UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ AN ASSESSMENT OF THE POTENTIAL INFLUENCE OF BEXAROTENE, A NOVEL RETINOID X RECEPTOR AGONIST, ON THE HEPATIC METABOLISM OF DOCETAXEL A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Division of Pharmaceutical Sciences of the College of Pharmacy 2005 by Purvi B. Shroff MBBS, University of Mumbai, India, 2000. Committee Chair: Dr. Pankaj B. Desai, Ph.D i ABSTRACT Background: Bexarotene, a novel RXR agonist, is used in the treatment of cutaneous T cell lymphoma and is under investigation for the treatment of solid tumors in combination with other chemotherapeutic agents including docetaxel. Both docetaxel and bexarotene are metabolized by CYP3A4 and in animal studies bexarotene has been shown to enhance the activity of CYP3A enzymes and increase its content. Thus, there exists a potential for interactions between these two agents, which was evaluated in this study. Method: Bexarotene from commercial capsules was dissolved in DMSO. Employing pooled human liver microsomes, we assessed the influence of bexarotene on the metabolism of docetaxel. Microsomal fractions (1mg protein) were incubated with bexarotene (0.1 – 10µM) and docetaxel (0.5 – 5µM) in the presence of NADPH (1 mM). Using liquid-liquid extraction, unreacted docetaxel was extracted and levels were measured using a validated HPLC method. Next, we employed primary human hepatocytes to assess the inductive effects of bexarotene. Hepatocytes were treated with bexarotene (1 – 50µM) for 72 hours. Docetaxel metabolism by microsomal fractions was then evaluated. Testosterone 6β-hydroxylation and CYP3A4- specific protein and mRNA levels were also measured to evaluate whether bexarotene acts as an inducer of CYP3A4. Results: Bexarotene, at clinically relevant concentrations (1-10µM) did not inhibit docetaxel metabolism. Also, incubation of primary human hepatocytes with bexarotene did not enhance docetaxel metabolism, which was consistent with the observation that the ii testosterone 6β-hydroxylation and CYP3A4 expression in bexarotene – treated cells were not statistically different than those from untreated cells. Conclusions: Our findings suggest that bexarotene is unlikely to alter hepatic metabolism of docetaxel. Bexarotene appears to exhibit species – specific differences in the induction of CYP3A and further studies are required to understand the mechanistic basis of these differences. iii iv ACKNOWLEDGEMENTS I take this opportunity to deeply thank my advisor, Dr. Pankaj B. Desai, who not only initiated this project but also gave me valuable guidance till its completion. I remain indebted to him for his support, encouragement, advice, affection and critique of my work. On a personal note, he has been a source of inspiration to me. His wife, Sangita and his two daughters, Shivani and Kiran, are an extended family to me! I would also like to thank Dr. Jane Pruemer and Dr. Sadanand Patil for serving on my Masters Thesis Committee and giving me valuable inputs on this project. I appreciate them for reviewing my thesis and for their support and critique of my work. None of this work would have been possible without my parents and my in-laws who were supportive throughout my stay here in Cincinnati. I express my sincere gratitude to my parents once again for giving me the strength to believe in myself and get through life with pride. I cannot thank my husband, Manoj, enough for being so cooperative and patient with me. I hope to make up for all the sacrifices that he made for me to be here and pursue graduate studies at the University of Cincinnati, USA. He has made me the person that I am today with his love and support. I would like to appreciate Rucha, my senior in the laboratory, for not only teaching me laboratory skills but also being a great friend. I wish to thank her for all the wonderful moments that we shared while working together. I also thank Murad for his help in guiding me with HPLC techniques. I have enjoyed working with Niresh and Fang in the laboratory and I thank all of them for the healthy working environment that we have in the laboratory. v I wish to thank the faculty at UC for being such wonderful teachers and for all the guidance, encouragement and challenges they provided us during the classes. I appreciate that they participated in so many extracurricular activities and gave us a chance to interact with them outside the academic settings. I thank the Department of Pharmaceutical Sciences at the College of Pharmacy for their academic, fiscal and administrative support and for making our stay here in Cincinnati so comfortable. I deeply appreciate my friend, Hitesh Deshmukh, for all his patience, help, affection and encouragement. I cannot thank him enough for being such a kind human being that he is. I would also like to acknowledge all my friends and family here in the US and back in India for their love and support. Lastly, I would like to acknowledge my darling son, Taaraksh, who makes everything worthwhile in my life! With this I seek Lord Ganesha’s blessings and thank Him for all that He has showered upon me! vi TABLE OF CONTENTS ABSTRACT……………………………………………………………………………....ii ACKNOWLEDGEMENTS……………………………………………………………....iv TABLE OF CONTENTS…………………………………………………………………vi LIST OF FIGURES……………………………………………………………………….x LIST OF TABLES……………………………………………………………………...xiii 1. INTRODUCTION……………………………………………………………….....1 1.1. ADVERSE DRUG REACTIONS (ADRs)………………………………………1 1.2. DRUG – DRUG INTERACTIONS……………………………………………...2 1.3. DRUG INTERACTIONS IN ONCOLOGY…………………………………….2 1.4. TYPES OF DRUG-DRUG INTERACTIONS………………………………......3 1.5. NON-SMALL CELL LUNG CARCINOMA (NSCLC)………………………...5 1.6. BEXAROTENE………………………………………………………………….8 1.6.1. Chemistry 1.6.2. Mechanism of action of retinoids 1.6.3. Clinical Indications 1.6.4. Pharmacokinetics of bexarotene 1.6.5. Metabolism of bexarotene: role of CYP3A4 1.7. DOCETAXEL…………………………………………………………………..14 1.7.1. Chemistry 1.7.2. Mechanism of action of docetaxel 1.7.2.1. Background on microtubule structure vii 1.7.2.2. Mechanism of action of docetaxel 1.7.3. Clinical Indications 1.7.4. Pharmacokinetics of docetaxel 1.7.5. Metabolism of docetaxel: role of CYP3A4 1.8. CYTOCHROME P450 FAMILY…………………………………………….21 1.8.1. The CYP3A subfamily 1.8.2. CYP3A4 Inhibition 1.8.3. CYP3A4 Induction 1.8.4. Regulation of CYP3A4 2. RATIONAL AND HYPOTHESIS ………………………………………………28 2.1 SPECIFIC AIMS 2.1.1. Aim I 2.1.2. Aim II 3. MATERIALS AND METHODS…………………………………………………30 3.1. Experimental Strategy …………………………………………….............30 3.2. Human liver microsomal metabolism of docetaxel in the presence and in the absence of bexarotene…………………………………………………33 3.2.1. Chemicals 3.2.2. Reaction optimization 3.2.3. Assessment of the potential influence of bexarotene on docetaxel metabolism in human liver microsomal reactions 3.2.4. HPLC analysis of docetaxel viii 3.3. Assessment of the potential induction of docetaxel metabolism by bexarotene in primary human hepatocytes………………………….......38 3.3.1. Hepatocyte culture 3.3.2. Drug treatments 3.3.3. Assay of CYP3A4 in human hepatocytes 3.3.4. Docetaxel metabolism by human hepatocytes 3.3.5. Western blot analysis of immunoreactive CYP3A4 protein levels in human hepatocytes 3.3.6. Real-time PCR to assess the levels of CYP3A4 mRNA in human hepatocytes 3.4. Statistical Analysis………………………………………………………..42 4. RESULTS……………………………………………………………………......43 4.1. Human liver microsomal metabolism of docetaxel in the presence / absence of bexarotene………..…………………………………………..43 4.1.1. Michaelis-Menten plot of docetaxel metabolism kinetics 4.1.2. – 4.1.5. Human liver microsomal metabolism of docetaxel (0.5 – 5 µM) in the presence and the absence of bexarotene (0.1 – 10 µM). 4.2. Assessment of the potential induction of docetaxel metabolism by bexarotene in primary human hepatocytes……………………………….53 4.2.1. Typical chromatogram showing effect of rifampicin and bexarotene treatment of hepatocytes on CYP3A4 activity measured by testosterone 6β-hydroxylation as compared to untreated controls. 4.2.2. Effect of rifampicin and bexarotene treatment on the CYP3A4 activity as compared to untreated controls. 4.2.3. Effect of rifampicin and bexarotene treatment on docetaxel metabolism by human hepatocytes as compared to untreated controls. ix 4.2.4. Western blot analysis of rifampicin and bexarotene treatment on immunoreactive CYP3A4 protein levels as compared to untreated controls. 4.2.5. RT – PCR analysis of the effect of rifampicin and bexarotene treatment on the CYP3A4 specific mRNA levels as compared to untreated controls. 5 DISCUSSION……………………………………………………………………63 6 REFERENCES…………………………………………………………………..70 x LIST OF FIGURES 1.6.1. Chemical structure of bexarotene ……………………………………………….9 1.6.2. Mechanism of action of retinoids ..………………………………………………11 1.7.1. Chemical structure of docetaxel…..………………………………………………15 1.7.2. Biotransformation of docetaxel. The structural modifications of docetaxel are highlighted……………………………………………………………………….20 3.1.1. Diagrammatic representation of the experimental strategy to assess the inductive effects of