% , 1,/)1%"0&011%""!& )+&3"/0&16,#&"++ #,/,1&+&+$1%" !"*& !"$/"" 2*&11"!6 " 2-"/3&0,/? +&3@/,#@/@ /)! @&11" "+1"/#,/%60&,),$6+!%/* ,),$6 +01&121",#%/* ,),$6 "!& )+&3"/0&16,#&"++ 8%/&+$"/01/00"LN=LKTK&"++=201/& &"++=KLCMKLR && Declaration I, Felix Paul Anton Mayer, declare that the current thesis entitled “Unravelling the mechanism of action of new psychoactive substances and their phase 1 metabolites“ was conducted under supervision of Prof. Dr. Harald H. Sitte at the Institute of Pharmacology, Medical University of Vienna. Except for the manuscripts embedded herein, the thesis at hand has not been submitted or published elsewhere. Further, I declare that this thesis was written in accordance with the guidelines for good scientific practice of the Medical University of Vienna. With respect to the publication embedded, entitled “Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters”, I declare that I had the original idea for the study, i.e. testing the phase 1 metabolites of mephedrone for their bioactive properties. Further, I performed and analysed uptake inhibition and release experiments in HEK293 cells. Together with Prof. Dr. Harald H. Sitte and Dr. Michael H. Baumann, I planned the design of the study and wrote the initial version of the manuscript. Dr. Laurin Wimmer synthesized the metabolites in the laboratory of Prof. Dr. Marko D. Mihovilovic. The release assays in rat brain synaptosomes, in vivo microdialysis and behavioural pharmacology studies were planned, designed and conducted in the laboratory of Dr. Michael H. Baumann. With respect to the publication embedded, entitled “Pharmacological characterization of fluorinated phenmetrazine “research chemicals””, I declare that I performed and analysed uptake inhibition and release studies in HEK293 cells. Dr. Simon D. Brandt provided the fluorinated phenmetrazine research chemicals. Release studies in rat brain synaptosomes were performed in the laboratories of Dr. Michael H. Baumann and Dr. Bruce E. Blough. Together with Prof. Harald H. Sitte, I planned the design of the study. I wrote the first draft of the manuscript, which was corrected and approved by Prof. Dr. Harald H. Sitte. We received significant input from all other co-authors, especially Dr. Michael H. Baumann. With respect to the book chapter embedded in this thesis, entitled “Application of a combined approach to identify new psychoactive drugs and decipher their mechanisms at monoamine transporters”, I declare that I wrote the first draft of the book chapter, which was then corrected and approved by Prof. Dr. Harald H. Sitte. &&& We received significant input from all other authors, especially Dr. Rainer Schmid and the editors Dr. Michael H. Baumann and Prof. Dr. Richard A. Glennon. The work embedded in this thesis was supported by a DOC-fellowship of the Austrian Academy of Sciences and the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung, FWF grant W1232 DK "Molecular Drug Targets" and F3506 SFB35 "Transmembrane Transporters in Health and Disease"). Vienna, January 2017 Felix P.A. Mayer &3 Abstract Psychostimulant abuse constitutes a growing problem on a global scale, with no effective treatments for psychostimulant addiction being available at present. In addition to well-characterized and regulated stimulants, such as cocaine or 3,4- methylenedioxymethamphetamine (MDMA, “ecstasy”), the drug markets are flooded with new psychoactive substances (NPS). NPS are often referred to as “bath salts” or “research chemicals” and are sold as legal alternatives to scheduled substances. Due to chemical modifications, NPS bypass regulations and result in an overwhelming variety of substances with unknown pharmacology. The rewarding, stimulating and addictive properties of psychostimulants arise from their ability to elevate the extracellular concentrations of the monoamines dopamine, norepinephrine and serotonin. This is achieved by disrupting the function of the transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Under physiological conditions, DAT, NET and SERT mediate the reuptake of exocytically-released monoamines and tightly regulate the strength of monoaminergic signaling. Despite similarities in their ability to enhance monoaminergic transmission, psychostimulants differ in their specific mechanisms of action. Cocaine-like drugs act as non-transported inhibitors, whereas amphetamine- like releasers, including some types of NPS, cause efflux of monoamines through the transporters. Furthermore, releasers act as substrates of DAT, NET and SERT and gain access to the cytosol. In the cytosol, releasers may disrupt the vesicular storage pools of monoamines and can exert neurotoxic effects. Thus, understanding how NPS act on DAT, NET and SERT is crucial to establishing the mechanism of action of this new class of compounds. During my thesis, I tested whether the phase 1 metabolites of 4-methylmethcathinone (mephedrone) possess psychoactive properties similar to the phase 1 metabolites of MDMA, and thus might contribute to the effects of mephedrone. Mephedrone became famous as part of the group of compounds called “bath salts” and is still abused as an alternative to MDMA. Results obtained from radiotracer-flux experiments showed that the phase 1 metabolites of mephedrone act as releasers at DAT, NET and SERT. In vivo studies in rats identified one metabolite that elevated extracellular dopamine and serotonin in the Nucleus accumbens and triggered locomotion upon systemic administration. Future studies shall investigate the pharmacokinetics of mephedrone and its metabolites in brain and plasma to estimate the overall contribution of the metabolites to the effects 3 of mephedrone. The second study embedded in this thesis provides a pharmacological characterization of the NPS 3-fluorophenmetrazine (3-FPM) and its positional isomers 2-FPM and 4-FPM. The chemical structure of 3-FPM is based on the scheduled drug phenmetrazine. 2-, 3- and 4-FPM were identified as releasers at DAT, NET and SERT. The marked affinity of each FPM at DAT and NET suggests addictive properties and enhanced likelihood for abuse. In conclusion, the projects embedded in this thesis reveal that the metabolites of mephedrone might contribute to the overall-effects of mephedrone in vivo and provide a pharmacological characterization of potential future drugs of abuse. Further, the techniques applied herein may serve as a guideline for unraveling the mode of action of psychostimulants at monoamine transporters. 3& Zusammenfassung Missbrauch von Psychostimulanzien stellt ein globales und im Wachsen begriffenes Problem dar. Die gleichzeitige Abwesenheit von effektiven Behandlungsstrategien, um die Abhängigkeit von diesen Substanzen zu behandeln, verleiht diesem Problem zusätzliche Brisanz. Neben den etablierten und intensiv beforschten Psychostimulanzien, wie etwa Cocain oder 3,4-Methylenedioxymethylamphetamin (MDMA; „ecstasy“), drängen neue psychoaktive Substanzen (NPS) auf die globalen Drogenmärkte. Diese auch als „Badesalze“ und „Research Chemicals“ bezeichneten Substanzen werden als legale Alternativen zu den regulierten und illegalen Stimulanzien konsumiert. Aufgrund von chemischen Modifikationen unterwandern NPS die Gesetzgebung und bringen eine Vielfalt an Substanzen hervor, deren pharmakologisches und toxikologisches Profil gänzlich unbekannt ist. Die stimulierende und belohnende Wirkung von Psychostimulanzien basiert auf deren Fähigkeit, die extrazelluläre Konzentration der Monoamine Dopamin, Serotonin und Noradrenalin zu erhöhen. Unter physiologischen Bedingungen bewerkstelligen hochaffine Transportproteine die Wiederaufnahme dieser Neurotransmitter und terminieren somit deren Wirkung an pre- und postsynaptischen Rezeptoren. Die Transporter für Dopamin (DAT), Noradrenalin (NET) und Serotonin (SERT) stellen den Angriffspunkt für Psychostimulanzien dar, wobei diese unterschiedliche Wirkungen an den Transportproteinen entfalten: Cocain-artige Substanzen agieren als nicht-transportierbare Inhibitoren und hemmen somit die Wiederaufnahme von exozytotisch freigesetzten Monoaminen. Amphetamin-artige Stimulanzien kehren die Transportrichtung von DAT, NET und SERT um, wodurch eine nicht-exozytotische Freisetzung von Monoaminen ausgelöst wird. Zusätzlich gelangen Amphetamin- artige Substanzen als Substrate von DAT, NET und SERT in das Zytosol und können somit in einigen Fällen die vesikulären Monoaminspeicher depletieren, womit potentielle neurotoxische Effekte assoziiert sind. Daraus resultierend ergibt sich die Notwendigkeit, die Wirkung von stimulierend wirkenden NPS an DAT, NET und SERT zu entschlüsseln. Während meiner Dissertation wurde der Frage nachgegangen, ob -analog der Phase 1 Metaboliten von MDMA- die Phase I Metaboliten von 4-Methylmethcathinon (Mephedron) psychoaktive Eigenschaften besitzen und somit zu den subjektiven Effekten von Mephedron betragen könnten. Mephedron erlangte als „Badesalz“ Berühmtheit und wird Gegenwärtig als MDMA- Alternative konsumiert. Experimente mit radioaktiv markierten Substraten von 3&& Monoamintransportern konnten zeigen, dass die Phase 1 Metaboliten von Mephedron mit DAT, NET und SERT interagieren und als Amphetamin-artige Substanzen eine nicht-exozytotische Freisetzung von Monoaminen bewirken. Mittels in vivo Studien in Ratten konnte ein Metabolit identifiziert werden, welcher nach systemischer Administration Dopamin und Serotonin im