Modulation of Pharmacologic Effects of 5-Azacytidine by Ribonucleotide Reductase Antisense GTI-2040 Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Josephine Eki Aimiuwu, B.S.P.S Graduate Program in Pharmacy The Ohio State University 2011 Dissertation Committee: Kenneth K. Chan, Ph.D., Advisor Guido Marcucci, M.D., Co-advisor Robert Lee, Ph.D. Robert M. Snapka, Ph.D. ABSTRACT Cancer, like most human diseases is complex and the therapeutic approaches available to treat this disease have limited efficacy. Therefore, combination studies of two or more anticancer drugs is expected to be essential in achieving a better therapeutic response in patients and may also provide a cure for drug-resistant cancers. Leukemia is a cancer of the blood, which results from an uncontrolled proliferation of white blood cells, thereby inhibiting its functions. Important insights into the pathogenesis of this disease have led to the development of a number of anti-leukemia drugs, including nucleoside analogs and new antisense compounds, that intervene at the level of disease progression. 5- Azacytidine and decitabine are hypomethylating agents recently approved by the U.S Food and Drug Administration for the treatment of Myelodysplastic Syndrome (MDS) and are also in clinical trials for the treatment of hematological malignancies, such as acute myeloid leukemia (AML). These drugs are approved based on their ability to induce DNA demethylation, resulting in reactivation of hypermethylation-associated silencing of tumor suppressor genes. Aracytidine (Ara-C) is another nucleoside drug, widely used as antimetabolite for the treatment of acute myelogenous leukemia. Antisense GTI-2040 is a 20-mer oligonucleotide inhibiting the expression of ribonucleotide reductase subunit M2 (RRM2) mRNA, an enzyme that has been found to be over-expressed in most cancers. In this dissertation, investigations on pharmacodynamic studies of nucleoside analogs in combination with GTI-2040 were carried out. It has been demonstrated that inhibition of ii cellular RR, which subsequently decreases deoxyribonucleotide triphosphate (dNTP) pools, could enhance the anti-tumor activity of subsequently administered nucleoside analogs. We have in our studies, provided both in vitro and in vivo evidences to support the novel combination treatment of antisense GTI-2040 and 5-azaC, leading to a synergistic effect. In addition, GTI-2040 decreases RRM2 levels, and most notably, we discovered that 5-azaC modulates RRM2 for the first time and this result makes RR a novel target for 5-azaC. In addition, the biomarkers involved in the development of 5-azaC and decitabine (DAC) resistances were assessed in order to elucidate the potential mechanisms that contribute to the induction of resistance in cancer cells. In a phase II evaluation of GTI-2040 in combination with Ara-C in patients with AML at The James Cancer Hospital and Research Institute at The Ohio State University, clinical pharmacokinetic of GTI-2040 and the in vitro-in vivo pharmacodynamic analysis with Ara-C was established to assist in the exploration of their pharmacokinetic-pharmacodynamic (PK-PD) correlations in relation to clinical response. Finally, our studies of GTI-2040, 5-AzaC, DAC and Ara-C provide valuable insights into their clinical development as a single agent or in combination with other drugs. iii DEDICATION Dedicated to my family iv ACKNOWLEDGEMENTS I would like to thank my adviser, Dr. Kenneth K. Chan, for his intellectual guidance, encouragement and motivation, continuing support during my graduate studies. I sincerely appreciate his help in every aspect of this research and for his invaluable suggestions. I also would like to thank my co-adviser, Dr. Guido Marcucci for his support in the clinical study and his invaluable suggestions and comments on this project. I also thank Dr. Rebecca Klisovic, for the opportunity to work in the clinical trial. Appreciation also goes to my committee members, Dr. Robert Lee and Dr. Robert M. Snapka for their time, constructive suggestions and comments on this project. I would like to thank Dr. Duxin Sun’s support, sound advice and friendship during my graduate studies. Appreciation also goes to our collaborators in Dr. Marcucci’s laboratory. Special thanks go to Dr. Shujun Liu for the valuable scientific discussions on biological studies, and stimulating discussions on this project. Ms. Jiuxia Pang assistance on western blotting experiments and without both of their help and effort, my work would have undoubtedly been more difficult. I would also like to express my thanks to Ms. LeNguyen Huynh for the time she spent on collecting patient samples and help with real time PCR experiments. I would also like to thank Dr. Ping Chen for her valuable scientific discussions and the development of most of the assays used in our biochemical modulation experiments. I am v thankful for Dr. Zhongfa Liu’s suggestions and assistance earlier on in my studies. I benefited from his immense experience in analytical chemistry and help using mass spectrometers. My sincere thanks also go to Dr. Zhiliang Xie for the great help and assistance on sample preparation and analysis and Dr. Ming Chiu for his help in animal work. I also thank Dr. Hongyan Wang for stimulating discussion and assistance on this project and her immense friendship, Dr. Yonghua Ling for her support and warm friendship. I would like to thank all of my labmates and friends who directly or indirectly have allowed me to be where I am today. Thanks are also to Dr. Tracey Wei, Dr. Hao Cheng, Dr. Vijay Sarathi, Dr. Jiang Wang and Dr. Xianhua Cao for their assistance in various aspects. I am also very grateful to Ms. Joy Scott for her administrative help, encouragement and warm friendship and Ms. Kathy Brooks. I would also like to express my thanks to all of my friends and colleagues in the College of Pharmacy, especially Casey Hoerig for his help in computer related issues. Finally, my special thanks go to my family. I am deeply and forever indebted to my husband, Osagie Christopher for his love, support and encouragement throughout my studies and to my children, Izoduwa Maria and Osagie Kingsley for making my time in school so much easier than I could have imagined. I also like to thank my parents for their everlasting and unconditional love and for believing in me at all times. vi VITA June 2005 ....................................................... B.S. in Pharmaceutical Science, The Ohio State University 2005 to present ...............................................Graduate Research Associate, The Ohio State University PUBLICATIONS Research Publications 1. Eric H. Kraut, Christopher Rhoades, Yilong Zhang, Hao Cheng, Josephine Aimiuwu, Ping Chen, James Lang, Donn Young, Amit Agrawal, Janet Dancey, Kenneth Chan, Michael R Grever. Phase I and pharmacokinetic study of erlotinib (OSI-774) in combination with docetaxel in squamous cell carcinoma of the head and neck (SSCHN). Cancer Chemotherapy and Pharmacology, 2010, 1-8. 2. Guisheng Zhang, Lanyan Fang, Lizhi Zhu, Josephine Aimiuwu, Jie Shen, Hao Cheng, Mark Muller, Gun Lee, Duxin Sun and Peng Wang. Synthesis and Biological Activities of Disaccharide Daunorubics. J.Med.Chem. 2005, 48, 5269-5278. 3. Ping Chen, Zhongfa Liu, Shujun Liu, Zhiliang Xie, Josephine Aimiuwu, Jiuxia Pang, Rebecca Klisovic, William Blum, Michael Grever, Guido Marcucci and Kenneth K. Chan. A LC-MS/MS Method for the Analysis of Intracellular Nucleoside Triphosphate levels. Pharmaceutical Research. 2009, 26(6), 1504-1515. 4. Zhongfa Liu, Shujun Liu, Zhiliang Xie, Ryan E. Pavlovicz, Jiejun Wu, Ping Chen, Josephine Aimiuwu, Jiuxia Pang, Deepak Bhasin, Paolo Neviani, James R. Fuchs, Christoph Plass, Pui-Kai Li, Chenglong Li, Tim H-M Huang, Lai-Chu Wu, Laura Rush, Guido Marcucci, and Kenneth K. Chan. Modulation of DNA Methylation by a Sesquiterpene Lactone Parthenolide. Journal of Experimental Therapeutics. 2009, 329(2), 505-514. 5. Ping Chen, Josephine Aimiuwu, Zhiliang Xie, Xiaohui Wei, Shujun Liu, Rebecca Klisovic, Guido Marcucci, and Kenneth, K. Chan. Biochemical Modulation of Aracytidine (Ara-C) Effects by GTI-2040, a Ribonucleotide Reductase Inhibitor, in K562 vii Human Leukemia Cells. American Association Pharmaceutical Scientists Journal (In press). Research Abstracts: 1. Mitch A. Phelps, Danxin Wang, Josephine E. Aimiuwu, Sherri L. Starrett, Audrey C. Papp, Ahmed A. Ghany, William J. Hicks, Kavitha V. Kosuri, Gregory A. Otterson, Mark A. Socinski, Thomas E. Stinchcombe, Weiqiang Zhao, Larry J. Schaaf, Sanford H. Barsky, Kenneth K. Chan, Wolfgang Sadee, Miguel A. Villalona-Calero. Erlotinib in African Americans with lung cancer: a prospective, clinical, molecular, pharmacokinetic and pharmacogenetic characterization. American Association for Cancer Research, April 2009. 2. Josephine Aimiuwu, Ping Chen, Zhiliang Xie, Zhongfa Liu, Vijay Sarathi, Shujun Liu, Rebecca Klisovic, Guido Marcucci, Kenneth K. Chan. In vitro-in vivo Pharmacodynamic Analysis of GTI-2040 Combined with Ara-C in Acute Myeloid Leukemia. American Association for Cancer Research, April 2009. 3. Ping Chen, Ph.D., William Blum, MD, Josephine Aimiuwu, Vijayasarat Upadhyayula,Ph.D., Zhongfa Liu, Ph.D., Shujun Liu, Ph.D, Jiuxia Pang, Alison Walker, Rebecca B.Klisovic, MD, Ramiro Garzon, MD, Michael R. Grever, MD, Miguel Villalona-Calero, MD, John C. Byrd, MD, Kenneth K Chan, PhD and Guido Marcucci, MD. Quantification of Intracellular Decitabine-Triphosphate with A Novel, Highly Sensitive and Specific LC-MS/MS Assay in Acute Myeloid Leukemia Patients Treated with Low Dose Decitabine. The American Society of Hematology, December 2009. 4. Josephine Aimiuwu, Ping Chen, Zhiliang Xie, Zhongfa Liu Shujun Liu, Rebecca Klisovic, William Blum, Guido Marcucci, Kenneth K. Chan. Development and Characterization of 5-Azacytidine Resistance in Human Cancer Cell Lines. American Association of Pharmaceutical Scientist, November 2009. 5. M. J. Karpenko, Z. Liu, J. Aimiuwu, L. Wang, X. Wu, M. A. Villalona-Calero, D. Young, K. Chan, M. R. Grever, G. A. Otterson. Phase I study of 5-aza-2’- deoxycytidine in Combination with Valproic acid in Patients with NSCLC.