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University of Cincinnati UNIVERSITY OF CINCINNATI Date: August 29, 2006 I, Smita Chawla hereby submit this work as a part of the requirements of the degree of: Doctor of Philosophy (Ph. D.) in: Pharmaceutical Sciences It is entitled: Effect of deoxyArbutin and it’s second-generation derivatives on melanocyte viability and function. on Human Nail Permeabili ty . This work and its defense approved by: R. Randall Wickett, Ph.D., Chair Raymond E. Boissy, Ph.D. William Cacini, Ph.D. Prashiela Manga, Ph.D. Marty O. Visscher, Ph.D. EFFECT OF DEOXYARBUTIN AND ITS SECOND-GENERATION DERIVATIVES ON MELANOCYTE VIABILITY AND FUNCTION A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Division of Pharmaceutical Sciences of the College of Pharmacy 2006 by Smita Chawla B.S. in Pharmaceutical Sciences Mumbai University, Mumbai, India, 2001 Committee Chair: Randall Wickett, Ph.D. ABSTRACT Therapeutic treatment of skin pigmentary disorders such as melasma, solar lentigines, and post inflammatory hyperpigmentation has been challenging and seldom completely successful. The majority of these therapies target tyrosinase, a key enzyme required for pigment synthesis. The lack of success is due to the less than desired efficacy and safety of tyrosinase inhibitors marketed as skin lightening agents (i.e. hydroquinone, kojic acid and arbutin). We propose that the tyrosinase inhibitor deoxyArbutin (dA) and second-generation derivatives of dA, deoxyFuran, thio-dA, and fluoro-dA, have the potential to be effective inhibitors of skin melanization. In this study, we have analyzed the modulating effects of dA and its derivatives on melanocyte function and viability. At safe concentrations, these compounds reversibly down-regulated melanogenesis by competitively inhibiting the key enzyme, tyrosinase. Lineweaver-Burk plot analysis revealed that these compounds had higher competitive inhibitor potencies against tyrosinase as compared to hydroquinone (HQ). Concentrations of these compounds that did compromise viability of melanocytes resulted in an inhibition of cell proliferation (i.e., cytostatic) as opposed to the induction of apoptosis (i.e., cytotoxic) that was induced by HQ. Melanogenic enzymes; tyrosinase and TRP-1 mediated this cytostatic effect of dA and its derivatives. A comparative evaluation of oxidative stress demonstrated that a minimal amount of Reactive Oxygen Species (ROS) was generated upon treatment with dA and derivatives, in contrast to the dramatic amount induced by HQ. This increase in ROS triggered an increase in activity of the endogenous antioxidant catalase in treated melanocytes. Endogenous catalase was sufficient to protect cells against ROS generated by dA and its derivatives, but not HQ, since treatment with exogenous antioxidants conferred protection against HQ, but not dA and derivatives. Thus, dA and second-generation derivatives demonstrate great potential for therapeutic use in hyperpigmentation because they are effective tyrosinase inhibitors and less toxic relative to the current gold standard, HQ. Dedication This dissertation is dedicated to my father, Ramesh Chawla and my mother, Seema Chawla in the utmost appreciation for their unconditional love and support. ACKNOWLEDGEMENTS Foremost I would like to thank Dr. Raymond Boissy who gave me an opportunity to carry out research of my interest. For his constant advice and guidance. I could not have asked for a better guide and mentor for my graduate studies. At the same note, I would like to thank Dr. Prashiela Manga who has been more a friend than a supervisor. She not only guided me, but was a constant source of inspiration to me. Her advice and support constantly motivated me to work harder and I tried to adopt her perfectionist approach to my work. I really appreciate the concern and warmth she extended towards me throughout. I am very grateful to Dr. Randall Wicket for accepting me in the Cosmetic Science program and teaching me the fundamentals of hair and skin sciences. I want to thank him for his valuable inputs and encouragement that helped during these yrs. And for being there whenever I had a question. I want to thank Dr. Marty Visscher for taking me under her wing during the initial years and for her unwavering support, assistance along the way. I also thank her sincerely for the major financial support she offered during my PhD studies. I am grateful to Dr.William Cacini for being a part of my committee and his useful suggestions that have greatly benefited me in my research. I would like to acknowledge Mitch de long for synthesizing the compounds and his initial work with deoxyArbutin. My heart felt of thanks to Dr. Zalfa Abdel-Malek for her invaluable help, teachings and encouragement. I am extremely appreciative of Dr. Anna Luisa Kadekaro, Dr. Silva Terieva and Renny Starner. I found in them not only a source of knowledge and support but also caring friends. I would like to thank Amy Koshoffer who has been a constant help for the last three years right from ordering my supplies on time to providing me her technical expertise. I wish to thank Howard Epstein for being a supportive and helpful lab mate. Thanks to Bobbie Lambert and Joan Griggs for organizing all the wonderful parties and keeping the fun alive while working. In the Skin Sciences institute, I want to thank William Pickens and Penkanok Sriwiriyanont who took out time from their busy schedule to give me input when I was working on the HPLC machine. Thanks a lot for your assistance and your suggestions. To Lana Wade who was always a joy to talk and interact with, thanks a lot for being a great friend all these years. To Akira Hachiya and Yasuko Yashida for listening about my work n number of times during lab meetings and giving me constructive criticism and advice. I want to thank the faculty of College of Pharmacy especially Dr. Jerry Kasting and Dr. Adel Sakr for their teachings and the staff, Donna, Sue and Marcie for their special assistance. I also want to thank all my friends who made my stay in Cincinnati a pleasant and memorable experience. My friends back home, who even though were not physically present with me, have been my pillar of strength and support throughout. I really thank them for their never-ending belief in me. To Sandipto Banerjee, achieving this goal would not have been possible without his patience, love and support. I really thank him for being a constant part of my life. To my sister, Deepti Chawla for her endless love, sacrifice, and support that helped me achieve this important goal. Above all else the greatest source of inspiration have been my parents, without whose encouragement and blessings I would not have been able to make it this far. I am glad I could make them proud. TABLE OF CONTENTS PAGE Table of contents 1 List of Figures 6 List of Tables 8 CHAPTER 1. INTRODUCTION 9 1.1. Melanocyte Biology 10 1.1.1. Epidermal Melanin Unit 11 1.1.2. Melanosome Formation 12 1.2. Types of Melanin and Function 13 1.3. Melanin Synthesis 14 1.3.1. Eumelanin Synthesis 14 1.3.2. Pheomelanin Synthesis 18 1.4. Factors Influencing Melanogenesis 20 1.5. Melanogenic Enzymes 21 1.5.1. Tyrosinase 22 1.5.2. Tyrosinase-Related Protein 1 (Tyrp1) 23 1.5.3. Tyrosinase-Related Protein 2 (Tyrp2) 24 1.6. Correlation between Skin Color and Melanogenic Enzymes 25 1.7. Pigmentation Disorders 26 1.8. Mechanism of Depigmentation 27 1 PAGE 1.9. Properties of an Ideal Depigmenting Agent 28 1.10. Commercial Tyrosinase Inhibitors 28 1.10.1. Hydroquinone 28 1.10.2. Arbutin 29 1.10.3 Kojic Acid 30 1.11. QSAR of Tyrosinase Inhibitors 31 1.12. Introduction to deoxyArbutin (dA) and its second generation derivatives 32 1.13. Bibliography 36 CHAPTER 2. OBJECTIVES 51 2.1. Efficacy 52 2.2. Safety 52 Chapter 3. MECHANISM OF TYROSINASE INHIBITION OF DEOXYARBUTIN AND SECOND GENERATION 54 DERIVATIVES 3.1. Abstract 55 3.2. Introduction 56 3.3. Results 58 3.3.1. Effect of compounds on melanocyte cell number 58 3.3.2. Inhibition of human tyrosinase activity 60 2 PAGE 3.3.3. Inhibition of melanin synthesis in intact human melanocytes 65 3.3.4. Inhibition of mushroom tyrosinase activity 68 3.3.5. Determination of the kinetic parameters and inhibition mechanism by 70 dA and its derivatives of mushroom tyrosinase 3.3.6. Reversibility of inhibition of tyrosinase activity and melanin synthesis 72 by dA and its derivatives 3.3.7. Effect on melanogenic protein expression 77 3.4. Discussion 78 3.5. Conclusion 85 3.6. Material and Methods 86 3.6.1. Chemicals and test materials 86 3.6.2. Cell culture 86 3.6.3. Cell number assay 87 3.6.4. In situ (intact) tyrosine hydroxylase activity assay 88 3.6.5. Protein and melanin content assay 89 3.6.6. In vitro (lysate) tyrosine hydroxylase activity assay 90 3.6.7. Spectrophotometric assay of DOPAoxidase activity 91 3.6.8. SDS-PAGE and Western Blotting 91 3.6.9. Reversibility assay 92 3.6.10. Mushroom tyrosinase assay 93 3.6.11. Kinetic analysis 93 3 PAGE 3.6.12. Statistical analysis 94 3.7. Bibliography 95 Chapter 4. EVALUATION OF THE SAFETY PROFILE OF DEOXYARBUTIN AND ITS SECOND GENERATION 102 DERIVATIVES AS TYROSINASE INHIBITORS 4.1. Abstract 103 4.2. Introduction 104 4.3. Results 106 4.3.1. Effect of dA and its derivatives on melanocyte cell number 106 4.3.2. Cytostatic versus cytotoxic action of tyrosinase inhibitors 109 4.3.3. Involvement of melanogenic enzymes in cytostatic action 115 4.3.4.
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