Enhanced transdermal delivery with fatty acids and electroporation

93 5.1.2.6 Comparison between passive permeation with fatty acid-based formulations and electroporation with chemical drug vehicles .... ELECTROPORATION PROTOCOL IS 10 FIGURE 5.13 EFFECT

ACKNOWLEDGEMENTS

The road to getting a doctorate has been long and tortuous It is my pleasure to work with Dr Yang Yi Yan and Associate Professor Paul, Heng Wan Sia for the past three and a half years They have provided me with much support and guidance that made the completion of this thesis possible I also am grateful to Professor Neal Chung Tai Shung who first introduced me to research and inculcated the interest and desire to pursue a doctorate degree I would also like to express my heartfelt appreciation to my family members, close friends and fellow colleagues for the moral support and the numerous intellectual discussions that carried me through the journey Lastly and most importantly, I would like to acknowledge the generous funding from Agency of Science and Technology, Singapore, Institute of Bioengineering and Nanotechnology and Institute of Materials Research and Engineering and also providing an excellent environment for research with exposure to a fascinating array of research

ACKNOWLEDGEMENTS I LIST OF FIGURES V LIST OF TABLES X LIST OF INTERNATIONAL REFEREED PUBLICATIONS AND

CONFERENCE PAPERS XII LIST OF ABBREVIATIONS XIII

SUMMARY 1

1 INTRODUCTION 4

1.1 CONTROLLED DRUG DELIVERY 4

1.2 TRANSDERMAL AS A ROUTE OF ADMINISTRATION 5

1.3 STRUCTURE OF THE SKIN 6

1.4 TRANSPORT OF DRUG ACROSS THE SKIN AND FACTORS INVOLVED 7

1.5 STRATEGIES TO IMPROVE THE PERMEABILITY OF THE SKIN 10

1.5.1 Chemical enhancement 10

1.5.2 Physical enhancement 11

1.5.2.1 Iontophoresis 12

1.5.2.2 Electroporation 13

1.5.2.3 Sonophoresis 14

1.5.2.4 Microneedles 14

1.5.3 Synergy between chemical and physical permeation enhancement 15

1.6 FUTURE CHALLENGES IN TRANSDERMAL DELIVERY 16

2 LITERATURE REVIEW 17

2.1 STRUCTURE AND COMPOSITION OF THE STRATUM CORNEUM 17

2.2 DRUG TRANSPORT ACROSS SC 18

2.3 FATTY ACIDS AS PERMEATION ENHANCERS 19

2.4 ELECTROPORATION FOR TRANSDERMAL DELIVERY APPLICATION 20

2.4.1 Drugs delivered by electroporation 20

2.4.2 Enhancement mechanism 21

2.4.3 Skin recovery post electroporation 22

2.5 SYNERGY BETWEEN ELECTROPORATION AND CHEMICALS 24

2.6 BIOPHYSICAL TECHNIQUES 25

2.6.1 Fourier Transform Infrared Spectroscopy 25

2.7 TRANSDERMAL DELIVERY OF PHYSOSTIGMINE 27

2.8 TRANSDERMAL DELIVERY OF PEPTIDES AND PROTEINS 31

2.8.1 Transdermal delivery of cyclosporin A 33

2.8.2 Transdermal Delivery of Lutenising hormone releasing hormone 35

3 MOTIVATION AND RESEARCH OBJECTIVES 37

3.1 MOTIVATION 37

3.2 RESEARCH OBJECTIVES 40

4 MATERIALS AND METHODS 41

4.1 MATERIALS 41

4.2 METHODS 42

4.2.1 Skin and SC preparation 42

4.2.2 Lipid extraction of SC 42

4.2.3 Preparation of fatty acid containing PHY formulation 43

4.2.4 Preparation of CsA and [D-Ala 6 ]-LHRH formulation 43

4.2.5 1 H NMR spectroscopy for PHY and fatty acid 44

4.2.6 Conductivity measurement 44

4.2.7 Formulation treatment for SC sheets 44

4.2.8 Formulation/fatty acid uptake by SC 45

4.2.9 Partition coefficient between octanol and water 45

4.2.10 Apparent partition coefficient of PHY between IPM and vehicle 45

4.2.11 Fourier transform infrared spectroscopy (FTIR) 46

4.2.12 Permeation study and calculation of permeation parameters 47

4.2.13 High pressure liquid chromatography assay 49

4.2.14 Stability studies for CsA and [D-Ala 6 ]-LHRH 50

4.2.15 Statistical analysis 51

5 RESULTS AND DISCUSSION 52

5.1 TRANSDERMAL DELIVERY OF PHYSOSTIGMINE 52

5.1.1 Fatty acids-based formulations for delivery of physostigmine 52

5.1.1.1 1H NMR spectra 53

5.1.1.2 Conductivity measurement 55

5.1.1.3 Apparent partition coefficients 57

5.1.1.4 Formulation/fatty acid uptake by the SC 59

5.1.1.5 FTIR spectra of delipidised SC 63

5.1.1.6 Biophysical behaviour of SC lipids 65

5.1.1.7 Permeation profiles and parameters of PHY through full skin 70

5.1.1.8 Permeation profiles and parameters through tape stripped skin 74

5.1.1.9 Enhancement mechanisms of fatty acid-based formulations 77

5.1.2 Enhancement of PHY permeation using electroporation 85

5.1.2.1 Effect of electroporation protocol 85

5.1.2.2 Effect of using chemical drug vehicles after electroporation 87

5.1.2.3 Effect of oleic acid pretreatment before electroporation 91

5.1.2.4 Comparison between electroporation and tape stripping 92

5.1.2.5 Permeation parameters of PHY across electroporated skin 93

5.1.2.6 Comparison between passive permeation with fatty acid-based formulations and electroporation with chemical drug vehicles 96

5.1.2.7 Mechanisms of PHY permeation through electroporated skin 97

5.2 TRANSDERMAL DELIVERY OF CYCLOSPORIN A 105

5.2.1 Stability study of CsA 105

5.2.2 Partition coefficient of CsA between octanol and water 106

5.2.3 Solubility of CsA in various vehicles 107

5.2.4 Passive permeation of CsA 108

5.2.5 Permeation of CsA across electroporated skin 109

5.2.5.1 Effect of electrical protocol 109

5.2.5.2 Effect of drug vehicle 110

5.2.5.3 Effect of fatty acids in drug vehicle 111

5.2.5.4 Effect of oleic acid pretreatment 114

5.2.5.5 CsA permeation through tape stripped skin 116

5.2.5.6 CsA retention within skin 118

5.2.6 Elucidation of CsA permeation mechanisms post electroporation 119

5.3 TRANSDERMAL DELIVERY OF [D-ALA6]-LUTENISING HORMONE RELEASING HORMONE 125

5.3.1 Stability of [D-Ala 6 ]-LHRH 125

5.3.2 Partition coefficient of [D-Ala 6 ]-LHRH between octanol and water 126 5.3.3 Passive permeation of [D-Ala 6 ]-LHRH 127

5.3.4 Permeation of [D-Ala 6 ]-LHRH through electroporated skin 128

5.3.4.1 Effect of electroporation protocol 128

5.3.4.2 Effect of drug vehicle 130

5.3.4.3 Effect of fatty acids in drug vehicle 131

5.3.4.4 Effect of oleic acid pretreatment 134

5.3.4.5 [D-Ala6]-LHRH permeation through tape stripped skin 137

5.3.4.6 Enhancement mechanisms involved in [D-Ala6]-LHRH permeation 139 5.4 ELECTROPORATION AS A PERMEATION ENHANCEMENT TECHNIQUE 144

6 CONCLUSIONS 149

7 RECOMMENDATIONS 152

7.1 SKIN PERMEATION OF FATTY ACIDS 152

7.2 SKIN IRRITATION AND PAIN THRESHOLD STUDIES 152

8 REFERENCES 153

LIST OF FIGURES

FIGURE 1.1VARIOUS BLOOD DRUG LEVEL PROFILES FROM A ZERO ORDER DOSAGE

CONCENTRATION ADAPTED FROM ROBINSON AND LEE (1987) 5

FIGURE 1.2CROSS SECTIONAL VIEW OF SKIN ADAPTED FROM HOUSEL (2004) 7

FIGURE 1.3SCHEMATIC SHOWING THE DIFFERENCE BETWEEN THE DRUG RELEASE RATE FROM A TRANSDERMAL DEVICE AND THE RATE AT WHICH THE DRUG APPEARS IN THE BODY 9

FIGURE 1.4(A) E-TRANS® DEVELOPED BY ALZA CORPORATION FOR FENTANYL DELIVERY ADAPTED FROM ALZA.COM (2004) (B)IONTOPHORETIC DEVICE DEVELOPED BY VYTERIS FOR LIDOCAINE DELIVERY ADPATED FROM VYTERIS.COM (2004) 13

FIGURE 1.5ELECTROPORATION DEVICE FOR TRANSDERMAL DRUG DELIVERY DEVELOPED BY GENETRONICS INC ADAPTED FROM GENETRONICS.COM (2004) 14 FIGURE 1.6 A MICRONEEDLE ARRAY AND ITS APPLICATOR (MACROFLUX) DEVELOPED BY ALZA CORPORATION ADAPTED FROM ALZA.COM (2004) 15

FIGURE 2.1 POSSIBLE ROUTES OF DRUG PERMEATION ACROSS SC ADAPTED FROM BARRY (1997) 18

FIGURE 2.2 A TYPICAL FTIR SPECTRUM OF PORCINE SC 25

FIGURE 2.3DECOMPOSITION PATH OF PHY 30

FIGURE 2.4MOLECULAR STRUCTURE OF CYCLOSPORIN A 34

FIGURE 2.5 AMINO ACID SEQUENCE OF LHRH ANALOG USED 35

FIGURE 4.1SCHEMATIC DIAGRAM OF HORIZONTAL DIFFUSION CELL SETUP INVOLVING ELECTROPORATION 48

FIGURE 5.1 1H NMR SPECTRA OF ACETIC ACID, OLEIC ACID, PHY AND THEIR MIXTURES 54

FIGURE 5.2ANALYSIS OF PHY 1H NMR SPECTRUM 55

FIGURE 5.3CONDUCTIVITY MEASUREMENTS OF SOLVENT AND 0.5M FATTY ACID CONTAINING SOLVENT BEFORE AND AFTER THE ADDITION OF PHY 56

FIGURE 5.4EFFECT OF FATTY ACID AND SOLVENT USED ON THE PERCENTAGE WEIGHT

FIGURE 5.5PERCENTAGE WEIGHT UPTAKE OF FATTY ACIDS BY PORCINE SC (N=3,

FIGURE 5.6 REPRESENTATIVE FTIR SPECTRA OF THE SC BEFORE AND AFTER

PG (D) TREATED WITH OLEIC ACID IN MO THE LOCATION OF THE DASHED LINES CORRESPONDS WITH THE VIBRATION FREQUENCY OF THE ASYMMETRIC AND

FIGURE 5.7CHANGES IN CH2C-H STRETCHING VIBRATION FREQUENCIES AFTER FATTY

FIGURE 5.8PERCENTAGE CHANGES IN PEAK AREA OF INTERCELLULAR LIPIDS

FIGURE 5.9 CUMULATIVE PHY PERMEATED THROUGH AN AREA OF 1.77 CM2 OF THE

FIGURE 5.10 CUMULATIVE PHY PERMEATED THROUGH AN AREA OF 1.77CM2 OF THE

FIGURE 5.11 CUMULATIVE PHY PERMEATED THROUGH AN AREA OF 1.77 CM2 OF THE

SD) 76

FIGURE 5.12 EFFECT OF ELECTROPORATION VOLTAGE ON PHY PERMEATION THROUGH

PHY IN PBS) WAS USED FOR EACH SOLVENT ELECTROPORATION PROTOCOL IS 10

FIGURE 5.13 EFFECT OF NUMBER OF ELECTRICAL PULSES ON PHY PERMEATION

FIGURE 5.14 EFFECT OF CHEMICAL DRUG VEHICLES ON PHY PERMEATION THROUGH

100 MS DURATION AT INTERVAL OF 900 MS (B) ELECTROPORATION PROTOCOL IS

10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS 88

FIGURE 5.15 EFFECT OF OLEIC ACID CONCENTRATION IN ETHANOL ON PHY

500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS 90

FIGURE 5.16 EFFECT OF OLEIC ACID SKIN PRETREATMENT ON PHY PERMEATION

PBS 92

FIGURE 5.17 COMPARISON OF PHY PERMEATION BETWEEN TAPE STRIPPED SKIN AND

100/500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS.DRUG DONOR

FIGURE 5.18 EFFECT OF TEMPERATURE AND SKIN HYDROLYTIC ENZYMES ON CSA

FIGURE 5.19 PASSIVE PERMEATION OF CSA THROUGH 1.77 CM2 OF PORCINE EPIDERMIS

109

FIGURE 5.20 EFFECT OF ELECTROPORATION VOLTAGE ON CSA PERMEATION THROUGH

0.5M OLEIC ACID IN ETHANOL 110

FIGURE 5.21 EFFECT OF DRUG VEHICLE ON CSA SKIN PERMEATION AFTER

FIGURE 5.22 EFFECT OF CHAIN LENGTH OF FATTY ACID IN VEHICLE ON CSA

FIGURE 5.23 EFFECT OF CONCENTRATION OF OLEIC ACID IN VEHICLE ON CSA

FIGURE 5.24 EFFECT OF TYPE OF CARRIER FOR OLEIC ACID ON CSA PERMEATION

FIGURE 5.25 EFFECT OF OLEIC ACID PRETREATMENT ON CSA PERMEATION THOUGH

FIGURE 5.26 EFFECT OF VEHICLE NATURE ON CSA PERMEATION THROUGH TAPE

FIGURE 5.27 EFFECT OF TEMPERATURE AND SKIN HYDROLYTIC ENZYMES ON [D-ALA6

FIGURE 5.28 PASSIVE [D-ALA6]-LHRH SKIN PERMEATION IN DIFFERENT VEHICLES (N =

FIGURE 5.29 EFFECT OF ELECTRICAL PROTOCOL ON [D-ALA6]-LHRH SKIN

FIGURE 5.30 EFFECT OF DRUG VEHICLE ON [D-ALA6]-LHRH SKIN PERMEATION AFTER

FIGURE 5.31 EFFECT OF CHAIN LENGTH OF FATTY ACID ON [D-ALA6]-LHRH

500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS 131

FIGURE 5.32 EFFECT OF OLEIC ACID CONCENTRATION ON [D-ALA6]-LHRH SKIN

IS 50/50 ETHANOL/PG ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100

FIGURE 5.33 EFFECT OF CARRIER FOR OLEIC ACID ON [D-ALA6]-LHRH PERMEATION (N

= 3, MEAN ± SD) ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS

0.25M 133

FIGURE 5.34 EFFECT OF OLEIC ACID PRETREATMENT ON [D-ALA6]-LHRH PERMEATION

FIGURE 5.35 [D-ALA6]-LHRH PERMEATION THROUGH TAPE STRIPPED SKIN (N = 3,

LIST OF TABLES

TABLE 1.1 CHARACTERISTICS OF COMMERCIAL TRANSDERMAL PATCHES…….… 6

TABLE 5.1 APPARENT PARTITION COEFFICIENTS OF PHY BETWEEN IPM AND PG (N =

TABLE 5.2 CHANGES IN ν S(CH2) PEAK AREA/PEAK HEIGHT RATIOS AS A FUNCTION OF

TABLE 5.3 SUMMARY OF DRUG RELEASE PARAMETERS FOR PHY PERMEATION

SD) 74

TABLE 5.4 SUMMARY OF DRUG RELEASE PARAMETERS FOR PHY PERMEATION

TABLE 5.5 SUMMARY OF PERMEATION PARAMETERS FOR PHY PERMEATION

TABLE 5.6 PHY SOLUBILITY IN VARIOUS DRUG VEHICLES ……….…… … 101

TABLE 5.7 PARTITION COEFFICIENTS OF CSA BETWEEN OCTANOL AND WATER IN THE

TABLE 5.8 SOLUBILITY OF CSA IN VARIOUS DRUG VEHICLES (N =3, MEAN ±SD) 109

TABLE 5.9 CSA PERMEATION ENHANCEMENT AND SC BARRIER RATIO ……… 118

TABLE 5.10 AMOUNT OF CSA RETAINED IN SC AND EPIDERMIS AFTER PERMEATION

TABLE 5.11 SUMMARY OF CSA SKIN PERMEATION EXPERIMENTS (N =3, MEAN ±

……… 120

TABLE 5.12 PARTITION COEFFICIENTS OF [D-ALA6]-LHRH BETWEEN OCTANOL AND

TABLE 5.13 [D-ALA6]-LHRH PERMEATION ENHANCEMENT AND SC BARRIER

TABLE 5.14 SUMMARY OF [D-ALA6]-LHRH SKIN PERMEATION EXPERIMENTS (N =3,

LIST OF INTERNATIONAL REFEREED PUBLICATIONS AND CONFERENCE PAPERS

International Refereed Publications

physostigmine permeation through skin post electroporation, (Manuscript in

preparation)

cyclosporin A by using combination of chemicals and electroporation,

(Manuscript in preparation)

• M.Y Wang, Y.Y Yang and P W S Heng, Strategy to increase skin permeation

of peptides via combination of electroporation and fatty acids, (Manuscript in

preparation)

from fatty acids-based formulations: Evaluating the choice of solvent, (Accepted

for Inter J Pharm.)

between fatty acids-based formulations and lipids in porcine stratum corneum, J

Control Release, 94(2004) 207-216

Conference Papers

enhancements by fatty acids, Materials Research Society Fall Meeting 2003,

Boston, USA Oral presentation

of Physostigmine with Fatty Acids based formulations – Role of the Solvent, 30 th

Annual Meeting of the Controlled Release Society, 2003 Poster

LIST OF ABBREVIATIONS

νa(CH2) : CH2 asymmetric stretching mode

νs(CH2) : CH2 symmetric stretching modes

[D-Ala6]-LHRH : lutenising hormone releasing hormone analogue with D-alanine at

6th amino acid position

SUMMARY

In spite of numerous challenges and limitations, high patient compliance and the ability to deliver drug direct to the systemic circulation using the transdermal drug delivery continue to fuel growth of this niche market Challenges in transdermal drug delivery include the delivery of larger molecules such as peptides and proteins, reduction in lag time and reduction in patch size by improving skin permeation rate This thesis sought to tackle some of the challenges mentioned above by focusing on the development and understanding of skin permeation enhancement methods using fatty acids, electroporation and a combination of both

The first part of thesis dealt with elucidation of permeation enhancement mechanisms

of fatty acids in solvents with different polarities, using physostigmine as model drug

It was found that different enhancement mechanisms of fatty acids were involved when the solvent was altered The choice of solvent was critical to transport fatty acids into stratum corneum and the effect of fatty acids on partitioning of physostigmine into stratum corneum was reduced when a lipophilic solvent such as mineral oil was used as mineral oil imparted the necessary lipophilicity required to partition into stratum corneum Based on physostigmine permeation data, fatty acids showed greater enhancement capabilities in hydrophilic solvents such as propylene glycol This was attributed to higher permeation capacity due to the use of proposed intracellular pathway for physostigmine in propylene glycol-based formulations

Next, skin permeability post electroporation was investigated Physostigmine was used as a model drug and its permeation across electroporated skin was investigated

In spite of the lack of electrophoretic drug transfer across skin, physostigmine permeation across skin was improved and the lag time was shorter than that of passive permeation A study of physostigmine permeation parameters through electroporated skin revealed that higher diffusion coefficients were the major cause for improved permeation after skin electroporation However, partition coefficients were reduced after electroporation, due to a rise in water content within stratum corneum Synergy

of oleic acid-based formulation and electroporation was detected and physostigmine permeation was further improved This synergy was attributed to the ability of oleic acid to improve skin partitioning of physostigmine post electroporation

The second part of thesis utilised the synergy between chemical enhancers and electroporation to deliver larger molecules like cyclosporin A (CsA) and lutenising hormone releasing hormone analog ([D-Ala6]-LHRH) CsA and [D-Ala6]-LHRH have similar molecular weights but different hydrophilicity Higher permeation was achieved for both peptides as compared with passive permeation The choice of drug vehicle played an important role in determining CsA permeation across electroporated skin and CsA permeation was highest in ethanol-based formulation due to high CsA

tremendously after electroporation Although CsA and [D-Ala6]-LHRH had similar

much faster than that of CsA The synergy between fatty acids and electroporation was effective in increasing [D-Ala6]-LHRH permeation across the skin Surprisingly, [D-Ala6]-LHRH permeation in this protocol was quite comparable to that obtained

with iontophoresis as reported in literature, capable of delivering a therapeutic dose across skin

In short, this thesis has successfully shed some insights into the enhancement mechanisms of fatty acids and derived a skin permeation enhancement method based

on the synergy between fatty acids and electroporation to deliver large molecules such

as [D-Ala6]-LHRH at therapeutic levels, without the need of a device

1 INTRODUCTION

1.1 Controlled drug delivery

Controlled release systems provide drug release in a controlled manner in the body In general, controlled drug delivery attempts to sustain drug action at a predetermined rate by maintaining a relatively constant, effective drug level in the body with concomitant minimization of undesirable side effects associated with a sawtooth kinetic pattern In order to maintain a constant drug level in either plasma or target tissue, release rate from the controlled release system should be equal to the elimination rate from plasma or target tissue

Figure 1.1 shows comparative blood drug level profiles obtained from administration

of conventional, controlled as well as prolonged release dosage forms As long as the amount of drug is above the minimum effective concentration, a pharmacological response is observed Problems occur when the therapeutic window is very narrow in the case of potent drugs or when the peak is greater than the upper limit of the range Thus, one of the main purposes of controlled drug delivery is to improve safety and minimize side effects of the drug by reducing fluctuations in drug level in plasma

Therapeutic window (A)

(B)

Figure 1.1 Various blood drug level profiles from a zero order dosage form, a

sustained release dosage form and a conventional tablet (A) Toxic level (B) Minimum effective level Adapted from Robinson and Lee (1987)

1.2 Transdermal as a route of administration

Continuous intravenous infusion is a superior mode of drug administration not only to bypass hepatic “first-pass” metabolism but also to maintain a constant and prolonged drug level in the body These benefits can be duplicated by using the skin as a portal

of drug administration, coupled with improved patient compliance and an option to discontinue the drug therapy in the event of complications The drug applied topically will be adsorbed first into the blood circulation and then be transported to target tissues to achieve its therapeutic purposes

The future of transdermal patches looks promising with an annual US market of more than US $3 billion despite the limited number of drugs that can be used for

transdermal delivery [Prausnitz et al., 2004] Table 1.1 gives a summary of patches

available at present

The subsequent parts of this section will detail on the structure of skin, mechanism of drug transport across skin, obstacles in transdermal drug delivery, ways to enhance transdermal drug delivery and future challenges

Table 1.1

Characteristics of commercial transdermal patches

Adapted from Prausnitz et al (2004)

40 mAmin iontophoresis

Dermal anaesthesia Nicotine

Habitrol Nicoderm-CQ Nicotrol Prostep

0.15 – 0.20 mg O and 0.42 – 1.0 mg

N in 3 – 4 days

Hormone replacement

Testoderm

10 -328 mg in

1.3 Structure of the skin

The skin is the largest organ (total surface area of 2 m2) in the body, composed of three major tissue layers: the epidermis, the dermis and the hypodermic layers with stratum corneum forming the outermost layer of the epidermis, exposed to the

external environment (Figure 1.2) Hair follicles and sweat ducts occupy about 0.1%

of the human skin surface

Figure 1.2 Cross sectional view of skin Adapted from Housel (2004)

1.4 Transport of drug across the skin and factors involved

The transport of drug across the skin is believed to occur by a slow, passive diffusion through the stratum corneum, followed by rapid diffusion through the viable epidermis and papillary dermis and ultimately the microcirculation of the skin The physicochemical properties of the drug determine whether it is a suitable candidate for transdermal administration Based on properties of drugs that have been successfully delivered via the transdermal route passively, it was deemed that ideal drug candidate should have a molecular weight lower than 500 Daltons, log of octanol/water partition coefficient lies between 1 and 3, aqueous solubility greater than 1 mg/mL and dose deliverable is less than 10 mg/day These properties imply that only potent drugs that

are small in size and moderately lipophilic can be delivered via the transdermal route

with greater ease A predictive rule of thumb is 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 [Berner and Cooper, 1987]

Since the skin is a natural barrier to both the ingress of xenobiotics and egress of water, it would present a similar barrier in the context of drug delivery In 1924, Rein (1924) proposed that stratum corneum posed the major resistance to transdermal transport A detailed discussion of the properties of stratum corneum that gave rise to its barrier function is found in Section 2.1 After the drug reaches the viable tissue, it encounters a phase change It has to transfer from the predominantly lipophilic intercellular channels of the stratum corneum into the living cells of the epidermis, which will be largely aqueous in nature and essentially buffered at pH 7.4 For lipophilic drugs, transfer into the viable epidermis can be a slow process and one that will be influenced by a number of factors The magnitude of the partition coefficient will be important However, since many drugs possess ionisable groups, the partition coefficient between an appropriate lipid and an aqueous phase at pH 7.4 should be considered The log of octanol/water partition coefficient should be between 1 and 3

so that partitioning in stratum corneum is not hindered and yet able to partition into the aqueous epidermis The intrinsic solubility in the aqueous phase will be important

and may be modified by the presence of co-diffusing formulation components

When the penetrant has dissolved in the upper regions of the viable tissue, it will diffuse down a concentration gradient to the epidermal-dermal junction It is unlikely that the presence of any formulation constituents in this area will significantly alter the diffusional characteristics The viable tissue is much more metabolically active than stratum corneum and some transdermally delivered drugs may be metabolised

before they reach the systematic circulation When the drug reaches the base of the viable tissue, it is rapidly taken into the blood vessels and redistributed around the body

When a transdermal patch is applied to the skin, the steady state systematic dosage may not be reached for some time because of absorption of the drug in the skin For the majority of drugs, a plot of drug release from the device and drug absorption rate

at the site of action has the general shape shown in Figure 1.3 Initially, there is a difference between the drug release from the device curve and the drug systematic absorption curve as some drug is immobilized in the skin At saturation, the rate of drug release equals the rate of appearance in the blood When the device is removed, drug release from the device stops The release of drug absorbed in the skin will continue for some time Therefore the depot effect is a major factor to be taken into account in device design One way to minimise the loss of efficacy caused by the depot effect is to load up the skin quickly with excess drug

Figure 1.3 Schematic showing the difference between the drug release rate from a

transdermal device and the rate at which the drug appears in the body

1.5 Strategies to improve the permeability of the skin

Practical use of transdermal administration is limited because of the inherent barrier properties of the skin It is difficult to get drug to cross the skin at a sufficient rate to deliver a therapeutic dose even when the drug is potent Although physicochemical properties of drug, such as molecular weight and lipophilicity, affect its inherent permeation rate across the skin, there are enhancement strategies that can be employed to boost its permeation These strategies focused on mechanical disruption, electrical disruption and chemical modification of the skin and can be broadly classified into chemical (passive) and physical (active) enhancement A short discussion of each is described as follows

1.5.1 Chemical enhancement

Chemical enhancement in transdermal drug delivery involves the use of chemicals (termed as chemical penetration enhancers) to alter the barrier property of the stratum corneum These chemical penetration enhancers interact with the formulation applied

or the skin It is one of the oldest and yet the most common technique used as the enhancers can be easily incorporated into the drug formulation Since the stratum corneum act as the rate-limiting step, even a transient enhancement would yield a great improvement in drug uptake Ideally, the enhancer should be pharmacologically inert and should possess no action of itself at receptor sites in the skin or in body generally It should not be toxic, irritating or allergenic The onset of penetration enhancing action should be immediate on application The duration of the effect should be predictable and suitable Also, the change in barrier property of the skin should be transient and reversible after exposure to chemical enhancers

Although a vast array of chemicals has been used as penetration enhancers, none of them possess all the ideal attributes as described above Common penetration

enhancers include dimethyl sulfoxide, dimethyl formamide, N-methyl-2-pyrrolidone,

propylene glycol, azone, sodium lauryl sulphate, fatty acids, fatty alcohols and a variety of nonionic surfactant such as polyalkyl ethers or esters The modes of action

of these enhancers are complex and a good review is provided by Williams and Barry (2004) Briefly, these enhancers may increase the diffusion coefficient of the drug in stratum corneum by disrupting the lipid organization, increase the effective concentration of drug by acting as an anti solvent, improve the partitioning of the drug between stratum corneum and vehicle or allow the drug to travel through a less tortuous pathway across stratum corneum (Williams and Barry, 2004)

1.5.2 Physical enhancement

Physical enhancement of drug permeation across the skin involves the application of

an external driving force such as electric field (iontophoresis and electroporation), ultrasonic waves (sonophoresis), magnetic field (magnetophoresis) or the use of mechanical force to drive the drug across the skin in the case of jet injection and microneedle array With the exception of microneedles and jet injection, the other enhancement techniques involve the disruption of stratum corneum organization under the influence of the external driving force other than concentration difference Ideally, the stratum corneum organization should be restored once the external driving force is removed In reality, the recovery of skin is dependent on the application protocol and sometimes a complete recovery is not attained due to the application of a severe protocol applied A brief review of some physical techniques, namely

iontophoresis, electroporation, sonophoresis and microneedles, is detailed in the following section

1.5.2.1 Iontophoresis

Iontophoresis involves the application of a small electric potential across the skin to maintain a constant current that drives charged molecules across skin The amount of drug (charged species) delivered is directly proportional to the quantity of charge applied The factors controlling the delivery rate are amplitude of applied current, duration of current application and the area of the skin surface in contact with the active electrode compartment Other than an improved onset time and also a more rapid offset time, the main strength of iontophoresis over other techniques is its ability

to control the amount of drug delivered, which offers the option of pulsatile delivery while its main drawback is that the drug has to be charged for iontophoretic delivery

The main mechanisms behind iontophoretic delivery are electromigration and

electroosmosis A comprehensive review was provided by Kalia et al (2004), which

detailed the mechanisms behind iontophoresis and summarised the drugs that can be delivered by iontophoresis Commercially, two corporations have been active in the development of iontophoretic delivery devices, namely ALZA Corporation and Vyteris ALZA has developed E-TRANS® for fentanyl delivery, which is currently in Phase III clinical trials while Vyteris is expected to launch an iontophoretic system for lidocaine delivery in 2004 (Figure 1.4)

(A) (B)

Figure 1.4 (A) E-TRANS® developed by Alza Corporation for fentanyl delivery

Adapted from Alza.com (2004) (B) Iontophoretic device developed by Vyteris for

lidocaine delivery Adpated from Vyteris.com (2004)

1.5.2.2 Electroporation

Electroporation, an established technique used to permeabilise cell membrane, is a

relatively new enhancement technique in transdermal drug delivery It involves the

application of short, high voltage electrical pulses to create a transient

permeabilisation of the skin The principle involved is similar to that in cell

electroporation; the short pulsing can cause a sharp increase in the skin permeability,

permitting the entry of macromolecules such as heparin and even microbeads Unlike

iontophoresis, electroporation offers the possibility of delivering uncharged molecules

into the skin A more detailed review of electroporation is discussed in Section 2 The

successful transition for electroporation enhanced transdermal delivery into clinical

application would rely on the development of appropriate devices Currently,

Genetronics is developing a couple of electroporation devices for delivery of drugs

across membrane barriers and the one for transdermal drug delivery is shown in

Figure 1.5

Figure 1.5 Electroporation device for transdermal drug delivery developed by

Genetronics Inc Adapted from Genetronics.com (2004)

1.5.2.3 Sonophoresis

Sonophoresis is a relatively new technique that involves the application of ultrasonic energy to the skin, disrupting lipid bilayers within the stratum corneum, creating reversible micro channels in the skin, allowing the permeation of large molecules into the skin A number of macromolecules such as insulin and heparin (Mitrogotri and Kost, 2004) were able to permeate the skin with the use of sonophoresis A detailed description of enhancement mechanisms of sonophoresis was provided by Mitrogotri and Kost (2004)

1.5.2.4 Microneedles

The development of microneedle arrays as devices for transdermal drug delivery was made possible with the advent of microfabrication technology in the 1990s Unlike other penetration enhancement strategies, microneedles literally break the stratum corneum barrier through the insertion of microstructured projections (Figure 1.6) into the skin, allowing the direct entry of drug molecules into the epidermis Due to the absence of nerves in stratum corneum and the small size of the projections, the microneedle patches caused little or no pain and are able to deliver drugs of virtually

any size and large molecules such as insulin, oligonucleotides and DNA were delivered to the epidermis using microneedles (Prausnitz, 2004) A primary concern with the use of microneedles is the large number of micro holes left in the skin after removal of microneedle patch Although these holes are much smaller than the hole

made by a hypodermic needle (Champion et al., 1998), the safety issue of these holes

requires further investigation and verification

Figure 1.6 A microneedle array and its applicator (Macroflux) developed by Alza corporation Adapted from Alza.com (2004)

1.5.3 Synergy between chemical and physical permeation enhancement

Both chemical and physical permeation enhancement techniques have individually shown to enhance transdermal drug transport Their combinations have been hypothesised to be more effective compared to each of them alone (Mitragotri, 2000)

In addition to increasing transdermal transport, a combination of enhancement techniques should also reduce the severity of the enhancers required to achieve the desirable drug flux Typically, the highest strength of the enhancers that can be applied on the skin is limited by its safety By combining two or more enhancers, one

can reduce the strength of individual enhancers required to achieve the desired enhancement

1.6 Future challenges in transdermal delivery

Traditional transdermal delivery involves the passive delivery of potent drugs with small molecular weight where chemical penetration enhancers are often employed to assist permeation Judging by the current developments in iontophoresis, electroporation, sonophoresis and microneedles, the next generation of transdermal delivery devices will be able to deliver a wider range of drugs across the skin at therapeutic doses

The therapeutic value with any drug therapy is a balance between tolerability and efficacy During development, patients in clinical trials are closely monitored by the physician and encouraged during frequent clinic visits During chronic therapy, the patient may perceive no benefits of therapy, only side effects Compliance then becomes problematic Thus the therapeutic value of a drug in the practical situation is also determined by factors affecting the patient's acceptance of the drug therapy

2 LITERATURE REVIEW

This section serves to provide the readers with a brief literature review of the various subjects of interest in this thesis These topics encompass the structure and composition of stratum corneum, use of fatty acids as permeation enhancers, biophysical techniques used to evaluate barrier property of SC, electroporation as permeation enhancer and the skin delivery of physostigmine, cyclosporin A and lutenising hormone releasing hormone

2.1 Structure and composition of the stratum corneum

In 1924, Rein (1924) proposed that stratum corneum (SC) posed the major resistance

to transdermal transport The SC is a multilayer composite of the corneocytes (terminally differentiated keratinocytes) and the secreted contents of the lamellar bodies (intercellular lipids) The lipids organization within SC was put forth in the domain mosaic model by Forslind (1994) In this model, islands of gel phase domains are separated by a continuous liquid crystalline domain In the crystalline domain, polar molecules would diffuse laterally along or close to the polar head regions and less polar molecules would diffuse laterally within the hydrophobic interior of the bilayer Besides the organization of the lipids, its unique composition also plays a role

in the SC barrier During epidermal terminal differentiation, the mixture of polar and neutral lipids is replaced by a more nonpolar mixture of ceramides, free sterols, free fatty acids, triglycerides and sterol esters The barrier property of SC is dependent on

nature of lipids present According to Grubauer et al (1989), the removal of nonpolar

lipids from SC only resulted in minor impairment of barrier function while removal of sphingolipids caused a major defect in barrier function of SC

2.2 Drug transport across SC

The exact route of permeation of drugs across the SC has been an issue of debate for years Figure 2.1 illustrates the possible routes of drug permeation across intact SC The transappendageal route, transports substances via the sweat glands and the hair follicles with their associated sebaceous glands, at a rate that is faster than through the intact area of the SC The transepidermal route across the continuous SC comprises

transport via intracellular and intercellular spaces

Figure 2.1 Possible routes of drug permeation across SC Adapted from Barry (1997)

According to Hadgraft (2004), under normal circumstances, the predominant route is through the intercellular space in the SC (intercellular route) However, there was also association between nature of drug and route of administration It is believed that

polar drugs transverse the SC through the transcellular route while non-polar drugs penetrate through the intercellular route According to Barry (1997), the polar molecules mainly diffuse through the polar pathway (transcellular) consisting of

“boundwater” within the hydrated SC, whereas the non-polar molecules dissolve and diffuse through the non-aqueous lipid matrix of the SC (intercellular)

2.3 Fatty acids as permeation enhancers

Fatty acids are one class of chemical permeation enhancers that have been successfully used to improve permeation fluxes of both hydrophilic and lipophilic

drugs such as salicylic acid, estradiol, progesterone and acyclovir (Aungst et al.,

1986; Aungst, 1989; Ogiso and Shintani, 1990) Both saturated and unsaturated fatty acids were employed to enhance transdermal drug permeation The major attraction of these compounds is that many of these materials are classified as Generally Regarded

As Safe (GRAS) by FDA Among the saturated fatty acids, alkyl chain length of around C10 to C12 seemed to result in the greatest permeation enhancement (Aungst, 1989) In contrast, the optimum permeation enhancement with unsaturated fatty acids was attained when the chain length of unsaturated fatty acid was around C18

Oleic acid has been extensively used and its enhancement mechanism has been extensively studied Oleic acid interacts with and modifies the lipid domains of the

SC, due to the presence of the double bond in alkyl chain At physiological temperatures, oleic acid molecules are in liquid state, and thus are phase separated

from the endogenous solid lipids in the SC (Francoeur et al., 1990; Ongpipattanakul

et al., 1991) More recently, electron microscopic studies have shown that a discreet lipid domain is induced within SC bilayer lipids on exposure to oleic acid (Tanojo et

al., 1997) The degree of permeation enhancement by fatty acids is dependent on the solvent According to Aungst et al (1986), the highest permeation enhancement was

attained when propylene glycol was used as a solvent for fatty acids

2.4 Electroporation for transdermal delivery application

Electroporation is a well established method for inducing cell permeabilisation through the application of short, high voltage pulses, permitting the entry of water and large molecules into the cell During electroporation, the cell membrane discharges by forming multiple transient pores and eventually returns to its initial state after electroporation ceases (Weaver and Chizmadzhev, 1996)

2.4.1 Drugs delivered by electroporation

Electroporation has enabled the delivery of macromolecules across the skin Molecules that have been successfully transported across the skin ranged from fluorescent, low molecular weight tracers such as lucifer yellow and cascade blue

(Chen et al., 1998) to larger compounds like oligonucleotides (Zehart et al., 1995), insulin (Sen et al., 2002), heparin (Prausnitz et al., 1995) and even naked DNA (Zhang et al., 2002) Unlike iontophoresis, permeation of neutral molecules across the skin could also be enhanced by electroporation (Vanbever et al., 1998)

Controversial results on the delivery of particles by electroporation across the SC

were reported by two research groups (Zhang et al., 1997; Chen et al., 1999) Zhang

et al (1997) reported that the delivery of Lupron Depot microspheres (2 - 20 µm) into

hairless mouse skin and human skin xenograft by pressure-mediated electroincorporation was successful The ratio of efficiency to deliver Lupron depot

microspheres across the SC between pulsing and control was a factor of 100 for human skin xenograft In the case of hairless mice, the ratio was a factor of 18 On the

other hand, Chen et al (1999) reported that charged polystyrene microbeads (14 nm -

2.14 µm) were not transported across the human SC in vitro by short high voltage pulses and it was concluded that the size of the aqueous pathways that form across the skin during high-voltage pulsing must be less than 14 nm

2.4.2 Enhancement mechanism

The application of electroporation to skin was first demonstrated by Prausnitz et al

(1993) Even though the SC contains multilamellar, intercellular lipid bilayers with few phospholipids and no living cells, the physical changes that occurred during electroporation were comparable to that of phospholipid membrane in a living cell In the skin, majority of electrical resistance derives from the presence of ∼ 100 lipid bilayer membranes in the SC and thus the transdermal voltage is often approximated

by the voltage drop across the SC It was reported by Weaver and Chizmadzhev (1996) that the threshold voltage for permeabilisation of a phospholipid bilayer during electroporation was around 1 V Therefore the 100 lipid bilayers in SC would require about a transdermal voltage of 100 V for permeabilisation

Indeed, significantly higher transport through the skin was observed when a

transdermal voltage of around 100 V is attained (Chen et al., 1998a; Chen et al.,

1998b) This sudden increase in transport across the skin was attributed to the

formation of localised transport regions (LTRs) within the SC (Pliquett et al., 1996a; Pliquett et al., 1996b; Chen et al., 1998; Weaver et al., 1998) and evidence for this

hypothesis was substantiated with real time imaging of fluorescent molecule transport

(Pliquett et al., 1996b; Pliquett et al., 2000) When a transdermal voltage of around

100 V is attained, water is electrically forced to enter the lipid environment, resulting

in new aqueous pathways Once a pathway on the order of 10 nm in radius exists, the local high current density yields Joule heating and the surrounding lipids are heated, increasing the probability of electroporation As the SC is permeabilised by a combination of electric field and Joule heating in the presence of an electric field,

charged molecules are transported by local electrophoresis via LTRs, which form during pulsing and are randomly distributed in the skin (Chen et al., 1998) Although

LTRs do not exist during low electroporation voltage, a small degree of enhanced transport was observed due to electrophoresis The mode of transport at low voltage is believed to occur through preexisting pathways such as sweat glands and hair follicles

in a manner similar to iontophoresis (Weaver et al., 1999)

2.4.3 Skin recovery post electroporation

One of the requirements of any skin permeation enhancement technique is the ability

to alter the skin in a reversible manner In the case of electroporation, the ability of the skin and more specifically the SC to recover completely post electroporation is found

to be dependent on the electrical protocol applied (Pliquett, 1999; Pliquett et al., 1995; Jadoul et al., 1999; Sen et al., 2002; Essa et al., 2003) The recovery of SC is judged

based on its resistance recovery At the onset of a high voltage pulse, the resistance of

SC dropped by orders of magnitude within less than 1 µs This fast drop in resistance

at high voltage was consistent with the electrical creation of aqueous pathways within

SC (Pliquett, 1999) The behaviour of skin resistance with applied voltage was investigated by Pliquett (1999) It was found that skin resistance dropped with increasing voltage and leveled off as the duration and strength of the electrical

stimulus increased The decrease in resistance during the pulse involved two phases The rapid decrease during the first 10 µs was attributed to the creation and expansion

of pores by electric field while the subsequent decrease arose from thermal effects caused by Joule heating

The resistance recovery of the skin was found to exhibit four phases (Pliquett et al.,

1995) Phase one consists of a 20 ms interval immediately after beginning of pulse and the skin resistance reached up to 60% of prepulsed value In phase two, the skin resistance increased up to 40-70% of prepulsed value during the next 0.4-0.8 s Skin recovery slowed down significantly in phase three for the next 10 s and resistance approached 90% of pre-pulsed level Finally, in phase four, the recovery process was very long and sometimes, incomplete recovery occurred Besides the use of resistance

as a gauge of skin recovery, biophysical techniques such as Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction and freeze fracture electron microscopy were also employed to determine the SC integrity

after electroporation (Jadoul et al., 1999) Significant water permeation was observed

after electroporation and increased SC hydration was observed by thermogravimetry FTIR spectra did not show a change in lipid fluidity after electroporation and DSC did not reveal a change in transition temperature of SC lipids However, there was a decrease in enthalpies of the lipid peaks after long electroporation pulses, suggesting that electroporation caused a generalised lipid disordering effect From X-ray diffraction, a disordering of the lipid lamellar stacking and of the lipid lateral stacking was observed after application of long, high voltage pulses Contrary to long pulses, short, high voltage pulses only altered the SC lamellar phases slightly

2.5 Synergy between electroporation and chemicals

Some research groups have reported the use of chemicals before or after

electroporation (Sen et al., 2002; Essa et al., 2003; Murthy et al., 2004, Weaver et al., 1997; Ilic et al., 1999; Zehart et al., 1999, Murthy et al., 2003) Sen et al (2002) have

reported that saturated anionic phospholipids were able to enhance skin permeation after electroporation by prolonging the existence of pathways formed during electroporation as these anionic phospholipids preferred loose layers or vesicular

rather than multilamellar forms On the other hand, Essa et al (2003) showed that

electroporation did not affect the skin penetration of estradiol containing phosphotidylcholine liposomes significantly as compared to passive permeation of those liposomes They further postulated that phosphatidylcholine could accelerate the skin barrier repair after electroporation In addition, molecules such as sodium

dodecyl sulphate (Murthy et al., 2004) and heparin (Weaver et al., 1997) were also

reported to enhance skin permeation after electroporation by prolonging the existence

of the aqueous pathways formed

Besides the stabilisation of the aqueous pathways during electroporation, use of keratolytic agents like sodium thiosulphate and urea can create transdermal pathways

during electroporation by disrupting the keratin in the SC (Ilic et al., 1999) The

recovery of the SC lipids post electroporation was also found to show a dependence

on pH and thereby affecting the skin permeability (Murthy et al., 2003) The recovery

of skin was found to be fastest at pH 5 At pH 5, the carboxylic groups of endogeneous fatty acids are protonated and thus the surface charge density of the lipid vesicles is reduced This initiates the fusion process that is crucial to lamellar recovery

2.6 Biophysical techniques

As mentioned in Section 2.1, SC lipids played an important role in the barrier property of the skin Thus several techniques, namely infrared spectroscopy, differential scanning calorimetry, X-ray diffraction and nuclear magnetic resonance, have been employed to study changes in conformation and composition of SC lipids

as well as their effects on subsequent drug permeation through the skin Specifically, the application of Fourier transform infrared spectroscopy to study conformation of

SC lipids is elaborated in the next section

2.6.1 Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FTIR) spectroscopy was extensively employed to study

phospholipid membranes (Casal et al., 1984) This technique was extrapolated to the study of SC lipids by Knutson et al (1987) and there were many similarities between

the spectra of phospholipids and that of SC A typical spectrum of porcine SC sheet is shown in Figure 2.2

Figure 2.2 A typical FTIR spectrum of porcine SC Adapted from Potts et al (1991)

The carbon-hydrogen (C-H) stretching vibrations give rise to bands in the spectral region 3100-2800 cm-1 and the strongest bands in the spectra of lipids correspond with the CH2 asymmetric (νa(CH2)) and symmetric (νs(CH2)) stretching modes at approximately 2920 and 2850 cm-1 respectively A shift in C-H stretching frequency was observed as the SC was heated through the gel to liquid crystalline transition

(Knutson et al., 1987) The molecular basis for the shift in C-H stretching frequency

was described below The wavenumber position of aliphatic C-H stretching bands has been attributed to conformation of the hydrocarbon chains The gel phase is characterised by the predominantly all planar trans conformation Increased flexing and twisting of the chains due to thermally enhanced mobility during the transition

result in the introduction of increased numbers of gauche conformers (Casel et al., 1980; Asher and Levin, 1977; Chapman, 1975; Casel et al., 1979; Levin et al., 1985)

The sensitivity of the C-H stretching frequency to various conformations of hydrocarbons results from changes in the interaction constants between adjacent CH2

groups as the relative orientation of these groups changes with the introduction of the gauche conformers Thus the infrared bands shift towards higher wavenumbers (Casel

et al., 1980; Asher and Levin, 1977; Chapman, 1975; Casel et al., 1979; Levin et al.,

1985) Therefore shifts in wavenumber positions of the bands can be used to determine the introduction of gauche conformers accompanying lipid phase transitions The magnitude of the shift is related to the relative numbers of gauche

conformers being introduced into the hydrocarbon chains (Casel et al., 1980)

A comprehensive study of the temperature induced mobility in SC lipids was reported

by Krill et al (1992) Since FTIR can probe the changes in conformational order in

SC lipids, it has been used to investigate the interaction of chemical penetration

enhancers with the SC lipids to understand the subsequent drug permeation enhancement One of the earliest penetration enhancer investigated was ethanol

Bommannan et al (1991) found a reduction in lipid content in human SC with

attenuated total reflectance infrared spectroscopy after exposure to pure ethanol for 30 min A shift in wavenumber position of CH2 asymmetric (νa(CH2)) and symmetric (νs(CH2)) stretching frequencies was not observed and the group further postulated that lipid removal played an important role in the skin permeability enhancement capability of ethanol Another study on the effect of short chain alcohols on SC structural changes, carried out by Goates and Knutson (1994), further revealed lipid and protein extraction was the main cause of increased mannitol permeation across the skin

Besides ethanol, the permeation enhancement mechanism of oleic acid was also partly

understood with the use of FTIR Golden et al (1987) have shown, by differential

scanning calorimetry and infrared spectroscopy, that skin permeability was proportional to physical changes in the SC lipids caused by isomers of octadecenoic acid The increase in skin permeability as reflected by shifts in νa(CH2) and νs(CH2) did correspond to enhanced skin permeation of both polar and nonpolar solutes

(Bhatia and Singh, 1998; Barry, 1987; Takeuchi et al., 1992) Thus, FTIR has proved

to be a useful tool in understanding the conformation of the SC lipids and proteins by looking at the shift in wavenumber frequencies and the intensity of absorption bands

2.7 Transdermal Delivery of Physostigmine

Physostigmine (PHY) is a lipophilic tertiary amine with a pKa value of 7.9 and is approximately 75% ionized at the pH of blood and brain (Triggle and Filler, 1998)