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
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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
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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
xxix
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
xxx
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
xxxix
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]
lxv
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.
lxx
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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