University of Mississippi eGrove Electronic Theses and Dissertations Graduate School 2012 Metabolism And Pharmacokinetics In The Development Of New Therapeutics For Cocaine And Opioid Abuse Pradeep Kumar Vuppala University of Mississippi Follow this and additional works at: https://egrove.olemiss.edu/etd Part of the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Vuppala, Pradeep Kumar, "Metabolism And Pharmacokinetics In The Development Of New Therapeutics For Cocaine And Opioid Abuse" (2012). Electronic Theses and Dissertations. 731. https://egrove.olemiss.edu/etd/731 This Dissertation is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. METABOLISM AND PHARMACOKINETICS IN THE DEVELOPMENT OF NEW THERAPEUTICS FOR COCAINE AND OPIOID ABUSE A Dissertation presented in partial fulfillment of requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences in the Department of Pharmaceutics The University of Mississippi by PRADEEP KUMAR VUPPALA April 2012 Copyright © 2012 by Pradeep Kumar Vuppala All rights reserved ABSTRACT Cocaine and opioid abuse are a major public health concern and the cause of significant morbidity and mortality worldwide. The development of effective medication for cocaine and opioid abuse is necessary to reduce the impact of this issue upon the individual and society. The pharmacologic treatment for drug abuse has been based on one of the following strategies: agonist substitution, antagonist treatment, or symptomatic treatment. This dissertation is focused on the role of metabolism and pharmacokinetics in the development of new pharmacotherapies, CM304 (sigma-1 receptor antagonist), mitragynine and 7-hydroxymitragynine (µ-opioid receptor agonists), for the treatment of drug abuse. The affinity of cocaine to sigma-1 receptors render the hypothesis that targeting sigma-1 receptors using antagonists will be an effective strategy in the development of novel medications for cocaine abuse. In this light, we screened several sigma-1 receptor antagonists using in vitro metabolism studies and selected CM304, (3-(2-(azepan-1-yl) ethyl)-6-(3-fluoropropyl) benzo[d]thiazol-2(3H)-one), as a lead compound. CM304 was further characterized by determining its physicochemical properties such as pKa, Log PO/W and Log D PBS, pH 7.4, solubility, protein binding, in vitro metabolic stability. We also investigated pharmacokinetic parameters and bioavailability of CM304 in rats using a validated UPLC-MS/MS method. Mitragyna speciosa Korth is a tropical plant indigenous to Southeast Asia. The leaves of the plant have been used as a traditional medicine to treat cough, fever, diarrhea, pain, hypertension and morphine addicts. Mitragynine and 7-hydroxymitragyne, corynanthine-like ii alkaloids, have been reported to be responsible for the opioid properties found in this plant. We have developed simple, sensitive and high throughput bioanalytical methods for the quantification of mitragynine and 7-hydroxymitragyne in rat plasma. Pharmacokinetic parameters were evaluated by conducting single dose in vivo pharmacokinetic studies in rats. The extent of brain penetration of 7-hydroxymitragyne in rats was determined by performing an in vivo brain to plasma ratio. The above in vitro and in vivo experiments discussed on this dissertation will lay the groundwork for further development of these compounds as pharmacotherapies for cocaine and opioid abuse. iii DEDICATION Dedicated to my parents, Padma Vuppala and Mettaiah Vuppala, and my wife Seshulatha Jamalapuram iv ACKNOWLEDGMENTS First and foremost, I would like to express my deep and sincere gratitude to my advisor, Dr. Bonnie A. Avery. I have been amazingly fortunate to have an advisor who gave me the freedom to explore on my own and at the same time the guidance to recover when my steps faltered. Under her supervision I have been motivated to strive for and achieve exceptionally high standards in research. Her patience and endless support helped me overcome many crisis situations and finish this dissertation. Especially, the support I got from her during last semester is unforgettable in my life. I wish to express my warm and sincere thanks to Dr. Christopher R. McCurdy, whose insightful comments and guidance at different stages of my research were thought-provoking and they helped me focus my ideas. I would like to thank Dr. Soumyajit Majumdar and Dr. Seongbong Jo for accepting to be on my dissertation committee and for providing valuable suggestions during my prospectus. I would also like thank Dr. Michael A. Repka, Dr. Stephen J. Cutler, Dr. John F. Wiginton, Dr. S. Narasimha Murthy and Dr. Christy Wyandt for their guidance and support. I greatly appreciate my past (Dr. Sai Prasad, Dr. Deepthi and Dr. Harsha) and present (Seshu) group members for their valuable suggestions and helping me in completion of various projects successfully. I also thank all the graduate students of pharmaceutics department for being supportive and friendly. v I would like to extend my sincere thanks to, Ms. Debbie P. King and Mrs. Candace G. Lowstuter for their help in all the departmental procedures. I thank Ms. Penni Bolton and Dr. Harry Fyke for their help in animal experiments. The years that I spent in US would not have been as wonderful without my close friends Dr. Vivek and Kiran. I owe my loving thanks to my mother (Padma Vuppala) and father (Mettaiah Vuppala) for allowing me to be as ambitious as I wanted to be and providing me with unending encouragement and support. I owe my special gratitude to my sister (Swathi Nalla), brother-in- law (Kasinath Nalla) and niece (Srisha) for their loving support. I would like to give a big thank you to my wife, Seshulatha Jamalapuram. Without her I would be a very different person today, and it would have been certainly much harder to finish my PhD. Last but not the least, I am ever grateful to God, the creator and the guardian, and to whom I owe my very existence. vi TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………. lll ACKNOWLEDGEMENT…………………………………………………………………….. Vl CHAPTER 1: INTRODUCTION ……………………………………………………………. 1 CHAPTER 2: PRECLINICAL CHARACTERIZATION OF CM304, A NOVEL AND HIGH AFFINITY SIGMA-1 RECEPTOR LIGAND WITH POTENT ANTI-COCAINE ACTIONS……………………………………………………………………………………… 22 2.1. INTRODUCTION…………………………………………………………………………. 22 2.2. EXPERIMENTAL…………………………………………………………………………. 24 2.3. RESULTS AND DISCUSSION…………………………………………………………… 35 2.4. CONCLUSION…………………………………………………………………………….. 64 CHAPTER 3: PHARMACOKINETICS OF MITRAGYNINE, A NATURALLY OCCURRING OPIOID AGONIST, IN RATS……………………………………………… 65 3.2. INTRODUCTION…………………………………………………………………………. 65 3.2. EXPERIMENTAL…………………………………………………………………………. 68 3.3. RESULTS AND DISCUSSION…………………………………………………………… 74 3.4. CONCLUSION…………………………………………………………………………….. 86 CHAPTER 4: PRECLINICAL PHARMACOKINETICS AND BRAIN TO PLASMA RATIO OF 7 -HYDROXYMITRAGYNINE, A µ-OPIOID AGONIST WITH POTENT ANTINOCICEPTIVE EFFECTS……………………………………………………………. 88 4.1. INTRODUCTION…………………………………………………………………………. 88 4.2. EXPERIMENTAL…………………………………………………………………………. 90 4.3. RESULTS AND DISCUSSION………………………………………………………….. 100 4.4. CONCLUSION…………………………………………………………………………… 113 CHAPTER 5: ANALYSIS OF KRATOM LEAVES AND MECONIUM SAMPLE RECEIVED FROM HOSPITAL IN KENTUCKY………………………………………... 117 vii 5.1. INTRODUCTION………………………………………………………………………... 117 5.2. EXPERIMENTAL………………………………………………………………………... 118 5.3. RESULTS AND DISCUSSION………………………………………………………….. 122 5.4. CONCLUSION………………………………………………………………………….... 126 CHAPTER 6: SUMMARY AND CONCLUSIONS……………………………………...... 127 BIOBLIOGRAPHY………………………………………………………………………….. 132 VITA………………………………………………………………………………………….. 146 viii LIST OF FIGURES FIGURE PAGE Figure 1.1: Classical mechanism of action of cocaine………………………………………… 6 Figure 1.2: A structural model for the sigma-1 receptor......................................................... 13 Figure 1.3: In vitro and in vivo experiments that are used to address common issues in potential small molecule drug discovery……………………………………………………. 21 Figure 2.1: Chemical structure of SN56………………………………………………………. 23 Figure 2.2: Chemical structure of CM304…………………………………………………….. 39 Figure 2.3: (a) Chemical structure and protonated molecular ion spectra of CM304, (b) Production spectra of CM304…………………………………………………….. 41 Figure 2.4: (a) Chemical structure and protonated molecular ion spectra of IS, (b) Production spectra of IS………………………………………………………………………. 42 Figure 2.5: pKa determination of CM304…………………………………………………….. 44 Figure 2.6: Metabolic stability profiles for CM304 in mouse, rat and human liver microsomes………………………………………………………………………… 45 Figure 2.7: In vitro half-life (t1/2) of CM304 in mouse, rat and human liver microsomes……. 46 Figure 2.8: (a) Chromatogram for blank plasma at m/z 337 > 237.5 (for CM304), (b) Chromatogram for blank plasma at m/z 318 > 219 (for internal standard), (c) Chromatogram for blank plasma spiked with 3 ng/mL CM304, (d) Chromatogram for blank plasma spiked with 100 ng/mL IS……………………………………… 48 ix Figure 2.9: Standard graph of CM304 in rat plasma………………………………………….. 49 Figure 2.10: Pharmacokinetic profile of CM304 after IV administration to rats at 2.5 mg/kg (n=8)………………………………………………………………………………. 54 Figure2.11: Mean concentration of CM304 in plasma after oral administration versus time profile (n=8)……………………………………………………………………..... 55 Figure 2.12: MetaboLynx output