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PHARMACOKINETICS, PHARMACODYNAMICS AND METABOLISM OF GTI-2040, A PHOSPHOROTHIOATE OLIGONUCLEOTIDE TARGETING R2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaohui Wei, M.S. * * * * * * The Ohio State University 2006 Approved by Dissertation Committee: Dr. Kenneth K. Chan, Adviser Adviser Dr. Guido Marcucci, Co-adviser Graduate Program in Pharmacy Dr. Thomas D. Schmittgen Dr. Robert J. Lee Co-Adviser Graduate Program in Pharmacy ABSTRACT Over the last several decades, antisense therapy has been developed into a promising gene-targeted strategy to specifically inhibit the gene expression. Ribonucleotide reductase (RNR), composing of subunits R1 and R2, is an important enzyme involved in the synthesis of all of the precursors used in DNA replication. Over- expression of R2 has been found in almost every type of cancer studied. GTI-2040 is a 20-mer phosphorothioate oligonucleotide targeting the coding region in mRNA of the R2 component of human RNR. In this project, clinical pharamcokinetics (PK), pharmacodynamics (PD) and metabolism of this novel therapeutics were investigated in patients with acute myeloid leukemia (AML). A picomolar specific hybridization-ligation ELISA method has been developed and validated for quantification of GTI-2040. GTI-2040 and neophectin complex was found to enhance drug cellular uptake and exhibited sequence- and dose-dependent down-regulation of R2 mRNA and protein in K562 cells. Robust intracellular concentrations (ICs) of GTI-2040 were achieved in peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from treated AML patients. GTI-2040 concentrations in the nucleus of BM cells were found to correlate with the R2 mRNA down-regulation and disease response. In treated patients ii from 18-60 years old, down-regulations of R2 protein were found in responders, while the non-responders were found to exhibit up-regulation of R2 proteins. Plasma PK of GTI-2040 in AML patients were characterized and found to fit a two-compartment infusion model with mean body clearance of 10.1 L/hr, mean t1/2α of 0.81 hr and mean t 1/2β of 27 hr. Population PK analysis of GTI-2040 identified BM cellularity, white blood cell counts and gender to be important covariates of PK parameters. A PK/PD model has been developed to characterize the dynamics of down- regulation of R2 mRNA following drug perturbation. The 3’ end chain-shortened metabolites of GTI-2040 were identified in various specimens from human and animals using a novel ion-pair reverse-phase high performance liquid chromatography/mass spectrometry (HPLC/MS) method. Sequential metabolism and protein binding were found to complicate the enzyme kinetics of GTI- 2040 in human liver microsomes. The results from these studies provided valuable insights to the evaluation and utilization of GTI-2040 in the clinics. iii Dedicated to my parents and to my friends. iv ACKNOWLEDGMENTS I would like to thank my adviser, Dr. Kenneth K. Chan, for his intellectual guidance, constant encouragement, stimulation and support during my graduate studies. I also would like to thank my co-adviser, Dr. Guido Macucci, for his support in the clinical study and his invaluable suggestions and comments on this project. Appreciation also goes to my committee members, Dr. Thomas D. Schmittgen, Dr. Rober J. Lee and Dr. William L. Hayton for their time, constructive suggestions and comments on this project. The work with GTI-2040 project would not be possible without the significant contribution from the laboratory of Dr. Guido Marcucci in the Division of Hematology- Oncology, College of Medicine. A special acknowledgement goes to Dr. Guowei Dai for his invaluable suggestions and help on the entire project. I also thank Dr. Shujun Liu for his assistance on western blotting, transfection experiments and stimulating discussions on this project. A special thank is also extended to Ms. LeNguyen Huynh for the many hours she spent collecting patients’ samples and her technical assistance in the extraction and quantitation of R2 mRNA. Additional thanks go to Dr. Kui Liu for his help in cloning R2 cDNA standards and Dr. Jin Sun for his assistance in flow cytometry v analysis. Thanks also goes to Dr. Zhongfa Liu for his help on operation of mass spectrometer and valuable scientific discussions on the metabolism studies, to Dr. Jim Xiao for his discussion of modeling and continuous encouragement, to Dr. Duxin Sun and his laboratory for their support of cell culture facilities and valuable comments on this project, and to Drs. Hao Cheng and Zhiliang Xie for their help on tissue handling and sample preparation in metabolism study and their friendship. I am also grateful to Ms. Joy Scott and Ms. Kathy Brooks for their administrative assistance, encouragement and friendship. I am further indebted to all of my fellow graduate students and colleagues in the College of Pharmacy, especially my roommate Ms. Yan Xin, for their warmest friendship, invaluable encouragement and scientific discussions. Finally, I would like to express special thanks to my parents and sisters who have been giving me unconditional support and have encouraged me to strive for excellence in this long and challenging endeavor. vi VITA 1989-1993…………………………...B.S. in Pharmacy Beijing University of Chinese Traditional Medicine 1993-1996…………………………...Instructor Guangxi University of Chinese Traditional Medicine 1996-1999……………………………M.S. in Pharmacy West China University of Medical Sciences 1999-2000……………………………Associate Researcher Institute of Materia Medica Chinese Academy of Medical Sciences 2000-Present…………………………Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Wei X, Chen W, Liu H. Evaluation of dissolution rate of baicalin in yinhuang tablet. Chinese Medicine Patent. 1996, 18(1): 3-6. 2. Liu H, Wei X, Tang D, Zhong D. Pharmacokinetics of baicalin in Yinzhi injection in rabbit. Journal of Guanxi Traditional Chinese Medical University. 1998, 1:21-25. 3. Yuan Y, Wei X, Yang J. Zhang L, Jiang X. Study on the optimum technology of extraction for Danqi Chinese medicine preparation. West China Journal of Pharmaceutical Sciences. 2002, 17(1):10-13. 4. Dai G, Wei X, Liu Z, Liu S, Marcucci, G, Chan KK. Characterization and vii quantification of Bcl-2 antisense G3139 and major metabolites in plasma and urine by ion-pair reverse phase HPLC coupled with electrospray ion-trap mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2005, 825(2): 201-13. 5. Otterson GA, Lavelle J, Villalone-Calero MA, Shah M, Wei X, Chan K, Fischer B, Zwiebel J, Grever M. A phase I clinical and pharmacokinetic study of fenretinide combined with paclitaxel and cisplatin for refractory solid tumors. Invest New Drugs. 2005, 23(6): 555-562. 6. Wei X, Dai G, Marcucci G, Lui S, Hoyt D, Chan KK. A specific picomolar hybridization-based ELISA assay for the determination of phosphorothioate oligonucleotides in plasma and cellular matrices. Pharmaceutical Research. In press, 2006 7. O’Connor OA, Smith EA, Toner LE, Feldstien J, Frankel S, Rolfe M, Wei X, Lui S, Marcucci G, Chan KK, Chanan-Khan A. The combination of the proteasome inhibitor Bortezomib and the Bcl-2 antisense molecule Oblimersen sensitizes human B-cell lymphomas to cyclophosphamide. Clinical Cancer Research. Accepted, 2006. FIELDS OF STUDY Major Field: Pharmacy/Pharmaceutics -with studies on bioanalytical method development, clinical pharmacokinetics, pharmacodynamics and drug metabolism. viii TABLE OF CONTENTS Page ABSTRACT.................................................................................................................. ii ACKNOWLEDGMENTS .............................................................................................v VITA........................................................................................................................... vii LIST OF TABLES.......................................................................................................xv LIST OF FIGURES .................................................................................................. xvii LIST OF SCHEMES.……………………………………………………………….xxiii CHAPTER 1 ....................................................................................................................................1 BACKGROUND AND INTRODUCTION...................................................................1 1.1 Background........................................................................................................1 1.2 Introduction........................................................................................................4 1.2.1 Principle of antisense therapy....................................................................4 1.2.1.1 Mechanisms of action of antisense oligonucleotides.............................4 1.2.1.2 Antisense oligonucleotides modifications and other anti-mRNA strategies ………………………………………………………………………….6 1.2.1.2.1 First generation of antisense oligonucleotides.................................7 1.2.1.2.2 The second generation of antisense oligonucleotides.......................8 1.2.1.2.3 Other anti-mRNA strategies ............................................................9 1.2.2 Pharmacokinetics and pharmacodynamics of phosphorothioate oligonucleotides.......................................................................................................11 1.2.2.1 Cellular uptake of
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