Methods development for the in vitro assessment of cutaneous drug metabolism Item Type text; Thesis-Reproduction (electronic) Authors Thohan, Sanjeev, 1962- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 25/09/2021 03:16:04 Link to Item http://hdl.handle.net/10150/277962 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. 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Contact UMI directly to order. University Microfilms International . A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 1345441 Methods development for the in vitro assessment of cutaneous drug metabolism Thohan, Sanjeev, M.S. The University of Arizona, 1991 U-M-I 300N.ZeebRd. Ann Arbor, MI 48106 Methods Development for the In Vitro Assessment of Cutaneous Drug Metabolism by Sanjeev Thohan Sanjeev Thohan 1991 A Thesis Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE WITH A MAJOR IN TOXICOLOGY In the Graduate College The UNIVERSITY OF ARIZONA 199 1 2 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interest of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED:, APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: Date Professor and Head of Pharmacology afid Toxicology 3 Dedication I dedicate this thesis to my parents, Tarsem Lall and Rama Thohan. It is with the constant love and support of my entire family that this research effort was brought to its fruition. I think Kabir said it very well in couplet VI.8: What a stone you have become, ever absorbed in intellectual pursuits not a drop of love has touched you. O Kabir, remember: Without love it is all worthless and dreary. 4 Acknowledgements I owe a great debt of gratitude to Dr. Inderjit Singh for allowing me, as an undergraduate, to participate in his research efforts at Johns Hopkins. I also am indebted to Dr. Julie Eiseman and Dr. Merrill Egorin at the University of Maryland Cancer Center (UMCC). It was through your guidance and support that I was able to make the right decisions to be where I am. As for the rest of the UMCC staff, thank you all for providing a very productive and pleasant work environment. Thanks for the many enjoyable lunches at the "Market" whenever I came home and keeping in touch with me. I would like to take this chance to thank my major advisor Dr. I. Glenn Sipes for not only providing me with a positive environment in which to conduct scientific research, but for his critical evaluations, guidance and patience over the duration of this work. I also like thank Dr. Dean E. Carter for his assistance in the direction of this thesis. Dean, your door was always open to me and / will be indebted to you for the many lessons I learned in my discussions with you. Dr. Klaus Brendel, I thank you for the many insightful discussions and assistance with the completion of this work. Dr. John Barr thanks for your friendship, guidance, tutelage and critical insight into my development as a scientist. John, you have been a friend in every sense of the word. I miss those discussions on philosophy and some of those obtuse questions that came up on sleepless 'Liver Nights'. You always saw the good in all situations and helped me to realize more than I would have done on my own. Thank you my friend. To my 'partners in crime': Kevin Divine, Timothy Harlan, and Jonathan Patrick Rigsby Francis Monteleone: "You were right, I don't know how I would have made it without you guys." Thanks for always being there and those incredible "Power Science Lunches." Sanjeev Sharma thanks for many an '!impromptu night." Also, Sanjeev you were there to help me to see the good in situations that I thought were bad. Latika, Michele, Nilima and Suresh, thank you for your support from 'home'. Suketu Desai, thanks for your friendship and unique viewpoint. Dr. Shana Azri, thanks for being there on the other end of the phone. Ms. Jeanette O'Hare, thank you for invaluable assistance with art and visual aids throughout this work. Ms. Rita Pedroza, thanks for not only your help and friendship but also for your unique understanding of life. Finalfy, 1 would like to thank Tariok (Kandi) Kandola and Harjinder Gill for their brotherly love and for providing me with a family atmosphere here in the desert. I consider myself to very fortunate to have had advisors and friends such as you in my time here. TABLE OF CONTENTS List of Figures 10 List of Tables 12 Abstract 14 Introduction .15 1.0 Drag Metabolism - General Introduction 16 1.1 The Mixed Function Oxidase (MFO) System 19 1.1.1 Brief History 19 1.1.2 Components of the MFO system 20 1.1.2.1 Cytochrome P-450 (P-450) "... 20 1.1.2.2 Multiplicity of P-450 20 1.1.2.3 NADPH Cytochrome P-450 reductase (reductase) — 22 1.1.2.4 Cytochrome b5 22 1.1.2.5 Lipid 23 1.1.3 Organization of the MFO System 24 1.1.4 Generalized Reaction Scheme for Microsomal Oxidations T...... 27 1.1.5 Generalized Scheme for Microsomal Reductive - Reactions 30 1.1.6 Rate Limiting Step 31 1.1.7 Induction 33 1.1.7.1 Induction of Rat liver P-450 Isozymes 34 1.2 Conjugation Reactions- Phase n . 38 1.2.1 Cassification of Phase n Reactions 38 1.2.1.1 Glucuronidation 41 1.2.1.2 Sulfation 43 2.0 Skin - General Introduction . 45 2.1 Anatomy 45 2.1.1 Epidermis 46 2.1.2 Dermis 46 2.1.3 Subcutaneous Tissue 47 2.1.4 Cutaneous Appendages 47 6 2.2 Metabolism in Skin 51 2.2.1 Carbohydrate Metabolism 51 2.2.2 Glucose Metabolism 53 _ 2.2.3 lipid Metabolism 53 2.2.4 Protein Metabolism ;.. 54 2.2.5 Steroid - Hormone Metabolism 55 2.2.6 Cutaneous Phase I Metabolism 57 2.2.7 Cutaneous Phase II Metabolism 60 2.2.7.1 Glucuronidation 60 2.2.7.2 Sulfation 60 2.2.7.3 Methylation 61 3.0 Purpose 62 4.0 Model Substrates 64 4.1 Alkoxycoumarins 64 4.2 Paranitrophenol Conjugates 65 Materials and Methods 66 1.0 Animals 67 2.0 Human Skin 68 3.0 Chemicals 68 4.0 Animal Pretreatment 68 5.0 Subcellular Fractionation 69 5.1 Skin Microsomal Preparation 70 5.2 Liver Microsomal Preparation 73 6.0 Cutaneous Culture Techniques 73 6.1 Short Term Culture: "Swirly Culture" 73 6.2 Long Term Culture: LTC 74 7 7.0 Assays 75 7.1 Protein Determination 75 7.2 Cytochrome P-450 Content 75 7.3 Cytochrome c Reductase Determination 76 7.3 O-Dealkylase Activity Determination 76 7.3.1 Metabolite Analysis 77 7.3.1.1 Microsomal Incubations 77 7.4 Paranitrophenol (PNP) Deconjugation Assay 80 8.0 Statistical Analysis 80 Results 81 1.0 Protein Determinations 82 1.1 Species Differences in Microsomal Protein Yield 85 2.0 Species Comparison - Microsomal Studies 87 2.1 Cytochrome P-450 Determination 87 2.2 NADPH Cytochrome c Reductase Determination 89 2.3 Alkoxycoumarin Metabolism 95 2.3.1 7-EC Metabolism 99 2.3.2 7-MC Metabolism 100 3.0 The Effects of Aroclor Pretreatment on Rat Microsomal Activities 102 3.1 Microsomal Protein Yield 102 3.2 Cytochrome P-450 Determination 103 3.3 NADPH Cytochrome c Reductase Determination 106 3.4 Alkoxycoumarin Metabolism 109 3.4.1 7-EC Metabolism Ill 3.4.2 7-MC Metabolism Ill 8 4.0 Culture of Rat Skin Explants 113 4.1 Xenobiotic Metabolism by Rat Skin Explants in Swirly Culture ..