INVESTIGATION OF NOVEL PENETRATION MODIFIERS AS ENHANCERS AND RETARDANTS AND HUMAN PHARMACOKINETICS AND PHARMACODYNAMICS OF ORALLY ADMINISTERED QUERCETIN BY DIKSHA KAUSHIK A Dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the degree of Doctor of Philosophy Graduate Program in Pharmaceutical Science Written under the direction of Professor Bozena B. Michniak-Kohn, Ph.D. And approved by ……………………………………………… ……………………………………………… …………………………………………….. ……………………………………………….. New Brunswick, New Jersey October 2010 ABSTRACT OF THE DISSERTATION INVESTIGATION OF NOVEL PERCUTANEOUS PENETRATION MODIFIERS AS ENHANCERS AND RETARDANTS AND HUMAN PHARMACOKINETICS AND PHARMACODYNAMICS OF ORALLY ADMINISTERED QUERCETIN BY DIKSHA KAUSHIK Dissertation Director: Professor Bozena B. Michniak-Kohn, Ph.D. The delivery of the actives through the outermost layer of the skin, the stratum corneum has posed a challenge for scientists for a very long time. There are compound called “enhancers” that have been developed to enhance drug delivery across skin and at the same time there are compounds referred to as “retardants” that prevent or retard the delivery of actives across the skin. Since both enhancers and retardants are believed to act by modifying the properties of the SC barrier, they are collectively referred to as “penetration modifiers”. The first part of the study aimed at investigating five penetration modifiers that are analogues of laurocapram and iminosulfurane. These penetration modifiers were evaluated initially for their enhancement/retardation potential by computer modeling and investigated for their physical attributes (solubility, lipophilicity, etc.) and formulation effects. The effect of permeation modifier formulations (prepared in commonly used pharmaceutical vehicles) on permeation of model permeants were evaluated and mechanistic studies were performed using thermal, spectral and ii microscopic analyses. The penetration modifiers were also assessed for their in vitro permeation and cytotoxicity using MTS assay. These investigations should lead to a better understanding of the properties, effects and mechanisms of action of penetration modifiers. The second part of the dissertation covers human pharmacokinetics and pharmacodynamics of quercetin. Quercetin is a naturally occurring flavonoid with antioxidant and anti-inflammatory potential. In order to assess some of benefits of quercetin, this investigation examined the pharmacokinetics and pharmacodynamics of orally administered formulations to human healthy volunteers. Initially, we developed and validated a quercetin assay in human plasma and urine followed by selection of suitable oral carrier for pharmacodynamics study based on pharmacokinetic profiles of three nutritional carrier systems. This was followed by determination of the effects of quercetin supplementation on improving maximal oxygen uptake, enhancing exercise performance during heat stress and relief of muscle soreness. We expect to make conclusions regarding effects of quercetin in overall improvement in work performance of human subjects that would in turn enable the U.S. Department of Defense to consider inclusion of quercetin in daily rations for soldiers to further improve their performance. iii Dedication To Almighty God To my husband, Ranajoy and our family Acknowledgements The writing of a dissertation can be a difficult experience which is obviously not possible without the personal and practical support of numerous people. It is impossible to express my gratitude to my Ph.D. advisor, Professor Bozena B. Michniak-Kohn. Without her inspirational guidance, her enthusiasm, her encouragements and her unselfish help, I could never finish my doctoral work in Rutgers University. For me, she is not only a teacher, but also a lifetime friend and advisor. My thanks go also to the members of my advisory committee, Professor Tamara Minko, Dr. Guofeng You and Dr. Nava Dayan for their time, guidance and helpful discussions. I express gratitude to Dr James Chapman (University of South Carolina, Columbia) for providing N-0915 and TBDOC. In addition, I acknowledge the assistance of Drs. A. Joy, D. Bolikal, P. Chandra, J. Khan, S. Murthy and J. Kohn from the NJ Center for Biomaterials for providing technical and professional support. My sincere thanks to Drs. P. Dunne (NSRDEC), K. Conca (NSRDEC), E. Zambraski (USARIEM), P. Clarkson (U. Massachusetts), S. Cheuvront (USARIEM), L. Armstrong (U. Connecticut) and M. Ganio (U. Connecticut) and Kevin O’Fallon for technical and professional assistance. I thank my seniors Drs. R. Thakur and P. Batheja and fellow students Brian Kilfoyle, Vishwas Rai, Sergio D’Silva, L. Hu and P. Zhang for their help and support in my research. iv Finally, my sincere gratitude goes to my husband, Ranajoy, daughter, Aneesha, parents, parents-in-law, sisters, friends and all my colleagues and superiors in the Ernest Mario School of Pharmacy and NJ Center for Biomaterials for their love, support, and patience over the last few years. v TABLE OF CONTENTS Abstract of the dissertation …………………………………………………………….. ii Dedication/Acknowledgement………………………………………………….…….... iv Abbreviations………………………………………………………………………….. xv List of Tables………………………………………………………………………….. xix List of Figures……………………………………………………………………...…xxiii List of Structures………………………………………………………………….…xxvii Part 1……………………………………………………………………………………..1 Chapter 1: Introduction………………………………………………………………………………2 Chapter 2: Background and significance………………………………………............5 2.1 Background.................................................................................................................... 5 2.2 Characteristics of ideal penetration modifiers..............................................................6 2.3 Theory............................................................................................................................ 8 2.4 Enhancement vs. retardation.......................................................................................10 2.4.1 Factors affecting performance of transdermal permeation modifiers.............10 2.4.1.1 Molecular shape and size..............................................................................10 vi 2.4.1.2 Hydrogen bonding potential and polarity.....................................................11 2.4.1.3 Chemical structure........................................................................................12 2.4.1.4. Formulation effect........................................................................................12 2.5 Characterization of penetration modifiers...................................................................14 2.6 Penetration modifiers in drug delivery........................................................................15 2.7 Significance.................................................................................................................. 18 Chapter 3: Specific aims……………………………………………………………….. 20 Chapter 4: Penetration modifiers and formulation effects………………………….. 22 4.1 Introduction..................................................................................................................22 4.2 Materials and methods................................................................................................. 24 4.2.1 Materials.......................................................................................................... 24 4.2.1.1 Chemicals...................................................................................................... 24 4.2.1.2 Skin membranes............................................................................................24 4.2.2 Methods............................................................................................................25 4.2.2.1 Preparation of formulations......................................................................... 25 4.2.2.2 Determination of solubility and available amounts of penetration modifiers in vehicles...................................................................................................... 25 4.2.2.3 Modeling and partition coefficient determination of penetration modifiers........................................................................................................25 vii 4.2.2.4 In vitro skin permeation study...................................................................... 26 4.2.2.5 Analysis of DEET and penetration modifiers............................................... 28 4.2.3 Data and statistical analysis............................................................................28 4.3. Results and discussion................................................................................................ 29 4.3.1 Determination of solubility and available amounts of penetration modifiers in vehicles.............................................................................................................. 29 4.3.2 Modeling and partition coefficient determination of penetration modifiers.............................................................................................................30 4.3.3 In vitro skin permeation study results.............................................................. 31 4.3.3.1 Effect of various formulations of laurocapram on permeation of DEET.............................................................................................................. 32 4.3.3.2 Effect of N-0915