i

Transdermal Delivery of Gabapentin and Glipizide:

Effects of Cosolvent Systems and Microemulsions

BY

NNADI, CHARLES OKEKE

PG/M.PHARM./09/50519

A THESIS PRESENTED TO THE DEPARTMENT OF PHARMACEUTICAL AND

MEDICINAL CHEMISTRY, FACULTY OF PHARMACEUTICAL SCIENCES OF THE

UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILMENT FOR THE

AWARD OF MASTERS DEGREE IN PHARMACEUTICAL AND MEDICINAL

CHEMISTRY

SUPERVISOR: DR. C. J. MBAH

MARCH, 2012

ii

CERTIFICATION

This is to certify that NNADI, CHARLES OKEKE, a postgraduate student in the Department

of Pharmaceutical and Medicinal Chemistry, with Registration Number:

PG/M.Pharm./09/50519 has satisfactorily completed the requirements for the award of Masters

Degree in Pharmaceutical and Medicinal Chemistry. The work embodied in this project is

original and has not been submitted in part or full for any other Diploma or Degree of this or any

other university.

________________________________ ______________________________

Dr. C. J. Mbah Dr. (Mrs.) N. J. Nwodo

(Supervisor) (Head of Department)

_________________________________

(External Examiner)

iii

DEDICATION

The work is dedicated to two most outstanding people in my life: my wife, Mrs. Chinenye

Juliana Charles-Nnadi and my daughter, Miss Chidera Chinenye Charles-Nnadi.

iv

ACKNOWLEDGEMENT

First and foremost, the Living God has to take all glory for me keeping alive and healthy all

these days. Even pressure from work could not hold me back from matching forward. He sees all

and knows all. Words cannot express my gratitude to Him.

To every achievement, there is always a motivator; history of this achievement cannot be

complete without the motivation and assistance of my supervisor, Pharm. Prof. Chika John Mbah

who, despite his tight schedules, has always been willing and ready to assist me throughout the

duration of this work. He was always willing to assist without compromising the standards and

the necessary skills required to be imparted at this stage. The good aspect of it all was his

understanding and respect for every category of human being. He was more than a project

supervisor to me; he has been a father too. My gratitude will always go to him.

The members of staff of Pharmaceutical Chemistry are also remembered here, especially the

Head of Department, Dr. (Mrs.) Ngozi Nwodo who has been on my neck to conclude this work

on the record time. The Dean of the Faculty, Pharm. Prof. (Mrs.) P. O. Osadebe who has been

playing the role of a mother has always been available and encouraging to my little effort. Mr.

Matthias Agbo helped in no little measure during the extraction of the coconut oil and its

analysis. His wealth of experience as a pure chemist made this work beautiful. Pharm. Dr. Edwin

Omeje and Dr. Willy Obonga gave me encouraging words when I needed them most. My other

colleagues in the Department, Pharmacists Uzor, Philip and Late David Kenechukwu Ernest

could have offered more if they had the opportunities to do so. The technical staffs of the

Department were also helpful; some of them are Pharm. Justus Nwoga, Mrs. Rose Anyaoha, Mr.

Ozor Alphonsus, Mr. Mike Ugwuoke, and many others whose names could not appear here

because of want of space.

I could not have gone this far without the support of my family members. My brothers and sisters

were always encouraging whenever I needed it most. My wife was supportive even when I had

to leave her and the young baby at home to accomplish this work. My little daughter, Chidera

missed my absence from the house but always understood that my absence from the house was

for good.

My friends and associates also helped a lot. Uche, Gadafy, Daniel and many others helped as

much as they could.

The typing of this work was skillfully done by my wife, Juliana Chinenye Charles-Nnadi. She

spent greater part of her time helping whenever it was demanded. You are indeed a companion!

v

TABLE OF CONTENT

TITLE PAGE - - - - - - - - - - i

CERTIFICATION - - - - - - - - - ii

DEDICATION - - - - - - - - - iii

ACKNOWLEDGEMENT - - - - - - - - iv

TABLE OF CONTENT- - - - - - - - - v

LIST OF ILLUSTRATIONS - - - - -- - - - ix

LIST OF FIGURES - - - - - - - - - x

LIST OF TABLES - - - - - - - - - xi

ABSTRACT - - - - - - - - - - xiii

CHAPTER ONE: INTRODUCTION - - - - - - 1

1.1 Transdermal drug Delivery systems - - - - - 1

1.1.1 Advantages and Disadvantages of Transdermal Drug Delivery Systems 1

1.1.2 Criteria of a Drug Candidate for Transdermal Delivery - - 3

1.1.3 Factors Affecting Transdermal Drug Delivery - - - 4

1.2 Microemulsion as a Vehicle for Transdermal Delivery of Drugs - 5

1.2.1 Applications of Pharmaceutical Microemulsions - - - 6

1.2.2 Formulation of Microemulsion - - - - - 7

1.2.3 Advantages of Microemulsion Based Systems - - - 9

1.2.4 Disadvantages of Microemulsion Based Systems - - - 11

1.3 Mechanisms of Skin Penetration Enhancement - - - 11

1.4 Skin as a Permeation Barrier - - - - - - 12

1.4.1 The Structure of the Skin - - - - - - 13

1.5 Gabapentin - - - - - - - - 14

1.5.1 Physicochemical Properties of Gabapentin - - - - 14

vi

1.5.2 Pharmacology of Gabapentin -- - - - - - 15

1.5.3 Pharmacokinetics of Gabapentin - - - - - 17

1.6 Glipizide - - - - - - - - 18

1.6.1 Physicochemical Properties of Glipizide - - - - 18

1.6.2 Pharmacology of Glipizide - - - - - - 18

1.6.3 Pharmacokinetics of Glipizide - - - - - 20

1.7 Objectives of the Study- - - - - - - 21

CHAPTER TWO: MATERIALS AND METHODS - - - - 22

2.1 Materials - - - - - - - - 22

2.2 Methods - - - - - - - - 22

2.2.1 Preparation of Phosphate Buffered Saline Solution - - - 22

2.2.2 Preparation of standard Solution of Drugs - - - - 23

2.2.2.1 Preparation of standard Solution of Glipizide - - - 23

2.2.2.2 Preparation of standard Solution of Gabapentin - - - 23

2.2.3 Extraction of Coconut Oil - - - - - - 24

2.2.4 Physical Characterization of Coconut Oil - - - - 24

2.2.5 Quantitative Characterization of Coconut Oil - - - 24

2.2.5.1 Determination of Saponification Value - - - - 24

2.2.5.2 Determination of Iodine Value - - - - - 25

2.2.5.3 Determination of acid Value - - - - - - 25

2.2.5.4 Viscosity Measurement of Coconut Oil - - - - 25

2.2.6 Determination of solubility of Drugs in Coconut Oil - - - 25

2.2.6.1 Solubility of Glipizide in Coconut Oil - - - - 25

2.2.6.2 Solubility of Gabapentin in Coconut Oil - - - - 26

2.2.7 Construction of Pseudo ternary Phase diagrams - - - 26

vii

2.2.8 Preformulation Stability Studies of Microemulsions - - - 27

2.2.9 Drug Loading of the Microemulsions - - - - - 27

2.2.9.1 Preparation of Microemulsion Loaded with Glipizide - - 27

2.2.9.2 Preparation of Microemulsion Loaded with Gabapentin - - 28

2.2.10 Post Formulation stability studies of Drug-Loaded Microemulsions - 28

2.2.11 Preparation of Rat Abdominal Skin - - - - - 28

2.2.12 In-Vitro Skin Permeation Studies - - - - - 29

2.2.13 Characterization of Optimized Microemulsions - - - 30

2.2.13.1 Dilution Test of the Microemulsions - - - - - 30

2.2.13.2 Determination of pH of Microemulsions - - - - 30

2.2.13.3 Viscosity Measurement of Microemulsions - - - - 30

2.2.13.4 Determination of Globule Size and Polydispersity Index - - 30

2.2.13.5 Skin Irritation Studies of Microemulsions - - - - 31

2.2.14 Preparation of Stratum Corneum for FTIR and DSC Studies- - 32

2.2.15 FTIR Spectroscopic Studies on Stratum Corneum - - - 32

2.2.16 DSC Studies on Stratum Corneum - - - - - 32

2.3 Data and Statistical Analysis - - - - - - 33

CHAPTER THREE: RESULTS- - - - - - - - 35

3.1 Preparation of Standard Solution of Gabapentin and Glipizide - 35

3.2 Extraction and Physical Characterization of Coconut Oil - - 36

3.3 Quantitative Characterization of Coconut Oil - - - 36

3.4 Determination of solubility of Drugs in Coconut Oil - - - 36

3.5 Pseudo ternary Phase Diagrams - - - - - 37

3.6 Preformulation Stability Studies of Microemulsion - - - 40

3.7 Post Formulation Stability Studies of Drug-Loaded Microemulsions 40

viii

3.8 In-Vitro Skin Permeation Studies of Vehicles and Microemulsion - 41

3.9 Characterization of the Optimized Microemulsion - - - 52

3.10 FTIR Spectroscopic Studies on Stratum Corneum - - - 54

3.11 DSC Studies on Stratum Corneum- - - - - 60

CHAPTER FOUR: DISCUSSION AND CONCLUSION- - - - 62

4.1 Preparation of Standard Solution of Gabapentin and Glipizide - 62

4.2 Quantitative and Qualitative Characterization of Coconut Oil - 62

4.3 Preparation of Coconut Oil-Based Microemulsions - - - 63

4.4 Permeation Studies of Gabapentin and Glipizide - - - 66

4.4.1 Permeation Studies of Gabapentin in Different Vehicles - - 66

4.4.2 Permeation Studies of Glipizide in Different Vehicles - - 69

4.5 Skin Irritation Test - - - - - - - 74

4.6 Biophysical Analysis of Treated and Untreated SC - - - 75

4.7 Conclusion and Prospects - - - - - - 78

4.7.1 Conclusion- - - - - - - - - 78

4.7.2 Prospects - - - - - - - - 79

REFERENCES - - - - - - - - - 80

ix

LIST OF ILLUSTRATIONS

1. Hypothetical Phase regions of Microemulsion Systems - - - 8

2. Chemical Structure of Gabapentin - - - - - - 15

3. Chemical Structure of Glipizide - - - - - - 18

x

LIST OF FIGURES/GRAPHS

1. Calibration Curve of Gabapentin in Phosphate Buffered Saline - - 35

2. Calibration Curve of Glipizide in Ethanol - - - - - 35

3. Pseudo ternary Phase Diagram of Surfactant: Cosurfactant ratio 1:1 - 39

4. Pseudo ternary Phase Diagram of Surfactant: Cosurfactant ratio 1:2 - 39

5. Pseudo ternary Phase Diagram of Surfactant: Cosurfactant ratio 2:1 - 39

6. Pseudo ternary Phase Diagram of Surfactant: Cosurfactant ratio 1:3 - 39

7. Pseudo ternary Phase Diagram of Surfactant: Cosurfactant ratio 3:1 - 40

8. Permeation Profile of Gabapentin in Different Strengths of Ethanol - - 42

9. Permeation Profile of Gabapentin in Different Strengths of Propylene Glycol 43

10. Permeation Profile of Gabapentin in Different Microemulsions - 44

11. Permeation Profile of Glipizide in Different Strengths of Ethanol - - 48

12. Permeation Profile of Glipizide in Different Strengths of Propylene Glycol - 49

13. Permeation Profile of Glipizide in Different Microemulsions- - 50

14. Photomicrograph of MCEa - - - - - - - 53

15. Photomicrograph of MCEd - - - - - - - 54

16. FTIR Spectra of Untreated Stratum Corneum (Control) - - - 55

17. FTIR Spectra of Stratum Corneum treated with Ethanol - - - 56

18. FTIR Spectra of Stratum Corneum treated with Propylene Glycol - - 57

19. FTIR Spectra of Stratum Corneum treated with MCEa - - - 58

20. FTIR Spectra of Stratum Corneum treated with MCEd - - - 59

21. DSC Thermogram of Untreated Stratum Corneum (Control) - - 60

22. DSC Thermogram of Stratum Corneum Treated with MCEd - - 60

23. DSC Thermogram of Stratum Corneum Treated with MCEa - - 61

xi

LIST OF TABLES

1 Quantitative and Qualitative Parameters of Coconut Oil - - - 36

2 Solubility Profile of Gabapentin and Glipizide in Coconut Oil - - 37

3 Microemulsions with Surfactant: Co-Surfactant ratio of 1:1 - - - 37

4 Microemulsions with Surfactant: Co-Surfactant ratio of 1:2 - - - 38

5 Microemulsions with Surfactant: Co-Surfactant ratio of 2:1 - - - 38

6 Microemulsions with Surfactant: Co-Surfactant ratio of 1:3 - - - 38

7 Microemulsions with Surfactant: Co-Surfactant ratio of 3:1 - - - 38

8 Compositions of the Microemulsions Selected from the Regions of

Micro-emulsification in the Pseudo ternary Phase Diagrams- - - 40

9 Results of Cumulative Amount of Gabapentin Permeated - - - 41

10 Permeation Parameters of Gabapentin from Different Cosolvents - - 45

11 Permeation Parameters of Gabapentin from Different Microemulsions - 45

12 Permeation Kinetics Parameters of Gabapentin in Cosolvents and Microemulsions 46

13 Results of Expected Transdermal Patch Sizes of Gabapentin from Cosolvents and

Microemulsions - - - - - - - - 46

14 Results of Cumulative Amount of Glipizide Permeated - - - 47

15 Permeation Parameters of Glipizide from Different Cosolvents- - 51

16 Permeation Parameters of Glipizide from Different Microemulsions - 51

17 Permeation Kinetics Parameters of Glipizide in Cosolvents and Microemulsions 52

18 Results of Expected Transdermal Patch Sizes of Glipizide from Cosolvents and

Microemulsions - - - - - - - - 52

19 Physicochemical Properties of MCEa - - - - - 53

20 Physicochemical Properties of MCEd - - - - - 54

21 Data for the Skin Irritation Test - - - - - - 74

xii

22 Effects of Microemulsions on DSC of Stratum Corneum - - - 75

23 Effects of Vehicles on FTIR Spectra of Stratum Corneum - - - 77

xiii

ABSTRACT

Background: Recent advances in drug delivery have led to search for routes of drug

administration that could deliver drugs to systemic circulation without compromising efficacy or

posing any threats to the patient. Transdermal route is known to by-pass some obvious

challenges encountered in traditional drug administration procedures like hepatic first pass

metabolism, gastrointestinal disturbances, low absorption, short half-life, high frequency of

administration and poor compliance. This alternative route requires careful selection of vehicles

that can ensure adequate solubility of the drugs and circumvent barrier properties of the stratum

corneum of the skin. Microemulsion and co-solvent systems are known to possess these

qualities.

Objective: The principal aim of this study is to examine the possibility of delivering gabapentin

and glipizide transdermally using some cosolvent systems, and pharmaceutically acceptable o/w

and w/o microemulsions with good rheological properties formulated with polyethoxylated

castor oil (as surfactant), ethanol (as cosurfactant), locally sourced coconut oil (as oil phase) and

distilled water (as aqueous phase) as vehicles.

Method: Different strengths (10, 20 and 30 % v/v) of ethanol and propylene glycol cosolvent

systems were prepared by homogenous mixing. Various oil-in-water and water-in-oil

microemulsions were prepared by aqueous titration method. The microemulsion areas were

identified by constructing pseudo ternary phase diagrams. The prepared microemulsions were

subjected to pre- and post-drug loading formulation stability tests. The microemulsion

formulations that passed stability tests were characterized for viscosity, pH, surface tension, and

droplet size (and polydispersity index). Transdermal permeation of gabapentin and glipizide (in

both cosolvent systems and microemulsions) through rat abdominal skin (surface area, 2.54 cm2)

was determined by modified Franz diffusion cell. The in-vitro skin permeation profiles of the

optimized formulations were compared with that of control. Biophysical characterization

(Fourier Transform Infrared Spectroscopy and Differential Scanning Calorimetry) of the stratum

corneum of the rat before and after treatment with the optimized systems was done. The safety of

the optimized microemulsions was confirmed by carrying out the skin irritation tests.

xiv

Result: The pre- and post-formulation stability studies revealed that all the microemulsions

formed from surfactant: cosurfactant ratio of 1:1, 1:2 and 2:1 were stable beyond 90 days with

excellent rheological properties. A significant increase (p<0.05) in permeability parameters such

as steady-state flux (Jss), permeability coefficient (Kp) and enhancement ratio (Er) were observed

in optimized microemulsion formulation, MCEa for gabapentin (which consists of cremophor Rh

40®/ethanol-1:1 20%, coconut oil 60% and water 20%) and MCEd for glipizide (which consists

of cremophor Rh 40®/ethanol-1:2 20%, coconut oil 20% and water 60%) compared with the

control. Glipizide showed Jss of 121.2±9.98 µg/cm2.h, Kp of (60.62±5.29) x 10

-3 cm

2.h and Er of

23.09±0.04 while gabapentin showed Jss, Kp and Er of 141.2±34.1 µg/cm2.h, (56.5±9.4) x 10

-3

cm2.h and 20.0±0.1 respectively for their respective optimized microemulsion systems. All

cosolvent systems showed significant increase (p<0.05) in permeability parameters of

gabapentin. Propylene glycol cosolvent system did not have significant effect on permeability

parameters of glipizide compared with the control. DSC study showed 17.5 % and 40 %

reduction of SC protein and lipids respectively by MCEa; and 30 % and 42.9 % reduction of SC

protein and lipids respectively by MCEd. FTIR study showed 64.56 % reduction of peak height

of asymmetrical –CH2 vibration at 2920-2850 cm-1

and 70.37 % reduction of peak height of

amide I stretching vibration at 1650-1550 cm-1

by MCEa. Similarly, 52.74 % and 85.60 %

reductions were obtained for MCEd. This showed that the mechanism of skin permeation could

be by disruption of the SC lipid architecture and/or denaturation of SC keratin by the cosolvent

systems and microemulsion respectively. The primary irritancy index (PII) for the optimized

systems was 0.33±0.58 (PII<2) which showed that the materials used for the vehicles are non-

toxic.

Conclusion: The result of this study shows that the optimized microemulsions for delivery of

gabapentin and glipizide are w/o and o/w respectively. Cosolvent of ethanol-water (3:7) is the

best cosolvent for transdermal delivery of both drugs from transdermal patches of acceptable

sizes. The study also shows the probable mechanisms of permeation of these drugs as disruption

of the SC lipid bilayer and denaturation of SC keratin. Based on these findings, it could be

suggested that cosolvent systems (ethanol-water 3:7) and microemulsions (surfactant:

cosurfactant of 1:1 and 1:2) are possible vehicles for the development of transdermal products of

both drugs investigated.

xv

CHAPTER ONE

INTRODUCTION

1.1 Transdermal Drug Delivery Systems

Controlled release medication may be defined as the permeation-moderated transfer of an active

material from a reservoir to a target surface to maintain a predetermined concentration or

emission level for a specified period of time. Transdermal drug delivery system can be defined as

the controlled release of drugs through intact skin. Controlled release technology has received

increasing attention in the face of a growing awareness that substances are frequently toxic and

sometimes ineffective when administered or applied by conventional means. The transdermal

route now ranks with oral treatment as the most successful innovative research area in drug

delivery, with around 40 % of the drug delivery candidate products under clinical evaluation

related to transdermal or dermal system [1]. The worldwide transdermal patch market

approaches £2 billion, based on only ten drugs including scopolamine, nitroglycine, clonidine,

estrogen, testosterone, fentanyl, and nicotine with a lidocaine patch soon to be marketed [2]. The

success of a dermatological drug to be used for systemic drug delivery depends on the ability of

the drug to penetrate through skin in sufficient quantities to achieve the desired therapeutic effect

[3]. Transdermal drug delivery is the administration of a therapeutic agent through intact skin for

systemic effect.

1.1.1 Advantages and Disadvantages of Transdermal Drug Delivery Systems

Topical application has been used for many centuries, mainly for the treatment of localized skin

complaints. Usually, the drug only penetrates the outer layers of skin and little or no systemic

absorption occurs. The transdermal delivery systems are specifically designed to enhance drug

xvi

permeation into systemic circulation and offer the following advantages over the conventional

route for controlled drug delivery [3]: avoidance of hepatic first pass metabolism and

gastrointestinal incompatibility, ability to discontinue administration by removal of the system,

ability to control drug delivery for a longer time than the usual gastrointestinal transit of oral

dosage forms, ability to modify the properties of the biological barrier to absorption, reduces side

effects due to optimization of the blood concentration-time profile, provides greater patient

compliance due to the elimination of multiple dosing schedules, enhances therapeutic efficacy,

minimises inter- and intra-patient variations, ensures ease of self administration. However,

transdermal systems can impart other important advantages to active agents that could be

sufficient to elevate many products to commercial successes. Based on the economic

consideration, the cost of developing new drug entities as well as the time it takes to bring such

drugs to marketplace has been continuously increasing [4]. In transdermal delivery, it may be

started with drug that is already approved, therefore, the risks, time to the marketplace, and the

research costs are all substantially reduced. On clinical improvements, transdermal delivery can

increase the therapeutic value of many drugs by obviating specific problems associated with the

drug. Such problems might include gastrointestinal irritation, low absorption, decomposition due

to hepatic first pass effect, formation of metabolites that cause side effects, and short half-life

necessitating frequent dosing [5]. In transdermal medication, the above problems can be

eliminated because the drug diffuses over a prolonged period of time directly into the blood

stream. Therefore, a gold mine might exist in the files of major drug companies in drug

substances discarded because of gastrointestinal irritation, low absorption or other specific

problems which can be bypassed by the use of transdermal medication [6].

xvii

Only a small percentage of drugs can be delivered transdermally due to three limitations;

difficulty of permeation through human skin, skin irritation and clinical need [7]. In addition to

its use as a physical barrier, the human skin functions as a chemical barrier to almost all drugs

and chemicals. Skin irritation of excipients and enhancers of the drug used to increase

percutaneous absorption is another limitation. Persistence contact irritant dermatitis could result

from direct toxic injury to cell membranes, cytoplasm or nuclei [8].

1.1.2 Criteria of Candidate Drug for Transdermal Delivery

Basically, not every drug or chemical is a candidate for transdermal drug delivery. Judicious

choice of drug is the most important decision in the successful development of a transdermal

product. The most important drug properties that affect its diffusion through the devices as well

as the skin include molecular weight, chemical functionality and melting point. It is generally

accepted that the best drug candidates for passive adhesive transdermal patches must be non-

ionic, low molecular weight (less than 500 Daltons), have adequate solubility in oil and water

(log P in the range of 1 to 3), a low melting point (less than 200 °C), short plasma half-life, and

are potent (dose is less than 50 mg per day, and ideally less than 10 mg per day) [9, 10]. Given

these operating parameters, the number of drug candidates for passive transdermal patches is

low, owing to the challenge of diffusing across the bilayers in the tortuous stratum corneum [11,

12]. But, many new opportunities still exist for novel passive transdermal patch products. The

new transdermal technologies that were introduced in the previous section challenge the

paradigm that there are only a few drug candidates for transdermal drug delivery. The table

below shows a summary of the TDD technologies and the types of molecules that these

technologies enable for transdermal delivery. With the active and micropore-creating

transdermal technologies, molecular size is not a limiting factor. The same applies for other

xviii

physiochemical drug properties, such as ionization state, melting point, and solubility. Finally,

the active and micropore-creating technologies also enable therapeutic delivery of drugs at doses

higher than 10 mg. Clearly, the opportunities for transdermal drug delivery have been greatly

expanded through the application of new formulation technologies and active delivery systems.

Now, a much wider set of drug compounds, including macromolecules, have the possibility to be

delivered transdermally at therapeutic levels than was possible just a decade ago. Of course, the

use of a TDD technology for any drug must be clinically beneficial [13].

1.1.3 Factors Affecting Transdermal Drug Delivery

Apart from minor factors such as individual variations, age, site of application, occlusion,

temperature, race, and disease states [14], there are other physical related factors that affect the

permeation of drugs through the skin as described in the Fick’s equation:

Where, is the rate of drug penetration, Þ is the partition coefficient between stratum corneum

and vehicle, C is the concentration of drug in the vehicle, D is the average diffusion coefficient,

A is the surface area of application of the drug, l is the thickness of the skin barrier.

(a) Partition Coefficient:

For an individual drug, this is measured as the octanol-water ratio (or log P). It is a measure of

lipophilicity verses hydrophilicity. In skin permeation studies, the steady-state rate of permeation

across the skin can be expressed by the equation below:

xix

Where Cd and Cr are, respectively, the concentration of drug in the donor compartment and in the

receptor compartment and Ps is the permeability coefficient of the skin defined by the equation

below:

Where Ks is the partition coefficient for the interfacial partitioning of the drug from the device

(vehicle) to the skin, Ds is the diffusivity of the drug through the skin, h is the thickness of the

skin.

(b) Diffusion

This is the process by which a substance moves from one area to another. It is driven by thermal

agitation and requires a concentration gradient [15]. In other words, the area that a substance is

going to must have a lower concentration of the drug than the area it is coming from. Lipophilic

substances diffuse easily through stratum corneum lipids, but have much more difficulty with the

aqueous layers below. If transport slows too much in any layer of tissue (example, stratum

corneum, epidermis, dermis) diffusion slows, causing a build up in the outer layers.

(c) Concentration

This is the amount of substance per unit volume of vehicle. The importance of solubility is the

reason a solvent carrier is typically used despite its reduction in partition coefficient [16]. For

example, corticosteroid’s partition coefficient is reduced twofold by the addition of 50 % ethanol

to saline, but its solubility is increased 100 fold, giving a 40 fold penetration enhancement. The

solubility issue can become a problem if the vehicle evaporates before the drug has fully

partition into the skin, causing precipitation [17]. Thus, it is necessary to also incorporate a small

xx

amount of a less volatile solvent such as fatty acid, terpenes, isopropyl myristate into a

transdermal formulation.

(d) Surface Area

Large surface area of contact between the drug formulation and the stratum corneum exposes

more drug molecules to the lipid skin layer and so increases the rate of drug permeation [18].

1.2 Microemulsion as a Vehicle for Transdermal Delivery of Drug

Microemulsions are clear, thermodynamically stable, isotropic liquid mixtures of oil, water and

surfactant, frequently in combination with a cosurfactant. The aqueous phase may contain salt(s)

and/or other ingredients, and the "oil" may actually be a complex mixture of different

hydrocarbons and olefins. In contrast to ordinary emulsions, microemulsions form upon simple

mixing of the components and do not require the high shear conditions generally used in the

formation of ordinary emulsions. The three basic types of microemulsions are direct (oil

dispersed in water, o/w), reversed (water dispersed in oil, w/o) and bicontinuous.

Microemulsions are excellent candidates as potential drug delivery systems because of their

improved drug solubilisation, long shelf life, and ease of preparation and administration [19].

1.2.1 Applications of Pharmaceutical Microemulsion in Topical Delivery

Topical administration of drugs can have advantages over other methods for several reasons, one

of which is the direct delivery and targetability of the drug to affected area of the skin or eyes

[21]. Both o/w and w/o microemulsions have been evaluated in a hairless mouse model for the

delivery of prostaglandin E1. The microemulsions were based on oleic acid or Gelucire 44/14 as

the oil phase and were stabilized by a mixture of Labrasol (C8 and C10 polyglycolysed

glycerides) and Plurol Oleique CC 497 as surfactant. Although enhanced delivery rates were

xxi

observed in the case of the o/w microemulsion, it was concluded that the penetration rates were

inadequate for practical use from either system. The use of lecithin/IPP/water microemulsion for

the transdermal transport of indomethacin and diclofenac has also been reported. Fourier

transform infra red (FTIR) spectroscopy and differential scanning calorimetry (DSC) showed the

IPP organogel had disrupted the lipid organization in human stratum corneum after a 1 day

incubation [22].

The transdermal delivery of the hydrophilic drug, diphenhydramine hydrochloride, from a w/o

microemulsion into excised human skin has been investigated [23]. The formulation was based

on combinations of Tween 80 and Span 20 with IPM. However two additional formulations were

tested containing cholesterol and oleic acid, respectively. Cholesterol increased drug penetration

whereas oleic acid had no measurable effect, but the authors clearly demonstrated that

penetration characteristics can be modulated by compositional selection.

1.2.2 Formulation of Microemulsion

(a) Conditions Necessary to Produce Microemulsions

Microemulsions are fascinating systems in that nature prefers to have a dispersed system of oil,

water and surfactant having large total interfacial area, rather than separate phases of oil and

water with much smaller interfacial area. In order to form microemulsions, three major factors

must be considered [23]. First, emulsifiers or surfactants must be carefully chosen so that an

ultra-low interfacial tension (< 0.001 mN/m) can be attained at the oil/water interface. The ultra-

low interfacial tension at the oil/water interface is a prime requirement to produce

microemulsions. It is this very low interfacial tension that leads to spontaneous emulsification of

oil in water or water in oil. The second requirement is that the concentration of emulsifiers or

xxii

surfactants must be high enough to provide the number of surfactant molecules needed to

stabilize the microdroplets produced by an ultra-low interfacial tension. Because microemulsions

are in the range of 100-1000 Å in diameter, 30 % of oil dispersed in water with 200 Å droplet

diameters will create 106 cm

2 of total interfacial area per ml of microemulsion; therefore, the

larger concentration (10-40 %) of surfactant is required to stabilize the newly created interface of

microemulsion droplets. The third major consideration in formulating microemulsion is the

flexibility or fluidity of the interface to promote the formation of microemulsions. Therefore,

short-chain alcohols (C4 to C7) are often added as cosurfactant in surfactant + water + oil systems

to produce microemulsions [24]. The penetration of short-chain alcohols into the interfacial film

produces a more fluid interface by allowing the long hydrophobic tails of the C16 or C18

surfactants to move freely at the interface.

(b) Phase Equilibria of Microemulsion Systems

Phase diagrams are useful in formulation studies as a means of delineating the area of existence

of the microemulsion region. The method used to construct such diagrams depends on the mutual

solubility of the components, but in general, it is convenient to use the titration method, which

allows a large number of compositions to be examined relatively quickly. The titration method

consists of weighing quantities of surfactants, cosurfactants and oil which are then mixed to form

a monophasic solution. The constantly-stirred mixture is then titrated with water at constant

temperature. After each addition of water, the container should be stoppered to minimize loss of

volatile component and the system examined for clarity, birefringence, flow properties and

stability. After coarse determination of the microemulsion region, a more detailed study of this

xxiii

region of the phase diagram is required to assess the long-term stability of the systems in this

region. [25].

Illustration 1: Hypothetical Phase Regions of Microemulsion Systems

From above figure, it can see that with high oil concentration surfactant forms reverse micelles

capable of solubilizing water molecules in their hydrophilic interior. Continued addition of water

in this system may result in the formation of w/o microemulsion in which water exists as droplets

surrounded and stabilized by interfacial layer of the surfactant / co-surfactant mixture. At a

limiting water content, the isotropic clear region changes to a turbid, birefringent one. Upon

further dilution with water, a liquid crystalline region may be formed in which the water is

sandwiched between surfactant double layers. Finally, as amount of water increases, this lamellar

structure will break down and water will form a continuous phase containing droplets of oil

stabilized by a surfactant / co-surfactant (o/w microemulsions).

(c) Dynamic Behaviour of Microemulsions

Microemulsions are dynamic, self-organizing solutions in which aggregation/disintegration

processes operate simultaneously. In this process, dynamic exchange of matter between

dispersed phases occurs continuously, resulting in an overall equilibrium. The dynamic process

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comprises: (i) the exchange of water between bound and free state; (ii) the exchange of

counterions between ionic head groups of the surfactant and core water; (iii) the exchange of

cosurfactant between the interfacial film, the continuous phase and the dispersed phase (if

soluble in this phase); and (iv) the exchange of surfactant between the interfacial film and the

aqueous phase [26].

(d) Solubilisation of Drug Molecules in Microemulsions

The size and region of existence of a single-phase microemulsion domain within the phase

diagram are strongly influenced by the presence of salts in the aqueous phase, the nature of the

polar group and hydrocarbon group of the surfactant, the solvent and the temperature. In general,

increasing the ionic strength of the aqueous phase reduces electrostatic interactions among the

surfactant polar groups, which results in more rigid interfaces, lower aggregation numbers, lower

intermicellar attractions, and in most cases reduction of maximum solubilisation capacity for the

aqueous phase at a given set of conditions [27].

1.2.3 Advantages of Microemulsion Based Systems

Microemulsions exhibit several advantages as a drug delivery system:

(a) Microemulsions are thermodynamically stable system and the stability allows self-

emulsification of the system whose properties are not dependent on the process followed.

(b) Microemulsions act as super solvents of drug. They can solubilize hydrophilic and

lipophilic drugs including drugs that are relatively insoluble in both aqueous and

hydrophobic solvents. This is due to existence of micro domains of different polarity

within the same single-phase solution.

xxv

(c) The dispersed phase, lipophilic or hydrophilic (oil-in-water, o/w, or water-in-oil, w/o

microemulsions) can behave as a potential reservoir of lipophilic or hydrophilic drugs,

respectively. The drug partitions between dispersed and continuous phase, and when the

system comes into contact with a semi-permeable membrane, the drug can be transported

through the barrier. Drug release with pseudo-zero-order kinetics can be obtained,

depending on the volume of the dispersed phase, the partition of the drug and the

transport rate of the drug.

(d) The mean diameter of droplets in microemulsions is below 0.22 mm; they can be

sterilized by filtration. The small size of droplet in microemulsions e.g. below 100 nm,

yields very large interfacial area, from which the drug can quickly be released into

external phase when absorption (in vitro or in vivo) takes place, maintaining the

concentration in the external phase close to initial levels.

(e) Some microemulsions can carry both lipophilic and hydrophilic drugs.

(f) Because of thermodynamic stability, microemulsions are easy to prepare and require no

significant energy contribution during preparation. Microemulsions have low viscosity

compared to other emulsions.

(g) The use of microemulsion as delivery systems can improve the efficacy of a drug,

allowing the total dose to be reduced and thus minimizing side effects.

(h) The formation of microemulsion is reversible. They may become unstable at low or high

temperature but when the temperature returns to the stability range, the microemulsion

reforms.

xxvi

1.2.4 Disadvantages of Microemulsion Based Systems

(a) Use of a large concentration of surfactant and co-surfactant necessary for stabilizing the

nanodroplets.

(b) Limited solubilizing capacity for high-melting substances

(c) The surfactant must be nontoxic for using pharmaceutical applications

(d) Microemulsion stability is influenced by environmental parameters such as temperature and

pH. These parameters change upon microemulsion delivery to patients.

1.3 Mechanisms of Skin Penetration Enhancement

An ideal penetration enhancer will disrupt the barrier function of the skin without compromising

its barrier effects on microorganisms and toxins and without damaging cells. There are three

primary mechanisms of penetration enhancement [28].

(a) Disruption of the highly ordered structure of stratum corneum lipid.

Many effective chemical enhancers disrupt the highly ordered bilayer structures of the

intracellular lipids found in stratum corneum by inserting amphiphilic molecules into these

bilayers to disorganize molecular packing or by extracting lipids using solvents and surfactants

to create lipid packing defects of nanometre dimensions. Hundreds of different chemical

enhancers have been studied, including off-the-shelf compounds and others specifically designed

and synthesized for this purpose, such as Azone (1-dodecylazacycloheptan-2-one) and SEPA (2-

n-nonyl-1,3dioxolane).

(b) Interaction with intercellular protein.

xxvii

The key to altering the polar pathway is to cause protein conformational change or solvent

swelling. The fatty acid enhancers increased the fluidity of the lipid protein portion of the

stratum corneum. Some enhancers act on both polar and non polar pathway by altering the multi

laminate pathway for penetration [29]. Enhancers can increase the drug diffusivity through skin

proteins. The type of enhancer employed has a significant impact on the design and development

of the product.

(c) Improved partition of the drug, co-enhancer or solvent into the stratum corneum.

Chemical enhancers can increase skin permeability and provide an added driving force for

transport by increasing drug partitioning into the skin (thereby increasing the concentration

gradient driving diffusion), but the difficulty of localizing their effects to the stratum corneum so

as to avoid irritation or toxicity to living cells in the deeper skin has severely constrained their

application.

1.4 Skin as a Permeation Barrier

Drug diffusion from transdermal delivery systems to the blood can be considered as passage

through a series of diffusion barriers. The drug has to pass first from the delivery system through

the stratum corneum, the epidermis and the dermis, each of which has different barrier

properties. Differences in composition of these layers cause them to display different

permeability to drugs, depending on molecular properties such as diffusion coefficient,

hydrophobicity and solubility.

The human skin consists of three anatomical layers- the epidermis (Non-viable epidermis and

viable epidermis), which is a thin, dry and tough outer layer; the dermis, which is the support

xxviii

system containing blood vessels, nerves, hair follicles, sebum, sweat glands; the subcutaneous fat

layer which acts both as an insulator and depot of calories.

1.4.1 The Structure of the Skin

(a) Non-viable Epidermis (Stratum Corneum)

Stratum corneum is the outer most layer of skin, which is the actual physical barrier to most

substance that comes in contact with the skin. The stratum corneum is 10 to 20 cell layer thick

over most of the body. Each cell is a flat, plate-like structure - 34-44 μm long, 25- 36 μm wide,

0.5 to 0.20 μm thick - with a surface area of 750 to 1200 μm2 stocked up to each other in brick

like fashion. Stratum corneum consists of lipid (5-15%) including phospholipids, glycosphingo

lipid, cholesterol sulphate and neutral lipid, protein (75-85%) which is mainly keratin. The most

superficial layer of the epidermis is the stratum corneum. They are formed and continuously

replaced by the basal layer of the stratum germinativum. The water content of the normal stratum

corneum is 15-20 % of its dry weight, but when it becomes hydrated it can contain up to 75 %

water. The epidermis forms a barrier to water, electrolyte and nutrient loss from the body, and at

the same time is also responsible for limiting the penetration of water and foreign substances

from the environment into the body. The skin contains least moisture at its surface, 10-25 %,

with a pH of 4.2-5.6. The lower epidermal layers contain up to 70 % water and the pH gradually

increases to 7.1-7.3. The isoelectric point of keratin is 3.7 - 4.5 and hence materials applied to the

skin should have a pH greater than this value.

(b) Viable Epidermis (Stratum Germinativum)

This layer of the skin resides between the stratum corneum and the dermis and has a thickness

ranging from 50- 100 μm. The structures of the cells in the viable epidermis are

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physiochemically similar to other living tissues. Cells are held together by tonofibrils. The

density of this region is not much different than water. The water content is about 90%.

(c) Dermis

Just beneath the viable epidermis is the dermis. It is a structural fibrin and very few cells are like

it can be found histologically in normal tissue. Dermis thickness range from 2000 to 3000 μm

and consists of a matrix of loose connective tissue composed of fibrous protein embedded in an

amphorphose ground substance.

(d) Subcutaneous Connective Tissue

The subcutaneous tissue or hypodermis is not actually considered a true part of the structured

connective tissue is composed of loose textured, white, fibrous connective tissue containing

blood and lymph vessels, secretory pores of the sweat gland and cutaneous nerves. Most

investigators consider drug permeating through the skin enter the circulatory system before

reaching the hypodermis, although the fatty tissue could serve as a depot of the drug.

Pathways of transdermal permeation occur by diffusion via: (a) Transdermal permeation, through

the stratum corneum. (b) Intercellular permeation through the stratum corneum and

transappendaged permeation via the hair follicle, sebaceous and sweat glands. Most molecules

penetrate through skin via intercellular micro-route and therefore many enhancing techniques

aim to disrupt or bypass its elegant molecular architecture.

1.5 Gabapentin

1.5.1 Physicochemical Properties of Gabapentin

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Gabapentin is a Class I drug according to the Biopharmaceutical Classification System (BCS)

crystalline white solid with the following properties:

Chemical Structure:

Chemical names 1-(Aminomethyl)-cyclohexaneacetic acid or 2-[1-(Aminomethyl) cyclohexyl]

acetic acid, Molecular formula C9H17NO2, Molecular weight 171.237, Plasma half life 5 to 7

hours, Melting point 162 to 166 °C, Solubility Freely soluble in water (4490 mg/l), alcohols,

Partition Coefficient 1.400 [30] and 1.25 [31] (Octanol-water), Serum concentration: less than

2 µg/litre, pKa value 3.68 for the carboxylic group and 10.70 for amine group.

1.5.2 Pharmacology of Gabapentin

Gabapentin is a cyclohexylacetic acid derivative, similar in structure to the neurotransmitter;

gamma aminobutyric acid, GABA but it is not believed to act on the same brain receptors [32]. It

is an anticonvulsant with unknown mechanism of action; crosses the blood brain barrier (BBB),

increases GABA concentration in the brain and reduces excitatory amino acids

neurotransmission, perhaps through its effects on voltage-gated calcium channels. It also exhibits

antinociceptive, anxiolytic, neuroprotective and antiepileptic effects [30].

xxxi

(a) Pharmacodynamics of Gabapentin

Gabapentin is an analogue of GABA used as an anticonvulsant to treat partial seizures,

amyotrophic lateral sclerosis (ALS) and painful neuropathies. The potential uses include

monotherapy of refractory partial seizure disorders, and treatment of spasticity in multiple

sclerosis, tremor, mood disorders and attenuation of disruptive behaviours in dermentia.

Gabapentin has low lipid solubility; is not metabolized by the liver; has no protein binding

capacity, and does not possess the usual drug-drug interactions [33].

(b) Mechanism of Action of Gabapentin

Gabapentin interacts with cortical neurons at auxillary subunits of voltage-sensitive calcium

channels. Gabapentin increases the synaptic concentration of GABA, enhances GABA responses

at non-synaptic sites in neuronal tissues, and reduces the release of monoamine

neurotransmitters. One of the mechanisms implicated in these effects of gabapentin is the

reduction of the axon excitability measured as an amplitude change of the presynaptic fibre

volley (FV) in the CA1 area of the hippocampo. This is mediated through its binding to

presynaptic N-methyl-D-aspartate (NMDA) receptors. Other studies have shown have shown

that the antihyperalgesic and antiallodymic effects of gabapentin are mediated by the descending

noradrenergic system, resulting in the activation of spinal α2-adrenergic receptors. Gabapentin

has also been shown to bind and activate the adenosine A1 receptors.

(c) Side effects and Toxicology of Gabapentin

Symptoms of overdose include ataxia, laboured breathing, ptosis, sedation, hypoactivity, and

excitation.

xxxii

(d) Indications of Gabapentin

Gabapentin is primarily used for the management of postherpetic neuralgia in adults and as

adjunctive therapy in the treatment of partial seizures with or without secondary generalization in

patients over 12 years of age with epilepsy. It is an anticonvulsant medication indicated in the

treatment of bipolar disorder and may be effective in reducing pain and spasticity in multiple

sclerosis. It has been used in treatment of anxiety disorders such as social anxiety disorder and

obsessive-compulsive disorder. Gabapentin is used in controlling the pain of trigeminal

neuralgia, post herpetic neuralgia, the pain of diabetic neuropathy and other neuritic pains such

as pain from nerve irritation due to spinal arthritis, cardiac disease and occipital neuralgia [33].

Gabapentin suppresses nausea and vomiting after laparoscopic cholecystectomy. It has seven key

indications summarized as analgesic, antianxiety, anticonvulsant, antimanic, antiparkinsonism,

calcium channel blocker and excitatory amino acid antagonist [30].

1.5.3 Pharmacokinetics of Gabapentin

(a) Absorption of Gabapentin

The absorption after oral administration is rapid. It is absorbed in part by the L-amino acid

transport system, which is a carrier-mediated, saturable transport system; as the dose increases,

the bioavailability decreases. The bioavailability ranges from approximately 60 % for a 900 mg

dose per day to approximately 27 % for a 4800 mg dose per day. Food has a slight effect on the

rate and extent of absorption of gabapentin (14 % increases) in AUC) [33 ].

(b) Metabolism of Gabapentin

xxxiii

All pharmacological actions following gabapentin administration are due to the activity of the

parent compound. Gabapentin is not appreciably metabolised in human beings. The volume of

distribution is 58 ± 6 litres. Less than 3 % of gabapentin circulates bound to plasma protein.

(c) Route of Elimination

Gabapentin is eliminated from the systemic circulation by renal excretion as unchanged drug.

Gabapentin is not appreciably metabolised in humans. The clearance is 190 ml/min.

1.6 Glipizide

1.6.1 Physicochemical Properties of Glipizide

According to the biopharmaceutical classification system, glipizide is a BCS class II drug. It is a

white solid crystalline compound with the following properties:

Chemical Structure:

Chemical Names 1-Cyclohexyl-3-[{p-(2-(5-methylpyrazine-

carboxamido)ethyl)phenyl}sulphonyl]urea or N-[2-

{4({[cyclohexylcarbamoyl]amino}sulphonyl)phenyl}ethyl]-5-methylpyrazine-2-carboxamide or

N-[4-{N-(cyclohexylcarbamoyl)sulfamoyl}phenethyl]-5-methylpyrazine-2-carboxamide,

Molecular formula C21H27N5O4S, Molecular weight 445.535 g per mole, Plasma half life 2 to

xxxiv

5 hours, Melting point 208 to 209 °C, Solubility profile Freely soluble in Dimethyl formamide,

insoluble in water, soluble in alcohols and 0.1 N sodium hydroxide. Water solubility is 37.2 mg/l

[34], Partition Coefficient: 2.5 (octanol-water), Serum concentration: less than 5 µg/litre, pKa

value 5.9

1.6.2 Pharmacology of Glipizide

Glipizide is an oral long- acting antidiabetic drug from the sulphonylurea class. It is classified as

a second generation sulphonylurea (It undergoes enterohepatic circulation). It is a second

generation sulphonylurea used with diet to lower blood glucose in patients with diabetes mellitus

type-2. The primary mode of action in experimental animals appears to be the stimulation of

insulin secretion from the beta cells of pancreatic islet tissue and is thus dependent on

functioning beta cells in the pancreatic islets. In human, glipizide appears to lower blood glucose

acutely by stimulating the release of insulin from the pancrease, an effect dependent upon

functioning beta cells in the pancreatic islets. In man, stimulation of insulin secretion by glipizide

in response to the meal is undoubtedly very essential. Fasting insulin levels are not elevated even

on long term glipizide administration, but the postprandial insulin response continues to be

enhanced after at least 6 months of treatment. Some patients fail to respond initially, or gradually

lose their responsiveness to glipizide including other sulphonylurea drugs [35].

(a) Mechanism of action of Glipizide

Insulin is a hormone that is made in the pancreas, that when released into the blood causes cells

in the body to remove sugar (glucose) from the blood and reduces the formation of glucose by

the liver. Patients with Type-2 diabetes have higher glucose levels in their blood because the

cells in their bodies are resistant to the effect of the insulin, and the liver produces too much

xxxv

glucose. In addition, in Type-2 diabetes the pancreas is unable to produce the increased amount

of insulin that is necessary to overcome the resistance. Glipizide reduces blood glucose by

stimulating the pancreas to produce more insulin [35]. Glipizide, however, is not a cure for

diabetes. Another possible mechanism of action is the blocking of potassium channels in the beta

cells of the islets of Langerhans. By partially blocking the potassium channels, it will increase

the time the cells spend in the calcium release stage of cell signalling, leading to an increase in

calcium. The latter effect will initiate more insulin release from each beta cell.

(b) Side Effects and Toxicology of Glipizide

The acute oral toxicity was extremely low in all species tested LD50>4 g/kg. Over dosage of

sulphonylureas including glipizide can produce hypoglycaemia.

(d) Indications of Glipizide

Glipizide is used as an adjunct to diet for the control of hyperglycemia and its associated

symptomatology in patients with Non Insulin Dependent Diabetes Mellitus (NIDDM), type-2,

formally known as maturity-onset diabetes, after an adequate trial of dietary therapy has proved

unsatisfactory. The larger cyclo- or aromatic group (R2 position) on the chemical structure when

compared to the first generation sulphonylureas provides once a day dosing regimen [36].

1.6.3 Pharmacokinetics of Glipizide

(a) Absorption of Glipizide

The gastrointestinal absorption of glipizide is uniform, rapid and complete [36]. The

bioavailability after oral administration is almost 100 % for regular formulation and 90 % for

extended release formulation. It is highly protein bound- 98 to 99 %, primarily to albumin.

xxxvi

(b) Metabolism of Glipizide

Metabolism is through hepatic hydroxylation. The major metabolites of glipizide are products of

aromatic hydroxylation and have no hypoglycaemic activity. A major metabolite (an

acetylaminoethylbenzine derivative) which accounts for less than 2 % of a dose is reported to

have one-tenth to one third as much hypoglycaemic activity as the parent compound [36].

Metabolism by cytochrome P450 2C9 isoenzymes forms 3-cis-hydroxyglipizide and 4-cis-

hydroxyglipizide [37].

(c) Route of Elimination of Glipizide

The primary metabolites are inactive hydroxylation products and polar conjugates and are

excreted mainly in urine. Renal and faecal excretion can also occur [36]. The volume of

distribution of glipizide is 11 litres.

1.7 Objectives of the Study

The physicochemical properties of the drug candidates reveal the feasibility of transdermal

delivery. Glipizide, a non-ionic biopharmaceutical classification scheme (BCS) class II lipophilic

agent, has molecular weight of 445.5 Daltons, favourable partition coefficient of 2.5, melting

point of 208.5 °C, short half-life of 2-5 hours and low serum concentration of less than 5 µg/ml

while gabapentin, a non-ionic BCS class I hydrophilic agent, has molecular weight of 171.237

Daltons, favourable partition coefficient of 1.40, melting point of 162-167 °C, short half-life of

5-7 hours and low serum concentration of less the 2 µg/ml. The study, therefore, is aimed at:

(a) Investigating their skin permeation behaviours in some solvent systems (cosolvents and

microemulsions).

xxxvii

(b) Characterizing the optimized microemulsions.

(c) Investigating the mechanism of permeation enhancement of the optimized

microemulsions by using DSC analysis and FTIR spectroscopy.

(d) Formulating dosage forms of low drug strengths since their peak plasma concentrations

obtainable with current dosage forms are very low, and

A survey of the literature showed no such transdermal studies on gabapentin and glipizide.

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

(a) Equipment/Apparatus

The following equipment were used for the study: UV/VIS spectrophotometer (Jeenway 6405,

England), Fourier Transform Infrared Spectrophotometer (Shimadzu 8400S, Japan), Differential

Scanning Calorimeter (NETZSCH 204 F1 Kent 7020, Germany), pH meter (Eutech, Japan),

Electronic weighing balance (Sauter, England), Du Nuoy tensiometer (Sr-elrit, China), Cone and

xxxviii

plate viscometer (UTV Gallenkamp, England), Magnetic stirrer with hot plate (Remi PVT India),

Modified Franz diffusion cell (Nigeria), Photomicrograph (Moticam, Japan), Albino rats

(Department of Veterinary Medicine, University of Nigeria, Nsukka).

(b) Reagents and Chemicals

Gabapentin (Pfizer Ltd, China), Glipizide (Generics Ltd, UK), Distilled water (Lion water,

Nigeria), Ethanol, Sodium chloride, Potassium chloride, Disodium hydrogen phosphate

dihydrate, Potassium dihydrogen phosphate, Sodium azide, Propylene glycol, Sodium hydroxide

(Sigma-Aldrich, USA), Cremophor RH 40 (BASF, Germany), Coconut oil (Nigeria), Trypsin,

(Sigma-Aldrich, USA).

All other chemicals and reagents were of analytical grade and were used without further

purification.

2.2 Methods

2.2.1 Preparation of 0.9 % (w/v) Phosphate Buffered Saline

The composition of Phosphate Buffered Saline is as follows:

Sodium chloride ---------------------------------- 8.00 g

Potassium chloride ------------------------------- 0.20 g

Disodium hydrogen phosphate dihydrate --- 1.44 g

Potassium dihydrogen phosphate ------------- 0.24 g

Distilled water to -------------------------------- 1000 ml

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The solution was prepared as described [38] by weighing the required quantities of ingredients,

and dissolving in 800 ml of distilled water. The solution was adjusted to a pH of 7.4 using 0.1 N

hydrochloric acid and was made up to 1000 ml with distilled water.

2.2.2 Preparation of Standard Solutions

2.2.2.1 Preparation of Standard Solution of Glipizide

A 10 mg of pure sample of glipizide was weighed using a sensitive electronic weighing balance

and dissolved in 50 ml of ethanol. 1 ml of the stock solution was diluted to 10 ml to prepare a 20

µg/ml solution. From this solution, seven different strengths of glipizide were prepared by serial

dilution method to obtain 2, 4, 8, 10, 12, 16 and 20 µg/ml solutions. The solutions were analysed

spectrophotometrically at wavelength of 276 nm, using ethanol as blank. The preparations and

measurements were carried out in triplicate.

2.2.2.2 Preparation of Standard Solution of Gabapentin

A 10 mg of pure sample of gabapentin was weighed using a sensitive weighing balance and

dissolved in 50 ml of 0.9 % (w/v) phosphate buffered saline. The solution was diluted to prepare

seven different concentrations of 5, 10, 20, 25, 50 and 100 µg/ml solutions. The solutions were

analysed spectrophotometrically at wavelength of 210 nm, using 0.9 % (w/v) phosphate buffered

saline as blank. The preparations and measurements were carried out in triplicate.

2.2.3 Extraction of Coconut Oil

100 g of pulverized fresh coconut seed was macerated in 2500 ml of n-heaxane with intermittent

shaking for 7 days. The mixture was filtered using muslin cloth and the oil recovered by

evaporation in a rotary evaporator.

xl

2.2.4 Physical Characterization of Coconut Oil

The pH of coconut oil was determined using pH meter (Eutech, India) at room temperature. The

pH meter was calibrated before use with buffered solution at pH 7.0. The pH of the oil was

determined in triplicate [39] at weekly interval for five weeks. The odour, colour, taste and

clarity were visually assessed. The filtration test was determined by filtering the oil through a

Whattman filter paper No. 42.

2.2.5 Quantitative Characterization of Coconut Oil

2.2.5.1 Determination of Saponification Value

Approximately 2.2 ml (2 g) was measured into a conical flask. 25 ml of alcoholic potassium

hydroxide was added to the coconut oil and attached to a condenser on a water bath. The set up

was boiled for 2 hours and the solution allowed to cool. Three drops of phenolphthalein indicator

was added, and titrated against 0.5 N hydrochloric acid until pink colour was observed. The

blank experiment was also performed.

2.2.5.2 Determination of iodine Value

Approximately 2.2 ml (2 g) of coconut oil was measured into a conical flask containing 10 ml of

chloroform, warmed and allowed to cool for 10 minutes. 25 ml of Wij’s solution (solution of 7.5

g of iodine tetrachloride and 8.5 g of resublimed iodine in glacial acetic acid) was added, mixture

shaken vigorously and allowed to stand for 30 minutes in the dark. 10 ml of 15 % w/v potassium

iodide solution was added and solution titrated against 0.1 N sodium thiosulphate until

xli

appearance of yellow colour. Thereafter, 1 ml of 1 % v/v starch indicator was added and solution

titrated against 0.1 N sodium thiosulphate solutions until disappearance of blue colour. The blank

titration was conducted following the same procedure without the coconut oil.

2.2.5.3 Determination of Acid Value

Approximately 2.2 ml (2 g) of coconut oil was measured into a conical flask containing 10 ml of

ethanol. Three drops of phenolphthalein indicator was added and the mixture was titrated with

0.1 N potassium hydroxide until pink colour was observed. The blank titration was also

performed.

2.2.5.4 Viscosity Measurement of Coconut Oil

The viscosity measurement was performed using Universal Torsion viscometer. The cup of the

viscometer was properly cleaned and the sample placed in it. The angle of rotation of the

viscometer spindle was noted and compared with viscosities on the instrument chart. All

determinations were carried out in triplicates.

2.2.6 Determination of the solubility of Drugs in Coconut Oil

2.2.6.1 Solubility of Glipizide in Coconut Oil.

Excess (50 mg) of glipizide was dispersed in 10 ml of coconut oil and was shaken on magnetic

stirrer continuously at room temperature for 24 hours. Upon saturation of the oil, 100 ml of

ethanol was added to the mixture to extract the undissolved drug. The mixture was shaken for 30

minutes and allowed to stand undisturbed until separation into two distinct layers and

subsequently separated in a separating funnel. The ethanol layer was diluted appropriately and

xlii

assayed for glipizide spectrophotometrically at 276 nm using ethanol-saturated with the oil as

blank. The solubility was determined by difference.

2.2.6.2 Solubility of Gabapentin in Coconut Oil

Excess (50 mg) of gabapentin was dispersed in 10 ml of coconut oil and was shaken on magnetic

stirrer continuously at room temperature for 24 hours. Upon saturation of the oil, 100 ml of

phosphate buffered saline solution was added to the mixture to extract the undissolved drug. The

mixture was shaken for 30 minutes and allowed to stand undisturbed until separation into two

distinct layers and subsequently separated in a separating funnel. The phosphate buffered saline

layer was diluted appropriately and assayed for gabapentin spectrophotometrically at 210 nm

using phosphate buffered saline-saturated with the oil as blank. The solubility was determined by

difference.

2.2.7 Construction of Pseudo Ternary Phase Diagram

The pseudo ternary phase diagrams were constructed using the water titration method [39].

Coconut oil was used as the oil phase, Cremophor® RH 40 as the surfactant and ethanol as the

co-surfactant. Phase diagrams were constructed with 9:1 to 1:9 v/v ratio of oil to surfactant and

various ratios of surfactant/co-surfactant (1:1, 1:2, 1:3, 2:1, and 3:1 v/v). For each phase diagram

at specific surfactant/co-surfactant, mixtures of the oil, the surfactant and the co-surfactant were

prepared, and the mixture was diluted with distilled water by titration. Distilled water was added

drop by drop while mixing on a magnetic stirrer at room temperature, and the samples were

marked as being optically clear or turbid. The microemulsion regions were identified as isotropic

mixtures. The percentages of three different phases- oil, water and the mixture of surfactant and

xliii

co-surfactant were calculated and used to construct ternary phase diagram using Sigma Plot®

Exact Graph and data Analysis Software.

2.2.8 Preformulation Stability Studies of the Microemulsions

Ten microemulsions (MCEa-MCEj), two each from the various ratios of surfactant/co-surfactant

were selected for stability studies. The chosen microemulsions were allowed to stand on the

laboratory shelf for 21 days, and observed visually for phase separation and/or creaming at day

1, day 2, day 3, day 7, day 14 and day 21. Microemulsions that remained stable beyond 14 days

were selected for further studies.

2.2.9 Drug Loading of the Microemulsions

2.2.9.1 Preparation of Microemulsions Loaded with Glipizide

Appropriate quantities of surfactant (Cremophor® RH 40), co-surfactant (ethanol), and oil

(coconut oil), were measured into a screw-capped plastic vial. Glipizide was dissolved in a

concentration of 0.25, 0.50, 0.75 and 1.0 % w/v in the coconut oil being used and the mixture of

surfactant and co-surfactant was added. The mixture was stirred with a magnetic bar on a

magnetic stirrer, at room temperature with continuous addition of measured amount of distilled

water, until the formation of a microemulsion system. The procedures were carried out for the

microemulsions (MCEa-MCEEf) that showed thermodynamic stability beyond 14 days and

formed from the various ratios of surfactant/co-surfactant.

2.2.9.2 Preparation of Microemulsions Loaded with Gabapentin

Appropriate quantities of surfactant (cremophor® RH 40), co-surfactant (ethanol), and oil

(coconut oil), were measured into a screw-capped plastic vial. Gabapentin was dissolved at a

xliv

concentration of 0.25, 0.50, 0.75 and 1.0 % w/v in the distilled water being used. The mixture of

surfactant/co-surfactant was added to the coconut oil and the mixture was stirred with a magnetic

bar on a magnetic stirrer, at room temperature with continuous addition of measured amount of

drug solution, until the formation of a microemulsion system. The procedures were carried out

for the microemulsions (MCEa-MCEf) that showed thermodynamic stability beyond 14 days and

formed from the various ratios of surfactant/co-surfactant.

2.2.10 Post Formulation Stability Studies of Drug-loaded Microemulsions

The different strengths of drug-loaded microemulsions were allowed to stand on the laboratory

shelf for 21 days, and observed visually for drug precipitation, phase separation and/or creaming

at day 1, day 2, day 3, day 7, day 14 and day 21. The drug-loaded microemulsions that remained

stable beyond 14 days were selected for further studies.

2.2.11 Preparation of Rat Abdominal Skin

The animals used for the preparation of skin were male albino rats (150-200 g) obtained from the

Animal House of the Department of Veterinary Medicine, University of Nigeria, Nsukka. They

were permitted free access to food and water until used for the study. All experiments and

protocols described in this study were approved by the institutional animal ethics committee. The

rats were euthaniased using chloroform asphyxiation. Dorsal hair was removed with a razor

blade and full thickness skin was surgically removed from each rat. The epidermis was prepared

by heat separation technique. The entire abdominal skin was soaked in water at 60 °C for 60

seconds, followed by careful removal of the epidermis. The epidermis was washed and stored in

a refrigerator until used in the in vitro permeability studies.

xlv

2.2.12 In-vitro Skin Permeation Studies

Modified Franz diffusion cells were used in the in-vitro permeation studies. The epidermis

prepared above was soaked in distilled water for 12 hours prior to use for the permeation studies

and was mounted in between the compartments of the diffusion cells with the stratum corneum

facing the donor compartment. The effective diffusional area was 2.54 cm2. The volume of the

receiver compartment was 25 ml. The various vehicles used for each of the drugs studied were as

follows:

Cosolvent systems: Glipizide and gabapentin were separately dispersed in the 10, 20 and 30 %

v/v of ethanol and propylene glycol cosolvent systems each at a concentration of 5 mg/ml. 1 ml

of the resulting drug solution or suspension was added to the donor compartment of the Franz

diffusion cells.

Microemulsions: 1 ml of each of the drug-loaded microemulsions (equivalent to 5 mg/ml) was

added to the donor compartment of the Franz diffusion cells.

Ethanol and water in the ratio of 70:30 v/v was added to the receiver compartment in order to

maintain sink conditions. The cells were maintained at 37±0.5 °C by a magnetic stirrer with

heater. The contents in the receiver compartment were stirred with a magnetic bar at 500 rpm. At

predetermined times (1, 2, 4, 8, 12 and 24 hours), 1 ml samples were withdrawn from the

receiver compartment and replaced with an equivalent quantity of drug-free solvent (70:30 v/v

ethanol-water) pre-warmed to the working temperature to maintain a constant volume. The

samples were assayed spectrophotometrically for glipizide and gabapentin at 276 nm and 210 nm

respectively against their appropriate blanks. All the procedures were carried out in triplicates.

xlvi

2.2.13 Characterization of the Optimized Microemulsions

Two drug-loaded optimized microemulsions that produced the best permeation parameters (drug

flux, permeation coefficient and enhancement ratio), one for each of the drugs, were selected for

further characterization.

2.2.13.1 Dilution Test of the Microemulsions

The type of microemulsion was determined by dilution test. Small amount of microemulsion was

placed on a clean glass slide. A drop of water was added to the microemulsion and was mixed

with the help of glass rod and their transparency was assessed visually [40].

2.2.13.2 Determination of pH of the Microemulsions

The pH of the microemulsions was determined using pH meter (Eutech, India) at room

temperature. The pH meter was calibrated before each use with buffered solution at pH 7.0 and

the pH of each microemulsion preparation was determined in triplicate [39].

2.2.13.3 Viscosity Measurement of Microemulsions

The viscosity measurements were performed using Universal Torsion viscometer. The cup of the

viscometer was properly cleaned and the sample placed in it. The angle of rotation of the

viscometer spindle was noted and compared with viscosities on the instrument chart. This was

repeated with each of the microemulsions. All determinations were carried out in triplicates.

2.2.13.4 Determination of Globule Size and Polydispersity Index

xlvii

The globule sizes of each of the microemulsions selected were determined using a

photomicrograph. A drop of the microemulsion was fixed on a photomicrograph slide and

viewed through the objective lens. The in-built calibration was then used to measure the particle

sizes of the globules at random. Polydispersity index was determined as the ratio of standard

deviation to mean droplet size. All determinations were carried out in triplicates.

2.2.13.5 Skin Irritation Studies

The skin irritation studies for microemulsions were carried out on male albino rats weighing 150-

180 grams according to the modified Draize method [41]. The animals were kept under standard

laboratory conditions and housed in plastic cages and acclamatized before the beginning of the

study. Animals were divided into four groups (n=3) as follows:

Group 1: No treatment (Negative control)

Group 2: Treatment with formalin (standard irritant)

Group 3: MCEa- microemulsion

Group 4: MCEd – microemulsion

Group 1 was taken as negative control and group 2 served as positive control. Group 3 and 4

were applied with the microemulsion formulations. The first group served as negative control (no

treatment), the second group received 0.5 ml of 0.8% (v/v) aqueous formalin solution as a

standard irritant, and the third and fourth groups received the optimized formulae. A dose of 0.5

ml of optimized formula or 0.5 mL of formalin solution was applied on a 5 cm2 area of the

shaved dorsal side of the rats daily for three consecutive days (42). The development of erythema

and edema were monitored daily for 3 days. Subjective scores of 0 to 4 were assigned to

xlviii

represent: no evidence of irritation, minimum erythema, visible erythema, definite and readily

visible erythema respectively depending on the level of irritation.

2.2.14 Preparation of Stratum Corneum for FTIR Spectroscopy and DSC Studies

The rat epidermis was incubated for 4 hours in a 1 % trypsin solution in phosphate buffered

saline (pH 7.4) at 37 °C. The tissue was then smoothed out a flat surface and the mushy

epidermis was removed by rubbing with a moistened-cotton-tipped applicator. The transparent

stratum corneum obtained was briefly bloated on water, blotted dry and used in the DSC and

FTIR studies.

2.2.15 FTIR Spectroscopic Studies on Stratum Corneum

Stratum corneum was cut into small circular discs. 0.9 % w/v solution of sodium chloride was

prepared and 0.01 % w/v sodium azide was added as antibacterial and antimycotic agent. 35 ml

of 0.9 % w/v of sodium chloride was placed in different conical flasks and stratum corneum of

approximately 1.5 cm diameter was floated over it for 3 days for hydration, these discs were

thoroughly blotted over filter paper, and one of the discs kept serving as control. The other discs

were dipped into the microemulsion formulations (MCEa and MCEd) separately, 30 % v/v

ethanol and 30 % v/v propylene glycol and were kept for a period of 24 hours (equivalent to the

skin permeation studies) at 37 °C. Each stratum corneum disc after treatment was washed blotted

dry and then air dried for 2 hours. All samples were kept under vacuum in desiccators for 15

minutes to remove traces of formulations and solvents completely. FTIR spectra of treated and

untreated stratum corneum discs were recorded. Attention was focused on characterizing the

occurrence of peaks near 2850 cm-1

and 2920 cm-1

which were due to –CH2 symmetric and

xlix

asymmetric stretching vibrations respectively and 1550 to 1650 cm-1

due to amide bond

stretching vibration.

2.2.16 DSC Studies on stratum Corneum

Approximately 15 mg of freshly prepared stratum corneum was taken and hydrated over

saturated potassium sulphate solution for 3 days. Then the stratum corneum was blotted to get

hydration between 20-25 %. Hydrated stratum corneum sample was cut into 3 pieces. One was

kept to serve as control; two other pieces were dipped into each of the optimized microemulsion

formulation for 24 hours (equivalent to the permeation studies) at 37 °C. After treatments,

stratum corneum was removed and blotted to attain hydration of 20-25 %, sealed in aluminium

hermatic pans and equilibrated for 1 hour before the DSC run. The stratum corneum samples

were scanned on a DSC at the rate of 5 °C/minutes over temperature range of 0-350 °C.

2.3 Data and Statistical Analysis

1. All experiments were performed in triplicates (n=3) for validity of statistical analysis.

Results were presented as mean ± SEM. Students t-test was performed on data sets

generated using SPSS.

2. The pseudo ternary phase diagrams were plotted using Sigma Plot® 11 Exact graph and

data analysis software.

3. The concentration of gabapentin and glipizide in the sink solution were calculated from

the equation of the line of the Beer-Lambert’s plots. (Figures 1 and 2 respectively)

4. The cumulative drug permeated (Qn) corresponding to the time of the nth

sample was

calculated from the following equation: [43]

- - - - - - - - -

equation 1

l

Where Cn and Ci are the drug concentrations of the receptor solution at time of the nth sample

and the i (the first) sample, respectively and VR and Vs are the volumes of the receptor solution

and the sample, respectively.

5. The permeation profiles of the drugs across rat skin from different vehicles were

constructed by plotting the total cumulative amount of the drug permeated per unit

surface area (Q, µg/cm2; Area, 2.54 cm

2) verses time (hour) as shown in Figures 8 to 13

6. The steady state flux (Jss6-24 h µg/cm2.h) was calculated as the slope of linear regression

line at the steady state phase for each experimental run.

7. Permeability coefficient (Kp) was calculated using the relation derived from Fick’s first

law of diffusion, - - - - - - - -

-equation 2

Where Co is the initial drug concentration in the donor medium [44]

8. The quantitative estimation of the enhancing effects of the vehicles compared with the

control (distilled water) was calculated in terms of the enhancement ratio, ER given by

the following equation:

- - - - - - - - -

equation 3

9. The apparent diffusion coefficient of the drugs Dapp, partition coefficient, P and the lag

time, TL were calculated from the following equations:

- - - - - - - -

equation 4

li

- - - - - - - -

equation 5

10. The surface area of the patch expected from the permeation fluxes (at steady-state) at a

given plasma concentration and clearance of the drug was calculated from the input rate

(IR) with the following equations:

Input rate (IR) = plasma concentration x clearance [45] - - - - -

equation 6

Patch size = Input rate/Experimental flux. - - - - - - -

equation 7

Other data were processed using Microsoft Excel software.

CHAPTER THREE

RESULTS

3.1 Preparation of Standard Solution of Gabapentin and Glipizide

Figures 1 and 2 show the calibration plots of gabapentin and glipizide respectively. Gabapentin

obeyed Beer- Lambert’s law (ʎmax 210 nm) within the concentration range of 5-100 µg/ml while

glipizide obeyed the law (ʎmax 276 nm) within the concentration range of 2-20 µg/ml.

lii

Figure 1: Calibration Curve of Gabapentin in Phosphate Buffered Saline at ʎmax of 210

nm.

Figure 2: Calibration Curve of Glipizide in Ethanol at ʎmax of 276 nm.

3.2 Extraction and Physical Characterization of Coconut Oil

Without further treatment/purification, a colourless clear liquid that has characteristic coconut

odour and left no residue on filtration with Whattman filter paper No. 42 was obtained (Table 1).

liii

The pH measurements (Table 1) show neutral oil with value ranging from 7.6 after extraction to

7.2 on storage for 90 days.

3.3 Quantitative Characterization of Coconut Oil

The saponification value, acid value and iodine value obtained are shown in Table 1.

Table 1: Qualitative and Quantitative Characteristics of Coconut Oil

Parameter Properties*

Colour/clarity colourless, transparent liquid

Odour coconut odour

Filtration test leaves no residue on filtration

Saponification number* 256 ± 17

Acid value* 1.20 ± 0.10

Iodine value* 8.30 ± 0.42

Viscosity* 185.0 ± 16.5 poise

Surface tension* 88.4 ± 5.16 dyn/cm

pH* 7.33 ± 0.36

*n=3

3.4 Determination of Solubility of Drugs in Coconut Oil

Table 2 shows the results of solubility determinations of gabapentin and glipizide in coconut oil.

The solubility of glipizide in coconut oil is 48.90 mg/ml while that of gabapentin is 0.45 mg/ml

at room temperature.

Table 2: Solubility Profile of Gabapentin and Glipizide in Coconut Oil

Drugs *Solubility ± SEM (mg/ml)

Gabapentin 0.45 ± 0.11

liv

Glipizide 48.90 ± 0.86

*n=3

3.5 Pseudo Ternary Phase Diagram

Tables 3-7 show the percentage composition of distilled water, coconut oil and at different ratios

of cremophor®/ethanol while Figures 3- 7 shows the areas of microemulsion formation of

different systems used in the study consisting surfactant/co-surfactant at different ratios as

plotted with Sigma Plot-II software. All the ratios of surfactant/co-surfactant studied showed

good emulsification as shown by the large emulsification areas.

Compositions of the Different Microemulsions

Table 3: Microemulsions with Surfactant:Co-Surfactant ratio of 1:1

______________________________________________________________________________

Microemulsions %* Su:CoS % oil % Distilled water

1 07.70 69.20 23.10

2 15.70 62.70 21.6

3 25.00 58.30 16.70

4 34.80 52.20 13.00

5 45.50 45.50 09.00

6 55.80 37.20 07.00

7 62.20 26.70 11.10

8 61.50 15.40 23.10

9 64.30 07.10 28.60

* Surfactant to Cosurfactant ratio

Table 4: Microemulsions with Surfactant:Co-Surfactant ratio of 1:2

______________________________________________________________________________

Microemulsions % *Su:CoS % oil % Distilled water

lv

1 16.30 65.30 18.40

2 24.00 56.00 20.00

3 30.20 45.30 24.50

4 38.50 38.50 23.00

5 47.10 31.40 21.50

6 58.30 25.00 16.70

7 69.60 17.40 13.00

8 81.80 09.10 09.10

* Surfactant to Cosurfactant ratio

Table 5: Microemulsions with Surfactant:Co-Surfactant ratio of 2:1

______________________________________________________________________________

___

Microemulsions % *Su:CoS % oil % Distilled water

1 17.00 68.10 14.90

2 25.00 58.30 16.70

3 34.80 52.20 13.00

4 45.50 45.50 09.00

5 55.80 37.20 07.00

6 65.10 27.90 07.00

7 76.20 19.00 04.80

8 87.80 09.80 02.40

Table 6: Microemulsions with Surfactant:Co-Surfactant ratio of 1:3

______________________________________________________________________________

Microemulsions % *Su:CoS % oil % Distilled water

1 22.20 51.90 25.90

2 42.80 42.80 28.60

3 34.50 34.50 31.00

4 43.60 29.10 27.30

5 53.80 23.10 23.10

6 64.00 16.00 20.00

7 75.00 08.30 16.70

Table 7: Microemulsions with Surfactant:Co-Surfactant ratio of 3:1

Microemulsions %* Su:CoS % oil % Distilled water

1 23.10 53.80 23.10

2 30.20 45.30 24.5

3 41.70 41.70 16.60

4 48.00 32.00 20.00

5 53.80 23.10 23.10

7 66.60 16.70 16.70

8 73.40 08.20 18.40

* Surfactant to Cosurfactant ratio

lvi

Figure 3: Pseudo ternary Phase Diagram of Surfactant: Cosurfactant Ratio 1:1

Su:CoS (%)0 10 20 30 40 50 60 70 80 90 100

Oil (%)

0

10

20

30

40

50

60

70

80

90

100

Water (%)

0

10

20

30

40

50

60

70

80

90

100

Figure 4: Pseudo ternary Phase Diagram of Surfactant: Cosurfactant Ratio 1:2

Su:CoS (%)0 10 20 30 40 50 60 70 80 90 100

Oil (%)

0

10

20

30

40

50

60

70

80

90

100

Water (%)

0

10

20

30

40

50

60

70

80

90

100

Figure 5: Pseudo ternary Phase Diagram of Surfactant: Cosurfactant Ratio 2:1

Su:CoS (%)0 10 20 30 40 50 60 70 80 90 100

Oil (%)

0

10

20

30

40

50

60

70

80

90

100

Water (%)

0

10

20

30

40

50

60

70

80

90

100

lvii

Figure 6: Pseudo ternary Phase Diagram of Surfactant: Cosurfactant Ratio 1:3

Su:CoS (%)0 10 20 30 40 50 60 70 80 90 100

Oil (%)

0

10

20

30

40

50

60

70

80

90

100

Water (%)

0

10

20

30

40

50

60

70

80

90

100

Figure 7: Pseudo Ternary Phase Diagram of Surfactant: Cosurfactant Ratio 3:1

Su:CoS (%)0 10 20 30 40 50 60 70 80 90 100

Oil (%)

0

10

20

30

40

50

60

70

80

90

100

Water (%)

0

10

20

30

40

50

60

70

80

90

100

Table 8: Compositions of the Microemulsions Selected from the Regions of

Microemulsification

in the Pseudoternary Phase Diagrams.

______________________________________________________________________________

Microemulsions (*Su:CoS) %* Su:CoS % Oil % Distilled water Emulsion Type

MCEa (1:1) 20.0 60.0 20.0 w/o

MCEb (1:1) 20.0 20.0 60.0 o/w

MCEc (1:2) 20.0 60.0 20.0 w/o

MCEd (1:2) 20.0 20.0 60.0 o/w

MCEe (2:1) 20.0 60.0 20.0 w/o

MCEf (2:1) 20.0 20.0 60.0 o/w

MCEg (1:3) 20.0 60.0 20.0 w/o

MCEh (1:3) 20.0 20.0 60.0 o/w

MCEi (3:1) 20.0 60.0 20.0 w/o

MCEj (3:1) 20.0 20.0 60.0 o/w

* Surfactant to Cosurfactant ratio

3.6 Preformulation Stability Studies of Microemulsions

lviii

The choice of the ten microemulsions was based on the possibility of forming both o/w and w/o

microemulsion. The stability profiles of the microemulsions after 21 days of standing

undisturbed were also examined. Microemulsions formed from high concentration of either

surfactant or co-surfactant (3:1 and 1:3) did not show good stability on prolonged standing.

3.7 Post Formulation Stability Studies of Drug-Loaded Microemulsions

The six microemulsions that remained stable beyond 14 days of standing were loaded separately

with each of the drugs. Assessment of the stability did not show any sign of drug precipitation or

phase separation at concentrations of 0.25-0.50 % w/v. However, drug precipitation and/or phase

separation were observed for the 0.75 and 1.0 % w/v.

3.8 In-vitro Skin Permeation Studies of Vehicles and Microemulsions.

Tables 9 and 14 show the cumulative amount of each drug permeated per unit area of skin

membrane in distilled water (control), ethanol, propylene glycol and microemulsion formulation.

Tables 10 and 15 show the calculation of skin permeation parameters such as drug flux, lag time,

apparent diffusion coefficient, permeation coefficient, and skin/vehicle partition coefficient and

enhancement ratio for all the vehicles studied. Figures 8, 9, 11 and 12 show the plot of

cumulative amount of each drug permeated per unit skin area against time of permeation for all

cosolvent systems studied. Figures 10 and 13 show the same plot for the microemulsions.

Table 9: Results of the Cumulative Amount (microgram) of Gabapentin Permeated in

Cosolvents Systems and Microemulsions.

Time (h) 30 % Ethanol 20 % Ethanol 10 % Ethanol Distilled water

1.0 198.3589 138.0091 7.017414 6.783500

2.0 600.6906 296.6027 62.68890 15.20440

4.0 1055.887 520.4582 138.7109 36.95838

8.0 1627.104 775.6581 282.5679 68.53674

12.0 2341.945 1139.628 494.4938 109.4717

lix

24.0 3353.388 1618.683 880.9194 166.3127

Time (h) 30% Pr. Glycol 20% Pr. Glycol 10% Pr Glycol Distilled Water

1.0 4.91219 6.549586 8.186983 6.783500

2.0 14.26874 29.94097 49.35581 15.204

4.0 35.78881 83.27331 110.1734 36.958

8.0 97.77597 149.0031 188.3006 68.53674

12.0 247.013 374.9638 482.7981 109.47

24.0 544.3174 713.671 911.3281 166.3127

Time (h) MCEa MCEb MCEc MCEd MCEe MCEf Water

1.0 375.6656 13.33309 152.9796 11.9296 133.7987 17.07571 6.783500

2.0 931.4447 36.95838 420.3431 26.90009 276.2522 38.59578 15.20440

4.0 1710.845 75.32024 783.1434 58.71236 598.1176 70.87588 36.95838

8.0 2881.818 129.3543 1321.379 97.07422 1112.026 107.3664 68.53674

12.0 4316.177 194.6163 2025.460 149.4709 1692.132 147.8335 109.4717

24.0 3479.936 265.9600 2866.847 227.3642 2376.564 191.1076 166.3127

lx

Figure 8: Permeation profile of Gabapentin in Different Strengths of Ethanol/water

cosolvent in Comparison with Distilled Water as Control.

lxi

Figure 9: Permeation Profile of Gabapentin in Different Strengths of Propylene

Glycol/Water Cosolvent in Comparison with Distilled Water as Control.

lxii

Figure 10: Permeation Profile of Gabapentin in Different Microemulsion Systems in

Comparison with Distilled Water as Control.

lxiii

Table 10: Permeation Parameters of Gabapentin through Abdominal Rat skin from

various Vehicles in Comparison with Distilled Water as Control.

Vehicles Jss(6-24 h) TL Dapp(x 10-3

) Kp (x 10-3

) P Er

(µg/cm2.h) (h) (cm

2/h) (cm

2.h) (x10

-2)

Distilled Water 07.02±0.03 0.94±0.04 3.33±1.01 1.41±0.06 2.36±0.90 1.0

10 % Ethanol 16.59±1.20 0.61±0.02 5.10±0.45 3.32±0.71 3.64±0.11 2.3

20 % Ethanol 29.10±3.22 *1.08±0.95 2.92±0.36 5.82±1.60 11.18±0.21 4.1

30 % Ethanol 61.60±2.11 *0.98±0.34 *3.19±0.98 12.32±2.90 21.60±1.32 8.8

10 % Pr Glycol 39.95±1.61 0.83±0.48 3.76±0.85 7.99 ±0.73 11.89±0.45 5.7

20 % Pr Glycol 31.44±5.23 0.77±3.28 4.09±1.21 6.29 ±0.02 8.60 ±2.27 4.5

30 % Pr Glycol 24.12±1.45 0.55±0.09 5.68±0.49 4.82 ±0.49 4.75 ±0.83 3.4

______________________________________________________________________________

Pr Glycol = Propylene Glycol

Jss(6-24 h)= Steady-State Flux between 6-24 h

TL = Lag Time.

Dapp= Apparent Diffusion Coefficient.

Kp = Permeability Coefficient.

P = Skin-Vehicle Partition Coefficient.

Er = Enhancement ratio

* statistically insignificant at p<0.05 compared with control (n=5)

Table 11: Permeation Parameters of Gabapentin through Abdominal Rat Skin from

various Microemulsion Formulations in Comparison with Distilled Water as Control.

Vehicles Jss(6-24 h) TL Dapp(x 10-3

) Kp (x 10-3

) P Er

(µg/cm2.h) (h) (cm

2/h) (cm

2.h) (x10

-2)

Distilled Water 07.0±1.4 0.94±0.23 3.33±0.95 1.41±0.6 2.36±0.4 1.0

MCEa 141.2±34.1 0.61±0.18 5.12±0.54 56.5±9.4 61.9±6.5 20

MCEb 10.9±1.2 0.41±0.34 7.57±1.52 2.2± 0.7 1.6±0.1 1.5

MCEc 116.9± 9.4 0.44±0.11 7.17±0.47 23.4±3.9 18.5±3.7 16

MCEd 9.3± 2.1 0.59±0.06 5.32±0.32 1.9± 0.4 2.0±0.4 1.3

MCEe 98.8±6.4 0.52±0.04 6.07±1.10 19.8±3.5 18.2±0.8 14

MCEf 07.3±0.7* 0.22±0.10 14.12±3.06 1.5± 0.3* 0.5±0.1 1.0

MCEa-f = Microemulsion Codes

Jss(6-24 h)= Steady-State Flux between 6-24 h

TL = Lag time. Dapp= Apparent Diffusion Coefficient.

Kp = Permeability Coefficient.

P = Skin-Vehicle Partition Coefficient.

Er = Enhancement ratio

*statistically insignificant compared with the control at p<0.05 (n=5)

lxiv

Table 12: Permeation Kinetics Parameters of Gabapentin from various Vehicles and

Microemulsion Formulations in Comparison with Distilled Water as Control

Vehicles Regression Equation Higuchi Model (r2) **Q24 h (µg/cm

2)

Distilled Water Q = 7.027t - 7.477 0.971 166.3127

10 % Ethanol Q = 16.59t – 26.98 0.988 800.9190

20 % Ethaol Q = 29.10t – 27.05 0.962 1618.683

30 % Ethanol Q = 61.62t – 62.74 0.935 3353.388

10 % Pr Glycol Q = 39.95t – 47.92 0.983 911.3281

20 % Pr Glycol Q = 31.44t – 41.03 0.985 713.6710

30 % Pr Glycol Q = 24.12t – 47.70 0.983 544.3174

MCEa* Q = 141.2t – 230.4 0.984 3479.936

MCEb* Q = 10.92t – 26.37 0.939 265.9600

MCEc* Q = 116.9t – 267.3 0.954 2866.847

MCEd* Q = 9.341t - 15.84 0.972 227.3642

MCEe* Q = 98.82t - 191.4 0.950 2376.564

MCEf* Q = 7.343t - 33.05 0.909 191.1076

**Cumulative amount of gabapentin permeated at 24 hours *Microemulsion codes

Table 13: Results of Expected Transdermal Patch Size of Gabapentin from Different

Cosolventss and Microemulsions.

Vehicles Jss(6-24 h) IR (µg/h) Patch Size (cm2)____

10 % Ethanol 16.59 3.6 216.998

20% Ethanol 29.10 3.6 123.711

30 % Ethanol 61.60 3.6 *58.442

10 % Prop. Glycol 39.95 3.6 *90.112

20 % Prop. Glycol 31.44 3.6 114.504

30 % Prop. Glycol 24.12 3.6 149.254

MCEa 141.2 3.6 *25.496

MCEd 9.30 3.6 387.097___________

*Practically significant patch size (Patch size < 100 cm2) [170]

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Table 14: Results of the Cumulative Amount (microgram) of Glipizide Permeated in

Cosolvent Systems and Microemulsions.

Time (h) 30 % Ethanol 20 % Ethanol 10 % Ethanol Distilled water

1.0 41.35596 14.78335 7.29811 6.830283

2.0 93.19126 36.02272 19.46163 16.28040

4.0 185.8211 62.50177 42.01092 29.47314

8.0 289.8660 99.08588 68.30283 49.68329

12.0 454.5413 166.7338 107.8810 74.85241

24.0 744.7815 296.1349 201.1482 129.1204

Time (h) 30% Pr Glycol 20% Pr Glycol 10% Pr Glycol Distilled Water

1.0 10.47934 10.10508 1.309917 6.830283

2.0 22.26859 21.33294 3.087662 16.28040

4.0 36.67768 34.61924 5.520366 29.47314

8.0 61.19185 53.51948 8.888724 49.68329

12.0 102.7349 87.76446 21.52007 74.85241

24.0 155.6930 134.7343 37.23908 129.1204

Time (h) MCEa MCEb MCEc MCEd MCEe MCEf Water

1.0 17.40319 70.45484 5.707497 459.8745 3.742621 30.59592 6.830283

2.0 40.4203 153.5410 13.19274 1183.604 8.420897 79.53069 16.2804

4.0 67.36717 270.3108 27.69539 2214.228 16.65466 142.4067 29.47314

8.0 98.80519 471.5702 45.37928 3485.316 27.32113 259.7379 49.68329

12.0 149.0499 699.2151 68.86422 5016.515 41.73022 455.9448 74.85241

24.0 204.1600 979.0696 95.53039 3042.938 61.56611 687.4259 129.1204

lxvi

Figure 11: Permeation profile of Glipizide in Different Strengths of Ethanol/water

cosolvent in Comparison with Distilled Water as Control.

lxvii

Figure 12: Permeation Profile of Glipizide in Different Strengths of Propylene

Glycol/Water Cosolvent in Comparison with Distilled Water as Control.

lxviii

Figure 13: Permeation Profile of Glipizide in Different Microemulsion Systems in

Comparison with Distilled Water as Control.

lxix

Table 15: Permeation Parameters of Glipizide through Abdominal Rat skin from various

Vehicles in Comparison with Distilled Water as Control.

Vehicles Jss(6-24 h) TL Dapp(x 10-3

) Kp (x 10-3

) P Er

(µg/cm2.h) (h) (cm

2/h) (cm

2.h) (x10

-2)

Distilled Water 05.25±0.34 0.81±0.01 3.86±0.83 1.05±0.11 1.52±1.12 1.0

10 % Ethanol 08.70±1.65 4.49±1.53 0.70±0.20 1.74±0.49 13.94±2.46 1.66

20 % Ethanol 12.10±3.25 1.26±0.24 2.50±0.21 2.42±0.81 5.42±1.02 2.31

30 % Ethanol 30.25±5.78 0.68±0.29 4.61±0.97 6.05±1.34 7.35±2.32 5.77

10 % Pr Glycol 1.60±0.07 2.46±0.93 1.27±0.73 0.32±0.10 1.41±0.04* 0.30

20 % Pr Glycol 5.38±0.49* 0.48±0.03 6.58±0.56 1.08±0.39* 0.92±0.02 1.03

30 % Pr Glycol 6.34±1.29 0.58±0.38 5.44±0.04 1.27±0.94 1.31±0.07 1.20

Jss(6-24 h)= Steady-State Flux between 6-24 h

TL = Lag Time.

Dapp= Apparent Diffusion Coefficient.

Kp = Permeability Coefficient.

P = Skin-Vehicle Partition Coefficient.

Er = Enhancement ratio

Pr glycol = Propylene Glycol

*statistically insignificant at p<0.05 compared with control (n=5)

Table 16: Permeation Parameters of Glipizide through Abdominal Rat Skin from various

Microemulsion Formulations in Comparison with Distilled Water as Control.

Vehicles Jss(6-24 h) TL Dapp(x 10-3

) Kp (x 10-3

) P Er

(µg/cm2.h) (h) (cm

2/h) (cm

2.h) (x10

-2)

Distilled Water 5.25±0.24 0.81±0.01 3.86±0.21 1.05±0.12 1.52±0.25 1.0

MCEa 7.91±1.10 0.27±0.03 11.46±2.34 1.58±0.98 7.74±1.13 1.51

MCEb 39.41±4.72 0.37±0.02 8.40±1.87 7.89±2.00 5.25±2.43 7.51

MCEc 3.89±0.19 0.40±0.10 7.78±2.23 0.78±0.11 0.56±0.21 0.74

MCEd 121.2±9.98 0.40±0.12 7.77±2.27 60.62±5.29 43.70±4.29 23.09

MCEe 2.50±0.41 0.47±0.23 6.67±1.19 0.50±0.03 0.42±0.12 0.48

MCEf 28.89±2.23 0.95±0.15 3.29±0.56 5.78±0.39 9.82±0.33 1.22

MCEa-f = Microemulsion Codes

Jss(6-24 h)= Steady-State Flux between 6-24 h

TL = Lag time. Dapp= Apparent Diffusion Coefficient.

Kp = Permeability Coefficient.

P = Skin-Vehicle Partition Coefficient.

Er = Enhancement ratio *statistically insignificant at p<0.05 compared with control.

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Table 17: Permeation Kinetics Parameters of Glipizide from Various Vehicles and

Microemulsion Formulations in Comparison with Distilled Water as Control

Vehicles Regression Equation Higuchi Model (r2) **Q24 h (µg/cm

2)

Distilled Water Q = 5.245t – 6.450 0.993 129.1204

10 % Ethanol Q = 8.695t – 1.937 0.998 210.1482

20 % Ethaol Q = 12.10t – 9.632 0.994 296.1349

30 % Ethanol Q = 30.25t – 44.46 0.986 744.7815

10 % Pr Glyco l Q = 1.597t – 0.648 0.979 37.23908

20 % Pr Glycol Q = 5.397t – 11.28 0.979 134.7343

30 % Pr Glycol Q = 6.336t – 10.98 0.977 155.6930

MCEa* Q = 7.914t – 28.92 0.945 204.1600

MCEb* Q = 39.41t – 105.6 0.952 979.0696

MCEc* Q = 3.892t – 9.646 0.946 95.53039

MCEd* Q = 121.2t – 300.3 0.968 3042.938

MCEe* Q = 2.50t – 5.318 0.966 61.56611

MCEf* Q = 28.89t – 30.34 0.973 687.4259

**Cumulative amount of glipizide permeated at 24 hours *Microemulsion codes

Table 18: Results of Expected Transdermal Patch Size of Glipizide from Different

Cosolventss and Microemulsions.

Vehicles Jss(6-24 h) IR (µg/h) Patch Size (cm2)____

10 % Ethanol 8.70 12.66 145.517

20% Ethanol 12.10 12.66 104.628

30 % Ethanol 30.25 12.66 *41.851

10 % Prop. Glycol 1.60 12.66 791.25

20 % Prop. Glycol 5.38 12.66 235.316

30 % Prop. Glycol 6.34 12.66 199.685

MCEa 7.91 12.66 160.051

MCEd 121.2 12.66 *10.446___________

*Practically significant patch size (Patch size < 100 cm2). [170]

3.9 Characterization of the Optimized Microemulsions

lxxi

The results of various parameters studied: viscosity, pH, mean globule size and polydispersity

index of the optimized microemulsions are shown in Tables 19 and 20 while the results of skin

irritation tests are shown in Table 21. The morphology of the microemulsions is shown in

Figures 14 and 15.

Figure 14: Photomicrograph of the Particle Sizes of Microemulsion, MCEa

Table 19: Physicochemical Properties of Microemulsion, MCEa

Parameters Values/Descriptions

Stability Thermodynamically stable, monophasic and transparent system

Composition Cremophor Rh 40/ethanol (1:1) ------------ 20 % v/v

Coconut oil ------------------------------------ 60 % v/v

Distilled water -------------------------------- 20 % v/v

Type water-in-oil microemulsion (w/o)

Droplet size 85.8 ± 0.2 nm (n=5)

Polydispersity index 0.0098

*Viscosity 150 ± 20 poise

*Surface tension 83.4 ± 1.2 dyn/cm

*pH 7.6 ± 0.2

Dilution with Water phase separation upon dilution

lxxii

*n=3

Figure 15: Photomicrograph of Particle Sizes of Microemulsion, MCEd.

Table 20: Physicochemical Properties of Microemulsion, MCEd

Parameters Values/Descriptions

Stability Thermodynamically stable, monophasic and transparent system

Composition Cremophor Rh 40/ethanol (1:2) ------------ 20 % v/v

Coconut oil ------------------------------------ 20 % v/v

Distilled water -------------------------------- 60 % v/v

Type oil-in-water microemulsion (o/w)

Droplet size 89.0 ± 0.1 nm (n=5)

Polydispersity index 0.0428

*Viscosity 176 ± 16 poise

*Surface tension 76.6 ± 0.9 dyn/cm

*pH 7.2 ± 0.4

Dilution with Water remained stable upon dilution

*n=3

3.10 FTIR Spectroscopic Studies on Stratum Corneum

lxxiii

The FTIR spectra of the untreated and treated stratum corneum (SC) are shown in Figures 16 to

20.

Figure 16: FTIR Spectra of Untreated SC (Control)

lxxiv

Figure 17: FTIR Spectra of SC Treated with Ethanol/Water Cosolvent System.

lxxv

Figure 18: FTIR Spectra of SC Treated with Propylene Glycol/Water Cosolvent System.

lxxvi

Figure 19: FTIR Spectra of SC Treated with Microemulsion, MCEa

lxxvii

Figure 20: FTIR Spectra of SC Treated with Microemulsion, MCEd.

lxxviii

3.11 DSC Studies on stratum Corneum

The DSC thermograms of the treated and untreated stratum corneum are shown in Figures 21-23.

Figure 21: DSC Thermogram of Untreated Stratum Corneum (Control)

lxxix

Figure 22: DSC Thermogram of Stratum Corneum Treated with Microemulsion, MCEd.

Figure 23: DSC Thermogram of Stratum Corneum Treated with Microemulsion, MCEa.

lxxx

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 Preparation of Standard Solution of Gabapentin and Glipizide

The calibration plots obtained for gabapentin and glipizide in phosphate buffered saline (PBS)

and ethanol respectively showed straight lines at concentration ranges of 5-100 µg/ml and 2-20

µg/ml respectively. High correlation coefficients were obtained for both plots showing the

linearity and accuracy of the determinations. The concentration range of gabapentin (5-100

µg/ml) compared to glipizide (2-20 µg/ml) is higher because of the lower maximum wavelength

of absorption of gabapentin in phosphate buffered saline solution. The solvent used in both

lxxxi

determinations had negligible effects on their absorbances due their low cut-off point in

ultraviolet region. The equations of the straight lines are A = 0.014[C] (r2 = 0.999), and A =

0.035[C] (r2 = 0.999) for gabapentin and glipizide respectively where A is the absorbance and C

the concentration in µg/ml.

4.2 Qualitative and Quantitative Characterization of Coconut Oil

The oil used for the study was extracted with n-hexane from fresh coconut fruits and was used

without further purification. The identity was confirmed by the formation of a translucent surface

on a white tile/paper which is a simple test for oil. Visual assessment showed that the oil is

transparent, colourless, has coconut odour and did not leave any residue on filtration with

Whatman filter paper No. 42 which are characteristics of other pure standard oil used in drug

delivery [46]. These properties of the oil buttress its emollient properties as oil for topical drug

delivery vehicle because it has no potential of staining the skin. The quantitative analysis shows

that the acid, iodine and saponification values of coconut oil are 1.2, 8.3 and 256 respectively.

The acid value or neutralization number is used to quantify the amount of acid (carboxylic acid

groups) present in a sample of oil. Degradation of oil forms free fatty acidic compound that

increases the acid value. Naturally occurring coconut oil has been found to contain different

acids. Freshly prepared coconut oil contains 45 % lauric acid, 20 % myristic acid, 5 % palmitic

acid, 3 % stearic acid, 6 % oleic acid, and traces amount of linolic and linoleic acids [47]. These

acids are responsible for the acid value of coconut oil and the saponification value of the oil. This

is because the saponification value also indicates the concentration of fatty acids liberated upon

hydrolysis. The saponification value is an indication of the amount free fatty acid present in oil.

On degradation, more oil may be converted to acids that increase the acidity level of the oil.

Therefore, the low saponification value obtained is an indication of the freshness of the oil used

lxxxii

for the study. The iodine number of an oil indicates the degree of unsaturation of the oil. It does

not show whether the unsaturation is as a result of the presence of triolein, trilinolin, trilinolenin

only or mixture of the three [47]. As the drying qualities depend mainly upon trilinolin and

trilinolenin, pharmaceutical formulation of transdermal products finds useful, the determination

of iodine value. All values obtained show that the coconut oil used in this study was pure and

fresh since the values within previously reported values for most of the commercially available

oils used in topical delivery of drugs [47]. The freshly extracted coconut oil has a neutral pH of

7.6. After 90 days of storage, the pH fluctuated between 7.6 and 7.2. This signifies the possibility

of little or no degradation of oil to liberate free acids that could make the oil rancid

4.3 Preparation of Coconut Oil-based Microemulsions

To develop microemulsion formulations for transdermal delivery of poorly water-soluble

glipizide and freely water soluble gabapentin, proper selection of oil is needed. The optimisation

of the components to be used in formulating microemulsion was decided based on the solubility

of glipizide and gabapentin in the cocnut oil, surfactant (cremophor) and co-surfactant (ethanol).

The results of the solubility determination of the drugs in coconut oil showed that glipizide is

more soluble in the coconut oil than gabapentin. This is expected from a class II

biopharmaceutical classification system (BCS) drugs which are known for their hydrophobicity,

low aqueous solubility, high permeability, and solvation rate- limited bioavailability.

Gabapentin, a BCS class I drug is characterized by high permeability, high aqueous solubility,

and high absorption profile.

The microemulsion existence region was determined by constructing phase diagrams. Figures 3-

7 describe the pseudo ternary phase diagram with various weight ratios of cremophor to ethanol

(1:1, 1:2, 2:1, 1:3 and 3:1). The translucent region presented in phase diagram reveals the

lxxxiii

microemulsion existence region. No distinct conversion from water-in-oil (w/o) to oil-in-water

(o/w) microemulsion was observed. The rest of region on the phase diagram represents the turbid

and conventional emulsions based on visual inspection.

The phase study clearly revealed that microemulsion existence region increased with increase in

the weight ratio of surfactant. The maximum proportion of oil was incorporated in weight ratio

1:1 of cremephor to ethanol. From the pseudo ternary phase diagrams, the 1:1 surfactant/co-

surfactant ratio gave the highest self-emulsification region followed by 2:1 and then 1:2. The 1:3

and 3:1 gave smaller regions of micro emulsification. However, microemulsion from regions

within all the ratios of surfactant/co-surfactant were selected for further studies because of

relatively wide and close regions of micro emulsifications observed for all the ratios as they have

potentials for forming stable formulation with good emulsification characteristics [48].

The components of the microemulsions play specific role in maintaining a thermodynamically

stable system. Thus, in the phase diagrams, it could be seen that the free energy of

microemulsion formation could be considered to depend on the extent to which the surfactant

lowers the surface tension existing at the oil-water interface and the change in dispersion entropy

[50] Thus, a negative free energy of formation is achieved when a large reduction in surface

tension is accompanied by significant favourable entropic changes. In such cases, microemulsion

formation is spontaneous and the resulting dispersions are thermodynamically stable [49, 50]

The surfactant/cosurfactant mixture, which is able to increase the dispersion entropy, reduce the

interfacial tension, increase the interfacial area, and thus lower the free energy of the system to a

very low value with the minimum concentration (weight ratio, 1:1), has the potential for the

transdermal drug delivery. Safety is a major determining factor in choosing a surfactant, since

certain amount of surfactants might cause skin irritation. Non-ionic surfactants are less toxic than

lxxxiv

ionic surfactants. An important criterion for selection of surfactants is that the required

hydrophilic- lipophilic balance (HLB) value (to form the o/w microemulsion) be greater than 10.

The correct blend of low and high HLB surfactants leads to the formation of a stable

microemulsion formulation [49]. In this study, cremophor was selected as a surfactant with an

HLB value of 15. Transient negative interfacial tension and fluid interfacial film are rarely

achieved by the use of single surfactant. This necessitated the addition of a cosurfactant

(ethanol). The presence of cosurfactant decreases the bending stress of interface and allows the

interfacial film sufficient flexibility to take up different curvatures required to form

microemulsions over a wide range of composition [53, 54]. The cosurfactant selected for the

study was ethanol, which has low HLB value. Optimum concentration of surfactant was

employed in the formulation because it is well known that many surfactants cause skin irritation

[50-52]. From pseudoternary phase diagrams, the formulations in which the amount of oil phase

completely solubilized the drug, accommodate the optimum quantity of surfactant/cosurfactant

and distilled water, and has the possibility of forming both oil-in-water (o/w) and water-in-oil

(w/o) microemulsions with very high safety profile were chosen for further studies.

One major advantage of a microemulsion over traditional emulsion is the ease of preparation,

especially with regard to large batch manufacturing. In general, several factors have to be

considered for a coarse emulsion, such as intensity and duration of mixing, emulsification time

(including rate and temperature), order of adding and mixing each ingredient, heating and

cooling rates, etc. Because microemulsion forms spontaneously with only gentle agitation, some

of these factors can be avoided. Another advantage is the physical stability of the formulation. In

a traditional emulsion system, the larger droplet size favors a decrease in the surface area, which

in turn favors a decrease in the free energy of the system [55]. However, a microemulsion system

lxxxv

has lower interfacial tension between water and oil due to the presence of surfactant and

cosurfactant resulting in very large surface area of the dispersed droplets [55,56]. The lower

interfacial tension compensates the dispersion entropy making the microemulsion system to

become thermodynamically stable (due to the low free energy of the system). The two

microemulsion systems selected from each of the five pseudo ternary phase diagrams were

subjected to pre-drug- loading stability studies. Excess of the surfactant that was unable to

emulsify limited oil-water concentration during the process of microemulsification (leading to

the precipitation of the such) could be responsible for the observed longer period of instability of

high surfactant/cosurfactant ratio. The microemulsions were loaded with increasing

concentration of drug to determine the optimum concentration of the drug in the microemulsion

systems. It was observed that the microemulsions remainned stable beyond 21 days for

concentrations at 0.25 and 0.50 % w/v respectively. This observation was sharp reversal of

stability of the systems selected from previous studies at concentration above 0.75 % w/v.

(precipitation and/or phase separation of the microemulsion systems). This could be attributed to

the interaction between the hydrophobic drug and the oil phase and that between hydrophilic

drug and the aqeous phase. Maximum interaction (hence maximum solubility) was observed at

concentration of 0.5 % w/v of the drugs. The maximum solubilty of 0.5 % w/v was, therefore,

selected as the loading drug concentration for further studies.

4.4 Permeation Studies of Gabapentin and Glipizide across Excised Rat skin.

4.4.1 Permeation Studies of Gabapentin in Different Cosolvents and Microemulsions.

The calibration curve established for gabapentin in phosphate buffered saline solution gave a

high coefficint of correlation value of 0.999 showing linearity and high accuracy of the

lxxxvi

determination. The equation of the line of regression is A = 0.014 [Concentration] from where

all the calculations were derived. The determination was carried out at maximum wavelength of

210 nm at a concentration range of 5 – 100 µg/ml against the phosphate buffered saline blank.

The linear relationship observed between the cummulative amount permeated and time for the

in-vitro skin permeation of gabapentin from cosolvents and microemulsion formulations (Figures

8, 9 and 10) indicate zero order permeation kinetics (r2 ≥ 0.909 – 0.988). It also shows that the

permeation of gabapentin was based on diffusion controlled mechanism [57]. The steady-state

flux was observed after a small lag time of 0.61 – 0.98 (ethanol), 0.55 – 0.83 (propylene glycol)

and 0.22 – 0.41 h (microemulsions). The results reveal significant increase in permeation flux,

partition coefficient, permeability coefficient and enhancement ratio of the drug with increasing

concentrations of ethanol (p<0.05) when compared with the control. The enhanced permeation

properties at different strengths of ethanol compared with the control could be ascribed to two

factors. (i) ethanol is a vehicle known to increase the permeation of drugs through the skin either

by attacking the dense barrier structure of the skin [58] or by augmenting the solubility and

partitioning of the drug in stratum corneum [59]. (ii) ethanol is penetration enhancer that acts by

the disruption of the lipid bilayer of the skin thereby increasing the permeation of drug through

the skin. Another factor that might have played a role in the permeation of the drug from ethanol

is the high solubility of gabapentin (hydrophilic drug) in ethanol. There was linear increase in the

steady-state flux and enhancement ratio in the permeation of the drug with increasing

concentration of ethanol. Ethanol is an effective permeation enhancer that promotes penetration

via reducing skin resistance to drug molecules or by increasing skin/vehicle partition coefficient.

Proposal by other researchers show that ethanol denatures the intercellular structural proteins of

the horny layer of the skin or promotes lipid fluidity or may alter the physical structure of the

lxxxvii

skin by disruption of the ordered structure of the lipid chains; extraction of lipids, lipoproteins

and nucleoproteins of the stratum corneum.

With propylene glycol, a general decrease in the permeation flux, partition coeficient, skin-

vehicle partition coefficient and enhancement ratio of gabapentin at increasing concentration of

propylene glycol was observed. The decreases were found to be significant (p<0.05) when

compared with the control. There was no significant decrease in lag time at 10 % proplene

glycol. No significant decrease of apparent diffusion coefficient at 10 and 20 % propylene glycol

was observed. This might be due to poor solubility of gabapentin in propylene glycol, greater

interaction with the drug, viscosity of the cosolvent or the dielectric constant of the cosolvent.

Glycols, like terpenes, increase percutaneous absorption of drugs by increasing diffusivity of the

compounds in stratum corneum or by disruption of the intercellular lipid barrier or by increasing

electrical conductivity of tissues thereby opening polar pathways through the stratum corneum

[60]. However, this study shows that propylene glycol failed to enhance the skin permeation of

gabapentin at the concentration levels studied.

With different microemulsion systems , statistically significant difference in all the permeation

parameters considered in comparison with the control at p <0.05 were observed. To explain the

results obtained with the microemulsions, the study examined the previously reported possible

mechanism of action of microemulsions with hydrophilic drugs [61]. The reports indicate that (i)

microemulsions act as drug reservoirs where loaded drug is released from the inner pseudophase

to the outer pseudophase and finally further into the skin. (ii) microemulsion droplets might

break down on the surface of the stratum corneum and then release their contents into the skin.

(iii) permeation of loaded drug occurs directly from the droplets to the stratum corneum without

microemulsion fusion at the stratum corneum. The last mechanism has been frequently supported

lxxxviii

by findings of other groups of researchers [62, 63] and indicates that the enhancement effect of

microemulsions is caused by the nano-sized droplets dispersed in the continous phase which can

move easily into the stratum corneum and carry the drug through the skin barrier. Based on the

results obtained, it could be said that gabapentin permeation from microemulsions could be as a

results of the mechanisms highlighted above and may also explain the findings in the present

skin permeation study.

To estimate the patch size of the transdermal delivery system for gabapentin, the input rate was

estimated from the total drug clearance and steady-state plasma concentration of the drug. The

results show that the expected patch sizes of 90, 58 and 25 cm2

could be obtained from the 10 %

propylene glycol cosolvent, 30 % ethanol cosolvent and the optimized microemulsion, MCEa

respectively (Table 13). These findings were in line with previous reports that for a drug to be

formulated for transdermal delivery and maintain a steady-state plasma concentration at certain

clearance rate, an estimated patch size of 100 cm2

or less is required [64]

4.4.2 Permeation Studies of Glipizide in Different Cosolventss and Microemulsions

A solution of glipizide in ethanol was scanned in UV/VIS spectrophotometer at a wavelength

range of 200 to 700 nm to establish the wavelength of maximum absorption. This was

established at 276 nm.The calibration curve established for glipizide in ethanol gave a high

coefficint of correlation value of 0.999 showing the linearity of the calibration curve and the

curve did not deviate significantly from the origin as indicated by by its zero intercept and high

accuracy of the determination. The equation of the line of regression was A = 0.035 [C] where A

is the absorbance and C the concentration in µg/ml from where all the calculations of the skin

permeation studies were derived. The determination was carried out at maximum wavelength of

276 nm at concentration of 2 – 20 µg/ml against the ethanol blank. Ethanol has no effect on the

lxxxix

absorption of the drug at 276 nm because the cut-off point of ethanol in UV spectroscopy is 205

nm.

The linear relationship observed between the cumulative amount permeated and time for the in-

vitro skin permeation of glipizide from cosolvents and microemulsion formulations (Figures 11,

12 and 13) indicate zero order permeation kinetics (r2 ≥ 0.945 – 0.998). It also shows that the

permeation of gabapentin was based on diffusion controlled mechanism [57]. The amount of

glipizide permeated shows a linear relationship with the square root of time (r2

> 0.9), therefore,

the permeation rate of the test drug followed Higuchi theoretical model [63]. The steady-state

flux was observed after a lag of 0.68 – 4.49 (ethanol), 0.48 – 2.46 (propylene glycol) and 0.27 –

0.95 h (microemulsions). 10 % v/v ethanol showed a lower cumulative amount (210 µg/cm2)

than 30 % v/v ethanol (744 µg/cm2) while 30 % v/v propylene glycol showed a cumulative

amount of 155µg/cm2 compared to 37 µg/cm

2 observed for 10 % v/v propylene glycol. MCEd

showed the highest cumulative amount permeated at 24 hours of 3042 µg/cm2 while MCEe

showed the lowest cumulative amount permeated of 61 µg/cm2. The permeation rates were

observed to be dependent on the vehicle used as microemulsions showed the highest rates,

followed by ethanol and the propylene glycol. However, there was no statistically significant

difference in the steady-state permeation fluxes of all the concentration of propylene glycol used

when compared with the control at p < 0.05. At lower concentration of the vehicles (10 %

ethanol and propylene glycol), there was no significant difference (p < 0.05) in the apparent

diffusion coefficient and partition coefficient of the drug compared with the control. The reason

may be inability of the low concentration of the vehicles to solubilise the drug which serves as a

driving force to partition the drug into the stratum corneum. All the strengths of ethanol

xc

statistically increased the skin-vehicle partition coefficient and enhancement ratio of the drug

compared with the control.

With microemulsions, significant increase in the apparent diffusion coefficient of the drug

compared with the control was observed. Similarly, microemulsions b and d increased the

partition coefficient, a, b, d, and f increased the skin-vehicle partition coefficient significantly

while b, d and f increased the enhancement ratio when compared with the control at p < 0.05. All

the microemulsions, with the exception of f reduced the lag time significantly. Glipizide is a

lipophilic drug which solubilises well in the core of microemulsion system that is lipophilic in

nature. Their solubility in such systems provides a driving force necessary for their favourable

partitioning into the lipophilic stratum corneum. According to the permeation of the drug-loaded

microemulsion droplets attributing to the permeation enhancement effect, the oil droplets of the

o/w type might permeate into the epidermis easier than the water droplets of the w/o type at the

same surfactant concentration owing to the lipophilic nature of the stratum corneum. The oil can

enter the hydrophobic tail of the stratum corneum bilayer, perturb it by creating separate

domains, and induce highly permeable pathways in the stratum corneum [65]. Since the

microemulsions contain the same amount of surfactant/cosurfactant (20 % v/v), it is believed that

all effects of the microemulsions are due the hydrophilic-hydrophobic constituents of the

microemulsion. As a result of the high permeation steady-state flux, MCEb, MCEd and MCEf

could have oil core that were able to solubilise the lipophilic drug in their domain more than the

other microemulsions because a hydrophobic drug is preferentially encapsulated in the oil

droplets of the highly drug loaded droplets favour partitioning into the epidermis, resulting in the

highest permeation flux observed for MCEd compared with the control. This phenomenon

confirms that the oil droplet nature of an o/w microemulsion is a crucial factor for flux of drugs,

xci

especially hydrophobic substances. To explain the results obtained, the study examined previous

reports. The results were in agreement with previous reports which indicated that the o/w

microemulsion provided higher membrane fluxes of diclofenac diethylamine [67] and

ketoprofene [68] than the w/o microemulsions while bicontinous microstructure hampered the

drug release. The results were also in agreement with previous reports [69] that maximum

fluconazole permeation and 1.5 fold improvement in drug release were achieved from

microemulsion prepared with jojoba oil. Another report [70] explained the mechanism by which

microemulsions enhance the percutaneous absorption of drugs on the basis of the combined

effect of both the lipophilic and hydrophilic domains of the microemulsion. The lipophilic

domain of the microemulsion can interact with the stratum corneum in many ways. The drug

dissolved in the lipid domain of a microemulsion can directly partition into the lipid of the

stratum corneum or lipid vesicles themselves can intercalate between the lipid chains of the

stratum corneum, thereby destabilizing its bilayer structure. These interactions will lead to

increased permeability of the lipid pathway to the drugs. On the other hand, the hydrophilic

domain of the microemulsion can hydrate the stratum corneum to a greater extent. When the

aqueous fluid of the microemulsion enters the polar pathway, it will increase inter lamellar

volume of the stratum corneum lipid bilayer, resulting in disruption of its interfacial structure.

Since, some lipid chains are covalently attached to corneocytes, hydration of these proteins will

also lead to the disorder of lipid bilayers. Similarly, swelling of the intercellular proteins may

also disturb the lipid bilayers; a lipophilic penetrant like glipizide, can then permeate more easily

through the lipid pathways of stratum corneum.

The results of the estimated size of transdermal patch of glipizide show that only 30 % ethanol

cosolvent and microemulsion, MCEd could deliver the plasma concentration of the drug at

xcii

convenient patch sizes of 41 and 10 cm2 respectively (Table 18). This is based on the

requirement that an estimated patch size of 100 cm2 or less is required for transdermal drug

delivery [64]. Based on these findings, the skin permeation study of glipizide was successful and

promising for further development.

The droplet size decreased with the increase in concentration of oil in the formulations. For

example, the droplet size of formulation MCEd (containing 20 % v/v coconut oil), was higher

(89.0 nm) than droplet size of formulation MCEa (85.8 nm) which contains higher

concentrations of oil. All the formulations had droplet sizes in the nano range, which is very well

evident from the low polydispersity values. Polydispersity is the ratio of standard deviation to

mean droplet size, so it indicates the uniformity of droplet size within the formulation. The

higher the polydispersity, the lower the uniformity of the droplet size in the formulation.

Although the polydispersity values of all formulations were very low, indicating uniformity of

droplet size within each formulation, the polydispersity of formulation MCEd was lowest

(0.0098). The increase in percutaneous absorption of drug might also be affected by the droplet

size of the microemulsion. As the droplet size is very small the number of vesicles that interact

on fixed area of stratum corneum also increases thereby increasing the efficiency of

percutaneous uptake. This could be the reason why microemulsion, MCEd whose particle sizes

were larger than that of microemulsion, MCEa showed relatively lower permeation rates for the

vehicles investigated. The result shows that the droplet diameter decreases with increasing ratio

of oil: surfactant/co-surfactant. These results are in accordance with the report that the addition

of surfactant to microemulsion system causes the interfacial film to condense and to be stable,

while the co surfactant causes the film to expand [71].

xciii

With the viscosity study of microemulsion, MCEa is lower than that of microemulsion, MCEd

due to the lower viscosity of coconut oil as external pseudophase in the w/o microemulsion

compared to that of water as external pseudophase in o/w microemulsion. This could explain the

observation that (w/o) form gave higher permeation rate. The result agreed with previous report

[71].

The pH of the microemulsions studied were considered to be ideal for the study because one of

the conditions for good transdermal drug delivery is that the pH of the vehicle or the formulation

must be higher than the isoelectric point of keratin (3.7 to 4.5). The isolectric point is the pH at

which solutions of proteins produce the least electrical conductivity, the least osmotic pressure,

and the least viscosity. This is the point at which the protein shows the least swelling and does

not undergo cataphoresis (no migration of particles to either positive or negative electric pole).

As other ions are at their maximum, proteins best coagulate and contain the least amount of

inorganic matter at their isoelectric point. At this point the osmotic pressure, viscosity,

conductivity, swelling, precipitability by alcohol, acid- and base-binding power, and migration in

an electrical field are at a minimum. The adherence of colloidal particles in suspension to the

skin would depend upon the pH of the dispersion medium, the charge that the skin assumed in

contact with the colloidal suspension, and the charge on the suspended particle. The implication

of this lies in the fact that in the local application of colloidal suspensions of a medicinal or

cosmetic nature to the thoroughly cleansed epidermis, the adherence depends greatly upon the

charge on the suspended particle and the charge that the skin assumes in contact with the

solution. This latter would depend upon the isoelectric point of the skin and the pH of the

colloidal suspension. Should the pH of the solution fall on the acid side of the isoelectric point,

the outer layer of the epidermis would be expected to assume a positive charge and positively

xciv

charged particles in suspension would be repelled and easily removed, while negatively charged

particles would be closely adhered to the surface and consequently removed with difficulty. On

the other hand, should the pH of the medium be on the alkaline side of the isoelectric point, the

skin would assume a negative charge and the opposite behavior with respect to charged particles

would be expected.

4.5 Skin Irritation Test

Skin irritation test was performed to confirm that the concentration of materials used for

microemulsion preparation was non -toxic.

Table 21: Data for Skin Irritation Test

Rats First Group(-ve control) Second group(+ve control) Third Group(MCEa) Fourth Group(MCEd)

Erythema Edema Erythema Edema Erythema Edema Erythema Edema

1 0.00 0.00 3 2 1 0.00 0 0.00

2 0.00 0.00 4 1 0 0.00 1 0.00

3 0.00 0.00 3 3 0 0.00 0 0.00

_____________________________________________________________________________

Mean 0.00 0.00 3.33 2.00 0.33 0.00 0.33 0.00

S.D 0.00 0.00 0.58 1.00 0.58 0.00 0.58 0.00

PII 0.00±0.00 5.33 ±1.58 0.33 ± 0.58 0.33 ± 0.58

PII = Primary Irritancy Index (Mean edema + Mean erythema) [58]

The results of the skin irritation test (Table XI) show that the microemulsions used for the study

showed a primary irritancy indices (PII) of lesser than 2 compared with the standard irritant

(formaldehyde) of PII 5.33. This implies that MCEa and MCEd were considered to be non-

irritant as PII was lesser than 2. Previous report [41] has shown that a value of the primary

irritancy index lesser than 2 is non-irritant to human skin.

4.6 Biophysical Analysis of Rat Skin Treated with Vehicles and Microemulsions

(a) Differential Scanning Calorimetry (DSC) Analysis

xcv

A normal DSC analysis of rat skin shows four main thermograms: T1, T2, T3 and T4 with melting

point temperatures of 34 ºC, 82 ºC, 105 ºC and 114 ºC respectively. The T1 represents the

melting point of sebaceous fluid, T2 represents the melting point of stratum corneum lipids, while

T3 and T4 represent the melting point of stratum corneum proteins. The mechanism of

permeation can be understood from the effect of the vehicles on these thermograms.

Table 22: Effects of Microemulsions on the DSC of Stratum Corneum of Rat

Vehicles/Microemulsion Melting Points (ºC) % Decrease in Melting Points

T2 T3 T4 T2 T3 T4

Control (untreated) 70.0 - 200.0 - - -

Microemulsion (MCEa) 42.0 - 165.0 40.0 - 17.5

Microemulsion (MCEd) 40.0 - 140.0 42.9 - 30.0

Percentage decrease in melting point = (Melting point of control – Melting point of treated) /

(Melting point of control) x 100. T = Melting point temperature. (Figures 21-23).

The two optimized microemulsions had similar and close effect on the stratum corneum of rat. A

reduction of the stratum corneum protein (17.5 %) and lipids (40.0 %) was observed with MCEa,

while a similar reduction of stratum corneum protein (30 %) and lipids (42.9 %) was seen with

MCEd. The decrease in melting point of stratum corneum lipids indicates the disruption of lipid

bilayers of the skin which poses a barrier to permeation of drugs through the skin. Similarly, the

decrease in melting point of stratum corneum proteins (keratin) suggests keratin denaturation and

possible intracellular permeation mechanism in addition to the disruption of lipid bilayer. These

findings are in agreement with the mechanisms of permeation enhancement by microemulsions

earlier suggested [61-63].

(b) Fourier Transform Infrared Spectroscopy (FTIR) Analysis

The complete FTIR spectra of stratum corneum show, among other bands, two important

stretching vibrations. First, the –CH2 asymmetrical and symmetrical vibrations of the long chain

xcvi

hydrocarbon of lipid bilayer occur at 2920 and 2850 cm-1

respectively. Similar vibrations are

observed at 3000 to 2700 (or 2600) cm-1

due to –CH stretching of the alkyl groups present in

both proteins and lipids. Specifically, 2955 cm-1

vibration is due to asymmetrical and 2870 cm-1

is as a result of symmetrical –CH3 vibrations. These narrow bands were attributed to the long

alkyl chains of fatty acids, ceramides, and cholesterol that are components of the stratum

corneum lipids. Second, two strong bands 1650 and 1550 cm-1

are due to the amide I and amide

II stretching vibrations of stratum corneum proteins. Amide I and amide II bands arise from –

C=O stretching vibration and –C-N bending vibration respectively. The amide I band represents

various secondary structure of keratin in the stratum corneum. The disruption of any of these

bands by the vehicles or microemulsion suggests a mechanism for the permeation of drug loaded

in the vehicle.

Table 23: Effects of Cosolvents and Microemulsions on FTIR of Stratum Corneum of Rat

xcvii

Vehicles Asymmetrical –CH Stretching Vibrations Amide I (C=O)Stretching Vibrations

(2859-2929) cm-1

(1550 -1650) cm-1

Height(mm) Area(mm2) %↓Height %↓Area Height(mm) Area(mm2 ) %↓Height %↓Area

Control 77.15 31.75 ____ _____ 61.82 6.08 ____ ____

30 % Ethanol 46.20 20.16 40.12 34.65 5.79 1.47 90.63 75.82

30% P.Glycol 69.23 29.80 10.27 6.14 6.75 3.64 89.08 40.13

MCEa 27.34 9.05 64.56 71.50 18.32 2.24 70.37 63.16

MCEd 36.46 11.10 52.74 65.04 8.68 2.80 85.60 53.95

P.Glycol = Propylene glycol. ↓ = decrease. MCE = Microemulsion. Percentage decrease (Peak

Height or Area) = (Peak height/area of control – Peak height/area of treated) / (Peak

height/area of control) x 100. Peak height = Base length (H) – Base length (L) (Figures 16-20).

The Table shows that there was clear difference in the FTIR spectra of untreated and

vehicles/microemulsions treated stratum corneum with prominent decrease in asymmetric and

symmetric –CH stretching peak height and peak area. The microemulsion treated stratum

corneum showed the highest percentage decrease in peak height and peak area in the

asymmetrical –CH stretching vibrations compared with vehicle treated stratum corneum.

Propylene glycol treated showed the least percentage decrease in peak height and peak area. In

the amide I (-C=O) stretching vibrations, propylene showed the lowest percentage decrease in

peak area. Similar decreases in peak height or area were observed for other frequency bands in

the spectra of the studied microemulsions.

The rate limiting step in transdermal delivery of drugs involves the lipophilic part of stratum

corneum in which lipids (ceramides) are tightly packed as bilayers due to the high degree of

hydrogen bonding. The amide I group of ceramides hydrogen is bonded to amide II group of

another ceramide and forming a tight network of hydrogen at the head of ceramides. This

hydrogen bonding makes stability and strength to lipid bilayers and thus imparts barrier property

xcviii

to stratum corneum. When treated with microemulsion or vehicle, ceramides got loosened

because of competitive hydrogen bonding leading to breaking of hydrogen bond networks.

4.7 Conclusion and Prospects

4..7.1 Conclusion

This study showed that coconut oil, cremophor Rh 40®, and distilled water were suitable to

formulate glipizide and gabapentin as a pharmaceutically acceptable microscaled emulsion for

transdermal delivery. The selected ratio (1:1 and 1:2 surfactant: cosurfactant) was capable of

containing the desired drug concentration employed transdermally. Investigations revealed that

drugs with lower molecular weight could permeate from vehicles and microemulsions into the

epidermis to a greater extent than those with higher molecular weight. In general, the drugs with

lower molecular weight have a higher drug mobility, thermodynamic activity or diffusion

coefficient and subsequently higher permeation rates through intact epidermis [72]. In this case,

therefore, gabapentin (molecular weight, 171.237 g/mole) produced higher cumulative amount of

permeation and permeation fluxes for all the strengths of vehicles and microemulsion studied

compared to glipizide (molecular weight, 445.535 g/mole) as shown in Tables 11 and16 which is

in agreement with the predictive rule that the maximum flux of drug through the skin should

decrease by a factor of 5 for an increase of 100 Daltons in molecular weight [73]. The FTIR and

DSC results show that the vehicles (microemulsion and cosolvent systems) have significant

effects on the SC of rat. This led to the proposition of the mechanism of permeation

enhancement of the vehicles studied. The skin toxicity test shows that the chemicals used in the

formulation of the optimized microemulsion systems are generally safe and non-toxic for

transdermal delivery of drugs.

xcix

On the clinical basis, it was observed that the optimized microemulsion systems could deliver a

therapeutic plasma concentration of the drugs studied at specified clearance levels of 42.2

ml/min for gabapentin (plasma concentration of 5.0 µg/litre) and 30.0 ml/min for glipizide

(plasma concentration 2.0 µg/litre) using a practically acceptable patch sizes lesser than 100 cm2.

Based on these findings, possible development of these systems into patches for transdermal

delivery is promising.

4.6.2 Prospects

Inasmuch as the result of the present study is promising, more effort is being made to develop the

optimized systems into transdermal patches such that self-administration for prolonged period of

time and compliance will be assured. The following areas should be further worked on:

a) Advanced characterization of the optimized systems using modern analytical tools such

as transmission electron microscopy (TEM), 1H-nmr spectroscopy, refractive index,

electrical coductivity, zetasizer zeta potential measurement, optical birefringence,etc.

b) Using more sensitive equipment, such as HPLC to sample the drug in the permeation

sink.

c) Carrying out animal studies in order to compare effectively the in-vitro/in-vivo

relationship in the behaviour of the drug permeation parameters.

c

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