Neuropharmacology and toxicology of novel amphetamine-type stimulants Bjørnar den Hollander Institute of Biomedicine, Pharmacology University of Helsinki Academic Dissertation To be presented, with the permission of the Medical Faculty of the University of Helsinki, for public examination in lecture hall 2, Biomedicum Helsinki 1, Haartmaninkatu 8, on January 16th 2015 at 10 am. Helsinki 2015 Supervisors Thesis committee Esa R. Korpi, MD, PhD Eero Castrén, MD, PhD Institute of Biomedicine, Pharmacology Neuroscience Center Faculty of Medicine University of Helsinki P.O. Box 63 (Haartmaninkatu 8) P.O. Box 56 (Viikinkaari 4) 00014 University of Helsinki, Finland 00014 University of Helsinki, Finland Esko Kankuri, MD, PhD Sari Lauri, PhD Institute of Biomedicine, Pharmacology Neuroscience Center and Faculty of Medicine Department of Biosciences/ Physiology P.O. Box 63 (Haartmaninkatu 8) University of Helsinki 00014 University of Helsinki, Finland P.O.Box 65 (Viikinkaari 1) 00014 University of Helsinki, Finland Reviewers Dissertation opponent Atso Raasmaja, Professor, PhD Prof David Nutt DM FRCP FRCPsych Division of Pharmacology and FMedSci Pharmacotherapy Edmond J. Safra Chair of Faculty of Pharmacy Neuropsychopharmacology P. O. Box 56 (Viikinkaari 5E) Division of Brain Sciences 00014 University of Helsinki, Finland Dept of Medicine Imperial College London Petri J. Vainio, MD, PhD Burlington Danes Building Pharmacology, Drug Development and Hammersmith Hospital Therapeutics Du Cane Road Institute of Biomedicine London W12 0NN, United Kingdom Faculty of Medicine Kiinamyllynkatu 10 C 20014 University of Turku, Finland The cover layout is done by Anita Tienhaara. The cover photo is by Edd Westmacott and shows a close-up of ecstasy tablets, photographed in Amsterdam in 2004. ISBN 978-951-51-0549-3 (paperback) ISBN 978-951-51-0550-9 (PDF, http://ethesis.helsinki.fi) ISSN 2342-3161 (print) and ISSN 2342-317X (online) Hansaprint Oy, Vantaa “…I feel absolutely clean inside, and there is nothing but pure euphoria. I have never felt so great, or believed this to be possible. The cleanliness, clarity, and marvelous feeling of solid inner strength continued throughout the rest of the day, and evening, and through the next day. I am overcome by the profundity of the experience…” - Alexander Shulgin, commenting on the subjective effects of 120 mg MDMA Table of contents Table of contents ................................................................................................................... 4 Original Publications .............................................................................................................. 7 Abbreviations......................................................................................................................... 8 Abstract ................................................................................................................................. 9 1. Introduction ..................................................................................................................... 10 2. Literature review ............................................................................................................. 11 2.1 Overview .................................................................................................................... 11 2.2 Defining amphetamine-type stimulants .................................................................... 11 2.3 History of amphetamines .......................................................................................... 14 2.3.1 Amphetamines in pre-industrial societies .......................................................... 14 2.3.2 Synthetic amphetamine and methamphetamine .............................................. 14 2.3.3 Rise of MDMA ..................................................................................................... 15 2.3.4 Substituted cathinones and the new drug market ............................................. 16 2.4 DA, 5-HT and NE systems .......................................................................................... 18 2.4.1 Neuroanatomy .................................................................................................... 18 2.4.2 Neurotransmitter biogenesis and metabolism .................................................. 21 2.5 Neuropharmacology of amphetamines .................................................................... 24 2.5.1 AMPH, METH and their isomers ......................................................................... 27 2.5.2 Action of AMPH at the plasmalemmal DAT ........................................................ 28 2.5.3 AMPH-mediated modulation of exocytotic DA release ..................................... 32 2.5.4 Effects of AMPH on secretory vesicles ............................................................... 33 2.5.5 Other AMPH targets ........................................................................................... 34 2.5.6 Action of substituted amphetamines and cathinones ....................................... 35 2.6 Evidence of amphetamine neurotoxicity .................................................................. 36 2.6.1 Preclinical studies ............................................................................................... 36 2.6.2 Human studies .................................................................................................... 40 2.7 Mechanisms of amphetamine neurotoxicity ............................................................ 44 2.7.1 ROS and oxidative stress .................................................................................... 45 2.7.2 Mitochondrial dysfunction ................................................................................. 48 2.7.3 Excitotoxicity....................................................................................................... 51 2.7.4 Hyperthermia...................................................................................................... 53 2.7.5 Other factors....................................................................................................... 54 2.8 Summary and rationale for the present study .......................................................... 55 3. Aims of the study ............................................................................................................. 56 4. Materials and methods ................................................................................................... 57 4.1 Study I: Effect of age on MDMA neurotoxicity .......................................................... 57 4.1.1 SPECT imaging in humans ................................................................................... 58 4.1.2 SERT binding studies in rats ................................................................................ 59 4.1.3 Statistics .............................................................................................................. 59 4.2 Study II: Long-term effects of 4-MMC and MDMC .................................................... 59 4.2.1 Measurement of monoamine levels .................................................................. 59 4 4.2.2 Measurement of DAT and SERT levels ............................................................... 60 4.2.3 Behavioral experiments ...................................................................................... 60 4.2.4 Statistics .............................................................................................................. 61 4.3 Study III: Effect of 4-MMC and METH on brain activity ............................................. 62 4.3.1 MEMRI procedure and data analysis .................................................................. 62 4.3.2 Behavioral experiments ...................................................................................... 63 4.3.3 Statistics .............................................................................................................. 63 4.4 Study IV: In vitro toxicity and redox reactivity .......................................................... 63 4.4.1 Cell culture .......................................................................................................... 64 4.4.2 Cytotoxicity assay ............................................................................................... 64 4.4.3 Cell proliferation and redox sensitivity assay ..................................................... 64 4.4.4 Analysis of reaction products ............................................................................. 64 4.4.5 Mitochondrial respiration................................................................................... 65 4.4.6 Mitochondrial complex I/II assay ....................................................................... 65 4.4.7 Statistics .............................................................................................................. 66 5. Results ............................................................................................................................. 67 5.1 Study I: Effect of age on MDMA neurotoxicity .......................................................... 67 5.1.1 Human SPECT neuroimaging .............................................................................. 67 5.1.2 Rat SERT binding ................................................................................................. 67 5.2 Study II: Long-term effects of 4-MMC and MDMC ...................................................