Acknowledgements 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. i Table of Contents 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 INTRODUCTION . 1.1 CONTROLLED DRUG DELIVERY . 1.2 TRANSDERMAL AS A ROUTE OF ADMINISTRATION 1.3 STRUCTURE OF THE SKIN 1.4 TRANSPORT OF DRUG ACROSS THE SKIN AND FACTORS INVOLVED 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 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 MOTIVATION AND RESEARCH OBJECTIVES 37 3.1 3.2 MOTIVATION . 37 RESEARCH OBJECTIVES 40 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 - ii - Table of Contents 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.2.9 4.2.10 4.2.11 4.2.12 4.2.13 4.2.14 4.2.15 Preparation of fatty acid containing PHY formulation 43 Preparation of CsA and [D-Ala6]-LHRH formulation . 43 H NMR spectroscopy for PHY and fatty acid . 44 Conductivity measurement 44 Formulation treatment for SC sheets 44 Formulation/fatty acid uptake by SC . 45 Partition coefficient between octanol and water . 45 Apparent partition coefficient of PHY between IPM and vehicle . 45 Fourier transform infrared spectroscopy (FTIR) . 46 Permeation study and calculation of permeation parameters 47 High pressure liquid chromatography assay . 49 Stability studies for CsA and [D-Ala6]-LHRH . 50 Statistical analysis 51 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 - iii - Table of Contents 5.3.1 Stability of [D-Ala6]-LHRH 125 5.3.2 Partition coefficient of [D-Ala6]-LHRH between octanol and water 126 5.3.3 Passive permeation of [D-Ala6]-LHRH . 127 5.3.4 Permeation of [D-Ala6]-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 CONCLUSIONS . 149 RECOMMENDATIONS 152 7.1 7.2 SKIN PERMEATION OF FATTY ACIDS 152 SKIN IRRITATION AND PAIN THRESHOLD STUDIES 152 REFERENCES 153 - iv - List of Figures LIST OF FIGURES FIGURE 1.1 VARIOUS BLOOD DRUG LEVEL PROFILES FROM A ZERO ORDER DOSAGE FORM, A SUSTAINED RELEASE DOSAGE FORM AND A CONVENTIONAL TABLET. (A) MAXIMUM EFFECTIVE CONCENTRATION (B) MINIMUM EFFECTIVE CONCENTRATION. ADAPTED FROM ROBINSON AND LEE. (1987) . FIGURE 1.2 CROSS SECTIONAL VIEW OF SKIN. ADAPTED FROM HOUSEL (2004) 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. 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 1.5 ELECTROPORATION 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 2.3 DECOMPOSITION PATH OF PHY. . 30 FIGURE 2.4 MOLECULAR STRUCTURE OF CYCLOSPORIN A 34 FIGURE 2.5 AMINO ACID SEQUENCE OF LHRH ANALOG USED . 35 FIGURE 4.1 SCHEMATIC 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 5.2 ANALYSIS OF PHY 1H NMR SPECTRUM . 55 FIGURE 5.3 CONDUCTIVITY MEASUREMENTS OF SOLVENT AND 0.5M FATTY ACID CONTAINING SOLVENT BEFORE AND AFTER THE ADDITION OF PHY . 56 v List of Figures FIGURE 5.4 EFFECT OF FATTY ACID AND SOLVENT USED ON THE PERCENTAGE WEIGHT UPTAKE OF FORMULATIONS BY PORCINE SC (N=3-5, MEAN ± SD). 60 FIGURE 5.5 PERCENTAGE WEIGHT UPTAKE OF FATTY ACIDS BY PORCINE SC (N=3, MEAN ± SD). EXPERIMENTS INVOLVING DECANOIC AND LAURIC ACID WERE NOT PERFORMED AS THEY EXIST IN SOLID FORM AT ROOM TEMPERATURE. . 61 FIGURE 5.6 REPRESENTATIVE FTIR SPECTRA OF THE SC BEFORE AND AFTER DELIPIDISATION, AS WELL AS AFTER FORMULATION TREATMENT. (A) TREATED WITH BLANK PG (B) TREATED WITH BLANK MO (C) TREATED WITH OLEIC ACID IN PG (D) TREATED WITH OLEIC ACID IN MO. THE LOCATION OF THE DASHED LINES CORRESPONDS WITH THE VIBRATION FREQUENCY OF THE ASYMMETRIC AND SYMMETRIC PEAKS OF THE LIPIDS IN THE SC BEFORE DELIPIDISATION . 64 FIGURE 5.7 CHANGES IN CH2 C-H STRETCHING VIBRATION FREQUENCIES AFTER FATTY ACID-CONTAINING SOLVENT TREATMENT (N=4-6, MEAN ± SD): (!) MO USED AS THE SOLVENT; ( ) PG USED AS THE SOLVENT.(A) νA(CH2) CHANGES. (B) νS(CH2) CHANGES 66 FIGURE 5.8 PERCENTAGE CHANGES IN PEAK AREA OF INTERCELLULAR LIPIDS MONITORED AT νS(CH2) (N=4-6, MEAN ± SD). ( ) MO USED AS SOLVENT; ( ) PG USED AS SOLVENT. 68 FIGURE 5.9 CUMULATIVE PHY PERMEATED THROUGH AN AREA OF 1.77 CM2 OF THE PORCINE SKIN FOR 34 H IN MO-BASED FORMULATIONS WITH (A) SATURATED FATTY ACIDS (B) UNSATURATED FATTY ACIDS. SATURATED PHY SOLUTIONS WERE USED. (N=3, MEAN ± SD) 71 FIGURE 5.10 CUMULATIVE PHY PERMEATED THROUGH AN AREA OF 1.77 CM2 OF THE PORCINE SKIN FOR 34 H IN PG-BASED FORMULATIONS (N=3, MEAN ± SD). SATURATED PHY SOLUTIONS WERE USED. 73 FIGURE 5.11 CUMULATIVE PHY PERMEATED THROUGH AN AREA OF 1.77 CM2 OF THE TAPE STRIPPED PORCINE SKIN FOR H IN (A) MO-BASED FORMULATIONS (B) PGBASED FORMULATIONS. SATURATED PHY SOLUTIONS WERE USED. (N=3, MEAN ± SD). 76 FIGURE 5.12 EFFECT OF ELECTROPORATION VOLTAGE ON PHY PERMEATION THROUGH ELECTROPORATED SKIN (N=3, MEAN ± SD). SATURATED DRUG DONOR (5 MG/ML PHY IN PBS) WAS USED FOR EACH SOLVENT. ELECTROPORATION PROTOCOL IS 10 PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS . 86 FIGURE 5.13 EFFECT OF NUMBER OF ELECTRICAL PULSES ON PHY PERMEATION THROUGH ELECTROPORATED SKIN (N=3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. DRUG DONOR USED WAS MG/ML PHY IN PBS. . 87 FIGURE 5.14 EFFECT OF CHEMICAL DRUG VEHICLES ON PHY PERMEATION THROUGH ELECTROPORATED SKIN (N=3, MEAN ± SD). DRUG DONOR USED WAS SATURATED SOLUTIONS OF PHY. (A) ELECTROPORATION PROTOCOL IS 10 100 V PULSES OF - - vi List of Figures 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 PERMEATION THROUGH ELECTROPORATED SKIN (N=3, MEAN ± SD). DRUG DONOR USED WAS SATURATED SOLUTIONS OF PHY. ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. . 90 FIGURE 5.16 EFFECT OF OLEIC ACID SKIN PRETREATMENT ON PHY PERMEATION THROUGH ELECTROPORATED SKIN (N=3, MEAN ± SD). PRETREATMENT WAS CARRIED OUT BY INCUBATING THE SC SURFACE WITH 0.5M OLEIC ACID IN PG FOR H BEFORE ELECTROPORATION OR DRUG DONOR (FOR PASSIVE PERMEATION) WAS APPLIED. ELECTROPORATION PROTOCOL IS 10 100/500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. DRUG DONOR USED WAS MG/ML PHY IN PBS. 92 FIGURE 5.17 COMPARISON OF PHY PERMEATION BETWEEN TAPE STRIPPED SKIN AND ELECTROPORATED SKIN (N=3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 100/500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. DRUG DONOR USED WAS SATURATED SOLUTIONS OF PHY IN PBS. 93 FIGURE 5.18 EFFECT OF TEMPERATURE AND SKIN HYDROLYTIC ENZYMES ON CSA STABILITY IN PBS FOR 24 H. STIRRING WAS CARRIED AT 600 RPM. CONCENTRATION OF ETHANOL USED WAS 50% V/V IN PBS. THE PREPARATION OF SKIN EXTRACT WAS DESCRIBED IN SECTION 4.2.14 . 106 FIGURE 5.19 PASSIVE PERMEATION OF CSA THROUGH 1.77 CM2 OF PORCINE EPIDERMIS IN DIFFERENT DRUG VEHICLES FOR 24 H (N = 3, MEAN ± SD). CSA WAS NOT DETECTED IN THE RECEPTOR AFTER 24 H WHEN PBS WAS USED AS DRUG VEHICLE. 109 FIGURE 5.20 EFFECT OF ELECTROPORATION VOLTAGE ON CSA PERMEATION THROUGH ELECTROPORATED SKIN (N = 3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. DRUG VEHICLE USED WAS 0.5M OLEIC ACID IN ETHANOL . 110 FIGURE 5.21 EFFECT OF DRUG VEHICLE ON CSA SKIN PERMEATION AFTER ELECTROPORATION (N = 3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. 111 FIGURE 5.22 EFFECT OF CHAIN LENGTH OF FATTY ACID IN VEHICLE ON CSA PERMEATION THROUGH ELECTROPORATED SKIN (N = 3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. CONCENTRATION OF FATTY ACID USED IS 0.5M AND DRUG VEHICLE USED IS ETHANOL. . 112 FIGURE 5.23 EFFECT OF CONCENTRATION OF OLEIC ACID IN VEHICLE ON CSA PERMEATION THROUGH ELECTROPORATED SKIN (N=3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. DRUG VEHICLE USED IS ETHANOL. . 113 - - vii List of Figures FIGURE 5.24 EFFECT OF TYPE OF CARRIER FOR OLEIC ACID ON CSA PERMEATION THROUGH ELECTROPORATED SKIN (N = 3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. CONCENTRATION OF OLEIC ACID IN EACH VEHICLE IS 0.5M. . 114 FIGURE 5.25 EFFECT OF OLEIC ACID PRETREATMENT ON CSA PERMEATION THOUGH ELECTROPORATED SKIN (N = 3, MEAN ± SD). (A) WITHOUT OLEIC ACID PRETREATMENT (B) WITH OLEIC ACID PRETREATMENT. PRETREATMENT INCLUDES THE INCUBATION OF SC SURFACE WITH 0.5M OLEIC ACID IN PG FOR H BEFORE THE ONSET OF ELECTROPORATION. ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS . 115 FIGURE 5.26 EFFECT OF VEHICLE NATURE ON CSA PERMEATION THROUGH TAPE STRIPPED SKIN (N = 3, MEAN ± SD) . 117 FIGURE 5.27 EFFECT OF TEMPERATURE AND SKIN HYDROLYTIC ENZYMES ON [D-ALA6]LHRH STABILITY IN PBS FOR 24 H. STIRRING WAS CARRIED AT 600 RPM. CONCENTRATION OF ETHANOL USED WAS 50% V/V IN PBS. THE PREPARATION OF SKIN EXTRACT WAS DESCRIBED IN SECTION 4.2.14 . 126 FIGURE 5.28 PASSIVE [D-ALA6]-LHRH SKIN PERMEATION IN DIFFERENT VEHICLES (N = 3, MEAN ± SD). [D-ALA6]-LHRH WAS NOT DETECTED IN THE RECEPTOR AFTER 24 H WHEN CARRIED IN PBS, ETHANOL OR PG 128 FIGURE 5.29 EFFECT OF ELECTRICAL PROTOCOL ON [D-ALA6]-LHRH SKIN PERMEATION (N = 3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. DRUG VEHICLE USED IS 0.25M OLEIC ACID IN 50/50 ETHANOL AND PG. 129 FIGURE 5.30 EFFECT OF DRUG VEHICLE ON [D-ALA6]-LHRH SKIN PERMEATION AFTER ELECTROPORATION (N = 3, MEAN ± SD). ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. 130 FIGURE 5.31 EFFECT OF CHAIN LENGTH OF FATTY ACID ON [D-ALA6]-LHRH PERMEATION (N = 3, MEAN ± SD). DRUG VEHICLE USED IS 50/50 ETHANOL/PG. BLANK REFERS TO PURE 50/50 ETHANOL/PG. ELECTROPORATION PROTOCOL IS 10 500 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 PERMEATION AFTER ELECTROPORATION (N = 3, MEAN ± SD). DRUG VEHICLE USED IS 50/50 ETHANOL/PG. ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS 132 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 DURATION AT INTERVAL OF 900 MS. CONCENTRATION OF OLEIC ACID USED WAS 0.25M. . 133 - - viii List of Figures FIGURE 5.34 EFFECT OF OLEIC ACID PRETREATMENT ON [D-ALA6]-LHRH PERMEATION THROUGH ELECTROPORATED SKIN (N = 3, MEAN ± SD) (A) WITHOUT OLEIC ACID PRETREATMENT (B) WITH OLEIC ACID PRETREATMENT. ELECTROPORATION PROTOCOL IS 10 500 V PULSES OF 100 MS DURATION AT INTERVAL OF 900 MS. SKIN WAS PRETREATED WITH 0.5M OLEIC ACID IN PG FOR H BEFORE ELECTROPORATION. 135 FIGURE 5.35 [D-ALA6]-LHRH PERMEATION THROUGH TAPE STRIPPED SKIN (N = 3, MEAN ± SD). SC THICKNESS WAS REDUCED BY TAPE STRIPPING 20 TIMES BEFORE ONSET OF PERMEATION STUDIES. . 137 - - ix List of Tables 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 = 3, MEAN ± SD…………… …………………………………….… .58 TABLE 5.2 CHANGES IN νS(CH2) PEAK AREA/PEAK HEIGHT RATIOS AS A FUNCTION OF FATTY ACIDS AND SOLVENT (N = 4-6, MEAN ± SD)………………… 69 TABLE 5.3 SUMMARY OF DRUG RELEASE PARAMETERS FOR PHY PERMEATION THROUGH THE PORCINE SKIN FOR 34 H (N = 3, MEAN ± SD) .74 TABLE 5.4 SUMMARY OF DRUG RELEASE PARAMETERS FOR PHY PERMEATION THROUGH TAPE STRIPPED PORCINE SKIN (N = 3, MEAN ± SD) 77 TABLE 5.5 SUMMARY OF PERMEATION PARAMETERS FOR PHY PERMEATION THROUGH ELECTROPORATED SKIN FOR 10 H (N = 3, MEAN ± SD) 94 TABLE 5.6 PHY SOLUBILITY IN VARIOUS DRUG VEHICLES …………….…… … .101 TABLE 5.7 PARTITION COEFFICIENTS OF CSA BETWEEN OCTANOL AND WATER IN THE PRESENCE OF FATTY ACID………………………………………… .107 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 STUDIES…………………………………… ………… 119 TABLE 5.11 SUMMARY OF CSA SKIN PERMEATION EXPERIMENTS (N = 3, MEAN ± SD)…………… ……………………………… ………………… 120 TABLE 5.12 PARTITION COEFFICIENTS OF [D-ALA6]-LHRH BETWEEN OCTANOL AND WATER IN PRESENCE OF FATTY ACID (N = 3, MEAN ± SD)………… 127 TABLE 5.13 [D-ALA6]-LHRH PERMEATION ENHANCEMENT AND SC BARRIER RATIOS……………………………………………………… 138 TABLE 5.14 SUMMARY OF [D-ALA6]-LHRH SKIN PERMEATION EXPERIMENTS (N = 3, MEAN ± SD)………………… ……………………………………… 143 x Conclusions electroporation, the degree of permeation enhancement was not as high as that observed for PHY. 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 solubility in ethanol. Nonetheless, CsA permeation rate through skin was not sufficient for application such as immunosuppression after organ transplantation. However, coupled with high CsA retention within skin, skin permeation of CsA may be suitable for topical treatment of psoriasis. Permeation of [D-Ala6]-LHRH through skin was improved tremendously after electroporation. Although CsA and [D-Ala6]-LHRH had similar molecular weights, [D-Ala6]-LHRH permeation through skin was 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. These promising findings indicated that therapeutic delivery of [D-Ala6]LHRH across the skin could be achieved without the application of an electrical driving force. It has been demonstrated here that the use of electroporation as a tool to permeabilise the skin was feasible. Coupled with the use of fatty acids, in particular oleic acid, greater permeation of drug across the skin could be achieved. Since electroporation was applied to the skin before the onset of drug delivery, this protocol could be integrated with existing transdermal technology to reduce the long lag time for small molecules and to allow permeation across the skin for larger molecules. 151 Recommendations RECOMMENDATIONS This thesis focused on the understanding of enhancement mechanisms of fatty acids in transdermal drug delivery and the development of electroporation as a tool to increase skin permeability. Combination of electroporation and fatty acids has shown promise in enhancing skin permeation of drug. However, there are some issues that need to be addressed before practical application. Further studies could be carried out to evaluate in vivo aspects. However, the focus of the thesis remains with the understanding of transdermal drug delivery using physicochemical principles involving excised skin and formulations. 7.1 Skin permeation of fatty acids For safety concerns, the permeation rate of fatty acids through skin needs to be determined. Knowledge of permeation rate of fatty acids through skin will aid in the design of the optimum drug formulation and also serves to provide a deeper insight into their permeation enhancement mechanisms. 7.2 Skin irritation and pain threshold studies The irritation potential of electroporation to skin and its associated damage remain to be determined. More importantly, fatty acids permeation across electroporated skin will be enhanced and this may raise potential skin irritation issues. 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Enhanced delivery of naked DNA to the skin by non-invasive in vivo electroporation. Biochim. Biophys. Acta 1572, 1-9. 166 [...]... 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... Yang and P W S Heng, Strategy to increase skin permeation of peptides via combination of electroporation and fatty acids, (Manuscript in preparation) • M.Y Wang, Y.Y Yang and P W S Heng, Skin permeation of physostigmine from fatty acids- based formulations: Evaluating the choice of solvent, (Accepted for Inter J Pharm.) • M.Y Wang, Y.Y Yang and P W S Heng, Role of solvent in interactions between fatty acids- based... (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. .. 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... acids- based formulations and lipids in porcine stratum corneum, J Control Release, 94(2004) 207-216 Conference Papers • M.Y Wang, Y.Y Yang and P W S Heng, New insights into skin permeation enhancements by fatty acids, Materials Research Society Fall Meeting 2003, Boston, USA Oral presentation • M.Y Wang, Y.Y Yang, P W S Heng and S Moochhala, Transdermal Delivery of Physostigmine with Fatty Acids based formulations... Summary 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... 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... Publications and Conference Papers LIST OF INTERNATIONAL REFEREED PUBLICATIONS AND CONFERENCE PAPERS International Refereed Publications • M.Y Wang, G.L Xu, Y.Y Yang and P W S Heng, Oleic acid enhanced physostigmine permeation through skin post electroporation, (Manuscript in preparation) • M.Y Wang, Y.Y Yang and P W S Heng, Enhanced skin permeation of cyclosporin A by using combination of chemicals and electroporation, ... 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. .. 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 . 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. 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. 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,