PHARMACOKINETICS, PHARMACODYNAMICS, METABOLISM, TRANSPORT, AND RESISTANCE STUDIES OF A NOVEL HISTONE DEACETYLASE INHIBITOR FK228 (FR901228, NSC630176) DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jin Xiao, M.S. ***** The Ohio State University 2004 Dissertation Committee: Approved by Dr. Kenneth K. Chan, Adviser Dr. John C. Byrd Dr. William L. Hayton ___________________ Adviser Dr. James T. Dalton Graduate program in Pharmacy ABSTRACT Depsipeptide FK228 (FR901228, NSC630176), a promising histone deacetylase (HDAC) inhibitor, is currently undergoing clinical evaluation against various malignancies. Pharmacokinetic study of FK228 in the rat was first conducted with a focus on its pharmacokinetic properties and dose recovery. FK228 was found to be removed rapidly from the circulation with a total body clearance higher than the rat cardiac output, suggesting extensive metabolism in the blood. Dose recovery of FK228 was low (<15%) and there was an involvement of glutathione in FK228 elimination. Based on these results, we conducted in vitro metabolism studies, which led to identification of four glutathione conjugates and two thiols from rat and human blood incubations. Purification of these major metabolites followed by HDAC inhibition assays indicated that FK228 is a prodrug, with three major metabolites being more potent HDAC inhibitors than FK228 itself. A clinical pharmacokinetic study in AML and CLL patients was conducted. A pharmacokinetic-pharmacodynamic correlation study showed that HDAC inhibitory activity was inversely correlated with FK228 systemic exposure. This provided the first clinical evidence that FK228 was a prodrug. ii FK228 transport and uptake was investigated. It was found that FK228 is a substrate for both MDR1 and MRP1 by transport and cytotoxicity studies using specific inhibitors of the membrane transporters. In order to study the roles of MDR1 and MRP1 in acquired FK228 resistance, four FK228 resistant cell lines were established and characterized. We found that upregulation of MDR1, but not MRP1 or other ABC transporters, was responsible for the acquired resistance. The maintenance of acquired FK228 resistance depended on continuous drug exposure. No deregulation or impairment of the histone acetylation machinery was found. The MDR1 upregulation was further found to be via a reversible induction procedure, in which FK228 first inhibited HDACs and then caused hyperacetylation at the MDR1 promoter region to form the euchromatin structure ready for transcription. Overall, the dissertation work, involving both preclinical and clinical studies, provided valuable information of pharmacokinetics, bioactivation, transport and uptake, and resistance of the novel anticancer drug FK228. iii DEDICATION To my dear wife Sunny For her love and support ii ACKNOWLEDGMENTS I would like to extend my gratitude to my adviser, Dr. Kenneth K. Chan, for his scientific guidance, constant encouragement and financial support. I would also like to thank my committee members, Drs. William Hayton, James Dalton, and John Byrd for their valuable discussions. I am indebted to my collaborators: Amy Foraker, Drs. Peter Swaan, Wolfgang Sadée, Ying Huang, Zunyan Dai, Guido Marcucci and Shujun Liu for their technique assistance and discussions. I am grateful to my labmates: Xiaohui Wei, Guowei Dai, Drs. Jin Liu, Hui Zheng, Zhongping Lin, Frank Hua, Zhongfa Liu, Yilong Zhang and Deping Cheng for all their help and friendship. Sincere thanks also go to Joy Scott, Kathy Brooks, and Karen Lawler for their timely assistance and friendship. I am grateful to my dear wife Sunny Chen, my parents, and my parents-in- law for their unconditional love, support and consideration. iii VITA December 25, 1973………………...Born in Beijing, P.R. China 1992 – 1996…………………………B.S. Pharmacy, Beijing Medical University 1996 - 1999…………………………..M.S. Pharmacy Institute of Materia Medica Chinese Academy of Medical Sciences 1999 - Present………………………Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Jim J. Xiao, John Byrd, Guido Marcucci, Michael Grever, and Kenneth K. Chan. Identification of thiols and glutathione conjugates of depsipeptide FK228 (FR901228), a novel histone protein deacetylase inhibitor, in the blood. Rapid Communications in Mass Spectrometry. 17:757-766, 2003. 2. Berg SL, Stone J, Xiao JJ, Chan KK, Nuchtern J, Dauser R, McGuffey L, and Blaney SM. Plasma and cerebrospinal fluid pharmacokinetics of depsipeptide (FR901228) in nonhuman primates. Cancer Pharmacol Pharmacol 54:85-88, 2004. 3. Sameek Roychowdhury, Robert A. Baiocchi, Srinivas Vourganti, Darshna Bhatt, Brad W. Blaser, Aharon G. Freud, Jason Chou, Ho-pi Lin, Jim Xiao, Mark Parthun, Ken K. Chan, Charles F. Eisenbeis, Amy Ferketich, Michael R. Grever, Ching-Shih Chen, Michael A. Caligiuri. Selective in vivo efficacy of depsipeptide (FR901228) against epstein-barr virus-positive Type III latency lymphoproliferative disorder. Journal of National Cancer Institute. 96:1447-57, 2004. ii 4. Zhongfa Liu, Heinz G. Floss, John M. Cassady, Jim J. Xiao and Kenneth K. Chan. An API LC/MS/MS quantification method for Ansamitocin P-3 (AP3) and its preclinical pharmacokinetics. Journal of Pharmaceutical and Biochemical Analysis 36:815-821, 2004. FIELDS OF STUDY Major Field: Pharmacy Studies in Pharmacokinetics, Drug Metabolism and Transport. iii TABLE OF CONTENTS ABSTRACT.......................................................................................................... II DEDICATION ....................................................................................................... II ACKNOWLEDGMENTS ..................................................................................... III VITA ..................................................................................................................... II LIST OF TABLES .............................................................................................VIII LIST OF FIGURES...............................................................................................X TABLE OF SCHEMES...................................................................................... XV LIST OF ABBREVIATIONS ............................................................................. XVI CHAPTER 1 ......................................................................................................... 1 INTRODUCTION .................................................................................................. 1 1.1 LITERATURE REVIEW................................................................................. 1 1.1.1 Identification of novel molecular targets in anticancer therapies ..... 4 1.1.2 Histone deacetylase inhibitors as anticancer drugs ...................... 24 1.2 DEPSIPEPTIDE FK228, A NOVEL HDAC INHIBITOR .................................... 36 1.2.1 Chemistry...................................................................................... 36 1.2.2 Pharmacology ............................................................................... 39 1.2.3 Analytical methodology ................................................................. 42 1.2.4 Preclinical pharmacokinetics and toxicity ...................................... 43 1.2.5 Clinical pharmacokinetics and toxicity........................................... 45 1.2.6 Transport and uptake .................................................................... 46 CHAPTER 2 ....................................................................................................... 47 PHARMACOKINETICS OF FK228 IN THE RAT ............................................... 47 2.1 ABSTRACT.............................................................................................. 47 2.2 INTRODUCTION ....................................................................................... 49 2.3 EXPERIMENTAL PROCEDURES .................................................................. 50 2.3.1 Drug and chemicals ...................................................................... 50 2.3.2 Animal protocols............................................................................ 51 2.3.3 Dosing and sample collection ....................................................... 53 2.3.4 Preparation of blood, urine, bile and organ tissue samples........... 55 2.3.5 HPLC/MS analysis ........................................................................ 56 2.3.6 Pharmacokinetic data analysis...................................................... 57 iv 2.4 RESULTS................................................................................................ 57 2.4.1 Plasma FK228 pharmacokinetics in the rat................................... 57 2.4.2 Dose recovery............................................................................... 64 2.5 DISCUSSION ........................................................................................... 64 2.5.1 Plasma pharmacokinetics of FK228.............................................. 64 2.5.2 FK228 recovery as the intact drug ................................................ 66 CHAPTER 3 ....................................................................................................... 68 METABOLISM OF FK228 IN THE BLOOD ....................................................... 68 3.1 ABSTRACT.............................................................................................. 68 3.2 INTRODUCTION