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. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. HQgher quality 6 ” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Infonnation Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 IN VITRO MECHANISTIC STUDIES OF PEROXISOME PROLIFERATION BY CHIRAL CLOFIBRATE ANALOGS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shamina M. Rangwala, B. Pharm. ***** The Ohio State University 1997 Dissertation Committee: Dr. Dennis R. Feller, Adviser Approved by Dr. Norman J. Uretsky, Co-Adviser Dr. Lane J. Wallace Adviser ' Dr. Bethany J Holycross College of Pharmacy ÜMI Number: 9801766 UMI Microform 9801766 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT The mechanism by which clofibrate causes peroxisome proliferation is not known. A series of chiral clofibrate-related 2-(4-chlorophenoxy)acetic acid (CPAA) analogs were selected to examine the effects of stereoselectivity on activation of peroxisome proiiferator-activated receptor (PPAR) and stimulation of peroxisomal activity. The abilities of these compounds to activate the receptor were examined using transactivation assays using exogenously added PPARa in CV-1 and endogenous PPAR in H4IIEC3 cells. In both systems, the 2-n-propyl and 2-phenyl-substituted CPAA analogs were highly stereoselective activators of PPARa, suggesting that these compounds act via a receptor. The effects of these analogs on peroxisomal acyl-CoA oxidase aciidty was determined. There exists a strong correlation between the ability of a compound to activate PPAR and stimulate fatty acyl-CoA oxidase (AGO) activity, implicating a causal relationship between these events. Small modifications in structure around the 2 position of CPAA can result in dramatic alterations in stereoselectivity and potency, e.g., changing the n-propyl substituent to the isopropyl substituent results in the loss of stereoselectivity, while the substitution of the n-hexyl group with the phenyl group at the 2 -position results in a decrease in activity, however, the stereoselectivity increases strikingly. Enlargement of the chlorophenoxy group to the chlorobenzyloxy group leads to an increase in activity of the compound; however, addition of one more phenyl ring to this side-chain decreased activity, but increased stereoselectivity. We showed that the rank order o f stereoselectivity observed for activating PPAR and stimulating ACO activity is not conserved for displacing tritiated oleic acid from L-FABP. This indicates that binding to liver fatty acid binding protein (L-FABP) is not predictive of the peroxisome proliferative effects of these compounds. However, for non-metabolizable fatty acids tested, such as 2- bromopalmitic acid and perfluorinated octanoic acid, L-FABP may play an important role in the signaling pathways for these molecules as compared to xenobiotic peroxisome proliferators. Ciprofibrate and 2-n-propyl- and 2-phenyl-substituted CPAA analogs bind to the Xenopus PPARa ligand binding domain, displacing leukotriene B$ from this binding site. Thus, the peroxisome proliferative effects of the fibrates are probably mediated by the direct binding of these compounds to the PPARa. in To my parents, Mohsin and Sehra Rangwala, without your encouragement, faith and support, I would have never come this far. With my love and thanks. IV ACKNOWLEDGMENTS I would like to thank; Dr. Dennis Feller, for his advice, support and constant enthusiasm throughout my graduate career. Dr. Norman Uretsky, my co-adviser at OSU, for his advice and help, especially in the past two years. Other members of my dissertation committee, Drs. Wallace and Holycross, for their assistance during the preparation of this dissertation. Dr. Dan Noonan, Michelle O’Brien and George Yuan, at the University of Kentucky, for their friendship, help and support throughout this research. Dr. David Pasco, Chuan-Li Xu, Vincent Siu and all the other members of the Pasco lab at Ole Miss for their assistance and friendship and for tolerating my constant questions while setting up some of the experimental methods! Dr. JefiF Lawrence and Dr. Patrick Eacho, Eli Lilly and Company, for their assistance. Rose Smith and Kathy Brooks at OSU for their cheerful, eflBcient personalities, which helped making the move to Ole Miss less stressful. Becky Drewery for her friendship and help, especially while this dissertation was being written. My friends, both at OSU and Ole Miss; Suzette, Joya, Marina, Subbu, Ratna, Anish, Edwin, David, Reshma, Grace and Vivek, for their companionship which always made me feel like I belonged; thank you for making my graduate school experience an entirely enjoyable one. My family, whose love, encouragement and support has sustained me throughout my graduate study. VI VITA June 13, 1971 ............................................... Born-Bombay, India 1992 ..............................................................Bachelor of Pharmaceutical Sciences, University of Bombay. 1992 - 1993 .................................................. University Fellow, The Ohio State University. 1993 - present ...............................................Graduate Teaching and Research Associate The Ohio State University PUBLICATIONS O'Brien ML, Rangwala SM, Henry KW, Weinberger C, Crick DC, Waechter CJ, Feller DR and Noonan DJ. Convergence of three steroid receptor pathways in the mediation of nongenotoxic hepatocarcinogenesis. Carcinogenesis 17: 185-190, 1996. Feller DR, O'Brien M, Rangwala SM, Tortorella V, Loiodice F and Noonan DJ. Structural requirements of chiral clofibric acid analogs for activation of the rat peroxisome proiiferator-activated receptora (rPPARa). Ann. N. Y. Acad. Sci. 804: 713-715, 1996. Rangwala SM, O'Brien ML, Loiodice F, Longo A, Tortorella V, Noonan DJ and Feller DR. Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proiiferator- activated receptor (PPARa) activation and peroxisomal fatty acid B-oxidation. Chirality 9: 37-47, 1997. vu Rangwala SM, O'Brien ML, Noonan DJ and Feller DR. Differential effects of statins on peroxisomal acyl CoA activity and the rat peroxisome proliferator activated receptor (rPPAR). FASEBJ. 9: A690, 1995. Rangwala SM, O'Brien M, Loiodice F, Tortorella V, Noonan DJ and Feller DR. Specificity of the effects of isomeric clofibric acid analogs on hepatic peroxisome proliferation. Toxicologist 3d'. 1\0, 1996 Rangwala SM, O’Brien M, Lawrence J, Eacho P, Tortorella V, Loiodice F, Longo A and Feller DR. Studies on the mechanism of peroxisome proliferation by clofibric acid analogs. Toxicologist 252, 1997. FIELD OF STUDY Major Field: Pharmacy Pharmacology vui TABLE OF CONTENTS Page Abstract................................................................................................................................ ii Dedication ............................................................................................................................ iv Acknowledgments ................................................................................................................ v Vita......................................................................................................................................vii List of Figures ....................................................................................................................xiii List of Tables .....................................................................................................................xvi Chapters: 1. Introduction ..............................................................................................................1 1.1 Peroxisomes ................................................................................................1 1.1.1 Morphology .......................................................................................1 1.1.2 Biogenesis ......................................................................................... 2 1.1.3 Biochemistry.....................................................................................2 1.2 Peroxisome