CHARACTERIZING THE PHARMACOLOGICAL PROFILE OF MEPHEDRONE AND DETERMINING THE ABUSE LIABILITY MECHANISMS A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Iman A. Saber December 2017 Examining Committee Members: Ellen Walker, Advisory Chair, Department of Pharmaceutical Sciences Wayne Childers, Department of Pharmaceutical Sciences Ellen Unterwald, Department of Pharmacology, Center for Substance Abuse and Research Sarah Jane Ward, Department of Pharmacology, Center for Substance Abuse and Research Scott Rawls, Department of Pharmacology, Center for Substance Abuse and Research © Copyright 2017 by Iman A. Saber All Rights Reserved ! ii! ABSTRACT Illicit drug use has been a growing concern over the past few decades. The rise in use of illegal drugs drove the government and law enforcement to aggressively tackle this problem and crackdown on the illicit use of drugs. However, this sparked a further interest in ‘legal highs.’ Before 2011, among the newly popular ‘legal highs’ was ‘Bath Salts.’ Cathinone is a monoamine alkaloid and the active ingredient found in the leaves of the khat plant. The psychoactive form of bath salts may contain a mixture of synthesized cathinones, including, 4-methyl-N-methcathinone (mephedrone), 3,4-methylenedioxy-N- methylcathinone (methylone) and methylenedioxypyrovalerone (MDPV). These three are commonly found in bath salts. One of the major psychoactive ingredients in bath salts is mephedrone. Mephedrone grew in popularity due to its low price, accessibility, and the shortage of MDMA, thus making mephedrone the prime drug to sell as a ‘legal high’ up until 2011 when it became banned in the United States. Before 2012, most of the studies focused on the identification and clinical case reports of mephedrone. During the recent years, other preclinical studies supported the notion that mephedrone might have strong abuse liability and may lead to addiction. The shortage of information about the pharmacological mechanism of the novel drug mephedrone present in preclinical observations inhibits the ability of law enforcement and health care personnel to tackle the problems of its misuse. With this in mind, the overarching intention of this dissertation is to characterize the pharmacological mechanism of mephedrone and further determine the mechanisms that are involved in its abuse liability. Specifically, we want to evaluate the contribution of dopamine and ! iii! serotonin to mephedrone’s discriminative and reinforcing effects and drug-seeking behavior in male Sprague Dawley rats. We first established two doses of mephedrone as discriminative stimuli in drug naïve rats and tested the capacity of various dopaminergic and serotonergic agonists to generalize to 0.5 mg/kg or 3.2 mg/kg mephedrone. We then examined the capacity of dopamine and serotonin receptor antagonists to attenuate the discriminative effects of the training doses 0.5 and 3.2 mg/kg mephedrone. We were able to successfully establish both a low and high dose of mephedrone as discriminative stimuli in male rats. 3,4- methylenedioxymethamphetamine (MDMA) was the only drug to fully substitute for both 0.5 and 3.2 mg/kg mephedrone, while cocaine, methamphetamine, d-amphetamine and, 2,5-dimethoxy-4-iodoamphetamine (DOI) fully substituted for the low training dose of 0.5 mg/kg mephedrone. The D1 receptor antagonist, SCH23390, was able to significantly attenuate the discriminative stimulus effects of 0.5 mg/kg mephedrone while significantly decreasing the response rates, and the D2/3 receptor antagonist, sulpiride, significantly attenuated the discriminative stimulus effects of 0.5 mg/kg mephedrone while significantly increasing the response rates. The 5-HT2C receptor antagonist, SB242084, significantly increased the response rates of 0.5 mg/kg mephedrone. SCH23390 significantly disrupted response rates for 3.2 mg/kg mephedrone and substitution effects could not be measured. Next, we examined the capacity of mephedrone to reinstate cocaine responding and support self-administration. We established a history of self-administering 0.375 mg/kg/infusion cocaine in rats for 14 days. Rats then went through a period of extinction and were reinstated with mephedrone, methamphetamine, cocaine, or saline. We then ! iv! determined if rats were able to develop a mephedrone self-administering behavior when a history of cocaine self-administration was already established. We also used a D1 receptor antagonist, SCH23390, to inhibit this mephedrone seeking behavior. Mephedrone and saline did not fully reinstate the extinguished cocaine-seeking behavior; however, methamphetamine and cocaine did. Mephedrone was able to substitute and maintain the cocaine-seeking behavior. A moderate dose of 0.01 mg/kg SCH23390, antagonized the reinforcing effect of cocaine, but not the reinforcing effects of mephedrone, although the responses of individual rats highly suggest a trend in that direction. Overall, the experiments presented here suggests that mephedrone has a pharmacological mechanism that is more similar to MDMA than cocaine. ! v! This work is dedicated to my sweet, sweet children. For all of the happiness they brought to my life, no matter how short-lived it was. So that my future children may know how hard their mother worked and to know that absolutely no dream is ever too big to achieve. Also to my husband, who always challenges me and pushes me to become just a little bit better than I was before. ! vi! ACKNOWLEDGMENTS The completion of this Ph.D. degree would not be possible without the support and guidance of specific people. I am and will be forever grateful to have Dr. Ellen Walker as an advisor and a mentor. Throughout this long and tiring journey, she has been very patient with me. Through her careful guidance, I attained the ability to be independent and the ability to objectively evaluate my research. Watching her over the years, I learned how to effectively and respectively maneuver through the scientific community as a woman. I am also thankful for her endless critique of my scientific writing. Throughout the years, she spent endless hours correcting my writings and consistently allowed anything that I wrote to be my own work. It has been such an honor to be the understudy of someone who is very respected within the field of behavioral pharmacology. Her immense wealth of knowledge will forever serve as a reservoir for me. I would like to thank my dissertation committee for all of their academic and scientific guidance. My research was highly influenced by the collaborative inputs of Dr. Ellen Walker, Dr. Wayne Childers, Dr. Sarah Jane Ward, Dr. Ellen Unterwald, and Dr. Scott Rawls. I always enjoyed the stimulating discussions we had after the presentation of my data and I actually looked forward to them. I would like to give a special thank you to Dr. Sarah Jane Ward for training me from day one. She was the first person who showed me how to properly handle both mice and rats. She also trained me in drug-discrimination and self-administration, the two behavioral assays that encompassed my graduate research. When I was performing the self-administration studies, she was always eager to answer any of my questions, correct ! vii! my many experimental design issues, and made me really excited about reviewing my results, no matter how confounded they were. I would like to thank my fellow graduate students and lab mates Dr. Harshini Neelakantan and Rajesh Sanku for being easy to work around and for being daily, encouraging reminders that graduate students are not robots and still have the ability to socialize. I have to especially thank Dr. Neelakantan for showing me around the lab early on and teaching me the basics of laboratory etiquette. Without knowing it, she taught me what it meant to be a graduate student and showed me how to be an independent and dedicated scientist. I would like to thank the previous animal caretaker of the Pharmacy School, Beatrice, for taking such good care of my research animals throughout the years. Also for our daily conversations and the laughs that we shared. I must acknowledge and thank the Pharmaceutical Department of the Pharmacy School. I could not have chosen a better department to be apart of and a better environment to flourish in. I would like to thank AlMira Cutler for always taking care of any administrative assistance that I needed and being willing to answer any question that I had, no matter how irrelevant it was; if she was not able to answer it, she always pointed me in the direction of the person that could. I would like to thank Dr. Daniel Canney of the Pharmacy School and Dr. Zebulon Kendrick of Temple University Graduate Department for financially supporting me over the years. I would like to thank my family and especially my dad, for always inquiring about how I am doing in school and for sacrificing so much of himself to ensure my success and happiness. I thank him for instilling in me, from an early age, the importance of a secured education. Most importantly, I would like to thank my husband, future Dr. David ! viii! Burgess, for being by my side through this whole journey. Getting married in the middle of my graduate career was not easy, but it was the best decision I have ever made. He unselfishly endured my long nights studying and me spending every weekend at the lab, without any complaint. He spent countless hours listening to me practice my presentations and explaining my work, even though he had work of his own to focus on. Over the years of me being a graduate student, he was the stone that made me sharper and the little hint of light than made me shine brighter. Lastly, I would have to give thanks to God, for without my faith, I would not have made it this far.