Formulation and Evaluation Of

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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. GhazyProfessor of Pharmaceutics

Faulty of PharmacyZagazig University

Dr. Mohamed A. Hammad Dr. Nagia A. El-MegrabAssistant Professor of Assistant Professor of

Pharmaceutics PharmaceuticsFaulty of Pharmacy Faulty of PharmacyZagazig University Zagazig University

Department of PharmaceuticsAnd Industrial Pharmacy

Faculty of PharmacyZagazig University

2010

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AACKNOWLEDGEMENTS

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 PProf. 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 DDr. 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 WORDGlz 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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Contents

List of Tables

List of Figures

Abstract ………………………………………………………………. i

General Introduction …………………………………………………. 1

Scope of work ………………………………………………………... 35

Part OneFormulation 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 TwoFormulation 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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List of Figures

Figure

Number Description

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

10

3 Schematic representation of types of external

medicines.

20

4 Structure of gliclazide 37

5 Diagrammatic representation of process of

solubilization

41

6 Phase diagram for eutectic system 55

7 Phase diagram for Discontinuous solid solutions 56

8 Substitutional crystalline solid solutions 57

9 Interstitial crystalline solid solutions. 58

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.

75

13 Calibration curve of gliclazide in phosphate buffer

(7.4)at �max 227 nm .

75

14 Phase solubility diagram of gliclazide in water at

25°C in presence of PEG 4000 and PEG 6000.

78

15 Phase solubility diagram of gliclazide in water

at 25°C in presence of glucose and urea.

78

16 Dissolution profile of gliclazide-PEG 6000 systems. 82

17 Dissolution profile of gliclazide-PEG 4000 systems. 84

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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.

92

21 FTIR spectra of gliclazide –PEG 6000 systems. 99

22 FTIR spectra of gliclazide –PEG 4000 systems. 100

23 FTIR spectra of gliclazide –glucose systems. 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.

125

34 In vitro release profile of gliclazide from different

topical preparations.

136

35 In vitro release profile of gliclazide and (8:92)

gliclazide –PEG 6000 solid dispersion from

different topical bases.

141

36 In vitro release profile of gliclazide and (1:10) gliclazide –glucose solid dispersion from different topical bases.

143

37 In vitro release profile of gliclazide and (8:92) gliclazide –PEG 4000 solid dispersion from different topical bases.

145

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38 In vitro release profile of gliclazide and (1:10)

gliclazide –urea solid dispersion from different

topical bases.

147

39 Release of gliclazide from different bases with

different solid dispersions.

148

40 Percent reduction in blood glucose levels after oral

and topical administration of gliclazide in normal

rats.

153

41 Percent reduction in blood glucose levels after oral

and topical administration of gliclazide in diabetic

rats.

156

42 Glibenclamide structure. 158

43 Techniques to optimize drug permeation across the

skin.

163

44 UV absorption spectra for glibenclamide in

methanol.

186

45 Calibration curve of glibenclamide in phosphate

buffer (7.4) at �max 227 nm.

188

46 Release profile of glibenclamide from different

topical bases.

192

47 Percentage drug released from different topical

bases.

194

48 Release profile of glibenclamide from water soluble

base containing different concentrations of

cetrimide.

199


base containing different concentrations of SLS.

201

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base containing different concentrations of Tween

80.

203

51 Release profile of glibenclamide from water

soluble base containing different concentrations of

labrafil.

205

52 Percentage drug released from water soluble base containing different concentrations of different surfactants

206


base containing different concentrations of oleic

acid.

209


base containing different concentrations of linoleic

acid.

211

55 Percentage drug released from water soluble base

containing different concentrations of fatty acids.

212



isopropyl myristate.

215



isopropyl palmitate .

217



Transcutol.

220

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59 . Percentage drug released from water soluble base

containing different concentrations of fatty acid

esters and Transcutol.

221

60 Percentage drug released from water soluble base

containing the best concentrations of different

penetration enhancers used.

222

61 Percent reduction in blood glucose levels after oral and topical administration of glibenclamide in normal rats.

231

62 Percent reduction in blood glucose levels after oral and topical administration of glibenclamide in diabetic rats.

234

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List of Tables

Table

Number

Description Page

Number

1 Methods for the characterization of solid

dispersion.

64

2 Types of carriers and their ratios in gliclazide solid

dispersions and physical mixtures.

69

3 Solubility enhancement data of gliclazide in various

carrier solutions at 25°C.

77

4 Effect of change in pH on the solubility of

gliclazide.

79

5 Dissolution parameters (±SD) of gliclazide in

distilled water from different gliclazide - PEG 6000

systems.

81


distilled water from different gliclazide - PEG 4000

systems.

83


distilled water from different gliclazide – glucose

systems.

86


distilled water from different gliclazide –urea

systems.

88

9 Collective data for dissolution of gliclazide

obtained from different carriers used.

91

10 FTIR spectra of gliclazide solid dispersions and

physical mixtures compared with individual

components.

95

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11 Fusion temperatures (Tc) and heat of fusion (��

of gliclazide solid dispersions and physical mixtures

compared with individual components.

105

12 �� º)

for some gliclazide solid dispersions and physical

mixtures compared with individual components.

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

140


glucose solid dispersion from different topical

bases.

142


PEG 4000 solid dispersion from different topical

bases.

144


urea solid dispersion from different topical bases.

146

21 Kinetic data of the release of gliclazide and its solid

dispersions from different topical bases.

149

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.

152

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topical application of gliclazide and 10:90

gliclazide- PEG 6000 solid dispersion in diabetic

rats. All values are expressed as mean ± sd.

155

24 Composition of different topical formulations. 180

25 Types of penetration enhancers and percentages used.

182

26 In vitro release of glibenclamide from different

topical bases.

27 In vitro release of glibenclamide from water soluble


cetrimide

198


base containing different concentrations of Sodium

lauryl sulphate (SLS).

200


base containing different concentrations of Tween

80.

202


base containing different concentrations of labrafil.

204


base containing different concentrations of oleic

acid.

208


base containing different concentrations of linoleic

acid.

210



Isopropylmyristate (IPM).

214

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Isopropylpalmitate (IPP).

216



Transcutol.

219

36 Kinetic data of the release of Glib from different

topical bases

224


topical application of glibenclamide and

glibenclamide with 1% oleic acid in normal rats.

227



glibenclamide with 1% cetrimide in normal rats

228



glibenclamide with 1% isopropyl myristate (IPM) in

normal rats..

229



glibenclamide with 5 % Labrafil in normal rats.

230



glibenclamide with 1% cetrimide in diabetic rats.

233

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AbstractPart One

Formulation and Evaluation of Topically Applied Gliclazide.

Chapter OneFormulation 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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ratios 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 from�absorption 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 introductionSkin 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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replenish 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

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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,

- 28 -

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

- 29 -

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).

- 30 -

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

- 31 -

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)

- 32 -

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.

- 33 -

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).

- 34 -

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

- 35 -

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.

- 36 -

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 :

- 37 -

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

- 38 -

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.

- 39 -

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).

- 40 -

- 41 -

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

- 42 -

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

- 43 -

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).

- 44 -

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:

- 45 -

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:

- 46 -

(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).

- 47 -

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,

- 48 -

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 –� !?� �� @� J�Q�� *��$� �� *� ��

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

- 49 -

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) :

TDDS are broadly classified into the following types (Chien,

1987):

- 50 -

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

- 51 -

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.

- 52 -

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 AntidiabeticsIf 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

- 53 -

thiazolidinediones appear to increase insulin sensitivity (Martindale,

1996).

Sulphonylurea1.Mode of action:

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).

:Uses and Administration. 2

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

- 54 -

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

- 55 -

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) .

- 56 -

- 57 -

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.

- 58 -

� 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.

- 59 -

- 60 -

Introduction

Gliclazide 1. Description:

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:

Glz is a white, crystalline, odourless powder and practically

without taste.

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

- 61 -

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:

As mentioned before under sulfonylureas.

5. Dosage and adminstration:

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:

An increased hypoglycaemic effect has occurred or might be

expected with ACE inhibitors, alcohol, allopurinol, some analgesics

(notably azapropazone, phenylbutazone, and the salicylates), azole

- 62 -

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).

- 63 -

- 64 -

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.

- 65 -

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.

- 66 -

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.

- 67 -

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):

:Particle size reduction. 3.1Particle 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.

- 68 -

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

- 69 -

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) .

- 70 -

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

- 71 -

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 ��Z��&��$�

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

- 72 -

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).

- 73 -

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

- 74 -

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).

:Method for preparation of solid dispersions. .43.7

:Melting method. .4.13.7

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.

- 75 -

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.

:Melt extrusion.4.1.23.7

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).

:elting methodHot spin m. .4.1.33.7

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).

:Solvent method. .4.23.7

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.

- 76 -

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.

:solvent method-Melting. .4.33.7

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.

- 77 -

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.

:Simple eutectic mixtures. .5.13.7

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.

- 78 -

Figure 5: Phase diagram for eutectic system

(reproduced from Castellan, 1983).

:Solid solutions. .5.23.7

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).

- 79 -

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.

:Discontinuous solid solutions. .5.2.23.7

The solubility of each of the components in the other is limited. A

typical phase diagram is shown in (Figure 6), \��^�� Z��

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).

:Substitutional crystalline solid solutions. .5.2.33.7

Classical solid solutions have a crystalline structure, in which the

solute molecules can either substitute for solvent molecules in the crystal

- 80 -

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)

:Interstitial crystalline solid solutions. .5.2.43.7

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.

- 81 -

(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)

:Glass solution and glass suspensions. .5.33.7

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).

- 82 -

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).

:Carriers for solid dispersions. .63.7

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.

- 83 -

:Polyethylene glycol. .6.13.7

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).

:pyrrolidonePolyvinyl..6.23.7

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

- 84 -

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).

:Urea. .6.33.7

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).

:Sugars. .6.43.7

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

- 85 -

problematic, and their solubility in most organic solvents is poor, making

it difficult to prepare co-evaporates.

:Chitosan. .6.53.7

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).

:Emulsifiers. .6.63.7

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).

:Other carriers. .6.73.7

- 86 -

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 testing2- 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).

- 87 -

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).

:Thermoanalytical methods. .7.23.7

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).

- 88 -

):XRD(ray diffraction -X. .7.33.7

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).

:Infrared spectroscopy. .7.43.7

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).

- 89 -

Experiment and methodology

1- Materials and supplies:

* 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).

- 90 -

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 solutions`concentrations 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

- 91 -

regressing the relative absorbances against the corresponding Glz

solutions`concentrations 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).

- 92 -

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:

- 93 -

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

- 94 -

(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.

- 95 -

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-}???� *�Z�� Z�� ~��\� ��@�

�� Z�� *��

of 0-80°.

- 96 -

Results and discussion1. UV scanning of Glz:

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

�� max�� max 273 nm is

0.825 to 0.034. So, measurements were done at 227 nm (Tadeusz et al.,

2005).

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.

��Wavelength

- 97 -

y = 0.0329x + 0.0233R2 = 0.9973R= 0.9986

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30 35

Concentration (ug/ml)

Abso

rban

ce

Figure 12: Calibration curve of Glz in methanol at �max 227 nm.

y = 0.0361x + 0.0296R2 = 0.9887R= 0.9943

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20 25 30

concentration (ug/ml)

Abs

orba

nce

Figure 13 : Calibration curve of Glz in phosphate buffer (7.4) at �max227 nm.

- 98 -

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).

- 99 -

Table 3: Solubility enhancement data of Glz in various carrier

solutions at 25°C.

Item Glz PEG-

4000

PEG-

6000

Glu UR

Phase solubility

diagram type

----- AL AL BS BS

Optimum

carrier

concentration

%(w/v)

------ 7% 7% 3% 5%

Solubility

(ug/ml)

42.13 80.23 67.63 54.3 48.19

Solubility factor a

------- 1.9 1.6 1.28 1.14

Solubility factor a = Total solubility / intrinsic solubility.

- 100 -

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8

Carrier % (w/v)

Solu

bilit

y (u

g/m

l)

PEG 4000PEG 6000

Figure 14: Phase solubility diagram of Glz in water at 25°C in presence of PEG 4000 and PEG 6000.

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 Carrier % (w/v)

Sol

ubilit

y (u

g/m

l)

Urea Glucose

Figure 15: Phase solubility diagram of Glz in water

at 25°C in presence of glucose and urea.

- 101 -

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:

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

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

- 102 -

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.

103

Tab

le 5

: Dis

solu

tion

para

met

ers (

±SD

) of g

licla

zide

in d

istil

led

wat

er fr

om d

iffer

ent g

licla

zide

- PE

G 6

000

s

yste

ms.

Com

posi

tion

(w/w

)ID

R(%

diss

olve

d/m

in)

PD20

(%)

PD60

(%)

DE *

100

(%)

Glic

lazi

de p

owde

r0

± 0

0 ±

013

.05

± 1

.23.

83 ±

0.0

6G

licla

zide

-to-P

EG 6

000

PM

10:

90SD

10:9

01.

12 ±

0.1

23.

27 ±

0.0

722

.58

± 0

.465

.53

± 1

.438

.08

± 0.

1972

.89

± 1.

524

.44

± 0

.64

63.5

4 ±

1.1

PM

8:9

2SD

8:92

1.12

± 0

.09

3.2

± 0

.04

22.5

9 ±

1.

964

.04

± 0

.87

36.8

9 ±

0.18

69.2

9 ±

1.76

25.3

0 ±

0.9

660

.83

± 0.

91

PM

5

:95

SD

5:9

51.

08 ±

0.0

83.

66 ±

0.0

421

.63

± 1

.673

.25

±

0.9

38.1

±

1.2

93.2

3 ±

0.42

24.8

2 ±

1.1

76.4

5 ±

0.3

0PM

1:

99SD

1

:99

1.79

± 0

.02

4.6

± 0

.07

35.7

9 ±

0.4

892

.36

±

1.5

41.1

9 ±

0.49

100.

49 ±

1.0

034

.76

± 0

.65

89.7

8 ±

3.4

8

I

DR

= In

itial

dis

solu

tion

rate

.PD

20 =

Ext

ent o

f diss

olut

ion

afte

r 20

min

.PD

60 =

Ext

ent o

f diss

olut

ion

afte

r 60

min

.D

E% =

Dis

solu

tion

effic

ienc

y af

ter 6

0 m

in.

104

0

20

40

60

80

10

0

12

0

02

04

06

08

01

00

12

0T

ime

(m

in)

% Drug dissolved

pla

in d

rug

10

:90

PM

10

:90

SD

8:9

2 P

M8

:92

SD

5:9

5 P

M5

:95

SD

1:9

9 P

M1

:99

SD

Figu

re16

: Dis

solu

tion

prof

ile o

f glic

lazi

de-P

EG

600

0 sy

stem

s.

105

Tab

le6:

Dis

solu

tion

para

met

ers (

±SD

) of g

licla

zide

in d

istil

led

wat

er fr

om d

iffer

ent g

licla

zide

- PE

G 4

000

s

yste

ms.

Com

posi

tion

(w/w

)ID

R(%

diss

olve

d/m

in)

PD20

(%)

PD60

(%)

DE *

100

(%)

Glic

lazi

de p

owde

r0

± 0

0 ±

013

.05

± 1

.23.

83 ±

0.0

6G

licla

zide

-to-P

EG 4

000

PM

10:

90SD

1

0:90

1.08

± 0

.032

1.62

± .

047

21.6

9 ±

0.6

532

.53

± 0

.45

38.3

9 ±

0.19

42.3

2 ±

1.5

25.3

8 ±

1.09

34

.58

± 0.

85PM

8

:92

SD

8:

921.

2 ±

0.0

132.

07

± 0

.028

24.1

3 ±

1.69

64.0

4 ±

0.5

736

.49

± 0.

1850

.85

± 1.

7626

.99

± 1.

742

.23

± 0.

28

PM

5:

95SD

5:95

1.02

± 0

.11

3.17

± 0

.04

20.7

5 ±

1.3

63.4

9 ±

0.8

933

.67

± 1.

264

.29

± 0.

4223

.26

± 1.

661

.81

± 0.

44PM

1

:99

SD

1:

991.

77 ±

0.0

043.

34

± 0

.075

35.4

1 ±

0.0

866

.94

±

1.5

43.9

8 ±

0.49

68.5

8 ±

1.56

36.0

7±

1.9

62.8

±1.

9

IDR

= In

itial

dis

solu

tion

rate

.PD

20 =

Ext

ent o

f diss

olut

ion

afte

r 20

min

.PD

60 =

Ext

ent o

f diss

olut

ion

afte

r 60

min

.D

E% =

Dis

solu

tion

effic

ienc

y af

ter 6

0 m

in.

106

0

10

20

30

40

50

60

70

80

90

10

0

02

04

06

08

01

00

12

0

Tim

e (

min

)

% Drug dissolved

pla

in d

rug

10

:90

PM

10

:90

SD

8:9

2 P

M8

:92

SD

5:9

5 P

M5

:95

SD

1:9

9 P

M1

:99

SD

Figu

re 1

7: D

isso

lutio

n pr

ofile

of g

licla

zide

-PE

G 4

000

syst

ems.

107

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.

108

Tab

le 7

: Dis

solu

tion

para

met

ers (

±SD

) of g

licla

zide

in d

istil

led

wat

er fr

om d

iffer

ent g

licla

zide

– g

luco

se

sys

tem

s.

Com

posi

tion

(w/w

)ID

R(%

diss

olve

d/m

in)

PD20

(%)

PD60

(%)

DE *

100

(%)

Glic

lazi

de p

owde

r0

± 0

0 ±

013

.05

± 1

.23.

83 ±

0.0

6G

licla

zide

-to-G

luco

sePM

1

:1SD

1

:11.

12 ±

0.0

381.

16 ±

.04

522

.51

± 0

.65

23.2

3 ±

0.4

532

.13

± 1.

0340

.1

± 1.

1822

.84

± 0

.75

25.5

2±

0.05

PM

1:2

SD

1:2

1.03

± 0

.067

1.36

±

0.0

2820

.66

± 1

.69

27.2

7 ±

0.5

734

.18

± 01

.15

43.5

1 ±

1.30

24.0

8 ±

1.4

29.5

1 ±

0.51

PM

1:3

SD

1:3

1.06

± 0

.025

1.37

± 0

.027

21.3

5±

1.3

27.5

8 ±

0.8

938

.53

± 0

.442

.16

± 0.

825

.74

± 0.

5432

.45

± 0

.25

PM

1:5

SD

1:5

1.42

± 0

.022

1.61

±

0.0

7528

.42

± 0

.08

32.3

8 ±

1.

542

.76

± 0

.81

46.5

8±

1.00

30.0

5 ±

0.4

335

.02

±0.

79PM

1:

10SD

1

:10

1.3

± 0

.006

1.

8 ±

0.0

04

26

.07

± 0

.13

36.0

4 ±

0.0

9 41

.68

± 1

.51

45.5

6 ±

1.0

631

.56

± 1

.29

37.0

5 ±

0.87

IDR

= In

itial

dis

solu

tion

rate

PD20

= E

xten

t of d

issol

utio

n af

ter 2

0 m

in.

PD60

= E

xten

t of d

issol

utio

n af

ter 6

0 m

in.

DE%

= D

isso

lutio

n ef

ficie

ncy

afte

r 60

min

.

109

0

10

20

30

40

50

60

70

80

90

100

020

40

60

80

100

120

Tim

e (

min

)

% Drug Released

Pla

in d

rug

1:1

PM

1:1

SD

1:2

PM

1:2

SD

1:3

PM

1:3

SD

1:5

PM

1:5

SD

1:1

0P

M1:1

0 S

D

Figu

re 1

8: D

isso

lutio

n pr

ofile

of g

licla

zide

-glu

cose

syst

ems.

110

T

able

8: D

isso

lutio

n pa

ram

eter

s (±S

D) o

f glic

lazi

de in

dist

illed

wat

er fr

om d

iffer

ent g

licla

zide

–ur

ea

sy

stem

s.

Com

posi

tion

(w/w

)ID

R(%

dis

solv

ed/m

in)

PD20

(%)

PD60

(%)

DE *

100

(%)

Glic

lazi

de p

owde

r0

± 0

0 ±

013

.05

± 1

.23.

83 ±

0.0

6G

licla

zide

-to-G

luco

sePM

1

:1SD

1

:10.

28 ±

0.0

80.

68 ±

0.0

55.

6

± 0

.60

13.5

6 ±

1.00

524

.53

± 1.

0524

.81

± 1.

1511

.85

± 1

.3

1

6.49

± 1.

03PM

1

:2SD

1

:20.

67 ±

0.0

160.

95

± 0

.13

13.6

5 ±

0.32

19.1

8 ±

1.6

25.8

± 0

.69

34.3

1 ±

1.9

16.2

5 ±

0.02

22.4

8 ±

1.6

PM

1:3

SD

1:3

0.74

± 0

.033

1.29

± 0

.07

14.8

8 ±

0.6

725

.99

± 1

.44

29.0

1 ±

0.4

536

.45

± 1

.417

.93

± 0.

6427

.11

± 1

.003

PM

1:5

SD

1:5

0.78

± 0

.01

1.28

±

0.0

715

.78

± 0

.38

25.7

0 ±

1.

529

.3 ±

0.2

935

.06

± 1.

0018

.66

± 0

.09

26.8

1 ±

1.7

9PM

1:

10SD

1

:10

1.09

± 0

.05

1.

42 ±

0.0

3

21

.96

± 1.

055

28.5

1 ±

0.7

4

34.9

± 0

.35

39.1

8 ±

0.2

425

.15

± 0

.52

31.2

2 ±

1.47

ID

R =

Initi

al d

isso

lutio

n ra

te.

PD20

= E

xten

t of d

issol

utio

n af

ter 2

0 m

in.

PD60

= E

xten

t of d

issol

utio

n af

ter 6

0 m

in.

DE%

= D

isso

lutio

n ef

ficie

ncy

afte

r 60

min

.

111

0

10

20

30

40

50

60

70

80

90

100

020

40

60

80

100

120

Tim

e (

min

)

% Drug Released

Pla

in d

rug

1:1

PM

1:1

SD

1:2

PM

1:2

SD

1:3

PM

1:3

SD

1:5

PM

1:5

SD

1:1

0P

M1:1

0 S

D

Fi

gure

19:

Dis

solu

tion

prof

ile o

f glic

lazi

de-u

rea

syst

ems.

112

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

113

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 % 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.

114

1:9

9 P

EG

6000

1:9

9 P

EG

4000

1:1

0 g

lucose

1:1

0 u

rea

0123456789

10

A / B

Figu

re 2

0: R

atio

bet

wee

n %

of g

licla

zide

dis

solv

ed fr

om (A

) dru

g in

diff

eren

t sol

id d

ispe

rsio

ns a

nd (B

) dru

g

1

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

2

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).

3

Table 10: FTIR spectra of Glz solid dispersions and physical


System Assignment �max (cm-1)

Glz

N – H (stretching)

C – H (aromatic)

C – H (aliphatic)

C=O

N-H (deformation band)

S=O (asymmetrical and

symmetrical band)

3274.2

3192.9 - 3113.2

2950 - 2867 - 2836

1709

1596

1350 -1164

PEG 6000

-O-H (stretching)

C-H (stretching)

C-O (ether)

O-H (bending)

3445.8

2887

1110.7

1344

Glz – PEG 6000

(PM)(10:90)

-O-H (stretching)

C-H (stretching)

C=O

C-O (ether)

O-H (bending)

3446

2888.2

1710.3

1110.9

1345.5

Glz – PEG 6000

(SD)(10:90)

-OH (stretching)

C-H (stretching)

C=O

C-O (ether)

O-H (bending)

3447

2886.8

1710.2

1112.3

1345.7

4

Cont. Table 10: FTIR spectra of Glz solid dispersions and physical



Glz


C – H (aromatic)

C – H (aliphatic)

C=O



symmetrical band)

3274.2

3192.9 - 3113.2

2950 - 2867 - 2836

1709

1596

1350 -1164

PEG 4000

-OH (stretching)

C-H (stretching)

C-O (ether)

O-H (bending)

3414.3

2887.6

1110.4

1344.7

Glz – PEG 4000

(PM)(10:90)

-OH (stretching)

C-H (stretching)

C=O

C-O (ether)

O-H (bending)

3447.2

2887.23

1710.3

1110.5

1345.2

Glz – PEG 4000

(SD)(10:90)

-O-H (stretching)

C-H (stretching)

C=O

C-O (ether)

O-H (bending)

3422.6

2888.0

1710.3

1112.1

1346.2

5




Glz


C – H (aromatic)

C – H (aliphatic)

C=O



symmetrical band)

3274.2

3192.9 - 3113.2

2950 - 2867 - 2836

1709

1596

1350 -1164

Glucose

-O-H (stretching)

(broad)

C-H (stretching)

O-H (bending)

3411.6 -3316.0

2944.1

1342.0

Glz – glu

(PM)(1:10)

-OH (stretching)

-NH (stretching)

C-H (stretching)

C=O

O-H (bending)

3410.4

3280.7

2943.4

1709.9

1346.7

Glz – glu

(SD)(1:10)

-O-H (stretching)

-NH (stretching)

C-H (stretching)

C=O

O-H (bending))

3408.8

3276.3

2941.5

1709.9

1348.0

6




Glz


C – H (aromatic)

C – H (aliphatic)

C=O



symmetrical band)

3274.2

3192.9 - 3113.2

2950 - 2867 - 2836

1709

1596

1350 -1164

UR

-N-H (stretching)

C=O

-C-N

3445.7- 3347.4

1678.8 – 1622.7

1152.4

Glz – UR

(PM)(1:10)

-N-H (stretching)

C=O

-C-N

3446.5-3347.6-

3277.9

1707.4-1682-

1622.8

1162.1

Glz – UR

(SD)(1:10)

-N-H (stretching)

C=O

-C-N

3445.7-3347.6-

3276.0

1708.1-1682.4-

1623.4

1162.4

7

Figure 21: FTIR spectra of Glz –PEG 6000 systems A) Glz ; B) pure

PEG 6000; C) PM (1:9) and D) SD (1:9).

D

C B

A

Wave number (cm-1)

8

Figure 22: FTIR spectra of Glz –PEG 4000 systems A) Glz ; B) pure

PEG 4000; C) PM (1:9) and D) SD (1:9).

D

C B

A

Wave number (cm-1)

9

Figure 23: FTIR spectra of Glz –glucose systems A) Glz ; B) pure

glu; C) PM (1:10) and D) SD (1:10).

D

C B

A

Wave number (cm-1)

10

Figure 24: FTIR spectra of Glz –UR systems A) Glz ; B) pure UR; C)

PM (1:10) and D) SD (1:10).

6. Differential scanning calorimetry:

D

C B

A

Wave number (cm-1)

11

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

12

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).

13

Table 11: Fusion temperatures (Tc) and heat of fusion (��Glz

solid dispersions and physical mixtures compared with individual

components.

System Fusion temperature

(Tc) (ºC)

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)

154

182.46

193.21

42.72

Glz – glu (SD)

(1:10)

153.21

183.65

194.19

58.26

UR 132.97 225.17

Glz – UR (PM)

(1:10)

131.12

192.52

176

110.56

Glz – UR (SD)

(1:10)

132.23

181.92

170.97

73.12

14

Figure 25: DSC spectra of Glz –PEG 6000 systems A) Glz ; B) pure

PEG 6000; C) PM (1:9) and D) SD (1:9).

D

C

B

A

Temp (c)

15

Figure 26: DSC spectra of Glz –PEG 4000 systems A) Glz ; B) pure

PEG 4000; C) PM (1:9) and D) SD (1:9).

D

C

B

A

Temp (c)

16

Figure 27: DSC spectra of Glz –glucose systems A) Glz; B) pure glu;

C) PM (1:5) and D) SD (1:5).

D

C

B

A

Temp(c)

17

Figure 28: DSC spectra of Glz –UR systems A) Glz ; B) pure UR; C)

PM (1:5) and D) SD (1:5).

D

C

B

A

Temp (c)

18

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).

19

Tab

le 1

2: In

tens

ities

at c

hara

cter

istic

diff

er�

��

��

!��º)

for

som

e gl

icla

zide

solid

disp

ersi

ons a

nd p

hysi

cal

mix

ture

s com

pare

d w

ith in

divi

dual

com

pone

nts.

Syst

em�!

��i

nten

sity

�!��

��int

ensi

ty�!

��

inte

nsity

�!��

��in

tens

ity

�!��

��in

tens

ity

�!��

��in

tens

ity

Glic

lazi

de14

.58

3

3.1

19.6

8

43.

120

.12

36

.920

.58

1

0022

.72

24

.928

.4

35

.5

PEG

600

019

8

6.5

23.1

2

100

Glic

-PEG

600

0

(10:

90) (

PM)

19.0

3

89.

123

.12

1

00

Glic

-PEG

600

0

(10:

90) (

SD)

19.0

3

100

23.2

1

97.6

PEG

400

019

.016

1

0023

.217

9

0.6

Glic

-PEG

600

0

(10:

90) (

PM)

19.1

99.

723

.26

10

0

Glic

-PEG

600

0

(10:

90) (

SD)

19.0

2

92.

423

.2

10

0

20

Con

t.Tab

le 1

2:�"

��

#�#�

��$

��

$��

#�#$

�&#��

��

��

��

!��º)

for

som

e gl

icla

zide

solid

disp

ersi

ons a

nd p

hysi

cal

mix

ture

s com

pare

d w

ith in

divi

dual

com

pone

nts.

Syst

em�!

��i

nten

sity

�!��

��int

ensi

ty�!

��

inte

nsity

�!��

��in

tens

ity

�!��

��in

tens

ity

�!��

��in

tens

ity

Glic

lazi

de

14.5

8

33.

119

.68

4

3.1

20.1

2

36.

920

.58

100

22.7

2

24.

928

.4

3

5.5

Glu

cose

20.5

2

10

025

.38

3

0.9

17.0

1

44

28.3

9

64.

2

Glic

-glu

cose

(1:1

0) (P

M)

20.7

4

3

7.2

14.3

9

92.

217

.055

5

7.7

10.7

4

10

018

.15

7

0.3

22.0

14

30.1

Glic

- glu

cose

(1:1

0) (S

D)

20.5

7

100

14.6

2

25.

217

.067

25.

528

.4

3519

.72

7.2

22.0

17

17.

1

Ure

a22

.044

1

0028

.4

35

Glic

-ure

a

(1:1

0) (P

M)

22.2

2

96.

322

45.

7

31

.47

100

35.4

7

36.

9

Glic

-ure

a

(1:1

0) (S

D)

22.1

5

100

35.1

7

5

7

21

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).

D

C

B

A

2-Theta-Scale

22

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).

D

C

B

A

2-Theta-Scale

23

Figure 31: X-ray spectra of Glz –glucose systems A) Glz ; B) pure

glu; C) PM (1:5) and D) SD (1:5).

D

C

B

A

2-Theta-Scale

24

Figure 32: X-ray spectra of Glz –UR systems A) Glz ; B) pure UR; C)

PM (1:5) and D) SD (1:5).

D

C

B

A

2-Theta-Scale

25

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.

26

27

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.

28


1- Materials and supplies:

* 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.

29

_ 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

30

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

31

-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

32

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.

33

Each experiment was done in triplicate, and the average was calculated.

The cumulative amount of the drug released was calculated as mentioned

before.

34

Figure 33: Diagrammatic representation of the drug diffusion

apparatus.

35

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��Q�� @

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��@

36

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 ).

t1/2 = A��@

(II) First order kinetics:

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

t1/2 = 0.693 / k.

(III) Higuchi diffusion model:

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� � �� Z� ��

diffusion (Martin, 1994). A simplified Higuchi diffusion

equation for drug released from topical preparation is.

M = Q = 2C��½

37

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 t½

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

38

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 cm2. 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).

39

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

40

method (El-Sayed et al., 1989) using a commercial kit according to the

supplied instructions as follows:

Principle:

Glucose present in the sample is determined according the following

reaction:

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 ------- ------- !?��

41

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 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 4cm2 of the skin.

42

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.


The obtained data were compared statistically using One-way

analysis of variance (ANOVA) test of significance on a computer



43

Results and Discussion1. Partition coefficient of Glz:

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

44

Table 15: In vitro release data of Glz from different topical

bases.

Glz released % ± (sd) Time (min)

WSB HPMC gel O/W cream

0 0 ± 0 0 ± 0 0 ± 0

30 0 ± 0 1.35 ± 0.12 0 ± 0

60 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 31.21 ± 0.48 4.90 ± 0.56

300 56.55± 0.07 37.86 ± 0.9 6.7 ± 0.9

360 62.10 ± 0.42 43.38 ± 2.2 8.43 ± 0.38

45

0102030405060708090100

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Released

WSB

HPM

C g

elO

/W c

ream

Figu

re 3

4: In

vitr

o re

leas

e pr

ofile

of g

licla

zide

from

diff

eren

t top

ical

pre

para

tion.

46

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.

47

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

48

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.

49

Tab

le17

: In

vitr

o re

leas

e of

glic

lazi

de a

nd (8

:92)

glic

lazi

de-P

EG

600

0 so

lid d

ispe

rsio

n fr

om d

iffer

ent t

opic

al

base

s.

Glic

lazi

de r

elea

sed

% ±

(sd)

Tim

e (m

in)

WSB

(glic

lazi

de)

WSB

(SD

)

HPM

C g

el

(glic

lazi

de)

HPM

C g

el

(SD

)

O/W

cre

am

(glic

lazi

de±)

O/W

cre

am

(SD

)

00

± 0

0 ±

00

± 0

0 ±

00

± 0

0 ±

0

300

± 0

3.03

± 0

.41.

35 ±

0.1

26.

70 ±

0.2

80

± 0

1.90

± 0

605.

70±

0.43

11.4

5 ±

16.

71 ±

0.7

013

.02

± 0.

350

± 0

3.30

± 0

9014

.90

± 0

19.7

5 ±

0.4

11.9

7 ±

0.74

19.4

8 ±

0.05

1.70

± 0

.34.

35 ±

0.4

120

22.1

5±0

.38

27.3

0 ±

0.28

16.8

0 ±

0.6

25.4

1 ±

0.21

2.44

± 0

.37.

16 ±

0.4

8

150

30.4

6±0.

3734

.22

± 0.

1121

.41

± 0.

931

.16

± 0.

982.

71 ±

0.4

8.38

± 0

.44

180

35.4

± 0

.41

39.2

0 ±

0.24

25.7

9 ±

0.57

39.6

2± 0

.84

4.08

± 0

.021

8.80

± 0

.59

240

47.6

1±0.

3951

.50

± 1.

431

.21

± 0.

4844

.47

± 1.

24.

90 ±

0.5

610

.37

± 0.

63

300

56.5

5±0.

0760

.90

± 1.

637

.86

± 0.

951

.47

± 1.

56.

7 ±

0.9

14.4

± 0

.62

360

62.1

0 ±

0.42

69.9

6 ±

2.5

43.3

8 ±

2.2

60.4

7 ±

1.5

8.43

± 0

.38

16.8

0 ±

0.37

% In

crea

se--

----

-11

.23

----

---

28.2

6--

----

-99

.28

50

0

10

20

30

40

50

60

70

80

90

100

050

100

150

200

250

300

350

400

Tim

e (

min

)

% Released

WS

B-

glic

WS

B-S

DH

PM

C g

el-g

licH

PM

C g

el- S

DO

/W c

rea

m-g

licO

/W c

rea

m-

SD

Figu

re 3

5: In

vitr

o re

leas

e pr

ofile

of g

licla

zide

and

(8:9

2) g

licla

zide

–PE

G 6

000

solid

dis

pers

ion

from

diff

eren

t top

ical

bas

es.

51

Tab

le 1

8: In

vitr

o re

leas

e of

glic

lazi

de a

nd (1

:10)

glic

lazi

de-g

luco

se so

lid d

ispe

rsio

n fr

om d

iffer

ent t

opic

al

Glic

lazi

de r

elea

sed

% ±

(sd)

Tim

e (m

in)

WSB

(glic

lazi

de)

WSB

(SD

)

HPM

C g

el

(glic

lazi

de)

HPM

C g

el

(SD

)

O/W

cre

am

(glic

lazi

de±)

O/W

cre

am

(SD

)

00

± 0

0 ±

00

± 0

0 ±

00

± 0

0 ±

0

300

± 0

1.14

± 0

.11.

35 ±

0.1

26.

3 ±

0.28

0 ±

01.

60±

0.24

605.

70±

0.43

9.68

± 0

.76.

71 ±

0.7

012

.4 ±

0.3

50

± 0

2.06

± 0

.4

9014

.90

± 0

17.8

8 ±

0.97

11.9

7 ±

0.74

18.7

2 ±

0.05

1.70

± 0

.33.

16 ±

0.7

120

22.1

5±0

.38

25.9

6 ±

0.8

16.8

0 ±

0.6

21.9

0 ±

0.20

2.44

± 0

.35.

8 ±

0.7

150

30.4

6±0.

3733

.43

± 1.

221

.41

± 0.

927

.47

± 0.

982.

71 ±

0.4

7.4

± 1.

1

180

35.4

± 0

.41

37.9

0 ±

1.5

25.7

9 ±

0.57

34.0

4± 0

.84

4.08

± 0

.021

8.65

± 0

.7

240

47.6

1±0.

3948

.73

± 1.

531

.21

± 0.

4839

.24

± 1.

24.

90 ±

0.5

69.

1±0.

37

300

56.5

5±0.

0758

.5±

0.96

37.8

6 ±

0.9

45.8

9 ±

1.5

6.7

± 0.

912

.12

± 0.

9

360

62.1

0 ±

0.42

67.2

2 ±

1.1

43.3

8 ±

2.2

55.6

9 ±

1.5

8.43

± 0

.38

14.7

7 ±

1.0

% In

crea

se--

----

7.61

----

-22

.1--

---

42.9

2

52

0102030405060708090100

050

100

150

200

250

300

350

400

Tim

e (m

in)

% ReleasedW

SB

-glic

WS

B-S

DH

PM

C g

el- g

licH

PM

C g

el-S

DO

/W c

ream

- glic

O/W

cre

am- S

D

Fi

gure

36:

In v

itro

rele

ase

prof

ile o

f glic

lazi

de a

nd (1

:10)

glic

lazi

de –

gluc

ose

solid

dis

pers

ion

from

diff

eren

t top

ical

ba

ses.

53

Tab

le 1

9: In

vitr

o re

leas

e of

glic

lazi

de a

nd (8

:92)

glic

lazi

de-P

EG

400

0 so

lid d

ispe

rsio

n fr

om d

iffer

ent t

opic

al

ba

ses.

Glic

lazi

de r

elea

sed

% ±

(sd)

Tim

e (m

in)

WSB

(glic

lazi

de)

WSB

(SD

)

HPM

C g

el

(glic

lazi

de)

HPM

C g

el

(SD

)

O/W

cre

am

(glic

lazi

de±)

O/W

cre

am

(SD

)

00

± 0

0 ±

00

± 0

0 ±

00

± 0

0 ±

0

300

± 0

1.4

± 0.

171.

35 ±

0.1

22.

55 ±

00

± 0

1.20

±0.

2

605.

70±

0.43

8.15

± 0

.21

6.71

± 0

.70

8.98

± 0

.12

0 ±

01.

64 ±

0.1

3

9014

.90

± 0

18.4

0 ±

0.9

11.9

7 ±

0.74

18.0

6 ±

0.35

1.70

± 0

.32.

42 ±

0.2

2

120

22.1

5±0

.38

24.7

1 ±

0.7

16.8

0 ±

0.6

23.9

5 ±

0.43

2.44

± 0

.32.

84 ±

0.2

150

30.4

6±0.

3732

.71

± 1.

221

.41

± 0.

927

.7 ±

0.4

32.

71 ±

0.4

4.44

± 0

.5

180

35.4

± 0

.41

38.2

6 ±

0.08

25.7

9 ±

0.57

33.6

± 0

.58

4.08

± 0

.021

5.5

± 0.

35

240

47.6

1±0.

3948

.48

± 1.

0231

.21

± 0.

4838

.1 ±

1.9

4.90

± 0

.56

8.16

±0.

57

300

56.5

5±0.

0758

.89

± 1.

537

.86

± 0.

944

.7 ±

1.6

6.7

± 0.

910

.99

± 1.

03

360

62.1

0 ±

0.42

65.1

± 1

.843

.38

± 2.

253

.7 ±

1.9

8.43

± 0

.38

11.7

1 ±

0.1

% In

crea

se--

----

--4.

60--

----

-19

.21

----

-28

.01

54

0102030405060708090100

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Released

WS

B- g

licW

SB

-SD

HP

MC

gel

- glic

HP

MC

gel

- SD

O/W

cre

am-g

licO

/W c

ream

-SD

Figu

re 3

7: In

vi

tro

rele

ase

prof

ile o

f glic

lazi

de a

nd (8

:92)

glic

lazi

de –

PEG

400

0 so

lid d

ispe

rsio

n fr

om

diff

eren

t top

ical

bas

es.

55

Tab

le 2

0: In

vitr

o re

leas

e of

glic

lazi

de a

nd (1

:10)

glic

lazi

de-u

rea

solid

disp

ersi

on fr

om d

iffer

ent t

opic

a

base

s.

Glic

lazi

de r

elea

sed

% ±

(sd)

Tim

e (m

in)

WSB

(g

licla

zide

)W

SB

(SD

)H

PMC

gel

(g

licla

zide

)H

PMC

gel

(SD

)O

/W c

ream

(glic

lazi

de±)

O/W

cre

am(S

D)

00

± 0

0 ±

00

± 0

0 ±

00

± 0

0 ±

0

300

± 0

1.54

± 0

.13

1.35

± 0

.12

3.92

± 0

.18

0 ±

00±

0

605.

70±

0.43

8.07

± 0

.47

6.71

± 0

.70

10.3

0 ±

0.30

0 ±

00.

99 ±

0

9014

.90

± 0

16.9

8 ±

0.37

11.9

7 ±

0.74

15.5

0 ±

0.27

1.70

± 0

.32.

28±

0.59

120

22.1

5±0

.38

24.3

7 ±

0.48

16.8

0 ±

0.6

21.0

5 ±

0.07

2.44

± 0

.33.

44 ±

0.6

3

150

30.4

6±0.

3731

.14

± 0.

1821

.41

± 0.

925

.1 ±

0.5

62.

71 ±

0.4

4.21

± 0

.44

180

35.4

± 0

.41

37.6

±1.

825

.79

± 0.

5729

.94±

0.0

64.

08 ±

0.0

215.

89 ±

0.6

2

240

47.6

1±0.

3947

.25

± 1.

131

.21

± 0.

4835

.15

± 0.

494.

90 ±

0.5

67.

20±

0.48

300

56.5

5±0.

0757

.37±

1.03

37.8

6 ±

0.9

41.8

0 ±

0.71

6.7

± 0.

98.

80 ±

0.3

7

360

62.1

0 ±

0.42

64.1

5 ±

1.4

43.3

8 ±

2.2

52.4

1 ±

1.1

8.43

± 0

.38

10.7

4 ±

0.4

% In

crea

se--

----

3.19

----

---

17.2

2--

----

-21

.5

56

0102030405060708090100

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Released

WS

B- g

licW

SB

- SD

HP

MC

gel

-glic

HP

MC

gel

- S

DO

/W c

ream

- glic

O/W

cre

am -S

D

Figu

re

38: I

n vi

tro

rele

ase

prof

ile o

f glic

lazi

de a

nd (1

:10)

glic

lazi

de –

urea

solid

dis

pers

ion

from

diff

eren

t top

ical

bas

es.

57

1 W2 HPMC

3 O/W cr

58

Dru

g

Dru

g

Dru

g

PEG

600

0

PEG

600

0

PEG

600

0

glu

cose

gluc

ose

glu

cose

PEG

400

0

PEG

400

0

PEG

400

0

ure

a

ure

a

ure

a

01020304050607080

12

3T

opic

al b

ases

% Released

Dru

g al

one

(8:9

2) P

EG 6

000

SD(1

:10)

glu

cose

SD

(8:9

2) P

EG 4

000

SD(1

:10)

ure

a SD

Figu

re 3

9: R

elea

se o

f glic

lazi

de fr

om d

iffer

ent b

ases

with

diff

eren

t sol

id d

ispe

rsio

ns.

59

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

dispersions from different topical bases.

Correlation coefficient (R)Topicalpreparation Zero First Diffusion

Observedorder

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

WSB

(1:10) Glz-URSD

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.MHPM

C g

el

(1:10) Glz-URSD

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 FirstO/W

cre

am

(1:10) Glz-URSD

0.9957 0.9967 0.9935 First

60

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

(p �?@?}��*�� 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 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

61

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.

62

Tab

le 2

2 :

Red

uctio

n in

blo

od g

luco

se le

vel a

fter

ora

l and

topi

cal a

pplic

atio

n of

glic

lazi

de a

nd 1

0:90

glic

lazi

de- P

EG

60

00 so

lid d

ispe

rsio

n in

nor

mal

rat

s. A

ll va

lues

are

exp

ress

ed a

s mea

n ±

sd.

*R

educ

tion

in b

lood

glu

cose

leve

l (m

g/dl

).

**

Perc

enta

ge re

duct

ion

in b

lood

glu

cose

leve

ls.

Red

uctio

n in

blo

od g

luco

se le

vel (

mg/

dl)

(Per

cent

age

redu

ctio

n in

blo

od g

luco

se le

vels

)G

roup

Abs

olut

e bl

ood

gluc

ose

leve

l (m

g/dl

)2h

r4h

r6h

r8h

r24

hrC

ontr

ol(1

ml g

um a

caci

a su

spen

sion

)88

.72

± 8

.682

.33

± 7.

7*(7

.14±

1.25

)**

76.4

5 ±

5.2

(12.

04 ±

0.9

9)

76.1

± 9

.2(1

3.44

±1.

44)

75.0

6 ±

8.4

(15.

46 ±

2.45

) 73

.36

± 9.

89(1

6.6

± 2.

09)

Ora

l glic

lazi

de(2

5mg/

kg)

89.8

5 ±

5.01

35.2

7 ±

6.0

7(6

0.64

± 6

.3)

39.9

7 ±

5.3

(55.

99 ±

4.41

)43

.3 ±

4.6

4(5

1.85

± 3

.5)

48.5

± 6

.2(4

6.89

±4.

93)

67.6

6 ±

4.1

(24.

83 ±

2.05

)

WSB

(Glic

lazi

de)

81.5

9 ±

8.9

58.2

2 ±

4.6

(28.

19 ±

4.5

)58

.4 ±

2.2

(30.

14 ±

5.9

)47

.92

± 5

.1(4

0.05

± 5

.4)

52.1

8 ±

3.8

(34.

34 ±

4.8

)56

.29

± 1

.4(3

2.71

± 6

.9)

WSB

(10:

90 g

licla

zide

-PE

G 6

000

SD)

82.1

4 ±

3.8

62.0

0 ±

3.2

(25.

5 ±

4.0

3)47

.05

± 2

.26

(43.

5 ±

3.0

5)38

.46

± 4

.6(5

2.62

± 6

.7)

45.3

8 ±

3.5

(44.

66 ±

4.51

)51

.13

± 3.

5(3

7.45

± 3

.6)

63

0

10

20

30

40

50

60

70

80

02

46

81

01

21

41

61

82

02

22

42

6

Tim

e (h

r)

% Reduction in blood glucose level(mg/dl)

Con

trol

oral

gli

claz

ide

WS

B (

gli

claz

ide)

WSB

(gl

icla

zide

-PE

G 6

000

SD

)

Figu

re 4

0: P

erce

nt r

educ

tion

in b

lood

glu

cose

leve

ls a

fter

ora

l and

topi

cal a

dmin

istr

atio

n of

glic

lazi

de in

nor

mal

rat

s.

64

*** 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).

65

Tab

le 2

3 :

Red

uctio

n in

blo

od g

luco

se le

vel a

fter

ora

l and

topi

cal a

pplic

atio

n of

glic

lazi

dean

d 10

:90

glic

lazi

de- P

EG

60

00 so

lid d

ispe

rsio

n in

dia

betic

rat

s. A

ll va

lues

are

exp

ress

ed a

s mea

n ±

sd.

.

Red

uctio

n in

blo

od g

luco

se le

vel (

mg/

dl)

(Per

cent

age

redu

ctio

n in

blo

od g

luco

se le

vels

)G

roup

Abs

olut

e bl

ood

gluc

ose

leve

l (m

g/dl

)2h

r4h

r6h

r8h

r24

hrC

ontr

ol(1

ml g

um a

caci

a su

spen

sion

)23

5.6

± 4

0.3

228.

4± 3

6.26

*(2

.89±

1.65

)**

232.

2 ±

38.4

6(1

.35

± 1.

06)

230.

6 ±

40.8

(2.1

6 ±1

.68)

224.

4 ±

38.8

(4.7

6±0.

89)

223.

4 ±

40.8

(5.2

2 ±

2.74

)

Ora

l glic

lazi

de(2

5mg/

kg)

577

± 48

.16

355.

1 ±

55.

32(3

8.28

± 5

.57)

327.

2 ±

41.5

(43.

18 ±

6.8)

293.

8 ±

43.

11(4

8.88

± 4

.9)

316.

6 ±

32.

5(4

4.84

±7.

03)

365.

6 ±

13.

63(3

6.35

±4.

42)

WSB

(10:

90 g

licla

zide

-PE

G 6

000

SD)

324.

6 ±

47

302.

4 ±

44.

89(6

.78

± 2

.2)

293

± 4

3.73

(9.7

± 1

.14)

284.

2 ±

37.

7(1

2.53

± 1

.47)

263.

2 ±

34.

98(1

8.69

±3.

08)

254

± 27

.21

(21.

33 ±

3.7

3)

*R

educ

tion

in b

lood

glu

cose

leve

l (m

g/dl

).

**

Perc

enta

ge re

duct

ion

in b

lood

glu

cose

leve

ls.

66

0102030405060

02

46

810

1214

1618

2022

2426

Tim

e (h

r)

% Reduction in the blood glucose level (mg/dl)C

ontr

olO

ral G

lzT

opic

al G

lz

Figu

re 4

1: P

erce

nt r

educ

tion

in b

lood

glu

cose

leve

ls a

fter

ora

l and

topi

cal a

dmin

istr

atio

n of

glic

lazi

de in

dia

betic

ra

ts.

67

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.

68

69

Introduction

Glibenclamide

1. Description

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).

70

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:


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) .

71

6. Precautions:


7. Adverse Effects:


8. Interactions:


9. Adverse Effects and Precautions


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.

72

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).

73

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:

dm/dt = J = D C0 P/ h

(Equation

1)

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.

74

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:

75

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

76

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

77

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).

78

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

79

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’.

80

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

81

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.

82

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)

83

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

84

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

85

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).

86

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 :

87

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).

88


1. Materials and supplies:

* 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

89

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 solutions`concentrations 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 gmPEG 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

90

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

91

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 gmTween 80 1 gmWater 70 gmHPMC 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

92

Table 24 : Composition of different topical formulations.

Gel baseType of base

Water soluble base (PEG base)

Absorpt-ion base

Oleaginou-s base

Emulsion base (O/W base)

HPMC Sod. Algina-te

Emulgel base (HPMC emulgel)

PEG 4000

30

PEG 400 70

Span 80 5

Soft paraffin

95 100

Propylene glycol

10

White bees wax

1

Sodium lauryl sulphate

2

HPMC 12 8 9

Tween 80 2

Liquid paraffin

20

Sodium alginateWater 88 92 69

93

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.

94

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 1 � �

2. Linoleic acid (LOA) 0.8 � �

(D) Fatty acid esters

1.Isopropyl myristate

(IPM)

0.5 � �

2. Isopropyl palmitate

(IPP)

0.2 � �

95

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:






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.

96

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:


��*� � - 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.

97

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 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.







98

Results and Discussion

1. UV scanning of Glib:

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).

Figure 44: UV absorption spectra for Glib in methanol.

Wavelength

100 μg/ml

20 μg/ml

227nm

299nm278nm

99

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 ªC 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.

100

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.

101

Tab

le 2

6: In

vitr

o re

leas

e of

glib

encl

amid

e fr

om d

iffer

ent t

opic

al b

ases

.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

Tim

e (m

in)

WSB

HPM

C e

mul

gel

Sodi

um a

lgin

ate

gel

HPM

C g

el

0 0

± 0

0 ±

0 0

± 0

0 ±

0

300.

68

± 0

0.54

± 0

0.65

± 0

.07

0.73

± 0

.016

601.

5 ±

0.0

91.

22 ±

0.0

6 0.

95 ±

0.0

51.

16 ±

0.0

6

901.

71

± 0.

25

2.

09 ±

0.1

41.

45 ±

0.0

81.

71 ±

0.1

3

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

± .0

622.

68 ±

0.1

6 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

.33.

99 ±

0.0

5

102

Con

t.Tab

le 2

6: In

vitr

o re

leas

e of

glib

encl

amid

e fr

om d

iffer

ent t

opic

al b

ases

.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

Tim

e (m

in)

O/W

cre

amA

bsor

ptio

n ba

seO

leag

inou

s bas

e

0 0

± 0

0 ±

00

± 0

300.

54 ±

00.

44 ±

0.0

20.

60 ±

0.0

13

600.

65±

0.03

0.49

± 0

0.89

± 0

.06

900.

97 ±

0.0

380.

97 ±

0.1

30.

91 ±

0.1

3

120

1.13

± 0

.06

1.05

5 ±

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.

091.

37 ±

0.9

1.51

± 0

.14

360

2.5

± 0.

11.

94 ±

0.3

81.

61 ±

0.1

7

103

01234567

050

100

150

200

250

300

350

400

Tim

e (m

in)

% drug releasesd

WS

B b

ase

HP

MC

gel

HP

MC

em

ulge

lS

odiu

m a

lgin

ate

gel

O/W

cre

amO

leag

ineo

us b

ase

Abs

orpt

ion

base

Figu

re 4

6: R

elea

se p

rofil

e of

glib

encl

amid

e fr

om d

iffer

ent t

opic

al b

ases

.

104

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).

105

WS

B

HP

MC

em

ulge

lS

od. a

lgin

ate

HP

MC

gel

O/W

cre

am

Abs

orpt

ion

base

Ole

agin

ous

base

01234567

1

% Drug released

WSB

HPM

C e

mul

gel

Sod.

alg

inat

eH

PMC

gel

O/W

cre

amA

bsor

ptio

n ba

seO

leag

inou

s ba

se

Figu

re 4

7: P

erce

ntag

e dr

ug r

elea

sed

from

diff

eren

t top

ical

bas

es.

106

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.

107

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:

"��*��#Z��?��#Z��?�� «��

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

108

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).

109

Tab

le 2

7: In

vitr

o re

leas

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

f cet

rim

ide.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

T

ime

(min

)0%

0.3%

0.

5%1%

2%0

0 ±

0 0

± 0

0 ±

00

± 0

0 ±

0

300.

68 ±

0

0.69

± 0

.2

0.7

± 0.

071.

09 ±

0.0

350.

6 ±

0.01

601.

5 ±

0.09

1.11

± 0

.12

2.02

± 0

.16

2.23

± 0

.11

1.1

± 0

901.

71 ±

0.2

5

2.

36 ±

0.1

43.

26 ±

0.3

73.

29 ±

0.1

81.

72±

0.01

120

2.25

± 0

.07

3.15

± 0

.14

4.22

± 0

.33

4.3

± 0.

072.

18 ±

0.0

4

150

2.68

± 0

.14

3.75

± 0

.19

5.16

± 0

.33

5.2

± 0.

512.

57 ±

0. 2

180

3.44

± 0

.12

4.41

± 0

.34

5.89

± 0

.12

6.1±

0.5

33.

22 ±

0.1

9

240

4.8

± 0.

255.

59 ±

0.1

76.

84 ±

0.1

27.

34 ±

0.4

83.

64 ±

0.0

07

300

5.29

± 0

.11

6.73

± 0

.18

7.87

± 0

.24

8.6

± 0.

164.

17 ±

0.1

9

360

5.94

± 0

.41

7.18

± 0

.19

8.75

± 0

.34

9.48

± 0

.56

5.05

± 0

.2

110

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

0.3%

cet

rim

ide

0.5%

cet

rim

ide

1% c

etri

mid

e2%

cet

rim

ide

Figu

re 4

9: R

elea

se p

rofil

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

fcet

rim

ide.

111

Tab

le 2

8: I

n vi

tro

rele

ase

of g

liben

clam

ide

from

wat

er s

olub

le b

ase

cont

aini

ng d

iffer

ent

conc

entr

atio

ns o

f So

dium

la

uryl

sulp

hate

(SL

S).

Glib

encl

amid

e r

elea

sed

% ±

(sd)

Tim

e (m

in)

0%0.

1%

0.4%

0.8%

0 0

± 0

0 ±

00

± 0

0 ±

0

300.

68 ±

0

0.5

± 0

1.96

± 0

.11

1.75

± 0

.014

601.

5 ±

0.09

1.42

± 0

.14

2.96

± 0

.07

2.59

± 0

.014

901.

71 ±

0.2

5

1.

81 ±

0.0

93.

66 ±

0.1

43.

38±

0.07

7

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.

194.

41 ±

0.1

54.

22 ±

0. 3

180

3.44

± 0

.12

3.37

± 0

.11

4.96

± 0

.17

4.85

± 0

.13

240

4.8

± 0.

254.

3 ±

0.46

6.56

± 0

.16

5.9

± 0.

17

300

5.29

± 0

.11

5.11

± 0

.35

7.26

± 0

. 46.

92 ±

0.1

8

360

5.94

± 0

.41

5.59

± 0

.41

7.95

± 0

.16

7.12

± 0

.18

112

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

0.1%

SLS

0.4%

SLS

0.8%

SLS

F

igur

e 50

: Rel

ease

pro

file

of g

liben

clam

ide

from

wat

er so

lubl

e ba

se c

onta

inin

g di

ffer

ent c

once

ntra

tions

of S

LS.

113

Tab

le 2

9: In

vitr

o re

leas

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

f Tw

een

80.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

Tim

e (m

in)

0%0.

3%

1%4%

5%

0 0

± 0

0 ±

00

± 0

0 ±

00

± 0

300.

68 ±

0

0.67

± 0

.16

0.95

± 0

.11.

72 ±

0.0

51.

1 ±

0.05

601.

5 ±

0.09

1.32

± 0

.11

1.72

± 0

.22.

47 ±

0.0

72.

12 ±

0.0

7

901.

71 ±

0.2

5

1.

95 ±

0.1

52.

17 ±

0.0

73.

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.

243.

89 ±

0. 0

7

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.

254.

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.

155.

81 ±

0.3

6

360

5.94

± 0

.41

5.66

± 0

.09

5.69

± 0

.08

9.05

± 0

.23

6.76

± 0

.24

114

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

0.3%

Tw

- 80

1% T

w- 8

04%

Tw

- 80

5% T

w- 8

0

Figu

re 5

1: R

elea

se p

rofil

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

f Tw

een

80.

115

Tab

le 3

0: In

vitr

o re

leas

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

f la

braf

il.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

T

ime

(min

)

0%3%

5%

7%0

0 ±

0 0

± 0

0 ±

00

± 0

300.

68 ±

0

1.68

± 0

1.

59 ±

0.2

7 0.

84 ±

0.0

19

601.

5 ±

0.09

3.01

± 0

.17

3.23

± 0

.21

1.97

± 0

.2

901.

71 ±

0.2

5

3.

56 ±

0.1

64.

02 ±

0.2

32.

46 ±

0.1

5

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.

372.

98 ±

0.2

1

180

3.44

± 0

.12

4.41

± 0

.18

5.97

± 0

.24

3.1

± 0.

17

240

4.8

± 0.

255.

86 ±

0.1

18.

46 ±

0.1

44.

13 ±

0.3

2

300

5.29

± 0

.11

6.72

± 0

.22

9.3

± 0.

264.

96 ±

0.2

5

360

5.94

± 0

.41

7.32

± 0

.17

10.0

3 ±

0.18

5.33

± 0

.22

116

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

Lab

3%La

b 5%

Lab

7%

Figu

re 5

2: R

elea

se p

rofil

e of

glib

encl

amid

e fr

om w

ater

sol

uble

bas

e co

ntai

ning

diff

eren

t con

cent

ratio

ns o

flab

rafil

.

117

118

SLS

0.4

%

Tw 8

0 4%

Lab

3%

Lab

5%

Lab

7%

Dru

gCet

rimid

e0.

3%

Cet

rimid

e0.

5%

Cet

rimid

e1%

Cet

rimid

e 2%

SLS

0.

1%

SLS

0.8

% Tw 8

0 0

.3%

Tw 8

0 1%

Tw 8

05%

024681012

1

% Drug released

Dru

g al

one

Cet

rim

ide

0.3%

Cet

rim

ide

0.5%

Cet

rim

ide

1%C

etri

mid

e 2%

SLS

0.1%

SLS

0.4%

SLS

0.8%

Tw

80

0.3%

Tw

80

1%T

w 8

0 4%

Tw

80

5%L

ab 3

%L

ab 5

%L

ab 7

%

Figu

re 5

3: P

erce

ntag

e dr

ug r

elea

sed

from

wat

er s

olub

le b

ase

cont

aini

ng d

iffer

ent

conc

entr

atio

ns o

f di

ffer

ent

surf

acta

nts.

119

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

120

Tab

le 3

1: In

vitr

o re

leas

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

fole

ic a

cid.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

T

ime

(min

)0%

0.5%

1%

2%3%

0 0

± 0

0 ±

00

± 0

0 ±

00

± 0

300.

68 ±

0

0.87

± 0

1.

59 ±

0.1

61.

15 ±

0.0

231.

31 ±

0.0

35

601.

5 ±

0.09

2.13

± 0

.05

2.7

± 0.

152.

71 ±

0.3

62.

08 ±

0.1

4

901.

71 ±

0.2

5

2.

95 ±

0.0

93.

89 ±

0.1

33.

74 ±

0.3

52.

81±

0.25

120

2.25

± 0

.07

3.44

± 0

.07

4.5

± 0.

394.

15 ±

0.1

73.

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±

0.4

53.

87 ±

0.2

4

240

4.8

± 0.

255.

05 ±

0.2

17.

09 ±

0.3

26.

82 ±

0.4

34.

98 ±

0.1

9

300

5.29

± 0

.11

5.64

± 0

.35

7.9

± 0.

457.

4 ±

0.15

5.38

± 0

.22

360

5.94

± 0

.41

6.19

± 0

.27

8.8

± 0.

47.

67 ±

0.1

96.

68 ±

0.2

1

121

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

0.5%

OA

1% O

A2%

OA

3% O

A

Fi

gure

54:

Rel

ease

pro

file

of g

liben

clam

ide

from

wat

er s

olub

le b

ase

cont

aini

ng d

iffer

ent

conc

entr

atio

ns o

f ol

eic

acid

.

122

Tab

le 3

2: I

n vi

tro

rele

ase

of g

liben

clam

ide

from

wat

er s

olub

le b

ase

cont

aini

ng d

iffer

ent

conc

entr

atio

ns o

f lin

olei

c ac

id.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

Tim

e (m

in)

0%0.

8%

1%2%

0 0

± 0

0 ±

00

± 0

0 ±

0

300.

68 ±

0

1.54

± 0

.024

1.

45 ±

0.0

9 0.

97 ±

0.1

601.

5 ±

0.09

2.42

± 0

.17

1.98

± 0

.16

1.95

± 0

.15

901.

71 ±

0.2

5

2.

9 ±

0.17

2.73

± 0

.23

2.51

± 0

.06

120

2.25

± 0

.07

3.33

± 0.

283.

27 ±

0.0

93.

22 ±

0.2

5

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.

354.

42 ±

0.4

4 4.

2 ±

0.26

240

4.8

± 0.

255.

89 ±

0.5

95.

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

123

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

0.8%

LO

A1%

LO

A2%

LO

A

F

igur

e 55

: Rel

ease

pro

file

of g

liben

clam

ide

from

wat

er so

lubl

e ba

se c

onta

inin

g di

ffer

ent c

once

ntra

tions

of l

inol

eic

acid

.

124

Dru

g al

one

OA

0.5

%

OA

1%

OA

2%

OA

3%

LOA

0.8

%

LOA

1%

LOA

2%

012345678910

1

% Drug released

Dru

g al

one

OA

0.5

%O

A 1

%O

A 2

%O

A 3

%LO

A 0

.8%

LOA

1%

LOA

2%

Fig

ure

56: P

erce

ntag

e dr

ug r

elea

sed

from

wat

er so

lubl

e ba

se c

onta

inin

g di

ffere

nt c

once

ntra

tions

of f

atty

aci

ds.

125

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.

126

Tab

le 3

3: I

n vi

tro

rele

ase

of g

liben

clam

ide

from

wat

er s

olub

le b

ase

cont

aini

ng d

iffer

ent

conc

entr

atio

ns o

fIs

opro

pylm

yris

tate

(IPM

).

Glib

encl

amid

e r

elea

sed

% ±

(sd)

Tim

e (m

in)

0%0.

5%

1%2%

0 0

± 0

0 ±

00

± 0

0 ±

0

300.

68 ±

0

1.36

± 0

.019

1.67

± 0

.05

1.55

± 0

.012

601.

5 ±

0.09

2.31

± 0

.17

3.45

± 0

.05

2.61

± 0

.13

901.

71 ±

0.2

5

2.

72 ±

0.0

44.

7 ±

0.13

3.51

± 0.

1

120

2.25

± 0

.07

3.17

± 0

.07

5.4

± 0.

243.

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.

254.

71 ±

0.2

38.

09 ±

0.4

15.

67 ±

0.0

5

300

5.29

± 0

.11

5.36

± 0.

519.

33 ±

0. 1

46.

07 ±

0.1

6

360

5.94

± 0

.41

6.11

± 0

.24

10.2

6 ±

0.4

6.82

± 0

.27

127

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

0.5%

IPM

1% I

PM

2% I

PM

Fig

ure

57:

Rel

ease

pro

file

of g

liben

clam

ide

from

wat

er s

olub

le b

ase

cont

aini

ng d

iffer

ent

conc

entr

atio

ns o

f is

opro

pyl m

yris

tate

.

128

Tab

le 3

4: I

n vi

tro

rele

ase

of g

liben

clam

ide

from

wat

er s

olub

le b

ase

cont

aini

ng d

iffer

ent

conc

entr

atio

ns o

fIs

opro

pylp

alm

itate

(IPP

).

Glib

encl

amid

e r

elea

sed

% ±

(sd)

T

ime

(min

)0%

0.2%

1%

2%0

0 ±

0 0

± 0

0 ±

00

± 0

300.

68 ±

0

0.86

± 0

.03

1.19

± 0

.11

0.89

± 0

.011

601.

5 ±

0.09

2.19

± 0

.09

2.15

± 0

.11

1.95

± 0

.11

901.

71 ±

0.2

5

3.

02 ±

0.0

43.

09 ±

0.2

82.

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.

256.

37 ±

0.3

55.

55 ±

0.1

45.

49 ±

0.2

4

300

5.29

± 0

.11

7.34

± 0.

046.

34 ±

0. 3

16.

02 ±

0.0

42

360

5.94

± 0

.41

8.39

± 0

.34

7.33

± 0

.47.

025

± 0.

31

129

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

0.2%

IPP

1% IP

P2%

IPP

Figu

re 5

8: R

elea

se p

rofil

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

f

is

opro

pyl p

alm

itate

(IPP

).

130

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).

131

Tab

le 3

5: In

vitr

o re

leas

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

f Tra

nscu

tol.

Glib

encl

amid

e r

elea

sed

% ±

(sd)

T

ime

(min

)

0%5%

8%

0 0

± 0

0 ±

00

± 0

300.

68 ±

0

1.37

± 0

.04

1.05

± 0

.07

601.

5 ±

0.09

2.38

± 0

.017

1.82

± 0

.07

901.

71 ±

0.2

5

3.

05 ±

0.1

42.

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±

0.1

84.

17 ±

0.0

7

240

4.8

± 0.

255.

65 ±

0.2

54.

95 ±

0.1

4

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

132

0123456789101112

050

100

150

200

250

300

350

400

Tim

e (m

in)

% Drug released

Dru

g al

one

5% T

c8%

Tc

Figu

re 5

9:R

elea

se p

rofil

e of

glib

encl

amid

e fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

fT

rans

cuto

l.

133

Dru

g al

one

IPM

0.5

%

IPM

1%

IPM

2%

IPP

0.2

%IP

P1%

IPP

2%

Tc 5

%

Tc 8

%

0123456789101112

Diff

eren

t enh

ance

rs w

ith d

iffer

ent c

once

ntra

tions

% Drug released

Dru

g al

one

IPM

0.5

%

IPM

1%

IPM

2%

IPP

0.2%

IPP1

%

IPP

2%

Tc

5%

Tc

8%

F

igur

e 60

: Per

cent

age

drug

rel

ease

d fr

om w

ater

solu

ble

base

con

tain

ing

diff

eren

t con

cent

ratio

ns o

f fat

ty a

cid

este

rs a

nd T

rans

cuto

l.

134

Cetr

imid

e 0

.5% SLS

0.4

%

Tw

80 4

%

Lab 3

%T

c 5%

IPM

2%

IPP

2%

OA

2%

Dru

g a

lone

LO

A 0

.8%

Ure

a 5

%

Labra

zol 5

%

012345678910

Dif

fert

ent

pen

etra

tion

en

han

cers

% Drug released

Dru

g al

one

Cet

rim

ide

0.5%

SL

S 0

.4%

Tw

80

4%

Lab

3%

Tc

5%

IPM

2%

IPP

2%

OA

2%

LO

A 0

.8%

Ure

a 5%

Lab

razo

l 5%

Figu

re 4

8: P

erce

ntag

e dr

ug r

elea

sed

from

wat

er so

lubl

e ba

se c

onta

inin

g di

ffer

ent c

once

ntra

tions

of d

iffer

ent

enha

ncer

s.

135

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).

136

Table 36: Kinetic data of the release of Glib from different topical

bases.

Cont. table 36: Kinetic data of the release of Glib from different

topical bases.

Correlation coefficient (R)Topical

preparation Zero First Diffusion

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

Top

ical

bas

es

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

WSB

-SL

S

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

WSB

-

Cet

rim

ide

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

WSB

-Tw

-80

5% Tw-80 0.9781 0.9804 0.9962 D.M

5% Tc 0.9806 0.9820 0.9859 D.M

WSB

-Tc

8% Tc 0.9836 0.9849 0.9881 D.M

137

Correlation coefficient (R)Topical

preparation Zero First Diffusion

Observed

order

3% lab 0.9063 0.9109 0.9676 D.M

5 % lab 0.9729 0.9685 �.9757 D.M

WSB

-

labr

afil

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

WSB

-ole

ic

acid 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

WSB

-lino

leic

acid 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

WSB

-IPP

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

WSB

-IPM

2% IPM 0.9556 0.9587 0.9918 D.M

138

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).

139

Tab

le 3

7: R

educ

tion

in b

lood

glu

cose

leve

l aft

er o

ral a

nd to

pica

l app

licat

ion

of g

liben

clam

ide

and

glib

encl

amid

e w

ith

1% o

leic

aci

d in

nor

mal

rat

s. A

ll va

lues

are

exp

ress

ed a

s mea

n ±

sd.

.

*R

educ

tion

in b

lood

glu

cose

leve

l (m

g/dl

).

**P

erce

ntag

e re

duct

ion

in b

lood

glu

cose

leve

ls.

Red

uctio

n in

blo

od g

luco

se le

vel (

mg/

dl)

(Per

cent

age

redu

ctio

n in

blo

od g

luco

se le

vels

)G

roup

Abs

olut

e bl

ood

gluc

ose

leve

l (m

g/dl

)2h

r4h

r6h

r8h

r24

hrC

ontr

ol(1

ml g

um

acac

ia

susp

ensi

on)

94.5

± 3

.97

93.2

5 ±

3.4*

(1.2

9±0.

4)**

84 ±

2.4

(11.

05±

2.49

) 84

.5 ±

2.0

8(1

0.53

±1.

84)

77.5

± 5

.74

(17.

59 ±

2.1)

71

± 2

.94

(24.

64 ±

1.5

)

Ora

l gl

iben

clam

ide

(5m

g/kg

)

103.

75 ±

8.9

43 ±

4.4

(58.

09 ±

1.5

)49

.75

± 5

.3(5

0.48

±6.

6)51

.75±

4.3

(49.

9 ±

4.0

6)59

.25

± 0

.95

(42.

53 ±

4.3)

69.5

± 4

.2(3

0.61

±3.

4)

WSB

(glib

encl

amid

e97

.75±

12.

1274

.25±

9.0

6(2

3.99

± 2.

84)

69.2

5 ±

8.5

3(2

9.13

± 1.

75)

66.0

0 ±

9.1

(32.

55±

2.14

)57

.25

± 8

.8(4

1.54

± 3

.6)

51 ±

6.2

7(4

8.91

± 4

.2)

WSB

(glib

encl

amid

e+1

% o

leic

ac

id)

94.5

± 1

3.1

69 ±

6.3

(26.

95 ±

5.7)

60.7

5 ±

5.9

(35.

26 ±

3.2

)59

.5 ±

6.6

(36.

68 ±

2.7

)56

.5 ±

7.3

(40.

04 ±

3.7)

40.7

5 ±

6.84

(56.

86 ±

3.8

)

140

Tab

le 3

8 :

Red

uctio

n in

blo

od g

luco

se le

vel a

fter

ora

l and

topi

cal a

pplic

atio

n of

glib

encl

amid

e an

d g

liben

clam

ide

with

1%

cet

rim

ide

in n

orm

al r

ats.

All

valu

es a

re e

xpre

ssed

as m

ean

± sd

.

. . *R

educ

tion

in b

lood

glu

cose

leve

l (m

g/dl

).

**P

erce

ntag

e re

duct

ion

in b

lood

glu

cose

leve

ls.

Red

uctio

n in

blo

od g

luco

se le

vel (

mg/

dl)

(Per

cent

age

redu

ctio

n in

blo

od g

luco

se le

vels

)G

roup

Abs

olut

e bl

ood

gluc

ose

leve

l (m

g/dl

)2h

r4h

r6h

r8h

r24

hr

Con

trol

(1m

l gum

ac

acia

su

spen

sion

)

94.5

± 3

.97

93.2

5 ±

3.4*

(1.2

9±0.

46)*

*84

± 2

.4(1

1.05

± 2

.49)

84

.5 ±

2.0

8(1

0.53

±1.

84)

77.5

± 5

.74

(17.

59 ±

2.1)

71

± 2

.94

(24.

64±

1.5)

Ora

l gl

iben

clam

ide

(5m

g/kg

)

103.

75±

8.9

43 ±

4.4

(58.

09 ±

1.5

)49

.75

± 5

.3(5

0.48

±6.

6)51

.75±

4.3

(49.

9 ±

4.0

6)59

.25

± 0

.95

(42.

53 ±

4.3)

69.5

± 4

.2(3

0.61

±3.

4)

WSB

(glib

encl

amid

e97

.75±

12

.12

74.2

5± 9

.06

(23.

99±

2.84

)69

.25

±8.

53(2

9.13

± 1

.75)

66.0

0 ±

9.1

(32.

55 ±

2.1

4)57

.25

± 8

.8(4

1.54

± 3

.6)

51 ±

6.2

7(4

8.91

± 4.

2)

WSB

(glib

encl

amid

e+1

%

cetr

imid

e)

115.

5± 7

.587

.25

±3.

3(2

4.32

±2.

8)70

.33

± 4

.9(3

9.07

± 2

.4)

60.7

5 ±

5.6

7(4

7.39

± 3

.3)

52.6

6 ±

6.8

(54.

54 ±

3.17

)45

.5 ±

6.3

(60.

9± 3

.33)

141

Tab

le 3

9 :

Red

uctio

n in

blo

od g

luco

se le

vel a

fter

ora

l and

topi

cal a

pplic

atio

n of

glib

encl

amid

ean

d gl

iben

clam

ide

with

1%

isop

ropy

l myr

ista

te (I

PM) i

n no

rmal

rat

s. A

ll va

lues

are

exp

ress

ed a

s mea

n ±

sd.

*R

educ

tion

in b

lood

glu

cose

leve

l (m

g/dl

).

**P

erce

ntag

e re

duct

ion

in b

lood

glu

cose

leve

ls.

Red

uctio

n in

blo

od g

luco

se le

vel (

mg/

dl)

(Per

cent

age

redu

ctio

n in

blo

od g

luco

se le

vels

)G

roup

Abs

olut

e bl

ood

gluc

ose

leve

l (m

g/dl

)2h

r4h

r6h

r8h

r24

hrC

ontr

ol(1

ml g

um

acac

ia

susp

ensi

on)

94.5

± 3

.97

93.2

5 ±

3.4*

(1.2

9±0.

46)*

*84

± 2

.4(1

1.05

± 2

.49)

84

.5 ±

2.0

8(1

0.53

±1.

84)

77.5

± 5

.74

(17.

59 ±

2.1)

71

± 2

.94

(24.

64 ±

1.

5)

Ora

l gl

iben

clam

ide

(5m

g/kg

)

103.

75 ±

8.9

43 ±

4.4

(58.

09 ±

1.5

)49

.75

± 5

.3(5

0.48

±6.

6)51

.75±

4.3

(49.

9 ±

4.0

6)59

.25

± 0

.95

(42.

53 ±

4.3)

69.5

± 4

.2(3

0.61

±3.

4)

WSB

(glib

encl

amid

e97

.75

± 1

2.12

74.2

5± 9

.06

(23.

99±

2.84

)69

.25

± 8

.53

(29.

13 ±

1.7

5)66

.00

± 9

.1(3

2.55

± 2

.14)

57.2

5 ±

8.8

(41.

54 ±

3.6

)51

± 6

.27

(48.

91 ±

4.

2)W

SB(g

liben

clam

ide

+1%

IPM

)97

± 1

7.9

93 ±

17.

75(4

.16

±0.

93)

78.2

5 ±

13.

37(1

9.13

± 3

.23)

69 ±

12.

83(2

8.82

± 2

.38)

62.7

5 ±

12.

89(3

5.26

±3.

2)50

.5 ±

6.5

(47.

52 ±

4.

2)

142

.Tab

le 4

0 :

Red

uctio

n in

blo

od g

luco

se le

vel a

fter

ora

l and

topi

cal a

pplic

atio

n of

glib

encl

amid

ean

d gl

iben

clam

ide

with

5 %

Lab

rafil

in n

orm

al r

ats.

All

valu

es a

re e

xpre

ssed

as m

ean

± sd

.

. *R

educ

tion

in b

lood

glu

cose

leve

l (m

g/dl

).

**P

erce

ntag

e re

duct

ion

in b

lood

glu

cose

leve

ls.

Red

uctio

n in

blo

od g

luco

se le

vel (

mg/

dl)

(Per

cent

age

redu

ctio

n in

blo

od g

luco

se le

vels

)G

roup

Abs

olut

e bl

ood

gluc

ose

leve

l (m

g/dl

)2h

r4h

r6h

r8h

r24

hrC

ontr

ol(1

ml g

um

acac

ia

susp

ensi

on)

94.5

± 3

.97

93.2

5 ±

3.4*

(1.2

9±0.

46)*

*84

± 2

.4(1

1.05

± 2

.49)

84

.5 ±

2.0

8(1

0.53

±1.

84)

77.5

± 5

.74

(17.

59 ±

2.1)

71

± 2

.94

(24.

64 ±

1.

5)

Ora

l gl

iben

clam

ide

(5m

g/kg

)

103.

75 ±

8.9

43 ±

4.4

(58.

09 ±

1.5

)49

.75

± 5

.3(5

0.48

±6.

6)51

.75±

4.3

(49.

9 ±

4.06

)59

.25

± 0

.95

(42.

53 ±

4.3)

69.5

± 4

.2(3

0.61

±3

.4)

WSB

(glib

encl

amid

e97

.75

± 1

2.12

74.2

5± 9

.06

(23.

99±

2.84

)69

.25

± 8

.53

(29.

13 ±

1.7

5)66

.00

± 9

.1(3

2.55

± 2

.14)

57.2

5 ±

8.8

(41.

54 ±

3.6

)51

± 6

.27

(48.

9± 4

.2)

WSB

(glib

encl

amid

e+5

% L

abra

fil)

102.

75±

8.5

96.7

5 ±

6.3

(5.7

±1.

9)95

± 6

.6(6

.9 ±

2.5

)95

± 5

.9(7

.41

± 2

.14)

83.7

5 ±

9.7

(18.

017

±2.5

)75

± 6

.97

(27.

0±

2.96

)

143

.

010203040506070

02

46

810

1214

1618

2022

2426

Tim

e (h

r)

% Reduction in blood glucose level

Con

trol

Ora

l glib

encl

amid

eTo

pica

l glib

encl

amid

e1%

OA

1% C

etrim

ide

1% IP

M5%

Lab

rafil

Figu

re 6

2: P

erce

nt r

educ

tion

in b

lood

glu

cose

leve

ls a

fter

ora

l and

topi

cal a

dmin

istr

atio

n of

glib

encl

amid

e in

nor

mal

ra

ts.

144

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).

145

Tab

le 4

1 :

Red

uctio

n in

blo

od g

luco

se le

vel a

fter

ora

l and

topi

cal a

pplic

atio

n of

glib

encl

amid

ean

d gl

iben

clam

ide

with

1%

cet

rim

ide

in d

iabe

tic r

ats.

All

valu

es a

re e

xpre

ssed

as m

ean

± sd

.

.

Red

uctio

n in

blo

od g

luco

se le

vel (

mg/

dl)

(Per

cent

age

redu

ctio

n in

blo

od g

luco

se le

vels

)G

roup

Abs

olut

e bl

ood

gluc

ose

leve

l (m

g/dl

)2h

r4h

r6h

r8h

r24

hr

Con

trol

(1m

l gum

ac

acia

su

spen

sion

)

235.

6 ±

40

.322

8.4

± 36

.26*

(2.8

9 ±

1.65

)**

232.

2 ±

38.4

6(1

.35

± 1.

06)

230.

6 ±

40.8

(2.1

6 ±1

.68)

224.

4 ±

38.8

(4.7

6±0.

89)

223.

4 ±

40.8

(5.2

2 ±

2.74

)

Ora

l gl

iben

clam

ide

(5m

g/kg

)

485

±

79.5

642

0.2

± 6

2.81

(15.

63 ±

3.0

9)28

3 ±

33.5

4(4

1.1

±5.2

5)31

8 ±

45.

06(3

4.02

± 5

.67)

342

± 51

.63

(29.

25

±3.7

5)

364.

8± 4

9.93

(24.

53 ±

3.74

)

WSB

(glib

encl

amid

e+1

%

cetr

imid

e)

319±

23.

230

5± 2

1.37

(4.2

8± 2

.4)

286.

7± 3

5.12

(9.6

± 4

.4)

277.

2 ±

30.

82(1

3.28

± 3

.5)

267.

5± 2

5.99

(16.

16 ±

2.7)

236.

8 ±

23.5

3(2

5.7

± 4.

69)

. *R

educ

tion

in b

lood

glu

cose

leve

l (m

g/dl

).

**P

erce

ntag

e re

duct

ion

in b

lood

glu

cose

leve

ls.

146

05101520253035404550

02

46

810

1214

1618

2022

2426

Tim

e (h

r)

% Reduction in blood glucose level(mg/dl)

Con

trol

Ora

l glib

encl

amid

eTo

pica

l glib

encl

amid

e

Figu

re 6

3 : P

erce

nt r

educ

tion

in b

lood

glu

cose

leve

ls a

fter

ora

l and

topi

cal a

dmin

istr

atio

n of

glib

encl

amid

e in

di

abet

ic r

ats.

147

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).

148

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).

149

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.

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