic)
C=O
N-H (deformation band)
S=O (asymmetrical and
symmetrical band)
-N-H (stretching)
C=O
-C-N
-N-H (stretching)
C=O
-C-N
-N-H (stretching)
C=O
-C-N
6
3274.2
3192.9 - 3113.2
2950 - 2867 - 2836
1709
1596
1350 -1164
3445.7- 3347.4
1678.8 – 1622.7
1152.4
3446.5-3347.6-
3277.9
1707.4-1682-
1622.8
1162.1
3445.7-3347.6-
3276.0
1708.1-1682.4-
1623.4
1162.4

Wave number (cm -1 )
Figure 21: FTIR spectra of Glz –PEG 6000 systems A) Glz ; B) pure
PEG 6000; C) PM (1:9) and D) SD (1:9).
7
D
C
B
A

Wave number (cm -1 )
Figure 22: FTIR spectra of Glz –PEG 4000 systems A) Glz ; B) pure
PEG 4000; C) PM (1:9) and D) SD (1:9).
8
D
C
B
A

Figure 23: FTIR spectra of Glz –glucose systems A) Glz ; B) pure
glu; C) PM (1:10) and D) SD (1:10).
Wave number (cm -1 )
9
D
C
B
A

Figure 24: FTIR spectra of Glz –UR systems A) Glz ; B) pure UR; C)
PM (1:10) and D) SD (1:10).
Wave number (cm -1 )
6. Differential scanning calorimetry:
10
D
C
B
A

It was the general aim to prepare dispersions in which the drug was
dispersed in as near a molecular state as possible to provide a thermo
energetic state of the drug of high aqueous solubility once the carrier
dissolved. Thermal analysis, especially DSC, had a powerful tool
evaluating the drug – carrier interactions (Nour, 1993). DSC is
particularly useful in determining the solubility of the drug in a polymeric
and is capable of detecting polymorphic modifications. Interactions in the
samples are derived or deduced from DSC by changes in thermal events
such as elimination of an endothermic or exothermic peak or appearance
of a new peak (Ford and Timmins, 1989). In order to get evidence on
the possible interaction between Glz and the investigated carriers, DSC
studies were performed on the prepared physical mixtures, solid
dispersions as well as various individual components. The DSC
thermograms of Glz containing systems are shown in (Figures 25-28).
The heat of fusion and fusion temperature values for the raw materials
and binary systems are represented in (Table 11). The DSC curves of
pure Glz exhibited a sharp endothermic peak at 166.2, which corresponds
to its melting point.
The DSC themograms of Glz-PEG 4000 and Glz-PEG 6000 solid
dispersions and corresponding physical mixtures showed no Glz
endothermic peak but did exhibit the endothermic peaks due to the fusion
of the carriers. This result indicated that Glz might be in an amorphous
state. Yakou et al., 1984, studied the physicochemical characteristics of
phenytoin – PEG 4000 solid dispersion; they observed the disappearance
of sharp endothermic peak corresponds to phenytoin melting point with
predominance of that corresponds to PEG 4000 melting point. They
concluded that phenytoin was uniformly dispersed in an amorphous state
in a solid matrix of PEG 4000. The absence of a drug melting
endothermic peak could also have been due to its dissolution in the
11

melted carrier. Mura et al., 1999, studied the DSC scans of solid
dispersion of naproxen in binary systems with different molecular
weights, they observed the disappearance of the drug melting peak which
indicated the dissolution of the naproxen in the melted carrier. A slight
change occurs in the shape of PEGs endothermic peaks which appeared
broadend in solid dispersions.
In case of UR, no differences were apparent between DSC scans of
the (PM) and the (SD) (Figure 28). In fact, the two systems displayed
two endothermic peaks corresponding to the carrier fusion, whereas drug
endothermic effect was not detected and this may be due to its dissolution
in the melted carrier.
(Figure 27) illustrates the DSC thermograms of Glz –glucose
systems. The absence of of Glz peak and the predominance of glucose
peaks. This suggests that Glz is completely soluble in liquid phase of
glucose (Domian et al., 2000).
12

Table 11: Fusion temperatures (Tc) and heat of fusion (Glz
solid dispersions and physical mixtures compared with individual
components.
System Fusion temperature
(Tc) (ºC)
13
Heat of fusion
(
Glz 166.2 135.38
PEG 6000 60.52 184.49
Glz – PEG 6000
(PM)(10:90) 60.02 169.52
Glz – PEG 6000
(SD)(10:90) 59.42 180.55
PEG 4000 60.52 192.58
Glz – PEG 4000
(PM)(10:90) 60.38 162.52
Glz – PEG 4000
(SD)(10:90) 59.97 167.82
Glu 156.13 223.18
Glz – glu (PM)
(1:10)
Glz – glu (SD)
(1:10)
154
182.46
153.21
183.65
193.21
42.72
194.19
58.26
UR 132.97 225.17
Glz – UR (PM)
(1:10)
Glz – UR (SD)
(1:10)
131.12
192.52
132.23
181.92
176
110.56
170.97
73.12

Temp (c)
Figure 25: DSC spectra of Glz –PEG 6000 systems A) Glz ; B) pure
PEG 6000; C) PM (1:9) and D) SD (1:9).
14
D
C
B
A

Temp (c)
Figure 26: DSC spectra of Glz –PEG 4000 systems A) Glz ; B) pure
PEG 4000; C) PM (1:9) and D) SD (1:9).
15
D
C
B
A

Figure 27: DSC spectra of Glz –glucose systems A) Glz; B) pure glu;
C) PM (1:5) and D) SD (1:5).
Temp(c)
16
D
C
B
A

Figure 28: DSC spectra of Glz –UR systems A) Glz ; B) pure UR; C)
PM (1:5) and D) SD (1:5).
Temp (c)
17
D
C
B
A

7. X-ray diffraction:
The x-ray diffractuion patterns of Glz, PEG 4000, PEG 6000, glu,
UR, physical mixtures and solid dispersions were illustrated in (Figures
29-32). Their characteristic peaks and intensities are presented in (Table
12)
Glz was a highly crystalline powder with characteristic diffraction
addition there were some other peaks of lower intensity.
In case of untreated PEG 6000, there were sharp peaks at 19° and
23.12°, while in case of PEG 4000 the diffraction peaks were traced at
19.016° and 23.217°. The diffraction patterns of PEG 6000 and PEG
4000 solid dispersions and physical mixtures are nearly identical to that
of untreated ones. The peaks of Glz were completely missed thus
indicating that Glz was in amorphous form. This was in line with our
findings from FTIR analysis where interactions might be present between
the drug and either of these two carriers.
Glucose and urea in pure form revealed high degree of crystallinity.
X-ray patterns of glucose solid dispersions and physical mixtures showed
the superimposed diffraction peaks of both drug and carrier with
reduction in their intensities. On other hand , in case of urea solid
dispersion and physical mixture, the diffraction peaks of Glz was not
observed whereas the diffraction peaks of urea was noted. This indicated
that Glz was in amorphous state (Okonogi et al., 1997).
18

Table 12: Intensities at characteristic differº) for some gliclazide solid dispersions and physical
mixtures compared with individual components.
System intensity intensity intensity intensity intensity intensity
Gliclazide 14.58 33.1 19.68 43.1 20.12 36.9 20.58 100 22.72 24.9 28.4 35.5
PEG 6000 19 86.5 23.12 100
Glic-PEG 6000
(10:90) (PM) 19.03 89.1 23.12 100
Glic-PEG 6000
(10:90) (SD) 19.03 100 23.21 97.6
PEG 4000 19.016 100 23.217 90.6
Glic-PEG 6000
(10:90) (PM) 19.1 99.7 23.26 100
Glic-PEG 6000
(10:90) (SD) 19.02 92.4 23.2 100
19

Cont.Table 12:º) for some gliclazide solid dispersions and physical
mixtures compared with individual components.
System intensity intensity intensity intensity intensity intensity
Gliclazide 14.58 33.1 19.68 43.1 20.12 36.9 20.58 100 22.72 24.9 28.4 35.5
Glucose 20.52 100 25.38 30.9 17.01 44 28.39 64.2
Glic-glucose
(1:10) (PM) 20.74 37.2 14.39 92.2 17.055 57.7 10.74 100 18.15 70.3 22.014 30.1
Glic- glucose
(1:10) (SD) 20.57 100 14.62 25.2 17.067 25.5 28.4 35 19.72 7.2 22.017 17.1
Urea 22.044 100 28.4 35
Glic-urea
(1:10) (PM) 22.22 96.3 22 45.7 31.47 100 35.47 36.9
Glic-urea
(1:10) (SD) 22.15 100 35.17 57
20

2-Theta-Scale
Figure 29: X-ray spectra of Glz –PEG 6000 systems A) Glz ; B) pure
PEG 6000; C) PM (1:9) and D) SD (1:9).
21
D
C
B
A

2-Theta-Scale
Figure 30: X-ray spectra of Glz –PEG 4000 systems A) Glz ; B) pure
PEG 4000; C) PM (1:9) and D) SD (1:9).
22
D
C
B
A

Figure 31: X-ray spectra of Glz –glucose systems A) Glz ; B) pure
glu; C) PM (1:5) and D) SD (1:5).
2-Theta-Scale
23
D
C
B
A

Figure 32: X-ray spectra of Glz –UR systems A) Glz ; B) pure UR; C)
PM (1:5) and D) SD (1:5).
2-Theta-Scale
24
D
C
B
A

Conclusion:
1- The preparation of Glz solid dispersions was examined with
different carriers.
2- The proportion and properties of the carrier used present an important
influence on the properties of the resulting soild dispersions
3- PEG 4000, PEG 6000, glucose and UR were used as carriers, led to an
increase in the dissolution rate of Glz .
4- FTIR, DSC and XRD diffraction revealed an interaction between
Glz and PEG 4000 and PEG 6000, with possibility of a
polymorphic change in Glz for all systems used.
25

Introduction
Percutaneous penetration involves drug dissolution in the vehicle,
diffusion of the solubilized drug from the vehicle to the surface of the
skin and drug penetration through skin layers. Selection of the
appropriate vehicle and modification of drug characteristics may improve
penetration (Mario et al., 2005).
Permeation of the drug from prepared systems in donor
compartment through a semipermeable membrane involves three
consecutive processes: first, dissolution of the solid dispersed particles,
then diffusion of the drug across the dissolution media, and finally its
permeation through the membrane. All three processes make a
contribution to the overall diffusion rate (Mario et al., 2005).
To improve the release rate of the drug, solid dispersions were
incorporated into the topical bases. The effectiveness of incorporation of
solid dispersions in topical formulations on the release of the Glz was
determined by comparing the percent of the drug released after six hours
in presence and absence of solid dispersions.
27

1- Materials and supplies:
Experiment and methodology
* Hydroxy propylmethyl cellulose 50 cp (HPMC) (Sigma
Chemical, St.Louis, MO, USA)
* White soft paraffin, wool fat, cetyl alcohol, propylene glycol,
sodium lauryl sulfate (SLS), polyethylene glycol 400, liquid
paraffin, hard paraffin and borax (El-Nasr CO. Cairo, Egypt).
* Octanol , span 80 (Merk Sharp and Dohmn, Germany)
* White beeswax, gum acacia (El-Gomhouria Co.,Egypt).
* Glucose- LS, GOD-PAP, Modern Laboratory Chemicals, Egypt.
* Streptozotocin (Sigma Chemical Company,USA).
* Other materials were mentioned previously in chapter one.
2- Equipment:
* Diffusion glass cell, this is composed of an open ends glass
tube with 2.9 cm as external diameter, 2.6 cm as internal
diameter and length of 30 cm. Semipermeable cellophane
membrane was stretched over one open end of glass tube and
made watertight by a rubber band.
* Viscometer (Fungi lab S.A, Spain).
* Eppendorf Centrifuge 5415 C (maximum speed 14000 min -1 ), West
Germany.
* UV/VIS Spectrophotometer (Jenway, 6105).
* PH meter (Cole-Parmer Instrument Co USA).
* On Call EZ Blood Glucose Meter (San Diego, CA 92121, USA).
* Other equipments were mentioned previously in chapter one.
3- Software:
Microsoft Office XP, Microsoft Corporation, USA.
28

SPSS statistics Package, SPSS Institute Inc., Cary, USA.
4. Methods:
4.1. Determination of partition coefficient of Glz:
*** Preparation of saturated solution of the drug:
An excess of the Glz (10 mg) was placed into 25-ml glass vial
containing 10 ml distilled water. The glass vials was closed with stopper
and cover-sealed with cellophane membrane to avoid solvent loss .The
content of the suspension was equilibrated by shaking in a
thermostatically controlled water bath at 25°C for 7 days. After
attainment of equilibrium, the content of the vial was then filtered
through a double filter paper (Whatman 42).The filtrate was assayed
spectrophotometrically at 227 nm to measure the amount of the drug.
*** Method:
In glass vials 5 ml of saturated solution of the drug were added to 5
ml of n-octanol. The vials were placed in a thermostatically controlled
water bath at 25°C for 24 hrs. The aqueous phase was separated from the
oily phase by the separating funnel and the amount of the drug in aqueous
phase was assayed spectrophotometerically at 227 nm using distilled
water as blank. The concentration of the drug was obtained from a
previously constructed calibration curve. Partition coefficient of Glz in
octanol/water system was determined according to the following equation
(El-Nahas, 2001):
Conc. of Glz in oily phase
Partition coefficient = --------------------------------------------
Conc. of Glz in aqueous phase
29

4.2. Preparation of solid dispersions:
Solid dispersions of Glz with each of PEG 6000, PEG 4000, urea
and glucose were prepared at weight ratios of 8:92 (drug:carrier) for
PEGs (SDs) and 1:10 (drug:carrier) for urea and glucose SDs. The
amount of SDs introduced was adjusted to maintain the drug
concentration at 1% in the formulations.
4.3. The methods of preparation of topical preparations:
The following formulae were selected in which 10 mg of Glz, or its
equivalent of (SDs) was incorporated in each one gram of the topical
formula. In case of urea and glucose, (SDs) that demonstrated the best
dissolution properties, (1:10) drug to carrier ratio, were used. However in
case of PEG 4000 and PEG 6000, (SDs) of (8:92) drug to carrier ratio
were used because the ratio of (1:99) that gave the highest dissolution
was not practically suitable for incorporation into the base due to higher
powder content.
4.3.1. Water soluble base:
Polyethylene glycol base :( U.S.P. XXII).
- PEG 4000 40 gm
- PEG 400 60 gm
Preparation:
PEG 4000 was melted at 60° C on a water bath. Then PEG 400
containing the drug or the solid dispersion was added. The mixture was
continuously stirred until congealed and packed in a plastic jar and stored
at ambient temperature until used.
4.3.2. Absorption base (B.P. 1963):
- Wool fat 5 gm
-Cetyl alcohol 5 gm
-Hard paraffin 5 gm
30

-White soft paraffin 85 gm.
Preparation:
Accurate amount of the drug or the solid dispersion was weighed,
levigated and incorporated into the melted base with continuous stirring
until congealed then packed into plastic jar until used.
4.3.3. Emulsion bases:
O/W emulsion base (Beeler’s base) (Ezzedeen et al., 1986).
-White bees wax 1 gm
-Cetyl alcohol 15 gm
-Propylene glycol 10 gm
-Sodium lauryl sulphate 2 gm.
-Water 72 gm.
W/O emulsion base: (Ezzedeen et al., 1986).
-Liquid paraffin 45 gm
-White bees wax 10 gm
- Wool fat 2 gm
- Borax 8 gm
-Water 41 gm
- Span 80 1 gm
Preparation:
The aqueous phase and the oil phase were placed in separate
containers and heated at 70°C .The drug was dissolved in the oily phase.
Then the aqueous phase was added to the oil phase at the same
temperature with continuous stirring until cool and congealed
31

4.3.4. Hydroxy propyl methylcellulose gel (Sobati, 1998):
- HPMC 12 gm
- Water 88 gm
Preparation:
The drug was dispersed in a quantity of water then the gelling agent
was added with continuous stirring, set aside for complete swelling and
the weight was adjusted by the addition of the water.
All the formulations mentioned previously were summarized in
(Table13)
4.4. In vitro release of Glz from different topical formulations:
The release study was determined using the simple dialysis
technique. In this method, 1 gm of the tested formulation containing (10
mg of the drug) was accurately weighed over the cellophane membrane
which previously soaked in the phosphate buffer pH 7.4 for 30 minutes,
the loaded membrane was stretched over the end of a glass tube of about
2.9 cm as external diameter, and 2.6 cm as internal diameter as shown in
(Figure 33) (Donor).
The diffusion cell was placed at the center of 1000 ml dissolution cell
containing 100 ml of phosphate buffer pH 7.4. The donor was suspended
in the acceptor in such a manner that the membrane was located just
below the surface of the sink condition. The stirring rate was 100 rpm and
the temperature was maintained at 37 ± 0.5 °C. At suitable time intervals
(30, 60, 90,120,150,180, 240,300 and 360 minutes), 2.5 ml sample was
withdrawn from the sink solution and replaced with an equivalent amount
of the fresh release medium kept at 37 °C, diluted with methanol and
assayed spectrophotometerically at 227 nm using a suitable blank.
32

Each experiment was done in triplicate, and the average was calculated.
The cumulative amount of the drug released was calculated as mentioned
before.
33

Figure 33: Diagrammatic representation of the drug diffusion
apparatus.
34

4.5. Effect of incorporation of solid dispersions in different topical
preparations:
Previously prepared solid dispersions were incorporated in the
topical formulations that demonstrated the best release results (water
soluble base, HPMC gel and O/W cream).In vitro release of these
preparation were done as mentioned above.
4.6. Detrmination of viscosity of topical different bases:
The viscosity of each of PEG bases, O/W cream and HPMC gel which
contains Glz :PEG 4000 (8:92)SD and Glz : glu (1:10) SD was
determined at room temperature, using spindle number 5 at 2 r.p.m (El-
Megrab et al., 2006).
4.7. Kinetic evaluation of the in vitro release data:
The data obtained from the experiments were analyzed to know the
mechanism of the release of the drug using the following kinetic
equations:
(I) Zero order kinetics:
A=A-k
Where A
A = drug concentration at time (t).
t = time interval.
k
When this linear equation is plotted with the percent of drug remained on
the vertical axis and (t) on the horizontal axis, a straight line would be
obtained with (R) correlation coefficient, a slope equal to (-k
intercept equal to (A
35

Half time: is the time required for a drug to decompose to one half of the
original concentration or it is the time at which A is decreased to 1 /2 A
(Martin, 1994 ).
(II) First order kinetics:
t1/2 = A
ln A = ln A- kt
log A = log A- kt/ 2.303
Where A
A = drug concentration at time (t).
t = time interval.
k
When this linear equation is plotted with the logarithm amount of percent
drug remained on the vertical axis and (t) on the horizontal axis, a straight
line would be obtained with (R) correlation coefficient, a slope equal to (-
kt/ 2.303) and an intercept equal to (log A(Martin, 1994 ).
The half life for first order kinetics equal to
(III) Higuchi diffusion model:
t1/2 = 0.693 / k.
i) The diffusion occurs in a direction opposite to that of increasing
concentration. That is to say, diffusion occurs in the direction of
decreasing concentration of diffusant, Fick
diffusion (Martin, 1994). A simplified Higuchi diffusion
equation for drug released from topical preparation is.
M = Q = 2C ½
36

Where:
M = Q = amount of the drug released into the receptor phase at time t.
C
t = time of release.
D = diffusion coefficient of the drug.
This equation describes drug release as being linear with the square root
of the time
Q = k t ½
4.8. In vitro permeation of Glz through abdominal rabbit skin:
4.8.1. Preparation of rabbit skin:
The abdomen of white male rabbits (Jia-You et al., 1996; Hosny et
al., 1998), weighing 2-3 Kg, were shaved by an electric hair clipper. The
rabbit was scarified; the skin then excised surgically, without injury. The
dermal side of the skin was carefully cleared of adhering blood vessels,
fats or subcutaneous tissues using fine-point forceps and surgical scissors
and washed with distilled water (Ceschel et al., 1999). The skin was
stored and frozen. The frozen skin was thawed prior to cutting into pieces
for experimental studies. The pieces of the skin were equilibrated by
soaking in sörensen’ phosphate buffer (pH 7.4) for about one hour before
the beginning of each experiment (Larrucea et al., 2001).
4.8.2. In vitro permeation studies:
In vitro permeation studies of Glz and Glz solid dispersions from
different topical formulations were carried out utilizing locally fabricated
diffusion cell. The excised rabbit skin was mounted on one end of the
37

vertical diffusion cell (internal diameter 2.6 cm) by a rubber band with
the sratum corneum side facing the donner compartment and the dermal
side facing the receptor compartment, and the total area available for
penetration was 5.3 cm 2 . Several drug concentrations ranging from 10 to
50 mg of Glz per one gram of the formulation were applied on the
stratum corneum. The diffusion cell was hanged into the center of the
glass beaker, containing 100 ml of sörensen’ phosphate buffer (pH 7.4)
(Mura et al., 2000; Ghazy et al., 2004) in such way that, the dermal
surface was just flushed to the permeation fluid (Fang et al., 2003).The
permeation fluid was maintained at 37°C ± 0.5°C and stirred at 100 rpm
in thermostatically controlled water bath (Tehrani and Mehramizi,
2000).To avoid evaporation, the beaker was kept covered during the
experiment. Four-millimeter samples were withdrawn from the receptor
phase at specified time intervals and immediately replaced by an equal
volume of fresh buffer solution (pH 7.4) at the same temperature (37°C ±
0.5°C) to maintain the volume of the receptor phase constant during the
experiment . The samples were analyzed spectrophotometrically at 227
nm against sörensen’ phosphate buffer (pH 7.4) as a blank (Larrucea et
al., 2001). Each experiment was performed three times and the average
was calculated. The cumulative amount of the drug permeated was
calculated as mentioned before.
4.8.3. Statistical analysis:
Data were expressed as mean of three experiments ± the standard
error (S.E.). The obtained data were compared statistically using One-
way analysis of variance (ANOVA) test of significance on a computer
statistical SPSS analysis program. A p-value of 0.05 or less was
considered to be significant (Suwanpidokkul et al., 2004).
38

4.9. In vivo studies:
4.9.1. Animals:
The animals used for the anti-diabetic and hypoglycemic activity
study were white adult albino rats weighing between 200-250 gm. The
animals were housed under standard laboratory conditions.
4.9.2. Hypoglycemic activity in normal rats:
The hair on the backside of the rats was removed with an electric hair
clipper on the previous day of the experiment. The oral doses were given
using a round tipped stainless steel needle attached to 1 ml syringe.
Following an overnight fast, rats were divided into 4 groups (n=5) and
treated as follows:
Group I (Control) – 1ml gum acacia suspension was given orally.
Group II - 25 mg/kg Glz in mucilage of gum acacia was given orally
(Stetinova et al., 2007).
Group III –1 gm water soluble ointment base containing 25 mg of the
Glz was applied on 4cm 2 of the skin. Many trials on rats were done to
find a suitable topical formula. The dose of 25 mg drug was selected by
conducting a series of experiments with graded doses ranging between 10
to 50 mg. The application site was covered with a non-occlusive dressing
and wrapped with a semi-occlusive bandage.
Group IV –1 gm water soluble ointment base containing certain amount
of Glz - PEG 6000 solid dispersion (10:90) equivalent to 25 mg Glz was
applied on 4cm 2 of the skin.
Blood samples were collected in eppendorf predose (0 hr) and 2, 4, 6, 8
and 24 hours post dose from the orbital sinuses; the serum glucose
concentrations were assayed based on the standard glucose oxidase
39

method (El-Sayed et al., 1989) using a commercial kit according to the
supplied instructions as follows:
Principle:
reaction:
Glucose present in the sample is determined according the following
Glucose + O2 + H2O2 glucose oxidase enzyme gluconic acid + H2O2
------------------>
2 H2O2 + phenol amino-4-antipyrine peroxidase enzyme quinoneimine+4
H2O
Sample preparation:
----------------->
The collected blood samples were left for 15 min in the refrigerator,
then the serum was separated using a centrifuge operating at 4000 rpm for
20 min, then the serum was taken by a syringe in a test tube.
Procedure of measurement:
The amounts of samples and standard used are summarized in (Table 14).
Table 14: Amounts of sample and standard used.
Blank Standard Sample
Reagent 1.0 ml 1.0 ml 1.0 ml
Standard
reagent
(100mg
glu/dl)
------- -------
Sample ------- -------
40

Samples and reagent were mixed and incubated for 10 min at 37°C.
The absorbance of sample (Asample) and standard (Astandard) against reagent
blank were measured. The intensity of the developed pink colour was
measured spectrometrically at 500 nm against a blank solution.
Calculation:
(Asample)
Glucose concentration (mg/dl) = ----------- x 100
(Astandard)
4.9.3. Anti-diabetic activity in diabetic rats:
4.9.3.1. Induction of diabetes mellitus:
The overnight fasted rats were made diabetic by a single
intraperitoneal injection of streptozotocin (STZ) (50 mg/kg; i.p) dissolved
in citrate buffer (pH 4.5). The blood glucose was measured after 24 hrs
and animals with blood glucose levels >250 mg/dL were selected
(Sridevi et al., 2000).
4.9.3.2. Anti-diabetic activity in diabetic rats:
The anti diabetic activity of the prepared topical preparation was
evaluated in overnight fasted diabetic rats.
Diabetic rats were divided into 3 groups (n=5). The rats were treated as
following:
Group I (Control) – 1ml gum acacia suspension was given orally.
Group II - Glz 25 mg/kg was given orally (Stetinova et al.,2007).
Group III –1 gm water soluble ointment base containing certain amount
of Glz - PEG 6000 solid dispersion (10:90) equivalent to 25 mg Glz was
applied on 4cm 2 of the skin.
41

At time intervals between 2-24 h after treatment, blood was collected
from orbital sinuses; blood glucose levels were determined using the
glucometer.
The results obtained from the measurement of blood glucose level by
both glucometer and standard glucose oxidase method were nearly the
same.
4.9.4. Statistical analysis:
The obtained data were compared statistically using One-way
analysis of variance (ANOVA) test of significance on a computer
statistical SPSS analysis program. A p-value of 0.05 or less was
considered to be significant (Suwanpidokkul et al., 2004).
42

1. Partition coefficient of Glz:
Results and Discussion
In the present study, the partition coefficient of Glz was found
to be 1.79 (log octanol/ water =0.25).
2. In vitro release of Glz from different topical formulations:
Glz was chosen to be formulated in topical bases, to demonstrate its
expected action from different topical preparations, such as an ointment,
cream and gel. It is important that the vehicle is able to release the active
ingredient which it carries. Selection of different topical bases as vehicle
for Glz depends on several factors such as polarity, viscosity, and
homogeneity. For this purpose traditional classes of topical bases were
investigated which include water soluble bases, emulsion bases,
absorption bases and gel bases. The emulsion bases included O/W
emulsion and W/O emulsion.
The partition coefficient of the drug is considered as one of the
important parameters for the estimation of the interaction of that drug
with the vehicle and the receiving medium (Celebi et al., 1993).
As general rule in ointment formulations is that, if the drug is held
firmly by the vehicle the rate of the release of the drug is slow (Barr,
1962)
The release of the drug from ointments can be altered by modifying
the composition of the vehicle (Idson, 1983). A greater release of the
drug is expected when there is less affinity of the drug for the base.
(Table 15) and (Figure 34) showed the release of Glz from different
topical bases.
From the data obtained it is clear that the percentage amount of drug
released from water soluble base and gel base are greater than that
released
43

Table 15: In vitro release data of Glz from different topical
Time (min)
bases.
Glz released % ± (sd)
WSB HPMC gel O/W cream
0 0 ± 0 0 ± 0 0 ± 0
30 0 ± 0
60
44
1.35 ± 0.12 0 ± 0
5.70± 0.43 6.71 ± 0.70 0 ± 0
90 14.90 ± 0 11.97 ± 0.74 1.70 ± 0.3
120 22.15 ±0.38 16.80 ± 0.6 2.44 ± 0.3
150 30.46± 0.37 21.41 ± 0.9 2.71 ± 0.4
180 35.4 ± 0.41 25.79 ± 0.57 4.08 ± 0.021
240 47.61± 0.39
300 56.55± 0.07
31.21 ± 0.48 4.90 ± 0.56
37.86 ± 0.9 6.7 ± 0.9
360 62.10 ± 0.42 43.38 ± 2.2 8.43 ± 0.38

WSB HPMC gel O/W cream
100
90
80
70
60
50
40
% Released
30
20
10
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 34: In vitro release profile of gliclazide from different topical preparation.
45

from other bases. The rate of drug release can be arranged in the
following descending order:
Water soluble base (62.1 %) > HPMC gel (43.38 %) > O/W emulsion
base (8.43 %).
There was no drug release from the absorption base. This may be
attributed to composition of the absorption base which contains white soft
paraffin with several additional lipoidal constituents which favor the
retention of the drug in the base ( Furia, 1972).
Also, there was no drug release from W/O emulsion base. This
finding can be explained on the bases that in case of W/O emulsion bases,
the presence of an oily vehicle as an external phase will result in
formation of an occlusive film on the membrane surface, which will
result in retardation of the permeation of the drug molecules across the
membrane, into the sink solution (Ismail et al., 1990; Khitworth and
Stephenson, 1976).
On the other hand, the higher release of Glz from O/W emulsion
base than from W/O emulsion base may be due to the formation of a
continuous contact between the external phase of the O/W emulsion and
the buffer (Nakano et al., 1971), however, the lower release of the drug
from O/W emulsion base than from water soluble base and HPMC base
may be due to the greater solubility of the drug in the internal oily phase
which may cause a decrease in the rate of release of the drug.
Due to the high lipid solubility of Glz, this may explain the slow
release of the drug that is observed from these bases.
The high diffusion rate of Glz from water soluble base that contains
mainly polyethylene glycol may be due to diffusion of the buffer solution
through the cellophane membrane and formation of water-PEG solution
which increase the solubility and accordingly the rate and extent of Glz
release.
46

The high release of the Glz from HPMC gel is considered to be due
to a high miscibility of this base with the release medium.
3. Viscosity determination:
As shown in (Table 16), water soluble base showed the highest
viscosity followed by O/W cream and finally HPMC gel. It is noted that
all bases included (8:92) PEG 4000 SD have higher viscosity than that
included (1:10) glu SD.
Table 16: Viscosity of different topical bases.
Preparation Viscosity (poise)
(8:92) PEG 4000 SD (1:10) glu SD
WSB .9 2271.4
O/W cream 2251.07 2071.02
HPMC gel 985.90 816.90
4. Effect of incorporation of solid dispersions in different topical
preparations:
(Tables 17-20) and (Figures 35-38) showed that all solid
dispersions increased the overall Glz diffusion by increasing the amount
of diffusible species in the donor phase by enhancing drug solubility.
Therefore, Glz solid dispersions increased the Glz concentration gradient
over the membrane, which resulted in increase in Glz diffusion (Mario et
al., 2005). Incorporation of clotrimazole solid dispersion in O/W cream
improved the antifungal activity of clotrimazol (Madhusudhan et al.,
1999). It was found that formulations containing Rifampicin in the form
of solid dispersion with PEG have shown the best release characteristics
of the antibiotic from oleaginous bases containing Tween 80 (Youssef, et
47

al., 1988). In this study PEG 6000 solid dispersion (8:92) drug to carrier
ratio had the most influential effect on the rate and the extent of release of
Glz , followed by glucose solid dispersion (1:10), PEG 4000 solid
dispersion (8:92) and finally urea solid dispersion (1:10). This is in
agreement with the dissolution results with exception that the dissolution
efficiency of (8:92) PEG 4000 SD (42.23% ± 0.28) was greater than that
of glucose solid dispersion (37.05 ± 0.87) and this may be due to the
higher viscosity of topical bases containing PEG 4000 solid dispersion
than that containing glucose solid dispersion as shown in Table 16. All
data are summarized in Figure 39.
48

Table 17: In vitro release of gliclazide and (8:92) gliclazide-PEG 6000 solid dispersion from different topical
bases.
Gliclazide released % ± (sd)
O/W cream
O/W cream
HPMC gel
HPMC gel
WSB
Time (min) WSB
(SD)
(gliclazide±)
(SD)
(gliclazide)
(SD)
(gliclazide)
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0 ± 0 3.03 ± 0.4 1.35 ± 0.12 6.70 ± 0.28 0 ± 0 1.90 ± 0
60 5.70± 0.43 11.45 ± 1 6.71 ± 0.70 13.02 ± 0.35 0 ± 0 3.30 ± 0
90 14.90 ± 0 19.75 ± 0.4 11.97 ± 0.74 19.48 ± 0.05 1.70 ± 0.3 4.35 ± 0.4
120 22.15 ±0.38 27.30 ± 0.28 16.80 ± 0.6 25.41 ± 0.21 2.44 ± 0.3 7.16 ± 0.48
150 30.46± 0.37 34.22 ± 0.11 21.41 ± 0.9 31.16 ± 0.98 2.71 ± 0.4 8.38 ± 0.44
180 35.4 ± 0.41 39.20 ± 0.24 25.79 ± 0.57 39.62± 0.84 4.08 ± 0.021 8.80 ± 0.59
240 47.61± 0.39 51.50 ± 1.4 31.21 ± 0.48 44.47 ± 1.2 4.90 ± 0.56 10.37 ± 0.63
300 56.55± 0.07 60.90 ± 1.6 37.86 ± 0.9 51.47 ± 1.5 6.7 ± 0.9 14.4 ± 0.62
360 62.10 ± 0.42 69.96 ± 2.5 43.38 ± 2.2 60.47 ± 1.5 8.43 ± 0.38 16.80 ± 0.37
% Increase ------- 11.23 ------- 28.26 ------- 99.28
49

WSB- glic WSB-SD HPMC gel-glic
HPMC gel- SD O/W cream-glic O/W cream- SD
100
90
80
70
60
50
40
% Released
30
20
10
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 35: In vitro release profile of gliclazide and (8:92) gliclazide –PEG 6000 solid dispersion from
different topical bases.
50

Table 18: In vitro release of gliclazide and (1:10) gliclazide-glucose solid dispersion from different topical
Gliclazide released % ± (sd)
O/W cream
O/W cream
HPMC gel
HPMC gel
WSB
Time (min) WSB
(SD)
(gliclazide±)
(SD)
(gliclazide)
(SD)
(gliclazide)
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0 ± 0 1.14 ± 0.1 1.35 ± 0.12 6.3 ± 0.28 0 ± 0 1.60± 0.24
60 5.70± 0.43 9.68 ± 0.7 6.71 ± 0.70 12.4 ± 0.35 0 ± 0 2.06 ± 0.4
90 14.90 ± 0 17.88 ± 0.97 11.97 ± 0.74 18.72 ± 0.05 1.70 ± 0.3 3.16 ± 0.7
120 22.15 ±0.38 25.96 ± 0.8 16.80 ± 0.6 21.90 ± 0.20 2.44 ± 0.3 5.8 ± 0.7
150 30.46± 0.37 33.43 ± 1.2 21.41 ± 0.9 27.47 ± 0.98 2.71 ± 0.4 7.4 ± 1.1
180 35.4 ± 0.41 37.90 ± 1.5 25.79 ± 0.57 34.04± 0.84 4.08 ± 0.021 8.65 ± 0.7
240 47.61± 0.39 48.73 ± 1.5 31.21 ± 0.48 39.24 ± 1.2 4.90 ± 0.56 9.1± 0.37
300 56.55± 0.07 58.5± 0.96 37.86 ± 0.9 45.89 ± 1.5 6.7 ± 0.9 12.12 ± 0.9
360 62.10 ± 0.42 67.22 ± 1.1 43.38 ± 2.2 55.69 ± 1.5 8.43 ± 0.38 14.77 ± 1.0
% Increase ------ 7.61 ----- 22.1 ----- 42.92
51

WSB-glic WSB-SD HPMC gel- glic
HPMC gel-SD O/W cream- glic O/W cream- SD
100
90
80
70
60
50
40
% Released
30
20
10
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 36: In vitro release profile of gliclazide and (1:10) gliclazide –glucose solid dispersion from different topical
bases.
52

Table 19: In vitro release of gliclazide and (8:92) gliclazide-PEG 4000 solid dispersion from different topical
bases.
Gliclazide released % ± (sd)
O/W cream
O/W cream
HPMC gel
HPMC gel
WSB
Time (min) WSB
(SD)
(gliclazide±)
(SD)
(gliclazide)
(SD)
(gliclazide)
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0 ± 0 1.4 ± 0.17 1.35 ± 0.12 2.55 ± 0 0 ± 0 1.20± 0.2
60 5.70± 0.43 8.15 ± 0.21 6.71 ± 0.70 8.98 ± 0.12 0 ± 0 1.64 ± 0.13
90 14.90 ± 0 18.40 ± 0.9 11.97 ± 0.74 18.06 ± 0.35 1.70 ± 0.3 2.42 ± 0.22
120 22.15 ±0.38 24.71 ± 0.7 16.80 ± 0.6 23.95 ± 0.43 2.44 ± 0.3 2.84 ± 0.2
150 30.46± 0.37 32.71 ± 1.2 21.41 ± 0.9 27.7 ± 0.43 2.71 ± 0.4 4.44 ± 0.5
180 35.4 ± 0.41 38.26 ± 0.08 25.79 ± 0.57 33.6± 0.58 4.08 ± 0.021 5.5 ± 0.35
240 47.61± 0.39 48.48 ± 1.02 31.21 ± 0.48 38.1 ± 1.9 4.90 ± 0.56 8.16± 0.57
300 56.55± 0.07 58.89 ± 1.5 37.86 ± 0.9 44.7 ± 1.6 6.7 ± 0.9 10.99 ± 1.03
360 62.10 ± 0.42 65.1 ± 1.8 43.38 ± 2.2 53.7 ± 1.9 8.43 ± 0.38 11.71 ± 0.1
% Increase -------- 4.60 ------- 19.21 ----- 28.01
53

WSB- glic WSB-SD HPMC gel- glic
HPMC gel- SD O/W cream-glic O/W cream-SD
100
90
80
70
60
50
40
% Released
30
20
10
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 37: In
vitro release profile of gliclazide and (8:92) gliclazide –PEG 4000 solid dispersion from
different topical bases.
54

Table 20: In vitro release of gliclazide and (1:10) gliclazide-urea solid dispersion from different topica
bases.
Gliclazide released % ± (sd)
Time (min) WSB WSB HPMC gel HPMC gel O/W cream O/W cream
(gliclazide) (SD) (gliclazide) (SD) (gliclazide±) (SD)
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0 ± 0 1.54 ± 0.13 1.35 ± 0.12 3.92 ± 0.18 0 ± 0 0± 0
60 5.70± 0.43 8.07 ± 0.47 6.71 ± 0.70 10.30 ± 0.30 0 ± 0 0.99 ± 0
90 14.90 ± 0 16.98 ± 0.37 11.97 ± 0.74 15.50 ± 0.27 1.70 ± 0.3 2.28± 0.59
120 22.15 ±0.38 24.37 ± 0.48 16.80 ± 0.6 21.05 ± 0.07 2.44 ± 0.3 3.44 ± 0.63
150 30.46± 0.37 31.14 ± 0.18 21.41 ± 0.9 25.1 ± 0.56 2.71 ± 0.4 4.21 ± 0.44
180 35.4 ± 0.41 37.6± 1.8 25.79 ± 0.57 29.94± 0.06 4.08 ± 0.021 5.89 ± 0.62
240 47.61± 0.39 47.25 ± 1.1 31.21 ± 0.48 35.15 ± 0.49 4.90 ± 0.56 7.20± 0.48
300 56.55± 0.07 57.37± 1.03 37.86 ± 0.9 41.80 ± 0.71 6.7 ± 0.9 8.80 ± 0.37
360 62.10 ± 0.42 64.15 ± 1.4 43.38 ± 2.2 52.41 ± 1.1 8.43 ± 0.38 10.74 ± 0.4
% Increase ------ 3.19 ------- 17.22 ------- 21.5
55

WSB- glic WSB- SD HPMC gel -glic
HPMC gel - SD O/W cream- glic O/W cream -SD
100
90
80
70
60
50
40
% Released
30
20
10
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure
38: In vitro release profile of gliclazide and (1:10) gliclazide –urea solid dispersion from different topical bases.
56

57
1 W
2 HPMC
3 O/W cr

80
PEG 6000
glucose
PEG 4000
70
urea
PEG 6000
glucose
PEG 4000
Drug
60
urea
Drug alone
(8:92) PEG 6000 SD
(1:10) glucose SD
(8:92) PEG 4000 SD
(1:10) urea SD
50
Drug
40
% Released
30
PEG 6000
glucose
PEG 4000
20
urea
Drug
10
0
1 2 3
Topical bases
Figure 39: Release of gliclazide from different bases with different solid dispersions.
58

5. Kinetic analysis of release data:
As shown in (Table 21) the data of Glz and solid dispersions
released from different topical formulations followed first order kinetics
while that obtained from HPMC gel followed diffusion controlled
mechanism or Higuchi model.
Table 21: Kinetic data of the release of Glz and solid
WSB
HPMC gel
O/W cream
dispersions from different topical bases.
Topical
Correlation coefficient (R) Observed
preparation Zero First Diffusion order
Drug 0.9883 0.9987 0.9965 First
(8:92) Glz- PEG
6000 SD
0.9933 0.9984 0.9982 First
(8:92) Glz- PEG
4000 SD
0.9889 0.9993 0.9981 First
(1:10) Glz-glu SD 0.9912 0.9990 0.9987 First
(1:10) Glz-UR
SD
0.9906 0.9996 0.9980 First
Drug 0.9908 0.9981 0.9987 D.M
(8:92) Glz- PEG
6000 SD
0.9884 0.9959 0.9963 D.M
(8:92) Glz- PEG
4000 SD
0.9904 0.9959 0.9967 D.M
(1:10) Glz-glu SD 0.9863 0.9949 0.9965 D.M
(1:10) Glz-UR
SD
0.9930 0.9941 0.9945 D.M
Drug 0.9931 0.9933 0.9810 First
(8:92) Glz- PEG
6000 SD
0.9912 0.9915 0.9837 First
(8:92) Glz- PEG
4000 SD
0.9897 0.9899 0.9654 First
(1:10) Glz-glu SD 0.9857 0.9870 0.9815 First
(1:10) Glz-UR
SD
0.9957 0.9967 0.9935 First
59

6. In vitro permeation of Glz through abdominal rabbit skin:
Skin permeation studies indicated that Glz permeation through
hairless rabbit skin was negligible. The possible reasons for this result
may be i) Glz , a lipophilic drug, was retained within the stratum corneum
with no partioning into the viable epidermis or ii) most of the drug was
used up to saturate the binding sites in the skin and the remaining drug
was probably insufficient to provide a significant concentration gradient
(Srini et al., 1998).
7. In vivo study:
The result of hypoglycemic activity of the topically applied
gliclazide and oral gliclazide (25 mg/kg; p.o.) in both normal and diabetic
rats are shown in (Table 22-23) and (Figure 40-41).
*** Studies in normal rats
Gliclazide (oral) produced a significant decrease of 60.64 % ± 6.3
(plevels at 2 hr and then the
blood glucose levels decreased. The percentage reduction in the blood
glucose levels at the end of 24 hr were only 24.83 ± 2.05. On other hand,
the blood glucose reducing response of gliclazide (topical) was gradual
and significant upto 24 h compared to control (p
blood glucose reducing response was observed after 6 hr and thereafter
remained stable up to 24 h. These results are in accordance with the
results obtained by (Mutalik and Udupa, 2005).
As shown in (Figure 40), the blood glucose reducing activity of
ointment contained (10:90) gliclazide –PEG 6000 solid dispersions was
significantly more when compared to ointment contained gliclazide alone.
This is in agreement with the results of Madhusudhan et al., 1999 who
60

found that Incorporation of clotrimazole solid dispersion in O/W cream
improved the antifungal activity of clotrimazol.
Topical route effectively maintained normoglycemic level in
contrast to the oral group which produced remarkable hypoglycemia.
61

Table 22 : Reduction in blood glucose level after oral and topical application of gliclazide and 10:90 gliclazide- PEG
6000 solid dispersion in normal rats. All values are expressed as mean ± sd.
Reduction in blood glucose level (mg/dl)
(Percentage reduction in blood glucose levels)
Absolute
blood glucose
level (mg/dl)
Group
2hr 4hr 6hr 8hr 24hr
73.36 ± 9.89
(16.6 ± 2.09)
75.06 ± 8.4
(15.46 ±2.45)
76.1 ± 9.2
(13.44 ±1.44)
76.45 ± 5.2
(12.04 ± 0.99)
88.72 ± 8.6 82.33 ± 7.7*
(7.14±1.25)**
Control
(1ml gum acacia
suspension)
67.66 ± 4.1
(24.83 ±2.05)
48.5 ± 6.2
(46.89 ±4.93)
43.3 ± 4.64
(51.85 ± 3.5)
39.97 ± 5.3
(55.99 ±4.41)
89.85 ± 5.01 35.27 ± 6.07
(60.64 ± 6.3)
Oral gliclazide
(25mg/kg)
56.29 ± 1.4
(32.71 ± 6.9)
52.18 ± 3.8
(34.34 ± 4.8)
47.92 ± 5.1
(40.05 ± 5.4)
58.4 ± 2.2
(30.14 ± 5.9)
81.59 ± 8.9 58.22 ± 4.6
(28.19 ± 4.5)
WSB
(Gliclazide)
51.13 ± 3.5
(37.45 ± 3.6)
45.38 ± 3.5
(44.66 ±4.51)
38.46 ± 4.6
(52.62 ± 6.7)
47.05 ± 2.26
(43.5 ± 3.05)
82.14 ± 3.8 62.00 ± 3.2
(25.5 ± 4.03)
WSB
(10:90 gliclazide-
PEG 6000 SD)
* Reduction in blood glucose level (mg/dl). ** Percentage reduction in blood glucose levels.
62

80
Control oral gliclazide WSB ( gliclazide) WSB (gliclazide-PEG 6000 SD)
70
60
50
40
30
20
% Reduction in blood glucose level(mg/dl)
10
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time (hr)
Figure 40: Percent reduction in blood glucose levels after oral and topical administration of gliclazide in normal rats.
63

*** Studies in diabetic rats:
Results obtained from the diabetic rats after application of ointment
base containing certain amount of Glz - PEG 6000 solid dispersion
(10:90) equivalent to 25 mg Glz and oral gliclazide administration are
shown in (Figure 41) and (Table 23).
Oral and topical groups showed significant hypoglycemic activity up
to 24 h (p
effect produced by the topical gliclazide was significantly less when
compared to oral administration. The topical and the oral drug produced a
decrease of 36.35 % ± 4.42 and 21.33 % ± 3.73 respectively, in the blood
glucose level after 24 h.
Studies in diabetic rats showed small difference in the duration of
action between the oral and topical groups and this may be due to reduced
insulin level in diabetic models which impairs the principal metabolic
pathways of sulphonylurea which resulted in its prolonged action in
orally treated group (Strove and Belkina, 1989).
These results are in accordance with the results obtained by Sridevi
et al., 2000 who stated that the hypoglycemic activity of oral and topical
groups did not differ significantly in the two groups after 8 hrs. The
TDDS and the oral drug produced decrease of 61.9 ± 9.5% and 63.4 ±
3.3% respectively, in the blood glucose levels after 24 hrs.
Finally, the slow and sustained release of the drug from the
transdermal system might reduce manifestations like severe
hypoglycemia, sulphonylurea receptor down regulation and the risk of
chronic hyperinsulinemia (Faber et al., 1990 and Bitzen et al., 1992).
64

Table 23 : Reduction in blood glucose level after oral and topical application of gliclazide and 10:90 gliclazide- PEG
6000 solid dispersion in diabetic rats. All values are expressed as mean ± sd.
.
Reduction in blood glucose level (mg/dl)
(Percentage reduction in blood glucose levels)
Absolute
blood glucose
level (mg/dl)
Group
2hr 4hr 6hr 8hr 24hr
223.4 ± 40.8
(5.22 ± 2.74)
224.4 ± 38.8
(4.76±0.89)
230.6 ± 40.8
(2.16 ±1.68)
232.2 ± 38.46
(1.35 ± 1.06)
235.6 ± 40.3 228.4± 36.26*
(2.89±1.65)**
Control
(1ml gum acacia
suspension)
365.6 ± 13.63
(36.35 ±4.42)
316.6 ± 32.5
(44.84 ±7.03)
293.8 ± 43.11
(48.88 ± 4.9)
327.2 ± 41.5
(43.18 ±6.8)
577 ± 48.16 355.1 ± 55.32
(38.28 ± 5.57)
Oral gliclazide
(25mg/kg)
254 ± 27.21
(21.33 ± 3.73)
263.2 ± 34.98
(18.69 ±3.08)
284.2 ± 37.7
(12.53± 1.47)
293 ± 43.73
(9.7 ± 1.14)
324.6 ± 47 302.4 ± 44.89
(6.78 ± 2.2)
WSB
(10:90 gliclazide-
PEG 6000 SD)
* Reduction in blood glucose level (mg/dl). ** Percentage reduction in blood glucose levels.
65

Control Oral Glz Topical Glz
60
50
40
30
20
10
% Reduction in the blood glucose level (mg/dl)
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time (hr)
Figure 41: Percent reduction in blood glucose levels after oral and topical administration of gliclazide in diabetic
rats.
66

Conclusion:
From the previously demonstrated data the following results can be
concluded:
1- Glz has a lipophilic property.
2- The amount of Glz released from water soluble base (PEG base) and
HPMC gel base was found to be higher than that from other bases.
3- The amount of Glz released from O/W emulsion base was greater than
that released from W/O emulsion base.
4- No drug is released from the absorption base and W/O emulsion base.
5- The investigation showed the effect of incorporation of Glz solid
dispersions in different carriers such as PEG 4000, PEG 6000, glucose
and urea on the amount of Glz released from different topical bases
which can be summarized as follows in descending order:
(8:92) Glz-PEG 6000 SD > (1:10) Glz-glu SD > (8:92) Glz – PEG
4000 SD > Glz- UR SD.
6- The present study showed that gliclazide was absorbed through the
skin and lowered the blood glucose levels.
Topical preparations of Glz or its solid dispersions exhibited better
control of blood glucose level than oral Glz administration in rats as
topical route effectively maintained normoglycemic level in contrast to
the oral group which produced remarkable hypoglycemia.
The blood glucose reducing activity of ointment contained (10:90)
gliclazide –PEG 6000 solid dispersions was significantly more when
compared to ointment contained gliclazide alone.
Finally, the slow and sustained release of the drug from the
transdermal system might reduce manifestations like severe
hypoglycemia, sulphonylurea receptor down regulation and the risk of
chronic hyperinsulinemia.
67

1. Description
Introduction
Glibenclamide
1.1 Name, formula, molecular weight
Glib is 1-{4-[2-(5-chloro-2-methoxybenzamido) ethyl]
benzenesulphonyl}-3-cyclohexylurea
Figure 43: Glib structure.
C23 H28 Cl N3 O5 S
Molecular Weight = 494.0
1.2 Appearance, odour, colour:
Glib is a white, crystalline, odourless powder and practically without
taste (Pamela, 1981).
2. Physical properties
2.1 Melting point
172° to 174°
2.2 Solubility
Glib is virtually insoluble in water and ether; soluble in 330 parts of
alcohol, in 36 parts of chloroform, and in 250 parts of methanol (Pamela,
1981).
69

3.Pharmacokinetics:
Glib is readily absorbed from the gastrointestinal tract, peak plasma
concentrations usually occurring within 2 to 4 hours, and is extensively
bound to plasma proteins. Absorption may be slower in hyperglycaemic
patients and may differ according to the particle size of the preparation
used. It is metabolised, almost completely, in the liver, the principal
metabolite being only very weakly active. About 50% of a dose is
excreted in the urine and 50% via the bile into the faeces (Martindale,
1996) .
4.Mode of action:
As mentioned before under sulfonylureas.
5. Uses and Administration:
Glib is a sulfonylurea antidiabetic. It is given by mouth in the
treatment of type 2 diabetes mellitus and has a duration of action of up to
24 hours.
The usual initial dose of conventional formulations in type 2
diabetes mellitus is 2.5 to 5 mg daily with breakfast, adjusted every 7
days by increments of 2.5 or 5 mg daily up to 15 mg daily. Although
increasing the dose above 15 mg is unlikely to produce further benefit,
doses of up to 20 mg daily have been given. Doses greater than 10 mg
daily may be given in 2 divided doses. Because of the relatively long
duration of action of Glib, it is best avoided in the elderly (Martindale,
1996) .
70

6. Precautions:
As mentioned before under sulfonylureas.
7. Adverse Effects:
As mentioned before under sulfonylureas.
8. Interactions:
As mentioned before under sulfonylureas.
9. Adverse Effects and Precautions
As mentioned before under sulfonylureas.
10. Methods of analysis:
10.1. Polarography:
Procedures have been described for quantitative work, an automated
system, having a flow through micro cell used with silver- silver chloride
reference electrode, has been stated to give good reproducibility (Pamela,
1981).
10.2. Non-aqueous titration:
Tetramethylurea has been used as solvent for the titration of Glib
with 0.1 normal lithium methoxide in benzene-methanol. The end point
was determined potentiometrically or by using 0.2% azoviolet in toluene
as visual indicator (Pamela, 1981).
10.3. Chromatography:
Several procedures have been proposed for the identification of Glib
by thin-layer chromatography. Among the solvent systems described are
butanol-methanol-chloroform-25% ammonia, propanol-cyclohexane and
propanol-benzene-cyclohexane.
71

High-perfprmance liquid chromatography has been recommended for
quantitative determination of Glib in tablets. The column packing uesd
was 1% ethylene propylene copolymer on DuPont Zipax, with 0.01 M
sodium borate containing 27.5% v/v methanol as mobile phase.
Testosterone serves as internal standard (Pamela, 1981).
72

Introduction
There is considerable interest in the skin as a site of drug application
both for local and systemic effect. However, the skin, in particular the
stratum corneum, poses a formidable barrier to drug penetration thereby
limiting topical and transdermal bioavailability. Skin penetration
enhancement techniques have been developed to improve bioavailability
and increase the range of drugs for which topical and transdermal
delivery is a viable option (Heather, 2005).
Drug permeation across the stratum corneum obeys Fick’s first law
(equation 1) where steady-state flux (J) is related to the diffusion
coefficient (D) of the drug in the stratum corneum over a diffusional path
length or membrane thickness (h), the partition coefficient (P) between
the stratum corneum and the vehicle, and the applied drug concentration
(C0) which is assumed to be constant:
1)
73
dm/dt = J = D C0 P/ h
(Equation
Equation 1 aids in identifying the ideal parameters for drug diffusion
across the skin. The influence of solubility and partition coefficient of a
drug on diffusion across the stratum corneum has been extensively
studied. Molecules showing intermediate partition coefficients (log P
octanol/water of 1-3) have adequate solubility within the lipid domains of
the stratum corneum to permit diffusion through this domain whilst still
having sufficient hydrophilic nature to allow partitioning into the viable
tissues of the epidermis. The maximum permeability measurement being
attained at log P value 2.5, which is typical of these types of experiments.

Optimal permeability has been shown to be related to low molecular size
(Potts and Guy, 1992) (ideally less than 500 Da (Bos and Meinardi,
2000)) as this affects diffusion coefficient, and low melting point which is
related to solubility. When a drug possesses these ideal characteristics (as
in the case of nicotine and nitroglycerin), transdermal delivery is feasible.
However, where a drug does not possess ideal physicochemical
properties, manipulation of the drug or vehicle to enhance diffusion,
becomes necessary. The approaches that have been investigated are
summarised in (Figure 42) and discussed below.
Figure 42: Techniques to optimize drug permeation across the skin.
1. Penetration enhancement through optimization of drug and
vehicle properties:
1.1. Prodrugs and ion-pairs:
74

The prodrug approach has been investigated to enhance dermal and
transdermal delivery of drugs with unfavourable partition coefficients
(Sloan, 1992; Sloan and Wasdo, 2003). The prodrug design strategy
generally involves addition of a promoiety to increase partition
coefficient and hence solubility and transport of the parent drug in the
stratum corneum. Upon reaching the viable epidermis, esterases release
the parent drug by hydrolysis thereby optimising solubility in the aqueous
epidermis. The prodrug approach has been investigated for increasing
skin permeability of non-steroidal anti-inflammatory drugs (Davaran et
al., 2003; Thorsteinsson et al., 1999), naltrexone (Stinchcomb et al.,
2002)
Charged drug molecules do not readily partition into or permeate
through human skin. Formation of lipophilic ionpairs has been
investigated to increase stratum corneum penetration of charged species.
This strategy involves adding an oppositely charged species to the
charged drug, forming an ion-pair in which the charges are neutralised so
that the complex can partition into and permeate through the stratum
corneum. The ion-pair then dissociates in the aqueous viable epidermis
releasing the parent charged drug which can diffuse within the epidermal
and dermal tissues. (Megwa et al., 2000; Valenta et al., 2000).
(Sarveiya et al., 2004) recently reported a 16-fold increase in the steady-
state flux of ibuprofen ionpairs across a lipophilic membrane.
1.2. Chemical potential of drug in vehicle – saturated and
supersaturated solutions:
The maximum skin penetration rate is obtained when a drug is at its
highest thermodynamic activity as is the case in a supersaturated solution.
Supersaturated solutions can occur due to evaporation of solvent or by
mixing of cosolvents. These systems are inherently unstable and require
75

the incorporation of antinucleating agents to improve stability (Heather,
2005).
1.3. Eutectic Systems:
As previously described, the melting point of drug influences
solubility and hence skin penetration. According to regular solution
theory, the lower the melting point, the greater the solubility of a material
in a given solvent, including skin lipids. The melting point of a drug
delivery system can be lowered by formation of a eutectic mixture: a
mixture of two components which, at a certain ratio, inhibit the
crystalline process of each other, such that the melting point of the two
components in the mixture is less than that of each component alone.
EMLA cream, a formulation consisting of a eutectic mixture of
lignocaine and prilocaine applied under an occlusive film, provides
effective local anaesthesia for pain-free venepuncture and other
procedures (Ehrenstrom and Reiz, 1982).
1.4. Complexes:
Complexation of drugs with cyclodextrins has been used to enhance
aqueous solubility and drug stability. Cyclodextrin has a hydrophilic
exterior and lipophilic core in which appropriately sized organic
molecules can form non-covalent inclusion complexes resulting in
increased aqueous solubility and chemical stability (Loftsson and
Brewster, 1996). As flux is proportional to the free drug concentration,
where the cyclodextrin concentration is sufficient to complex only the
drug which is in excess of its solubility, an increase in flux might be
expected. However, at higher cyclodextrin concentrations, the excess
76

cyclodextrin would be expected to complex free drug and hence reduce
flux. Skin penetration enhancement has also been attributed to extraction
of stratum corneum lipids by cyclodextrins (Bentley et al., 1997).
1.5. Liposomes and Vesicles:
A variety of encapsulating systems have been evaluated including
liposomes, deformable liposomes or transfersomes, ethosomes and
niosomes.
Liposomes are colloidal particles formed as concentric biomolecular
layers that are capable of encapsulating drugs. The skin delivery of
triamcinolone acetonide was four to five times greater from a liposomal
lotion than an ointment containing the same drug concentration (Mezei
and Gulasekharam, 1980). The mechanism of enhanced drug uptake
into the stratum corneum is unclear. It is possible that the liposomes
either penetrate the stratum corneum to some extent then interact with the
skin lipids to release their drug or that only their components enter the
stratum corneum. It is interesting that the most effective liposomes are
reported to be those composed of lipids similar to stratum corneum lipids
(Egbaria et al., 1990), which are likely to most readily enter stratum
corneum lipid lamellae and fuse with endogenous lipids.
Transfersomes are vesicles composed of phospholipids as their
main ingredient with 10-25% surfactant (such as sodium cholate) and 3-
10% ethanol. The surfactant molecules act as “edge activators”,
conferring ultradeformability on the transfersomes, which reportedly
allows them to squeeze through channels in the stratum corneum that are
less than one-tenth the diameter of the transfersome (Cevc, 1996).
77

Ethosomes are liposomes with a high alcohol content capable of
enhancing penetration to deep tissues and the systemic circulation (Biana
and Touitou, 2003; Touitou et al., 2000).
Niosomes are vesicles composed of nonionic surfactants that have
been evaluated as carriers for a number of drug and cosmetic applications
(Shahiwala and Misra, 2002; Sentjurc et al., 1999). This area continues
to develop with further evaluation of current formulations and reports of
other vesicle forming materials.
1.6. Solid lipid Nanoparticles:
Solid lipid nanoparticles (SLN) have recently been investigated as
carriers for enhanced skin delivery of sunscreens, vitamins A and E,
triptolide and glucocorticoids (Santos Maia et al., 2002; Mei et al.,
2003). It is thought their enhanced skin penetration is primarily due to an
increase in skin hydration caused by the occlusive film formed on the
skin surface by the SLN.
2. Penetration enhancement by stratum cornium modification:
2.1. Hydration:
Water is the most widely used and safest method to increase skin
penetration of both hydrophilic (Behl et al., 1980) and lipophilic
permeants (McKenzie and Stoughton, 1962). The water content of the
stratum corneum is around 15 to 20% of the dry weight Additional water
within the stratum corneum could alter permeant solubility and thereby
modify partitioning from the vehicle into the membrane. In addition,
increased skin hydration may swell and open the structure of the stratum
corneum leading to an increase in penetration. Hydration can be increased
by occlusion with plastic films; paraffins, oils, waxes as components of
ointments and water-in-oil emulsions that prevent transepidermal water
78

loss; and oil-in-water emulsions that donate water. A commercial
example of this is the use of an occlusive dressing to enhance skin
penetration of lignocaine and prilocane from EMLA cream in order to
provide sufficient local anaesthesia within about 1 hour.
2.2. Penetration enhancers:
They are chemicals that interact with skin constituents to promote
drug flux. To-date, a vast array of chemicals has been evaluated as
penetration enhancers (or absorption promoters). Properties for
penetration enhancers acting within skin have been given by Barry, 1983
as follows:
• They should be non-toxic, non-irritating and non-allergenic.
• They would ideally work rapidly, and the activity and duration of effect
should be both predictable and reproducible.
• They should have no pharmacological activity within the body—i.e.
should not bind to receptor sites.
• The penetration enhancers should work unidirectionally, i.e. should
allow therapeutic agents into the body whilst preventing the loss of
endogenous material from the body.
• When removed from the skin, barrier properties should return both
rapidly and fully.
• The penetration enhancers should be appropriate for formulation into
diverse topical preparations, thus should be compatible with both
excipients and drugs.
• They should be cosmetically acceptable with an appropriate skin ‘feel’.
79

2.2.1. Sulphoxides and similar chemicals:
Dimethylsulphoxide (DMSO) is one of the earliest and most widely
studied penetration enhancers. It is a powerful aprotic solvent which
hydrogen bonds with itself rather than with water. it has been shown to
promote the permeation of, for example, antiviral agents, steroids and
antibiotics (Wiiliam and Barry, 2004).
Although DMSO is an excellent accelerant it does create problems.
The effects of the enhancer are concentration dependent and generally co-
solvents containing >60% DMSO are needed for optimum enhancement
efficacy. However, at these relatively high concentrations DMSO can
cause erythema and wheals of the stratum corneum and may denature
some proteins. Studies performed over 40 years ago on healthy volunteers
painted with 90% DMSO twice daily for 3 weeks resulted in erythema,
scaling, contact urticaria, stinging and burning sensations and several
volunteers developed systemic symptoms (Kligman, 1965). A further
problem with DMSO use as a penetration enhancer is the metabolite
dimethylsulphide produced from the solvent; dimethylsulphide produces
a foul odour on the breath.
Since DMSO is problematic for use as a penetration enhancer,
researchers have investigated similar, chemically related materials as
accelerants. Dimethylacetamide (DMAC) and dimethylformamide (DMF)
are similarly powerful aprotic solvents with structures akin to that of
DMSO. Also in common with DMSO, both solvents have a broad range
of penetration enhancing activities.
The mechanisms of the sulphoxide penetration enhancers and
DMSO in particular, are complex. DMSO is widely used to denature
proteins and on application to human skin has been shown to change the
intercellular keratin confirmation. DMSO has also been shown to interact
80

with the intercellular lipid domains of human stratum corneum. Further,
DMSO within skin membranes may facilitate drug partitioning from a
formulation into this “universal solvent” within the tissue.
2.2.2. Azone:
Azone was the first molecule specifically designed as a skin
penetration enhancer. The chemical has low irritancy, very low toxicity
(oral LD50 in rat of 9 g/kg) and little pharmacological activity although
some evidence exists for an antiviral effect. Thus, judging from the
above, Azone appears to possess many of the desirable qualities listed for
a penetration enhancer.
Azone enhances the skin transport of a wide variety of drugs
including steroids, antibiotics and antiviral agents. As with many
penetration enhancers, the efficacy of azone appears strongly
concentration dependent and is also influenced by the choice of vehicle
from which it is applied. Surprisingly, Azone is most effective at low
concentrations, being employed typically between 0.1% and 5%, often
between 1% and 3%.
Azone probably exerts its penetration enhancing effects through
interactions with the lipid domains of the stratum corneum.
Singh et al., 1993 reported that ephedrine patches containing azone
showed an increased flux of ephedrine through rat skin and epidermis
with a reduced time lag.
2.2.3. Pyrrolidones:
A range of pyrrolidones and structurally related compounds have
been investigated as potential penetration enhancers in human skin. They
apparently have greater effects on hydrophilic permeants than for
lipophilic materials. N-methyl-2-pyrrolidone (NMP) and 2-pyrrolidone
(2P) are the most widely studied enhancers of this group.
81

Pyrrolidones have been used as permeation promoters for numerous
molecules including hydrophilic (e.g. mannitol, 5-fluorouracil and
sulphaguanidine) and lipophilic (betamethasone-17-benzoate,
hydrocortisone and progesterone) permeants. As with many studies,
higher flux enhancements have been reported for the hydrophilic
molecules. Recently NMP was employed with limited success as a
penetration enhancer for captopril when formulated into a matrix type
transdermal patch (Park et al., 2001).
In terms of mechanisms of action, the pyrrolidones partition well
into human corneum stratum. Within the tissue they may act by altering
the solvent nature of the membrane and pyrrolidones have been used to
generate ‘reservoirs’ within skin membranes. Such a reservoir effect
offers potential for sustained release of a permeant from the stratum
corneum over extended time periods (Wiiliam and Barry, 2004).
2.2.4. Fatty acids:
Percutaneous drug absorption has been increased by a wide variety
of long chain fatty acids, the most popular of which is oleic acid. It
appears that saturated alkyl chain lengths of around C10–C12 attached to a
polar head group yields a potent enhancer. In contrast, for penetration
enhancers containing unsaturated alkyl chains, then C18 appears near
optimum. For such unsaturated compounds, the bent cis configuration is
expected to disturb intercellular lipid packing more so than the trans
arrangement, which differs little from the saturated analogue. Santoyo
and Ygartua, employed the mono-unsaturated oleic acid, polyunsaturated,
linoleic and linolenic acids and the saturated lauric acid enhancers for
promoting piroxicam flux (Santoyo and Ygartua, 2000). As with Azone,
oleic acid is effected at relatively low concentrations (typically less than
10%) and can work synergistically when delivered from vehicles such as
PG or ternary systems with dimethyl isosorbide (Aboofazeli et al., 2002)
82

Considerable efforts have been directed at investigating the mechanisms
of action of oleic acid as a penetration enhancer in human skin. It is clear
from numerous literature reports that the enhancer 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.
2.2.5. Alcohols:
Ethanol is the most commonly used alcohol as transdrmal
penetration enhancer, it enhances permeation by extracting large amounts
of stratum corneum lipids, it also increases the number of free sulphydryl
groups of keratin in the stratum corneum proteins (Sinha and Maninder,
2000). It increases permeation of ketoprofen from gel-spray formulation
(Porzio et al., 1998).
2.2.6. Propylene glycol (PG):
PG is widely used alone or as cosolvent for other enhancers. PG
increased the flux of heparin sodium (Bonina and Montenegro, 1992)
and ketoprofen, but at higher concentration it inhibited the flux of
ketoprofen. In combination with azone, PG increased the flux of
methotrexate (Chatterjee et al., 1997), cyclosporine A (Duncan et al.,
1990), and 5-fluouracil (Goodman and Berry, 1988). PG works by
solvating keratin of stratum corneum , occupying hydrogen bonding sites
and, thus reducing drug- tissue binding .
2.2.7. Urea (UR):
Urea is a hydrating agent (a hydrotrope) used in the treatment of
scaling conditions such as psoriasis, ichthyosis and other hyper-keratotic
skin conditions. Applied in a water in oil vehicle, urea alone or in
combination with ammonium lactate produced significant stratum
cornum hydration and improved barrier function when compared to the
vehicle alone in human volunteers in vivo (Gloor et al., 2001). Urea also
has keratolytic properties, usually when used in combination with
83

salicylic acid for keratolysis. The somewhat modest penetration
enhancing activity of urea probably results from a combination of
increasing stratum cornum water content (water is a valuable penetration
enhancer) and through the keratolytic activity.
2.2.8. Surfactant:
As with some of the materials described previously (for example
ethanol and PG) surfactants are found in many existing therapeutic,
cosmetic and agro-chemical preparations. Usually, surfactants are added
to formulations in order to solubilise lipophilic active ingredients, and so
they have potential to solubilise lipids within the stratum corneum.
Typically composed of a lipophilic alkyl or aryl fatty chain, together with
a hydrophilic head group, surfactants are often described in terms of the
nature of the hydrophilic moiety. Anionic surfactants include sodium
lauryl sulphate (SLS), cationic surfactants include cetyltrimethyl
ammonium bromide, the nonoxynol surfactants are non-ionic surfactants
and zwitterionic surfactants include dodecyl betaine. Anionic and cationic
surfactants have potential to damage human skin; SLS is a powerful
irritant and increased the trans epidemeral water loss in human volunteers
in vivo (Tupker et al., 1990) and both anionic and cationic surfactants
swell the stratum corneum and interact with intercellular keratin. Non-
ionic surfactants tend to be widely regarded as safe. Surfactants generally
have low chronic toxicity and most have been shown to enhance the flux
of materials permeating through biological membranes.
Surfactant facilitated permeation of many materials through skin
membranes has been researched, with reports of significant enhancement
of materials such as chloramphenicol through hairless mouse skin by
SLS, and acceleration of hydrocortisone and lidocaine permeating across
84

hairless mouse skin by the non-ionic surfactant Tween 80 (Sarpotdar
and Zatz,1986a, 1986b).
2.2.9. Gramicidin:
Gramicidin is a linear peptide –type cataionic ionophore that has no
charged or hydrophilic chains and its aqueous solubility is low.
Gramicidin increased the flux of benzoic acid through rat abdominal skin
by rearranging lipid barrier and increasing hydration of stratum corneum
(Chi and Choi, 2000).
2.2.10. Phospholipids:
Phosphatidyl glycerol derivative increased the accumulation of
bifonazole in skin and percutaneous penetration of tenoxicam;
phosphatidyl choline derivatives promoted the percutaneous penetration
of erythromycin (Yokomizo, 1996).
2.2.11. Lipid synthesis inhibitors :
The barrier layer consists of a mixture of cholesterol, free fatty
acids, and ceramides, and these three classes of lipids are required for
normal barrier function. Addition of inhibitors of lipid synthesis enhances
the delivery of some drugs like lidocaine and caffeine .Fatty acid
synthesis inhibitors like 5-(tetradecyloxy)-2-furancarboxilic acid (TOFA)
and the cholesterol synthesis inhibitors like fluvastatin (FLU) or
cholesterol sulfate (CS) delay the recovery of barrier damage produced by
prior application of penetration enhancers like DMSO, acetone, and like
causes a further boost in the transdermal permeation (Tsia et al., 1996).
85

2.2.12. Amino acid derivatives :
Various amino acid derivatives have been investigated for their
potential in improving percutaneous permeation of drugs. Application of
n-dodecyl-L-amino acid methyl ester and n-pentyl-N-acetyl prolinate on
excised hairless mouse skin 1 hour prior to drug treatment produced
greater penetration of hydrocortisone from its suspension (Fincher et al.,
1996).
2.2.13. Clofibric acid :
Esters and amides of clofibric acid were studied for their
permeation-enhancing property using nude mice skin. The best
enhancement of hydrocortisone-21-acetate and betamethasone-17-
valerate was observed with clofibric acid octyl amide when applied 1
hour prior to each steroid. Amide analogues are generally more effective
than ester derivatives of the same carbon chain length (Michniak et al.,
1993).
2.2.14. Dodecyl-N,N-dimethylamino acetate (DDAA):
DDAA increasesd the transdermal permeation of a number of
drugs,like propranolol HCl and timolol maleate.The permeability
enhancing effect was due to changes in lipid structure of stratum corneum
, like azone and oleic acid (Ruland et al ., 1994) and hydrating effect on
the skin (Fleeker et al., 1989).Its duration of action is shorter than that of
azone and dodecyl alcohol because of presence of hydrophilic groups
(Hirvonen et al., 1994), so there is faster recovery of the skin structure
and hence less irritation potential.
2.2.15. Enzymes :
86

Due to the importance of the phosphatidyl choline metabolism
during maturation of the barrier lipids, the topical application of the
phosphatidyl choline-depentent enzyme phospholipase c produced an
increase in the transdermal flux of benzoic acid,mannitol, and
testosterone . Triglycero hydrolase (TGH) increased the permeation of
mannitol, while phospholipase A2 increased the flux of both benzoic acid
and mannitol (Patil et al., 1996).
87

1. Materials and supplies:
Experiment and methodology
* Glibenclamide was kindly supplied by Egyptian International
Pharmaceutical Industries Company (EIPICO).
* Sodium alginate (El-Gomhouria Company, Eygypt).
* Tween 80 (Merk Sharp and Dohmn, Germany).
* Cetrimide (Searle Company, England).
* Transcutol, labrafil, oleic acid, linoleic acid, isopropylmyristate and
isopropylpalmitate (Sigma Chemical Co.St.Louis, USA).
* Other materials were mentioned previously in chapter two.
2. Equipment:
These were mentioned previously in chapter two.
3. Software:
These were mentioned previously in chapter two.
4. Methods:
4.1. UV scanning of Glib:
About 20 and 100 μg /ml of Glib in methanol were scanned
spectrophotometerically from 200-400 nm using methanol as blank.
4.2. Construction of calibration curve of Glib in sörensen’phosphate
buffer pH 7.4.
0.1 gram of Glib were dissolved in 100 ml methanol to obtain a
solution of concentration of 1mg/ml, 10 ml is diluted to 100 ml with
sörensen’ buffer pH 7.4 to produce a solution containing 100 μg /ml of
Glib . Aliquots of 0.5, 1, 1.5, 2, 2.5, and 3 ml were furtherly diluted to 10
88

ml with sörensen’ buffer pH 7.4. After dilution, the solution contained 10,
15, 20, 25, and 30 μg/ml of Glib respectively.
The calibration equation was constructed by regressing the relative
absorbances, against the corresponding Glib solutionsconcentrations at
227 nm using sörensen’ buffer pH 7.4 as blank.
4.3. Solubility measurements:
Solubility studies were carried out according to the method of
Higuchi and Connors, (1965) as mentioned before.
4.4. Determination of partition coefficient of Glib:
Partition coefficient of Glib in octanol/water system was determined
as mentioned before.
4.5. The methods of preparation of topical preparations:
The following formulae were selected in which 10 mg of Glib in
each 1 gm of the topical base were incorporated.
4.5.1. Water soluble base:
Polyethylene glycol base :( U.S.P. XXII).
PEG 4000 40 gm
PEG 400 60 gm
Preparation
Water soluble base was prepared as mentioned before.
4.5.2. Absorption base: (U.S.P.XXII)
White soft paraffin 95 gm.
Span 80 5 gm
89

4.5.3. Oleaginous base: (Ammar., et al 2007)
White soft paraffin 100 gm.
Preparation:
Accurate amount of the drug was weighed, levigated and
incorporated into the melted base with continuous stirring until congealed
then packed into plastic jar until used.
4.5.4. Emulsion base:
O/W emulsion base (Beeler’s base) (Ezzedeen et al., 1986):
White bees wax 1 gm
Cetyl alcohol 15 gm
Propylene glycol 10 gm
Sodium lauryl sulphate 2 gm.
Water 72 gm.
Preparation:
O/W emulsion base was prepared as mentioned previously.
4.5.5. Gel bases:
* Hydroxypropyl methylcellulose gel (Sobati, 1998):
HPMC 12 gm
water 88 gm
* Sodium alginate gel (Sobati, 1998):
Sodium alginate 8 gm
Water 92 gm
90

Preparation:
The drug was dispersed in a quantity of water then the gelling agent
was added with continuous stirring and was set aside for complete
swelling and the weight was adjusted by the addition of the water.
:
4.5.6. Hydroxypropyl methylcellulose emulgel (Gehan, 1999):
Liquid paraffin 20 gm
Tween 80 1 gm
Water 70 gm
HPMC 9 gm
Preparation:
-A mixture of the aqueous phase containing hydrophilic emulsifier was
added to the oily phase to form a primary O/W emulsion.
- Drug was suspended into the primary emulsion, then the specified
quantity of the gelling agent powder was sprinkled on the emulsion
surface and was left a side for complete swelling and formation of
emulgel.
All the formulations mentioned previously were summarized in Table 24
91

Table 24 : Composition of different topical formulations.
Type of
base
PEG
4000
Water
soluble
base
(PEG
base)
30
PEG 400 70
Span 80 5
Absorp
t-ion
base
Oleagino
u-s base
92
Emulsio
n base
(O/W
base)
Gel base
HPMC Sod.
Algin
a-te
Soft
paraffin
95 100
Propylene
glycol
10
White
bees wax
1
Sodium
lauryl
sulphate
2
HPMC 12 8 9
Tween 80 2
Liquid
paraffin
Sodium
alginate
20
Water 88 92 69
Emulg
el base
(HPM
C
emulge
l)

4.6. In vitro release of Glib from different topical formulation:
The release study was determined using the simple dialysis
technique as mentioned in part one. 1 gm of the tested formulation
containing (10 mg of the drug) was accurately weighted over the
cellophane membrane (Donor). The diffusion cell was placed at the center
of 1000 ml dissolution cell containing 100 ml of phosphate buffer pH 7.4
(Receptor). The stirring rate was 100 rpm and the temperature was
maintained at 37 ± 0.5 °C
At suitable time intervals 2.5 ml sample was withdrawn from the
sink solution assayed spectrophotometerically at 227 nm using a suitable
blank.A similar volume of buffer was added to mentain the volume of
receptor constant. Each experiment was done in triplicate, and the
average was calculated. The cumulative amount of the drug released was
calculated as mentioned in chapter one.
4.7. Penetration enhancers screening procedure:
In order to select penetration enhancers which lend themselves to a
more detailed investigation, the screening procedure were developed
based on the percentage of the drug released after six hours.
4.8. Effect of incorporation of different penetration enhancers in
water soluble base:
On the basis of results obtained in the previous screening, different
penetration enhancers with different concentrations were incorporated in
the topical formulation that demonstrated the best release results (water
soluble base) as shown in (Table 25 ). In vitro release of these
preparations was done as mentioned above.
Table 25 : Types of penetration enhancers and percentages used.
93

Penetration enhancers Percentages used
(A)Surfactants
1.Cationic surfactant
(cetrimide) 0.3 0.5 1 2
2.Anionic surfactant
(SLS) 0.1 0.4 0.8
3. Non ionic surfactant
i.Tween 80 (Tw-80) 0.3 1 4 5
ii. Labrafil (Lab) 3 5 7
B) Solubilizing agents
Transcutol (Tc) 5 8
(C) Unsaturated free
fatty acids
1. Oleic acid (OA)
0.5
94
1
2. Linoleic acid (LOA) 0.8
(D) Fatty acid esters
1.Isopropyl myristate
(IPM)
2. Isopropyl palmitate
(IPP)
0.5
0.2

4.9. Kinetic evaluation of the in vitro release data:
The data obtained from the experiments were analyzed to know the
mechanism of the release of the drug using the following kinetic
equations:
(I) Zero order kinetics: A=A-k
(II) First order kinetics: ln A = ln A- kt
log A = log A- kt/ 2.303
(III) Higuchi diffusion model:
M = Q = 2C ) ½
4.10. In vitro permeation of glibenclamide through abdominal rabbit
skin:
Preparation of the rabbit skin and in vitro permeation of Glib were
done by the same methods mentioned in part one.
4.11. Statistical analysis:
Data were expressed as mean of three experiments ± the standard
error (S.E.). The obtained data were compared statistically using One-
way analysis of variance (ANOVA) test of significance on a computer
statistical SPSS analysis program. A p-value of 0.05 or less was
considered to be significant (Suwanpidokkul et al., 2004).
4.12. In vivo studies:
4.12.1. Animals:
The animals used for the anti diabetic and hypoglycemic activity study
were white adult albino rats weighing between 200-250g. The animals
were housed under standard laboratory conditions.
95

4.12.2. Hypoglycemic activity in normal rats:
The hair on the backside of the rats was removed with an electric hair
clipper on the previous day of the experiment. The oral doses were given
using a round tipped stainless steel needle attached to 1 ml syringe.
Following an overnight fast, rats were divided into7 groups (n=5). The
rats were treated as follows:
Group I (Control) – 1ml gum acacia suspension was given orally.
- 5 mg/kg Glib in mucilage of gum acacia was given orally
(Mutalik and Udupa, 2005).
Group III - 5 mg drug incorporated in 1gm water soluble ointment base
was applied topically.
Group IV - 5 mg drug incorporated in 1gm water soluble ointment base
containing 1% oleic acid was applied topically.
Group V - 5 mg drug incorporated in 1gm water soluble ointment base
containing 1% cetrimide was applied topically.
Group VI - 5 mg drug incorporated in 1gm water soluble ointment base
containing 1% isopropylmyristate was applied topically.
Group VII - 5 mg drug incorporated in 1gm water soluble ointment
base containing 5% Labrafil was applied topically.
At time intervals between 2-24 h after treatment blood was collected
from orbital sinuses; blood glucose levels were determined using the
glucometer.
96

4.12.3. Hypoglycemic activity in diabetic rats:
4.12.3.1. Induction of diabetes mellitus:
The overnight fasted rats were made diabetic by a single
intraperitoneal injection of streptozotocin (STZ) (50 mg/kg; i.p) dissolved
in citrate buffer (pH 4.5). The blood glucose was measured after 24 hrs
and animals with blood glucose levels >250 mg/dL were selected
(Sridevi et al., 2000).
4.12.3.2. Anti-diabetic activity in diabetic rats:
The anti diabetic activity of the prepared topical preparation was
evaluated in overnight fasted diabetic rats.
Diabetic rats were divided into 3 groups (n=5). The rats were treated as
follows:
Group I (Control) – 1ml gum acacia suspension was given orally.
Group II -Glib 5 mg/kg was given orally (Mutalik and Udupa, 2005).
Group III –5 mg drug incorporated in 1gm water soluble ointment base
in presence of 1% cetrimide was applied topically.
At time intervals between 2-24 h after treatment blood was
collected from orbital sinuses; blood glucose levels were determined
using the glucometer.
4.12.4. Statistical analysis:
Data were expressed as mean of three experiments ± the standard
error (S.E.). The obtained data were compared statistically using One-
way analysis of variance (ANOVA) test of significance on a computer
statistical SPSS analysis program. A p-value of 0.05 or less was
considered to be significant (Suwanpidokkul et al., 2004).
97

1. UV scanning of Glib:
Results and Discussion
UV scanning of Glib in methanol was carried out (Figure44). Three
absorption maxima were observed at 299, 278 and 227 nm at
concentration of Glib of (100 μg/ml) and nearly the same wavelengths
were observed at concentration of Glib of (20μg/ml) with lower intensity.
The measurements were done at 227 nm (Siavoush et al., 2005).
20 μg/ml
227nm
100 μg/ml
278nm
Wavelength
Figure 44: UV absorption spectra for Glib in methanol.
98
299nm

2. Calibration curves of Glib in sörensen’s phosphate buffer pH (7.4):
(Figure 45) show a linear relationship between the absorbance and
the concentration of Glib in sörensen’s phosphate buffer pH 7.4 at the
max in the concentration range used.
3. Solubility measurements:
In the present study, the solubility of the Glib in distilled water and
in sörensen’s phosphate buffer pH 7.4 at 25 C was found to be 4.41
μg/ml and 16.19 μg/ml respectively.
4. Partition coefficient of Glib:
In the present study, the partition coefficient of Glib was found
to be 2.05 (log octanol/ water =0.312) and this is in agreement with that
observed by Srinivas and Nayanabhirama, 2005, who found that the
log octanol /buffer = 0.32.
99

5. Release of Glib from different topical bases:
The influence of the type of base on the in vitro release of Glib has
been studied. The bases investigated consisted of ointment (water soluble
base, emulsion base, absorption base, and oleaginous base), emulgel and
gel (HPMC and sodium alginate gels).
Results of release from different topical bases are summarized in
(Table 26) and graphically illustrated in (Figures 46-47).
Due to the high lipid solubility and low water solubility (4.41
μg/ml) of Glib, this may explain the slow release of the drug that is
observed from all bases.
From the data obtained it is clear that the percentage amount of drug
released from water soluble base, gel bases and emulgel base are greater
than that released from other bases. The rate of drug release can be
arranged in the following descending order:
Water soluble base (5.94 %) > HPMC emulgel (4.6 %) > sodium alginate
gel (4.38 %) > HPMC gel (3.99) >O/W emulsion base (2.5 %) >
absorption base (1.94%) > oleaginous base (1.61%).It is clear that, water
soluble base showed the highest release than that of emulsion, gels,
emulgel, oleaginous and absorption bases.
100

Table 26: In vitro release of glibenclamide from different topical bases.
Glibenclamide released % ± (sd)
Time (min) WSB HPMC emulgel Sodium alginate gel HPMC gel
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 0.54 ± 0 0.65 ± 0.07 0.73 ± 0.016
60 1.5 ± 0.09 1.22 ± 0.06 0.95 ± 0.05 1.16 ± 0.06
90 1.71 ± 0.25 2.09 ± 0.14 1.45 ± 0.08 1.71 ± 0.13
120 2.25 ± 0.07 2.19 ± 0.19 1.97 ± 0.12 2.36 ± 0.03
150 2.86 ± 0.14 2.46 ± 0.048 2.44 ± 0.26 2.46 ± 0.14
180 3.44 ± 0.12 2.71 ± .062 2.68 ± 0.16 2.5 ± 0.25
240 4.8 ± 0.25 3.39 ± 0.17 3.15 ± 0.28 2.91 ± 0.15
300 5.29 ± 0.11 3.86 ± 0.18 4.14 ± 0.31 3.40 ± 0.25
360 5.94 ± 0.41 4.6 ± 0.27 4.38 ± 0.3 3.99 ± 0.05
101

Cont.Table 26: In vitro release of glibenclamide from different topical bases.
Glibenclamide released % ± (sd)
Time (min) O/W cream Absorption base Oleaginous base
0 0 ± 0 0 ± 0 0 ± 0
30 0.54 ± 0 0.44 ± 0.02 0.60 ± 0.013
60 0.65± 0.03 0.49 ± 0 0.89 ± 0.06
90 0.97 ± 0.038 0.97 ± 0.13 0.91 ± 0.13
120 1.13 ± 0.06 1.055 ± 0.21 1.22 ± 0.12
150 1.97 ± 0.08 1.12 ± 0.08 1.27 ± 0.08
180 1.81± 0.04 1.27 ± 0.15 1.48 ± 0.15
240 1.92 ± 0.07 1.35 ± 0.56 1.34 ± 0.2
300 2.2 ± 0.09 1.37 ± 0.9 1.51 ± 0.14
360 2.5 ± 0.1 1.94 ± 0.38 1.61 ± 0.17
102

WSB base HPMC gel HPMC emulgel Sodium alginate gel
O/W cream Oleagineous base Absorption base
7
6
5
4
3
% drug releasesd
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 46: Release profile of glibenclamide from different topical bases.
103

The highest release may be attributed to the rapid dissolution of the base
in water and the possible solubilizing effect of the base components
(Moes, 1982; Anshel, 1976)
(Chakole et al., 2009) found that halobetasol propionate and Fusidic
acid ointment formulation containing water miscible base showed better
in-vitro release profile in comparison to oleaginous base.
(Dhavse and Amin, 1997) stated that norfloxacin formulations
containing polyethylene glycol and Carbopol gel base showed better in
vitro release profile in comparison to creams and ointment base
formulations.
The higher release of the Glib from emulgel and gel bases than O/W
emulsion base, oleaginous and absorption ointment bases is considered to
be due to the high miscibility of these bases with the dissolution medium.
The higher release of the Glib from emulgel than gel bases is
considered to be due to the presence of Tween80 which can facilitate the
release of drug.
The lower release of the drug from O/W emulsion base than from
water soluble base, emulgel and gels owing to its biphasic nature which
leading to partitioning of the drug in 2 phases, that results in slower
release of drug (Dhavse and Amin, 1997)
The higher release of the Glib from O/W emulsion base than from
absorbtion base and oleaginous base may be due to the formation of a
continuous contact between the external phase of the O/W emulsion base
and the buffer (Nakano et al., 1976).
104

7
WSB
6
WSB
HPMC emulgel
Sod. alginate
HPMC gel
O/W cream
Absorption base
Oleaginous base
5
HPMC emulgel
Sod. alginate
HPMC gel
4
3
O/W cream
% Drug released
Absorption base
Oleaginous base
2
1
0
1
Figure 47: Percentage drug released from different topical bases.
105

In case of oleaginous base and absorption base, the external phase is non-
polar and immiscible with the polar diffusion medium hence retardation
of drug release is expected. Also this low release may be attributed to the
closing of the cellophane membrane pores with the fatty base and
prevention of penetration of the acceptor medium through the membrane
to dissolve the drug (Habib and El-Shanawany, 1989).
6. Effect of incorporation of penetration enhancers:
The transdermal route has been recognized as one of the highly
potential routes of systemic drug delivery and provides the advantage of
avoidance of the first-pass effect, ease of use and withdrawal (in case of
side effects), and better patient compliance. However, the major
limitation of this route is the difficulty of permeation of drug through the
skin (Sinha and Maninder, 2000). Studies have been carried out to find
safe and suitable permeation enhancers to promote the percutaneous
absorption of Glib.
106

6.1. Effect of incorporation of surfactants:
The effect of surfactant on the release of Glib from the prepared
water soluble base is shown in (Tables 27- 30) and (Figures 49-53).
Anionic, cationic and non-ionic surfactants were used.
6.1.1. Anionic surfactants:
Incorporation of sodium lauryl sulphate (SLS) in concentrations of
0.4% and 0.8% increased the percentage of drug released from 5.94 % to
7.95% and 7.12% respectively. While incorporation of SLS in
concentration of 0.1% decreased the amount of drug released by (-1.06
fold) in comparison to control.
Nokhodchi et al., 2003 studied the enhancing effects of SLS on the
permeation of lorazepam through rat skin and he found that, sodium
lauryl sulphate at a concentration of 5% w/w (the highest concentration)
exhibited the greatest increase in flux of lorazepam compared with
control.
6.1.2. Cationic surfactants:
Incorporation of cetrimide (Cetylpyridiniumbromide) in
concentrations of 0.3%, 0.5% and 1% increased the percentage of drug
released from 5.94 % to 7.18%, 8.75% and 9.48% respectively. While
incorporation of cetrimide in concentration of 2% decreased the amount
of drug released by (-1.17 fold) in comparison to control.
6.1.3. Non-ionic surfactants:
5% increased the percentage of drug released from 5.94 % to 9.05% and
6.76% respectively. While incorporation of Tween 80 in concentration of
0.3% and 1% decreased the amount of drug released by (-1.04 fold) in
comparison to control. This is in accordance with (Ramadan, 2008) who
studied Enhancement factors for the penetration of miconazole through
cellulose barrier from different bioadhesive gels containing different
107

concentrations of Tween80 and she found that, 1% concentration of
enhancers used seems to be the optimum concentration at which the
maximum release and concentration of enhancers beyond the maximum
concentration would be responsible for permeability coefficient declined
and reducing of enhancement effect. Enhancer at high level showed a
lower tendency to solubilize the drug which may be attributed to complex
formation.
-6 glycerides) in
concentrations of 3% and 5% increased the percentage of drug released
from 5.94 % to 7.32% and 10.03% respectively. While incorporation of
labrafil in concentration of 7% decreased the amount of drug released by
(-1.11 fold) in comparison to control.
Choi et al., 2003 found that incorporation of 1.5% of labrafil in
50/50 buffer (pH 10)/ PG solvent mixture increased the permeability of
clenbuterol through hairless mouse skin approximately 8 folds more than
control without permeation enhancer.
Based on the above mentioned results, it is obvious that addition of
the surfactant into the ointment base could result in increased solubility of
the hydrophobic drug, leading to the increase in the drug release rate
(Sarisuta et al., 1999). In addition increasing the concentration of
surfactant may decrease drug release and this may be attributed to
miceller complexation which decreases thermodynamic activity of the
drug (Fergany, 2001).
108

Table 27: In vitro release of glibenclamide from water soluble base containing different concentrations of cetrimide.
Glibenclamide released % ± (sd)
Time (min)
0% 0.3% 0.5% 1% 2%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 0.69 ± 0.2 0.7 ± 0.07 1.09 ± 0.035 0.6 ± 0.01
60 1.5 ± 0.09 1.11 ± 0.12 2.02 ± 0.16 2.23 ± 0.11 1.1 ± 0
90 1.71 ± 0.25 2.36 ± 0.14 3.26 ± 0.37 3.29 ± 0.18 1.72± 0.01
120 2.25 ± 0.07 3.15 ± 0.14 4.22 ± 0.33 4.3 ± 0.07 2.18 ± 0.04
150 2.68 ± 0.14 3.75 ± 0.19 5.16 ± 0.33 5.2 ± 0.51 2.57 ± 0. 2
180 3.44 ± 0.12 4.41 ± 0.34 5.89 ± 0.12 6.1± 0.53 3.22 ± 0.19
240 4.8 ± 0.25 5.59 ± 0.17 6.84 ± 0.12 7.34 ± 0.48 3.64 ± 0.007
300 5.29 ± 0.11 6.73 ± 0.18 7.87 ± 0.24 8.6 ± 0.16 4.17 ± 0.19
360 5.94 ± 0.41 7.18 ± 0.19 8.75 ± 0.34 9.48 ± 0.56 5.05 ± 0.2
109

Drug alone 0.3% cetrimide 0.5% cetrimide 1% cetrimide 2% cetrimide
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 49: Release profile of glibenclamide from water soluble base containing different concentrations of cetrimide.
110

Table 28: In vitro release of glibenclamide from water soluble base containing different concentrations of Sodium
lauryl sulphate (SLS).
Glibenclamide released % ± (sd)
Time (min)
0% 0.1% 0.4% 0.8%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 0.5 ± 0 1.96 ± 0.11 1.75 ± 0.014
60 1.5 ± 0.09 1.42 ± 0.14 2.96 ± 0.07 2.59 ± 0.014
90 1.71 ± 0.25 1.81 ± 0.09 3.66 ± 0.14 3.38± 0.077
120 2.25 ± 0.07 2.22 ± 0.06 3.96 ± 0.16 3.83 ± 0.056
150 2.68 ± 0.14 2.7 ± 0.19 4.41 ± 0.15 4.22 ± 0. 3
180 3.44 ± 0.12 3.37 ± 0.11 4.96 ± 0.17 4.85 ± 0.13
240 4.8 ± 0.25 4.3 ± 0.46 6.56 ± 0.16 5.9 ± 0.17
300 5.29 ± 0.11 5.11 ± 0.35 7.26 ± 0. 4 6.92 ± 0.18
360 5.94 ± 0.41 5.59 ± 0.41 7.95 ± 0.16 7.12 ± 0.18
111

12
Drug alone 0.1% SLS 0.4% SLS 0.8% SLS
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 50: Release profile of glibenclamide from water soluble base containing different concentrations of SLS.
112

Table 29: In vitro release of glibenclamide from water soluble base containing different concentrations of Tween 80.
Glibenclamide released % ± (sd)
Time (min)
0% 0.3% 1% 4% 5%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 0.67 ± 0.16 0.95 ± 0.1 1.72 ± 0.05 1.1 ± 0.05
60 1.5 ± 0.09 1.32 ± 0.11 1.72 ± 0.2 2.47 ± 0.07 2.12 ± 0.07
90 1.71 ± 0.25 1.95 ± 0.15 2.17 ± 0.07 3.45 ± 0.2 2.72± 0.04
120 2.25 ± 0.07 2.44 ± 0.07 2.75 ± 0.16 4.33 ± 0.007 3.31± 0.13
150 2.68 ± 0.14 3.03 ± 0.06 3.16 ± 0.03 5.3 ± 0.24 3.89 ± 0. 07
180 3.44 ± 0.12 3.5 ± 0.15 3.68 ± 0.02 5.64± 0.26 4.37 ± 0.24
240 4.8 ± 0.25 4.25 ± 0.1 4.99 ± 0.19 7.05 ± 0.14 5.08 ± 0.33
300 5.29 ± 0.11 4.71 ± 0.09 4.97 ± 0.04 8.2 ± 0.15 5.81 ± 0.36
360 5.94 ± 0.41 5.66 ± 0.09 5.69 ± 0.08 9.05 ± 0.23 6.76 ± 0.24
113

Drug alone 0.3% Tw- 80 1% Tw- 80 4% Tw- 80 5% Tw- 80
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 51: Release profile of glibenclamide from water soluble base containing different concentrations of Tween 80.
114

Table 30: In vitro release of glibenclamide from water soluble base containing different concentrations of labrafil.
Glibenclamide released % ± (sd)
Time (min)
0% 3% 5% 7%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 1.68 ± 0 1.59 ± 0.27 0.84 ± 0.019
60 1.5 ± 0.09 3.01 ± 0.17 3.23 ± 0.21 1.97 ± 0.2
90 1.71 ± 0.25 3.56 ± 0.16 4.02 ± 0.23 2.46 ± 0.15
120 2.25 ± 0.07 4.68 ± 0.25 4.96 ± 0.14 2.69 ± 0.36
150 2.68 ± 0.14 5.43 ± 0.33 5.5 ± 0.37 2.98 ± 0.21
180 3.44 ± 0.12 4.41 ± 0.18 5.97 ± 0.24 3.1 ± 0.17
240 4.8 ± 0.25 5.86 ± 0.11 8.46 ± 0.14 4.13 ± 0.32
300 5.29 ± 0.11 6.72 ± 0.22 9.3 ± 0.26 4.96 ± 0.25
360 5.94 ± 0.41 7.32 ± 0.17 10.03 ± 0.18 5.33 ± 0.22
115

Drug alone Lab 3% Lab 5% Lab 7%
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 52: Release profile of glibenclamide from water soluble base containing different concentrations of labrafil.
116

117

Drug alone Cetrimide 0.3% Cetrimide 0.5% Cetrimide 1% Cetrimide 2%
SLS 0.1% SLS 0.4% SLS 0.8% Tw 80 0.3% Tw 80 1%
Tw 80 4% Tw 80 5% Lab 3% Lab 5% Lab 7%
12
Lab 5%
Cetrimide
1%
10
Tw 80 4%
Cetrimide
0.5%
SLS 0.4%
Lab 3%
Cetrimide
0.3%
8
Tw 80
5%
SLS 0.8%
Tw 80
1%
Tw 80
0.3%
SLS
0.1%
Drug
Lab 7%
Cetrimide
2%
6
% Drug released
4
2
0
1
Figure 53: Percentage drug released from water soluble base containing different concentrations of different
surfactants.
118

6.2. Effect of incorporation of fatty acids:
The effect of unsaturated fatty acids on the release of Glib from the
prepared water soluble base is shown in (Tables 31-32) and (Figures 54-
56).
increased the percentage of drug released from 5.94 % to 6.19% and
8.8%. However, concentrations of 2% and 3% increased the release of
Glib when compared with the control but didn't lead to a further increase
in permeation and this is with agreement with (Ammar., et al 2007) who
studied the effect of oleic acid on the transdermal delivery of aspirin and
he found that oleic acid enhanced aspirin permeation from CMC gel base
at a concentration of 5% or 10% However, a concentration of 20%
enhanced the permeation when compared with the control but didn't lead
to a further increase in permeation. This may be attributed to an increase
in the lipophilicity of the vehicle (Ammar., et al 2006).
2% increased the percentage of drug released from 5.94 % to 7.34%,
6.56% and 6.43% respectively.
Gwak and Chun, 2001 studied the effect of linoleic acid on
transdermal delivery of aspalatone and they found that, linoleic acid
(LOA) at the concentration of 5% was found to have the largest
enhancement factor. However, a further increase in aspalatone flux was
not found in the fatty acid concentration greater than 5%, indicating the
enhancement effect is in a bell-shaped curve
119

Table 31: In vitro release of glibenclamide from water soluble base containing different concentrations of oleic acid.
Glibenclamide released % ± (sd)
Time (min)
0% 0.5% 1% 2% 3%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 0.87 ± 0 1.59 ± 0.16 1.15 ± 0.023 1.31 ± 0.035
60 1.5 ± 0.09 2.13 ± 0.05 2.7 ± 0.15 2.71 ± 0.36 2.08 ± 0.14
90 1.71 ± 0.25 2.95 ± 0.09 3.89 ± 0.13 3.74 ± 0.35 2.81± 0.25
120 2.25 ± 0.07 3.44 ± 0.07 4.5 ± 0.39 4.15 ± 0.17 3.08± 0.37
150 2.68 ± 0.14 3.71 ± 0.08 4.96 ± 0.4 4.73 ± 0.24 3.5 ± 0. 32
180 3.44 ± 0.12 4.24 ± 0.13 5.86 ± 0.34 5.4± 0.45 3.87 ± 0.24
240 4.8 ± 0.25 5.05 ± 0.21 7.09 ± 0.32 6.82 ± 0.43 4.98 ± 0.19
300 5.29 ± 0.11 5.64 ± 0.35 7.9 ± 0.45 7.4 ± 0.15 5.38 ± 0.22
360 5.94 ± 0.41 6.19 ± 0.27 8.8 ± 0.4 7.67 ± 0.19 6.68 ± 0.21
120

Drug alone 0.5% OA 1% OA 2% OA 3% OA
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 54: Release profile of glibenclamide from water soluble base containing different concentrations of oleic
acid.
121

Table 32: In vitro release of glibenclamide from water soluble base containing different concentrations of linoleic
acid.
Glibenclamide released % ± (sd)
Time (min)
0% 0.8% 1% 2%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 1.54 ± 0.024 1.45 ± 0.09 0.97 ± 0.1
60 1.5 ± 0.09 2.42 ± 0.17 1.98 ± 0.16 1.95 ± 0.15
90 1.71 ± 0.25 2.9 ± 0.17 2.73 ± 0.23 2.51 ± 0.06
120 2.25 ± 0.07 3.33± 0.28 3.27 ± 0.09 3.22 ± 0.25
150 2.68 ± 0.14 4.31 ± 0.38 3.84 ± 0.18 3.81 ± 0. 09
180 3.44 ± 0.12 4.9 ± 0.35 4.42 ± 0.44 4.2 ± 0.26
240 4.8 ± 0.25 5.89 ± 0.59 5.2 ± 0.24 5.12 ± 0.28
300 5.29 ± 0.11 6.66 ± 0.52 6.45 ± 0.28 5.9 ± 0.42
360 5.94 ± 0.41 7.34 ± 0.48 6.56 ± 0.47 6.43 ± 0.37
122

Drug alone 0.8% LOA 1% LOA 2% LOA
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 55: Release profile of glibenclamide from water soluble base containing different concentrations of linoleic
acid.
123

10
Drug alone OA 0.5% OA 1% OA 2% OA 3% LOA 0.8% LOA 1% LOA 2%
OA 1%
9
OA 2%
8
LOA 0.8%
LOA 1%
OA 3%
7
LOA 2%
OA 0.5%
Drug alone
6
5
4
% Drug released
3
2
1
0
1
Figure 56: Percentage drug released from water soluble base containing different concentrations of fatty acids.
124

6.3. Effect of incorporation of fatty acid esters:
The effect of fatty acid esters namely isopropylpalmitate (IPP) and
isopropylmyristate (IPM) on the release of Glib from the prepared water
soluble base is shown in (Tables 33-34) and (Figures 57, 58 and 60).
increased the percentage of drug released from 5.94 % to 6.11%, 10.26%
and 6.82% respectively.
increased the percentage of drug released from 5.94 % to 8.39%, 7.33%
and 7.025% respectively.
Malay et al., 2006 investigated the effect of (10% W/W) IPP and
IPM on transdermal permeation of trazodone hydrochloride from matrix-
based formulations through the skin and he found that, the highest
enhancing effect was obtained with IPM followed by IPP. The
permeation of TZN in the presence of 10% w/w of IPM and IPP was 3.79
and 2.00 times greater, respectively, than that in absence of these
enhancers.
125

Table 33: In vitro release of glibenclamide from water soluble base containing different concentrations of
Isopropylmyristate (IPM).
Glibenclamide released % ± (sd)
Time (min)
0% 0.5% 1% 2%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 1.36 ± 0.019 1.67 ± 0.05 1.55 ± 0.012
60 1.5 ± 0.09 2.31 ± 0.17 3.45 ± 0.05 2.61 ± 0.13
90 1.71 ± 0.25 2.72 ± 0.04 4.7 ± 0.13 3.51± 0.1
120 2.25 ± 0.07 3.17 ± 0.07 5.4 ± 0.24 3.71 ± 0.3
150 2.68 ± 0.14 3.54 ± 0.16 6.33 ± 0.06 4.13 ± 0. 26
180 3.44 ± 0.12 3.95 ± 0.28 6.94 ± 0.33 4.84 ± 0.4
240 4.8 ± 0.25 4.71 ± 0.23 8.09 ± 0.41 5.67 ± 0.05
300 5.29 ± 0.11 5.36± 0.51 9.33 ± 0. 14 6.07 ± 0.16
360 5.94 ± 0.41 6.11 ± 0.24 10.26 ± 0.4 6.82 ± 0.27
126

Drug alone 0.5%IPM 1% IPM 2% IPM
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 57: Release profile of glibenclamide from water soluble base containing different concentrations of
isopropyl myristate.
127

Table 34: In vitro release of glibenclamide from water soluble base containing different concentrations of
Isopropylpalmitate (IPP).
Glibenclamide released % ± (sd)
Time (min)
0% 0.2% 1% 2%
0 0 ± 0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 0.86 ± 0.03 1.19 ± 0.11 0.89 ± 0.011
60 1.5 ± 0.09 2.19 ± 0.09 2.15 ± 0.11 1.95 ± 0.11
90 1.71 ± 0.25 3.02 ± 0.04 3.09 ± 0.28 2.75± 0.16
120 2.25 ± 0.07 3.51 ± 0.02 3.58 ± 0.02 3.41± 0.12
150 2.68 ± 0.14 3.94 ± 0.04 4.04 ± 0.06 3.84 ± 0. 29
180 3.44 ± 0.12 4.93 ± 0.04 4.65 ± 0.24 4.65 ± 0.09
240 4.8 ± 0.25 6.37 ± 0.35 5.55 ± 0.14 5.49 ± 0.24
300 5.29 ± 0.11 7.34± 0.04 6.34 ± 0. 31 6.02 ± 0.042
360 5.94 ± 0.41 8.39 ± 0.34 7.33 ± 0.4 7.025 ± 0.31
128

Drug alone 0.2% IPP 1% IPP 2% IPP
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 58: Release profile of glibenclamide from water soluble base containing different concentrations of
isopropyl palmitate (IPP).
129

6.4. Effect of incorporation of solubilizing agent (Transcutol):
The effect of Transcutol on the release of Glib from water soluble
base is shown in (Table 35) and (Figures 59- 60).
Transcutol (Tc) (Diethylene glycol monoethyl ether) is a powerful
solubilizing agent used in several dosage forms and it seems to be very
attractive as a penetration enhancer due to its non-toxicity,
biocompatibility with the skin, miscibility with polar and non polar
solvents and optimal solubilizing properties for a number of drugs
(Barthelemy et al., 1995).
Incorporation of Transcutol in concentrations of 5%and 8%
increased the percentage of drug released from 5.94 % to 7.25% and 6.5%
respectively. Mura et al., (2000) found that incorporation of 50% of
Transcutol in carbapol hydrogel increased clonazepam flux three times
more than control gel.
The enhancing mechanism of Transcutol may be due to its powerful
solubilizing ability and consequently drug leaching increased.
Mutalik and Udupa, 2003 studied the effect of some penetration
enhancers on in vitro permeation of Glib and glipizide through mouse
skin. Ethanol in various concentrations, N-methyl-2-pyrrolidinone,
Transcutol, propylene glycol and terpenes like citral, geraniol and
eugenol were used as penetration enhancers. The flux values of both
drugs significantly increased in the presence of all penetration enhancers,
except Transcutol and propylene glycol.
All data are summarized in (Figure 61).
130

Table 35: In vitro release of glibenclamide from water soluble base containing different concentrations of Transcutol.
Glibenclamide released % ± (sd)
Time (min)
0% 5% 8%
0 0 ± 0 0 ± 0 0 ± 0
30 0.68 ± 0 1.37 ± 0.04 1.05 ± 0.07
60 1.5 ± 0.09 2.38 ± 0.017 1.82 ± 0.07
90 1.71 ± 0.25 3.05 ± 0.14 2.350± 0
120 2.25 ± 0.07 3.49 ± 0.19 2.82 ± 0
150 2.68 ± 0.14 4.04 ± 0.28 3.29 ± 0. 03
180 3.44 ± 0.12 5.4± 0.18 4.17 ± 0.07
240 4.8 ± 0.25 5.65 ± 0.25 4.95 ± 0.14
300 5.29 ± 0.11 6.99 ± 0.15 5.39 ± 0.13
360 5.94 ± 0.41 7.25 ± 0.25 6.5 ± 0.33
131

Drug alone 5% Tc 8% Tc
12
11
10
9
8
7
6
5
% Drug released
4
3
2
1
0
0 50 100 150 200 250 300 350 400
Time (min)
Figure 59: Release profile of glibenclamide from water soluble base containing different concentrations of
Transcutol.
132

12
11
IPM 1%
10
Drug alone
9
IPM 0.5%
8
IPM 1%
Tc 5%
IPP 0.2% IPP1%
IPP 2%
IPM 2%
7
IPM 2%
Tc 8%
IPM 0.5%
Drug alone
6
IPP 0.2%
IPP1%
5
% Drug released
IPP 2%
4
Tc 5%
3
Tc 8%
2
1
0
Different enhancers with different concentrations
Figure 60: Percentage drug released from water soluble base containing different concentrations of fatty acid
esters and Transcutol.
133

10
Drug alone
Tw 80 4%
Cetrimide 0.5%
9
Cetrimide
0.5%
SLS 0.4%
SLS 0.4%
Urea 5%
LOA 0.8%
OA 2%
8
Lab 3% Tc 5%
Tw 80 4%
Labrazol 5%
IPM 2% IPP2%
7
Lab 3%
Drug alone
6
Tc 5%
5
IPM 2%
IPP2%
4
% Drug released
OA 2%
3
LOA 0.8%
2
Urea 5%
1
Labrazol 5%
0
Differtent penetration enhancers
Figure 48: Percentage drug released from water soluble base containing different concentrations of different
enhancers.
134

7. Kinetic analysis of release data:
As shown in (Table 36) the release data of Glib from all different
topical formulations followed diffusion controlled mechanism (Higuchi
model).
8. In vitro permeation of gliclazide through abdominal rat skin:
Skin permeation studies indicated that Glib permeation through
hairless rabbit skin was negligible. The possible reasons for this result
may be i) Glib , a lipophilic drug, was retained within the stratum
corneum with no portioning into the viable epidermis or ii) most of the
drug was used up to saturate the binding sites in the skin and the
remaining drug was probably insufficient to provide a significant
concentration gradient (Srini et al., 1998).
135

Table 36: Kinetic data of the release of Glib from different topical
bases.
Topical
Cont. table 36: Kinetic data of the release of Glib from different
topical bases.
Correlation coefficient (R)
preparation Zero First Diffusion
Topical bases
WSB-SLS
WSB-
WSB-Tw -80
WSB-Tc
Cetrimide
136
Observed
order
WSB 0.9833 0.9784 0.9842 D.M
HPMC gel 0.9356 0.9375 0.9694 D.M
HPMC emulgel 0.9622 0.9642 0.9849 D.M
Sod-alginate gel 0.9804 0.9814 0.9852 D.M
O/W cream 0.9196 0.9205 0.9527 D.M
Oleaginous base 0.8095 0.8102 0.8960 D.M
Absorption base 0.8859 0.8865 0.8904 D.M
0.1% SLS 0.9813 0.9881 0.9891 D.M
0.4% SLS 0.9784 0.9857 0.9991 D.M
0.8% SLS 0.9652 0.9674 0.9855 D.M
0.3% cetrimide 0.9742 0.9768 0.9910 D.M
0.5% cetrimide 0.9521 0.9573 0.9956 D.M
1% cetrimide 0.9766 0.9803 0.9981 D.M
2% cetrimide 0.9789 0.9804 0.9917 D.M
0.3% Tw-80 0.980 0.9818 0.9954 D.M
1% Tw-80 0.9801 0.9820 0.9974 D.M
4% Tw-80 0.9722 0.9761 0.9987 D.M
5% Tw-80 0.9781 0.9804 0.9962 D.M
5% Tc 0.9806 0.9820 0.9859 D.M
8% Tc 0.9836 0.9849 0.9881 D.M

Topical
preparation
WSB-
WSB-oleic
WSB-linoleic
WSB-IPP
WSB-IPM
labrafil
acid
acid
Correlation coefficient (R)
Zero First Diffusion
137
Observed
order
3% lab 0.9063 0.9109 0.9676 D.M
5 % lab 0.9729 0.9685 .9757 D.M
7 % lab 0.9668 0.9685 0.9778 D.M
0.5 % OA 0.9418 0.9458 0.9906 D.M
1% OA 0.9614 0.9659 0.9970 D.M
2% OA 0.9521 0.9553 0.9870 D.M
3% OA 0.9855 0.9808 0.9808 D.M
0.8 % LOA 0.9752 0.9774 0.9895 D.M
1 % LOA 0.9728 0.9755 0.9991 D.M
2% LOA 0.9773 0.9791 0.9897 D.M
0.2% IPP 0.9569 0.9594 0.9840 D.M
1% IPP 0.9793 0.9819 0.9979 D.M
2% IPP 0.9662 0.9692 0.9965 D.M
0.5% IPM 0.9834 0.9851 0.9950 D.M
1% IPM 0.9636 0.9688 0.9971 D.M
2% IPM 0.9556 0.9587 0.9918 D.M

9- In- vivo study:
The result of hypoglycemic activity of the topically applied glibenclamide
and oral glibenclamide (5 mg/kg; p.o.) in both normal and diabetic rats
are shown in (Table 37-41) and (Figures 61-62).
The blood glucose reducing effect was significant in oral and all
topically treated groups up to 24 h except groups treated with ointment
contained 5% Labrafil, compared with control group (p
*** Studies in normal rats
Glib (oral) produced a significant decrease of 58.09 % ± 1.5 (p
0.05 compared to control) in blood glucose levels at 2 hr and then the
blood glucose levels decreased. The percentage reduction in the blood
glucose levels at the end of 24 hr were 30.61 ± 3.4. On other hand, the
blood glucose reducing response of all topical formulation was gradual
and increased slowly up to 24 h.
The effect of OA and cetrimide in amount of 1% within the Glib
ointment on reducing the blood glucose level is shown in (Figure 62).
OA and cetrimide increased significantly the blood glucose reducing
activity of glib
OA is a popular penetration enhancer and penetrates into the stratum
corneum and decompresses this layer and hence reduces its' resistance to
drug penetration (Barry and Bennett, 1987). OA can also accumulate
within the lipid bilayers of stratum corneum cells and hence increase their
flowability and penetration ability (Goodman and Barry, 1988).
138

Table 37: Reduction in blood glucose level after oral and topical application of glibenclamide and glibenclamide with
1% oleic acid in normal rats. All values are expressed as mean ± sd.
.
Reduction in blood glucose level (mg/dl)
(Percentage reduction in blood glucose levels)
Absolute
blood
glucose level
Group
(mg/dl) 2hr 4hr 6hr 8hr 24hr
71 ± 2.94
(24.64 ± 1.5)
77.5 ± 5.74
(17.59 ±2.1)
84.5 ± 2.08
(10.53 ±1.84)
84 ± 2.4
(11.05± 2.49)
94.5 ± 3.97 93.25 ± 3.4*
(1.29±0.4)**
Control
(1ml gum
acacia
suspension)
69.5 ± 4.2
(30.61 ±3.4)
59.25 ± 0.95
(42.53 ±4.3)
51.75± 4.3
(49.9 ± 4.06)
49.75 ± 5.3
(50.48 ±6.6)
103.75 ± 8.9 43 ± 4.4
(58.09 ± 1.5)
Oral
glibenclamide
(5mg/kg)
51 ± 6.27
(48.91 ± 4.2)
57.25 ± 8.8
(41.54 ± 3.6)
66.00 ± 9.1
(32.55± 2.14)
69.25 ± 8.53
(29.13± 1.75)
97.75± 12.12 74.25± 9.06
(23.99± 2.84)
WSB
(glibenclamide
40.75 ± 6.84
(56.86 ± 3.8)
56.5 ± 7.3
(40.04 ±3.7)
59.5 ± 6.6
(36.68 ± 2.7)
60.75 ± 5.9
(35.26 ± 3.2)
94.5± 13.1 69 ± 6.3
(26.95 ±5.7)
WSB
(glibenclamide
+1% oleic
acid)
* Reduction in blood glucose level (mg/dl). **Percentage reduction in blood glucose levels.
139

Table 38 : Reduction in blood glucose level after oral and topical application of glibenclamide and glibenclamide
with 1% cetrimide in normal rats. All values are expressed as mean ± sd.
. .
Reduction in blood glucose level (mg/dl)
(Percentage reduction in blood glucose levels)
Group
2hr 4hr 6hr 8hr 24hr
Absolute
blood
glucose
level
(mg/dl)
71 ± 2.94
(24.64± 1.5)
77.5 ± 5.74
(17.59 ±2.1)
84.5 ± 2.08
(10.53 ±1.84)
84 ± 2.4
(11.05 ± 2.49)
94.5 ± 3.97 93.25 ± 3.4*
(1.29±0.46)**
Control
(1ml gum
acacia
suspension)
69.5 ± 4.2
(30.61 ±3.4)
59.25 ± 0.95
(42.53 ±4.3)
51.75± 4.3
(49.9 ± 4.06)
49.75 ± 5.3
(50.48 ±6.6)
103.75± 8.9 43 ± 4.4
(58.09 ± 1.5)
Oral
glibenclamide
(5mg/kg)
51 ± 6.27
(48.91± 4.2)
57.25 ± 8.8
(41.54 ± 3.6)
66.00 ± 9.1
(32.55 ± 2.14)
69.25 ± 8.53
(29.13 ± 1.75)
74.25± 9.06
(23.99± 2.84)
97.75±
12.12
WSB
(glibenclamide
45.5 ± 6.3
(60.9± 3.33)
52.66 ± 6.8
(54.54 ±3.17)
60.75 ± 5.67
(47.39 ± 3.3)
70.33 ± 4.9
(39.07 ± 2.4)
115.5± 7.5 87.25 ±3.3
(24.32 ±2.8)
WSB
(glibenclamide
+1%
cetrimide)
*
Reduction in blood glucose level (mg/dl). **Percentage reduction in blood glucose levels.
140

Table 39 : Reduction in blood glucose level after oral and topical application of glibenclamide and glibenclamide
with 1% isopropyl myristate (IPM) in normal rats. All values are expressed as mean ± sd.
Reduction in blood glucose level (mg/dl)
(Percentage reduction in blood glucose levels)
Absolute
blood glucose
level (mg/dl)
Group
2hr 4hr 6hr 8hr 24hr
71 ± 2.94
(24.64 ±
1.5)
77.5 ± 5.74
(17.59 ±2.1)
84.5 ± 2.08
(10.53 ±1.84)
84 ± 2.4
(11.05 ± 2.49)
94.5 ± 3.97 93.25 ± 3.4*
(1.29±0.46)**
Control
(1ml gum
acacia
suspension)
69.5 ± 4.2
(30.61 ±3.4)
59.25 ± 0.95
(42.53 ±4.3)
51.75± 4.3
(49.9 ± 4.06)
49.75 ± 5.3
(50.48 ±6.6)
103.75 ± 8.9 43 ± 4.4
(58.09 ± 1.5)
Oral
glibenclamide
(5mg/kg)
51 ± 6.27
(48.91 ±
4.2)
57.25 ± 8.8
(41.54 ± 3.6)
66.00 ± 9.1
(32.55 ± 2.14)
69.25 ± 8.53
(29.13 ± 1.75)
97.75 ± 12.12 74.25± 9.06
(23.99± 2.84)
WSB
(glibenclamide
50.5 ± 6.5
(47.52 ±
4.2)
62.75 ± 12.89
(35.26 ±3.2)
69 ± 12.83
(28.82± 2.38)
78.25 ± 13.37
(19.13 ± 3.23)
97± 17.9 93 ± 17.75
(4.16 ±0.93)
WSB
(glibenclamide
+1% IPM)
*
Reduction in blood glucose level (mg/dl). **Percentage reduction in blood glucose levels.
141

.Table 40 : Reduction in blood glucose level after oral and topical application of glibenclamide and glibenclamide
with 5 % Labrafil in normal rats. All values are expressed as mean ± sd.
Reduction in blood glucose level (mg/dl)
(Percentage reduction in blood glucose levels)
Absolute
blood glucose
level (mg/dl)
Group
2hr 4hr 6hr 8hr 24hr
71 ± 2.94
(24.64 ±
1.5)
77.5 ± 5.74
(17.59 ±2.1)
84.5 ± 2.08
(10.53 ±1.84)
84 ± 2.4
(11.05 ± 2.49)
94.5 ± 3.97 93.25 ± 3.4*
(1.29±0.46)**
Control
(1ml gum
acacia
suspension)
69.5 ± 4.2
(30.61
±3.4)
59.25 ± 0.95
(42.53 ±4.3)
51.75± 4.3
(49.9 ± 4.06)
49.75 ± 5.3
(50.48 ±6.6)
103.75 ± 8.9 43 ± 4.4
(58.09 ± 1.5)
Oral
glibenclamide
(5mg/kg)
51 ± 6.27
(48.9± 4.2)
57.25 ± 8.8
(41.54 ± 3.6)
66.00 ± 9.1
(32.55 ± 2.14)
69.25 ± 8.53
(29.13 ± 1.75)
97.75 ± 12.12 74.25± 9.06
(23.99± 2.84)
WSB
(glibenclamide
75 ± 6.97
(27.0±
2.96)
83.75 ± 9.7
(18.017 ±2.5)
95 ± 5.9
(7.41 ± 2.14)
95 ± 6.6
(6.9 ± 2.5)
102.75± 8.5 96.75 ± 6.3
(5.7 ±1.9)
WSB
(glibenclamide
+5% Labrafil)
. *
Reduction in blood glucose level (mg/dl). **Percentage reduction in blood glucose levels.
142

.
Control Oral glibenclamide Topical glibenclamide
1% OA 1% Cetrimide 1% IPM
5% Labrafil
70
60
50
40
30
20
% Reduction in blood glucose level
10
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time (hr)
Figure 62: Percent reduction in blood glucose levels after oral and topical administration of glibenclamide in normal
rats.
143

Cetrimide is a cataionic surfactant. It has a potential to solubilise
lipids within the stratum corneum, swell the stratum corneum and interact
with intercellular keratin so increase the permeation of the drug
(Williams and Barry, 2004).
Incorporation of both 1% IPM and 5% Labrafil did not show any
enhancement in the blood glucose reducing activity (compared to Glib
without enhancer).
*** Studies in diabetic rats:
Oral and topical groups showed significant hypoglycemic activity
upto 24 hrs. The hypoglycemic effect produced by ointement containing
Glib and 1% cetrimide in the animals is significantly less when compared
to oral administration.
Glib (oral) produced a significant decrease of 41.1 ± 5.25 (p
compared to control) in blood glucose levels at 4 hr and then the blood
glucose levels increased. On other hand, the blood glucose reducing
response of topical formulation was gradual and increased slowly up to
24 h.
The results did not differ significantly in oral and topical groups
after 24 hrs. The topically applied Glib and the oral drug produced
decrease of 24.53 ± 3.74 and 25.7 ± 4.69 respectively, in the blood
glucose levels after 24 hrs. This may be due to reduced insulin levels in
diabetic models impairs principal metabolic pathways of sulphonylurea
which resulted in its prolonged action in orally treated group (Stroev and
Belkina, 1989).
144

Table 41 : Reduction in blood glucose level after oral and topical application of glibenclamide and glibenclamide
with 1% cetrimide in diabetic rats. All values are expressed as mean ± sd.
.
Reduction in blood glucose level (mg/dl)
(Percentage reduction in blood glucose levels)
Group
2hr 4hr 6hr 8hr 24hr
Absolute
blood
glucose
level
(mg/dl)
223.4 ± 40.8
(5.22 ± 2.74)
224.4 ± 38.8
(4.76±0.89)
230.6 ± 40.8
(2.16 ±1.68)
232.2 ± 38.46
(1.35 ± 1.06)
228.4 ± 36.26*
(2.89 ± 1.65)**
235.6 ±
40.3
Control
(1ml gum
acacia
suspension)
364.8± 49.93
(24.53 ±3.74)
342 ± 51.63
(29.25
±3.75)
318 ± 45.06
(34.02± 5.67)
283 ± 33.54
(41.1 ±5.25)
420.2 ± 62.81
(15.63 ± 3.09)
485 ±
79.56
Oral
glibenclamide
(5mg/kg)
236.8 ± 23.53
(25.7 ± 4.69)
267.5± 25.99
(16.16 ±2.7)
277.2 ± 30.82
(13.28± 3.5)
286.7± 35.12
(9.6 ± 4.4)
319± 23.2 305± 21.37
(4.28± 2.4)
WSB
(glibenclamide
+1%
cetrimide)
. * Reduction in blood glucose level (mg/dl). **Percentage reduction in blood glucose levels.
145

50
Control
Oral glibenclamide
Topical glibenclamide
45
40
35
30
25
20
15
10
% Reduction in blood glucose level(mg/dl)
5
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time (hr)
Figure 63 : Percent reduction in blood glucose levels after oral and topical administration of glibenclamide in
diabetic rats.
146

Conclusion:
From the previously demonstrated data the following results can be
concluded:
1- Glib has a lipophilic property.
2- The percentage amount of drug released from water soluble base, gel
bases and emulgel base are greater than that released from other bases.
The rate of drug release can be arranged in the following descending
order:
Water soluble base (5.94 %) > HPMC emulgel (4.6 %) > sodium alginate
gel (4.38 %) > HPMC gel (3.99) >O/W emulsion base (2.5 %) >
absorption base (1.94%) > oleaginous base (1.61%).It is clear that,
water soluble base showed the highest release than that of emulsion,
gels, emulgel, oleaginous and absorption bases.
3- The investigation showed the effect of addition of penetration
enhancers on the amount of Glib released from different topical bases
in vitro can be arranged in the follwing descending order:
1% IPM > 5% Lab > 1% Cetrimide > 4% Tw-80 > 1% OA > 0.2%
IPP > 0.8% LOA > 0.4% SLS > 5% Tc.
4- Topically applied glibenclamide exhibited better control of
hyperglycemia and more effectively reversed the glibenclamide side
effects than oral glibenclamide administration in both normal and
diabetic rats. Slow and sustained release of the drug from the
transdermal system might reduce
manifestations like severe hypoglycemia, sulphonylurea receptor down
regulation and the risk of chronic hyperinsulinemia ( Mutalik and
Udupa, 2004).
147

Ointments contained 1% cetrimide and 1% OA enhanced the blood
glucose reducing activity of glibenclamide . While addition of 1% IPM
and 5% Lab did not show any enhancement in the blood glucose reducing
activity (compared to glib without enhancer).
148

General Conclusion
The preceding study was an attempt to evaluate the potential of
pharmaceutical formulation of certain sulfonylureas namely, gliclazide
and glibenclamide in different bases for topical application.
In case of gliclazide, the amount of drug released from topical bases
incorporating solid dispersions can be arranged in the following
descending order. Topical preparations containing (8:92) PEG 6000 >
(1:10) glucose > (8:92) PEG 4000 > (1:10) urea solid dispersions > pure
drug.
The blood glucose reducing activity of ointment contained (10:90)
gliclazide –PEG 6000 solid dispersions was significantly more when
compared to ointment contained gliclazide alone.
In case of glibenclamide, the presence of various penetration
enhancers increase the amount of drug released from the topical base in
vitro. The maximum release was obtained by using IPM (1%).
Ointments contained 1% cetrimide and 1% oleic acid enhanced the
blood glucose reducing activity of glibenclamide . While addition of 1%
isopropyl myristate and 5% Labrafil did not show any enhancement in the
blood glucose reducing activity of glibenclamide.
In conclusion, it was demonstrated that sulfonylureas were absorbed
through the skin and lowered the blood glucose levels. The results
suggest the possibility of transdermal administration of sulfonylureas for
the treatment of NIDDM.
149

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