An investigation into the antidyskinetic and reinforcing effects of UWA-101 in Sprague Dawley rats Angela Estelle McLelland, B.Sc. (Hons), M.D. This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia School of Medicine and Pharmacology Pharmacology and Anaesthesiology Unit 2019 1 2 Thesis Declaration I, Angela McLelland, certify that: This thesis has been substantially accomplished during enrolment in this degree. This thesis does not contain material which has been submitted for the award of any other degree or diploma in my name, in any university or other tertiary institution. In the future, no part of this thesis will be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint-award of this degree. This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text and, where relevant, in the Authorship Declaration that follows. This thesis does not violate or infringe any copyright, trademark, patent, or other rights whatsoever of any person. The research involving animal data reported in this thesis was assessed and approved by The University of Western Australia Animal Ethics Committee. Approval RA/3/100/1165, RA/3/100/1182, RA/3/100/1178. The research involving animals reported in this thesis followed The University of Western Australia and national standards for the care and use of laboratory animals. This thesis contains published work and/or work prepared for publication, some of which has been co-authored. Signature: Date: 16/08/19 3 4 Abstract Long-term treatment of people with Parkinson’s disease (PD) with the gold-standard drug, L-3,4-dihydroxyphenylalanine (L-DOPA), often results in debilitating excessive involuntary movements known as L-DOPA induced dyskinesia (LID). There is limited treatment for LID, with only one drug, amantadine, currently licensed for this purpose, and its long-term efficacy is questionable. Research into pharmacological treatment of LID has included the investigation of MDMA-analogue UWA-101, which has affinity for the transporters of dopamine and serotonin, but not noradrenaline. The first aim of this thesis was to investigate the antidyskinetic and reinforcing effects of increasing doses of UWA-101 in Sprague-Dawley rats in three separate studies. Two of these studies involved the reserpinised rat model, and a second aim was to determine the potential of this model as an early screening procedure for LID drug discovery, prior to more extensive lesion models. This procedure serves to induce PD-like symptoms such as akinesia in a short time-frame through the administration of reserpine, which depletes monoamine neurotransmitter stores via vesicular monoamine transport 2 (VMAT2) inhibition. In this model, it has been proposed that rat vertical movements are analogous to human dyskinesia, whereas reductions in horizontal activity (akinesia) reflect the loss of dopamine and mimic PD symptoms. UWA-101 is an analogue of the illicit drug MDMA ('ecstasy'), so the third aim was to determine abuse liability or aversive subjective effects of UWA-101 that may reduce its treatment utility with three separate measurements: the conditioned place preference test, as a 5 measure of the reinforcing or aversive effects of a drug,; the elevated plus maze test, as a measure of anxiogenic effects of a drug; and ultrasonic vocalisations (USV), as a measure of both hedonic (50 kHz) and aversive (20 kHz) effects of a drug. Using custom-made locomotion detection equipment, UWA-101 (1 and 3 mg/kg) significantly reduced L-DOPA-induced hyperkinesia and behavioural dyskinesia correlates in reserpinised rats. Yohimbine, riluzole, and rimonabant, antidyskinetic compounds used to assess the validity of the reserpinised rat model, also significantly reduced L-DOPA-induced hyperkinesia and behavioural dyskinesia correlates. It can be concluded that the reserpinised rat may serve as an early and relatively quick screening model prior to more extensive lesion models. Treatment of reserpinised rats with UWA-101 (3 mg/kg) was then preceded by treatment with drugs of known pharmacological affinities to assess the underlying mechanism of the anti-hyperkinetic effect of UWA-101. Fluoxetine (a serotonin transporter [SERT] inhibitor), WAY-100635 (a 5-HT1A receptor antagonist) and riluzole (a glutamate release inhibitor) all blocked UWA-101 reversal of L-DOPA-induced hyperkinesia and dyskinesia correlates. These observations indicate that UWA-101 reversal of L-DOPA induced hyperkinesia may occur through reversal of SERT function, as does MDMA, and through activation of the 5-HT1A receptor, a process that is also dependent on glutamate release. UWA-101 (3 mg/kg) did not produce conditioned place preferences or aversions in the CPP test, changes in USVs or anxiolytic effects in the EPM test. In contrast, dexamphetamine (1.5 mg/kg) as a positive control, induced a robust place preference, increased 50 kHz USVs and increased open arm activity in the EPM test. Therefore, UWA- 101 did not appear to have abuse liability or to produce aversive side-effects at this dose. 6 UWA-101 reduced L-DOPA induced hyperkinesia in line with earlier lesion studies in rats and primates. Similar effects were induced by other drugs shown to have antidyskinetic activities in non-human lesioned animal models, including yohimbine, riluzole and rimonabant, further supporting the utility of the reserpinised model. These findings support the view that multiple neurotransmitters other than dopamine may be involved in the development or expression of L-DOPA induced dyskinetic behaviour. Furthermore, the mechanisms underlying the anti-hyperkinetic effect of UWA-101 seem to involve the serotonergic system. In conclusion, the reserpinised rat model is useful as an early screen for antidyskinetic compounds, prior to more expensive and time-consuming lesion studies. The findings with UWA-101 support earlier research in such lesion studies indicating its potential utility as a therapeutic agent for LID without evidence of abuse liability or aversive side-effects, and provides the first evidence that UWA-101, like MDMA, reverses the SERT. 7 8 Table of Contents Abstract……………………………………………………………………………………….5 List of Tables………………………………………………………………………………...15 List of Figures……………………………………………………………………………….17 List of Abbreviations………………………………………………………………………..21 Acknowledgements………………………………………………………………………….23 Authorship declaration: Co-authored publications………………………………..……..25 Statement of candidate contribution………………………………………………………27 Chapter 1 – General Introduction…………………………………………………………29 1. Introduction..................................................................................................................29 1.1 Development of L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) ......................................................................................................................31 1.1.1 Overview of the basal ganglia....................................................................31 1.1.2 Factors involved in LID development........................................................36 1.1.2.1 Hypothesis of LID emergence..............................................................36 1.1.2.2 Delaying the onset of LID....................................................................38 1.1.2.3 Modifications of L-DOPA in established LID.....................................41 1.2 Pharmacological treatment of LID.........................................................................43 1.2.1 Dopamine receptors....................................................................................43 1.2.1.1 D1-like receptors...................................................................................43 1.2.1.2 D2-like receptors...................................................................................45 1.2.2 Serotonin receptors.....................................................................................53 1.2.2.1 5-HT1A/b receptors...............................................................................54 1.2.2.2 5-HT2A receptors..................................................................................58 1.2.3 Monoamine transporters.............................................................................60 1.2.4 Glutamate receptors....................................................................................64 1.2.4.1 N-methyl-D-aspartate (NMDA) receptors...........................................64 1.2.4.2 Alpha-amine-3-hydroxyl-methyl-4-isoxazleproprionic acid (AMPA) receptors...............................................................................................67 1.2.4.3 Metabotropic receptors.........................................................................69 1.2.5 Adrenergic receptors..................................................................................73 1.2.6 Cannabinoid receptors................................................................................77 1.3 Animal models of LID...........................................................................................80 1.3.1 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated primate........................................................................................................81