Serotonin- and dopamine-mediated neurotransmission in the ... · ii Serotonin- and...

Serotonin- and dopamine-mediated neurotransmission in the pathophysiology and treatment of

Parkinson’s disease

by

Philippe Huot

A Thesis submitted in conformity with the requirements

for the degree of Philosophiae Doctor (PhD)

Graduate Department of Medical Science

University of Toronto

© Copyright by Philippe Huot 2012

ii

Serotonin- and dopamine-mediated neurotransmission in the pathophysiology and treatment of Parkinson’s disease

Philippe Huot, Philosophiae Doctor (PhD), Institute of Medical Science, University of Toronto (2012)

Abstract

Dopamine deficiency in the striatum is a central feature of Parkinson’s disease (PD). Symptomatic therapy

with L-3,4-dihydroxyphenylalanine (L-DOPA) aims at restoring physiological dopaminergic neurotransmission

within the brain. Unfortunately, current treatment paradigms fail to achieve this goal, which leads to the emergence

of motor complications, secondary to long term L-DOPA administration, including dyskinesia and wearing-OFF,

and non-motor symptoms related to disease progression, including neuropsychiatric symptoms such as psychosis.

However, degenerative changes in PD are not limited to the dopaminergic system, but also affect the serotonergic

system. There is increasing evidence suggesting an involvement of the serotonergic system in the pathophysiology

of both motor and non-motor complications of PD. The work presented in this Thesis has investigated the

serotonergic and dopaminergic systems in PD, by performing post mortem studies in the brains of PD patients and

of parkinsonian non-human primates (NHPs), and by performing behavioural studies in the parkinsonian rat and

NHP models of PD. The main conclusions presented are that: 1) serotonergic type 1A (5-HT1A) and 2A (5-HT2A)

levels are altered in the brains of dyskinetic parkinsonian NHPs, suggesting abnormal 5-HT1A- and 5-HT2A-mediated

neurotransmission in dyskinesia; 2) 5-HT2A receptor levels are altered in the brains of PD patients with visual

hallucinations (VH), suggesting abnormal 5-HT2A-mediated neurotransmission in VH; 3) some of the anti-dyskinetic

actions attributed to stimulating 5-HT1A or antagonising 5-HT2A receptors might in fact be due to an antagonist

action at D4 receptors, as antagonising D4 receptors significantly alleviates L-DOPA-induced dyskinesia in rat and

NHP models of PD; 4) concurrent inhibition of the serotonin and dopamine transporters (SERT and DAT,

respectively) enhances duration of L-DOPA-induced ON-time in the parkinsonian NHP. However, the ratio of

SERT/ DAT inhibition appears crucial in determining the quality of this extra ON-time; SERT > DAT inhibition

exacerbates the severity of L-DOPA-induced dyskinesia, whereas SERT = DAT and DAT > SERT inhibition do not

worsen the severity of L-DOPA-induced dyskinesia. Together these data extend our knowledge of the interaction

between serotonin and dopamine, specifically as they relate to symptoms and side effects of dopamine replacement

therapy in PD and highlight potential novel therapeutic approaches to PD.

iii

Acknowledgements

This candidate would like to express his gratitude to the people under whose supervision he has done his

PhD degree: Dr Robert Chen, Dr Anthony Lang, and Dr William Hutchison and especially Dr Jonathan Brotchie and

Dr Susan Fox, whose supervision and mentorship have been invaluable. This candidate would also like to warmly

thank the members of Dr Jonathan Brotchie’s laboratory: Dr Tom Johnston and Dr James Koprich, Ms Gabriela

Reyes, Ms Maria Espinosa and Ms Donna Pires. This candidate would also like to thank former members of Dr

Brotchie’s laboratory: Ms Sherri Thiele and Ms Sherry Sun. This candidate would also like to express his gratitude

to collaborators from the University of Western Australia: Dr Matthew Piggott, Dr Katie Lewis and Dr Michael

Gandy. This candidate would also like to thank people responsible for animal care in Toronto and Suzhou: Dr

Alyssa Goldstein, Dr Melissa Madden, Mr Robert Lopez, Mr Mario D’Souza, and Mr Taojian. This candidate would

also like to thank Mr Perry Chong and Mr Neil Amos, from the Radiation Safety Office of the University Health

Network. This candidate would also like to thank Mr Walter Schmanda from the library of the Toronto Western

Hospital. This candidate would like to express his gratitude to the organisations which fund him during his PhD: the

Edmond J Safra Philanthropic Foundation, the Parkinson Society Canada and the Canadian Institutes of Health

Research. Lastly, this candidate would like to thank his parents, Réjean and Carmen, and his sister Marie-Christine

for their continuous presence and their unconditional support.

iv

Contribution of the authors of each Chapter to the work presented in the Thesis

v

Chapter I: Regulation of cortical and striatal 5-HT1A receptors in the MPTP-lesioned macaque

Authors: Philippe Huot, Tom H Johnston, James B Koprich, Lieke Winkelmolen, Susan H Fox, Jonathan M Brotchie

Research project

Conception: PH, THJ, SHF, JMB; Organisation: PH, THJ, JBK, SHF, JMB; Execution: PH, LK

Statistical Analysis:

Design: PH, SHF, JMB; Execution: PH; Review: PH, SHF, JMB; Critique: PH, SHF, JMB

Manuscript Preparation

Writing of the first draft: PH; Review and critique: PH, THJ, JBK, SHF, JMB

Chapter II: 5-HT2A receptors increase in MPTP-lesioned macaques treated chronically with L-DOPA

Authors: Philippe Huot, Tom H Johnston, Lieke Winkelmolen, Susan H Fox, Jonathan M Brotchie

Research project

Conception: PH, THJ, SHF, JMB; Organisation: PH, THJ, SHF, JMB; Execution: PH, LK


Design: PH, SHF, JMB; Execution: PH; Review: PH, SHF, JMB; Critique: PH, SHF, JMB


Writing of the first draft: PH; Review and critique: PH, THJ, SHF, JMB

Chapter III: Increased 5-HT2A receptors in the temporal cortex of parkinsonian patients with visual hallucinations

Authors: Philippe Huot, Tom H Johnston, Tayyeba Darr, Lili-Naz Hazrati, Naomi P Visanji, Donna Pires, Jonathan

M Brotchie, Susan H Fox

Research project

Conception: PH, THJ, NPV, JMB, SHF; Organisation: PH, THJ, LN, NPV, DP, JMB, SHF; Execution: PH, TD


Design: PH, JMB, SHF; Execution: PH; Review: PH, JMB, SHF; Critique: PH, JMB, SHF


Writing of the first draft: PH; Review and critique: PH, THJ, SHF, JMB

Chapter IV: The D4 receptor antagonist L-745,870 reduces the severity of L-DOPA-induced dyskinesia in the MPTP-

lesioned macaque

Authors: Philippe Huot, Tom H Johnston, James B Koprich, Susan H Fox, Ahmed Aman, Jonathan M Brotchie

Research project

Conception: PH, THJ, SHF, AA, JMB; Organisation: PH, THJ, SHF, AA, JMB; Execution: PH, THJ, JBK, AA


Design: PH, SHF, AA, JMB; Execution: PH, AA; Review: PH, SHF, AA, JMB Critique: PH, SHF, AA, JMB


vi

Writing of the first draft: PH; Review and critique: PH, THJ, JBK, SHF, AA, JMB

Chapter V: The D4 receptor antagonist L-745,870 reduces the expression and de novo development, of abnormal

involuntary movements in the 6-OHDA-lesioned rat

Authors: Philippe Huot, Tom H Johnston, James B Koprich, Donna Pires, Maria Espinosa, M Gabriela Reyes, Susan

H Fox, Jonathan M Brotchie

Research project

Conception: PH, THJ, SHF, JMB; Organisation: PH, THJ, DP, SHF, JMB; Execution: PH, GR, ME


Design: PH, THJ, SHF, JMB; Execution: PH; Review: PH, THJ, SHF, JMB; Critique: PH, SHF, JMB


Writing of the first draft: PH; Review and critique: PH, THJ, JBK, SHF, JMB

Chapter VI: Characterisation of 3,4-methylenedioxymethamphetamine (MDMA) enantiomers in vitro and in the

MPTP-lesioned primate: R-MDMA reduces severity of dyskinesia whereas S-MDMA extends duration of ON-time

Authors: Philippe Huot, Tom H Johnston, Katie D Lewis, James B Koprich, M Gabriela Reyes, Susan H Fox,

Matthew J Piggott, Jonathan M Brotchie

Research project

Conception: PH, THJ, SHF, MJP, JMB; Organisation: PH, THJ, KDL, SHF, MJP, JMB; Execution: PH, THJ,

KDL, JBK, MGR


Design: PH, THJ, SHF, MJP, JMB; Execution: PH, KDL; Review: PH, THJ, SHF, MJP, JMB; Critique: PH,

SHF, MJP, JMB


Writing of the first draft: PH; Review and critique: PH, THJ, JBK, SHF, MJP, JMB

Chapter VII: The monoamine re-uptake inhibitor UWA-0101 improves motor fluctuations in the parkinsonian

marmoset



Research project


KDL, JBK, MGR


vii


SHF, MJP, JMB


Writing of the first draft: PH; Review and critique: PH, THJ, JBK, SHF, MJP, JMB

Chapter VIII: UWA-0121, a mixed dopamine and serotonin re-uptake inhibitor, enhances L-DOPA anti-

parkinsonian action without worsening dyskinesia or psychosis-like behaviours in the MPTP-lesioned common

marmoset



Research project


KDL, JBK, MGR



SHF, MJP, JMB


Writing of the first draft: PH

Review and critique: PH, THJ, JBK, SHF, MJP, JMB

This way of showing the contribution of the authors to each Chapter is based on the Wiley Online Library style and

can be found at: http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1531-8257/homepage/ForAuthors.html.

http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1531-8257/homepage/ForAuthors.html

viii

Table of contents

Abstract ........................................................................................................................................................................ ii

Acknowledgements .................................................................................................................................................... iii

Contribution of the authors of each Chapter to the work presented in the Thesis ............................................... iv

Table of contents ...................................................................................................................................................... viii

List of Tables ........................................................................................................................................................... xxiv

List of Figures ........................................................................................................................................................ xxvii

List of Appendices ................................................................................................................................................... xxx

List of abbreviations ............................................................................................................................................... xxxi

Thesis summary ........................................................................................................................................................... 1

Introduction ................................................................................................................................................................. 3

History ...................................................................................................................................................................... 4

L-DOPA-induced motor complications and visual hallucinations in PD: clinical description ................................. 4

Pathophysiology of L-DOPA-induced motor complications .................................................................................... 5

The classic model of the basal ganglia and dyskinesia ........................................................................................ 5

Table Int.1: changes in the circuitry of the basal ganglia in the dyskinetic state ........................................ 6

The dopaminergic system and dyskinesia ............................................................................................................ 7

Striatal dopamine denervation and dyskinesia ................................................................................................ 7

Pulsatile dopaminergic therapy and dyskinesia ............................................................................................... 7

Dopamine receptors and dyskinesia ................................................................................................................ 8

Table Int.2: important concepts involving dopamine receptors in dyskinesia ............................................ 9

The serotonergic system and dyskinesia ............................................................................................................ 10

The glutamatergic system and dyskinesia .......................................................................................................... 10

NMDA receptors and dyskinesia .................................................................................................................. 10

AMPA receptors and dyskinesia ................................................................................................................... 11

Metabotropic glutamate receptors and dyskinesia ........................................................................................ 11

Pharmacological modulation of the glutamatergic system and dyskinesia ................................................... 12

Altered synaptic plasticity and dyskinesia .................................................................................................... 12

Morphological alterations of glutamatergic synapses and dyskinesia ........................................................... 13

Table Int.3: the glutamatergic system and dyskinesia .............................................................................. 13

The GABAergic system and dyskinesia ............................................................................................................. 14

ix

Table Int.4: important concepts involving the GABAergic system in dyskinesia .................................... 14

The opioidergic system and dyskinesia .............................................................................................................. 14

Table Int.5: important concepts involving the opioidergic system in dyskinesia ..................................... 15

The adenosine system and dyskinesia ................................................................................................................ 16

Table Int.6: important concepts involving the adenosine system in dyskinesia ....................................... 16

Alpha-adrenergic neurotransmission and dyskinesia ......................................................................................... 16

Beta-adrenergic neurotransmission and dyskinesia ........................................................................................... 17

Histaminergic neurotransmission and dyskinesia .............................................................................................. 17

Cannabinoid neurotransmission and dyskinesia ................................................................................................. 18

Gene regulation and intracellular signalling in dyskinesia ................................................................................. 19

Table Int.7: important concepts involving gene regulation and intracellular signalling in dyskinesia ..... 20

Synaptic transmission and dyskinesia ................................................................................................................ 20

Pharmacological treatment of motor complications ............................................................................................... 21

Dyskinesia .......................................................................................................................................................... 21

Wearing-OFF ..................................................................................................................................................... 21

Non-motor manifestations of dopaminergic therapy and disease progression ....................................................... 23

Hypotheses and aims of each Chapter of the Thesis ............................................................................................... 25

Chapter I: Regulation of cortical and striatal 5-HT1A receptors in the MPTP-lesioned macaque ..................... 29

Chapter summary .................................................................................................................................................... 30

Abstract .................................................................................................................................................................. 32

Introduction ............................................................................................................................................................ 33

Materials and methods ............................................................................................................................................ 34

Animals .............................................................................................................................................................. 34

High performance liquid chromatography analysis for biogenic amines ........................................................... 34

Dopamine transporter autoradiography .............................................................................................................. 35

5-HT1A receptor autoradiography ....................................................................................................................... 35

Autoradiographic binding analysis .................................................................................................................... 35

Statistical analysis .............................................................................................................................................. 36

Results .................................................................................................................................................................... 36

Assessment of parkinsonian and dyskinetic states prior to termination ............................................................. 36

Extent of dopaminergic denervation in the striatum of normal and parkinsonian macaques ............................. 36

5-HT1A binding levels in the brains of normal and MPTP-lesioned macaques .................................................. 36

Discussion ............................................................................................................................................................... 38

Technical considerations .................................................................................................................................... 38

5-HT1A receptor levels in the premotor and motor cortex .................................................................................. 39

5-HT1A receptors in the striatum ........................................................................................................................ 40

5-HT1A receptors in the striosomes ............................................................................................................... 40

5-HT1A receptors in the matrix ...................................................................................................................... 40

x

Concluding remarks ................................................................................................................................................ 41

Figures and legends ................................................................................................................................................ 42

Figure I.1: 5-HT1A binding levels in vehicle-vehicle, MPTP-vehicle, MPTP-L-DOPA-acute, and MPTP-L-

DOPA-chronic macaques ....................................................................................................................................... 45

Figure I.2: 5-HT1A binding levels in the hippocampal formation ........................................................................... 46

Figure I.3: 5-HT1A binding levels in the premotor and motor cortex ...................................................................... 47

Figure I.4: 5-HT1A binding levels in the striatum ................................................................................................... 48

Figure I.5: 5-HT1A binding levels in the striosomes and the matrix ...................................................................... 49

Chapter II: 5-HT2A receptors increase in MPTP-lesioned macaques treated chronically with L-DOPA ......... 50

Chapter summary .................................................................................................................................................... 51

Abstract .................................................................................................................................................................. 53

Introduction ............................................................................................................................................................ 54


Animals .............................................................................................................................................................. 55

High performance liquid chromatography analysis ........................................................................................... 55

Dopamine transporter autoradiography .............................................................................................................. 56

5-HT2A receptor autoradiography ....................................................................................................................... 56

Autoradiographic binding analysis .................................................................................................................... 57


Results .................................................................................................................................................................... 57

Assessment of parkinsonian and dyskinetic states prior to termination ............................................................. 57

Dopamine transporter levels in the striatum of control and parkinsonian macaques ......................................... 58

High performance liquid chromatography for monoamines in the striatum of control and parkinsonian

macaques ............................................................................................................................................................ 58

5-HT2A receptor levels in the brains of control and parkinsonian macaques ..................................................... 59

Discussion ............................................................................................................................................................... 59


The relationship between chronic L-DOPA therapy and the emergence of dyskinesia in the MPTP-lesioned

macaque ............................................................................................................................................................. 61

Increased 5-HT2A receptor levels in the striatum and motor cortex of MPTP-lesioned macaques following

chronic L-DOPA therapy. .................................................................................................................................. 61

Concluding remarks ........................................................................................................................................... 62

Figures and legends ................................................................................................................................................ 64

Figure II.1: brain areas in which 5-HT2A receptor levels were determined ............................................................ 66

Figure II.2: dopamine transporter binding levels .................................................................................................... 67

Figure II.3: autoradiograms of [3H]-ketanserin binding across the different brain areas studied ........................... 68

Table II.1: Dopamine and metabolite levels in the striatum of control and parkinsonian macaques ...................... 69

Table II.2: 5-HT2A receptor levels across the studied brain areas ........................................................................... 70

xi

Chapter III: Increased 5-HT2A receptors in the temporal cortex of parkinsonian patients with visual

hallucinations ............................................................................................................................................................. 71

Chapter summary .................................................................................................................................................... 72

Abstract .................................................................................................................................................................. 74

Introduction ............................................................................................................................................................ 75


Tissue collection ................................................................................................................................................ 75

[3H]-Ketanserin autoradiographic binding ......................................................................................................... 76


Results .................................................................................................................................................................... 77

Patient characteristics ......................................................................................................................................... 77

[3H]-Ketanserin binding in human post mortem brains ..................................................................................... 77

Discussion ............................................................................................................................................................... 78


Patient characteristics ......................................................................................................................................... 78

Abnormal 5-HT2A-mediated neurotransmission in parkinsonian patients with visual hallucinations ................ 79

Increased 5-HT2A receptor levels in the infero-lateral temporal cortex of parkinsonian patients with visual

hallucinations ..................................................................................................................................................... 80

Increased 5-HT2A receptor levels in the motor cortex of parkinsonian patients ................................................. 80

Figure legends ........................................................................................................................................................ 82

Figure III.1: brain areas in which 5-HT2A receptor levels were determined ........................................................... 83

Figure III.2: 5-HT2A binding levels in normal brains ............................................................................................. 84

Figure III.3: 5-HT2A binding levels in parkinsonian brains .................................................................................... 85

Table III.1: Clinical characteristics of study patients ............................................................................................. 86

Table III.2: 5-HT2A receptor levels across the studied brain areas ......................................................................... 89

Chapter IV: The D4 receptor antagonist L-745,870 reduces the severity of L-DOPA-induced dyskinesia in the

MPTP-lesioned macaque .......................................................................................................................................... 90

Chapter summary .................................................................................................................................................... 91

Abstract .................................................................................................................................................................. 94

Introduction ............................................................................................................................................................ 95


Animals .............................................................................................................................................................. 96

Induction of parkinsonism and dyskinesia in the cynomolgus macaque ............................................................ 96

Administration of L-745,870 in combination with L-DOPA to MPTP-lesioned macaques .............................. 96

Behavioural assessment of L-745,870 in the MPTP-lesioned macaque............................................................. 97


Results .................................................................................................................................................................. 100

L-745,870 does not impair L-DOPA anti-parkinsonian action ........................................................................ 100

xii

L-745,870 reduces the severity of L-DOPA-induced dyskinesia and extends duration of “good quality” ON-

time .................................................................................................................................................................. 101

Discussion ............................................................................................................................................................. 103

Technical considerations .................................................................................................................................. 103

Effect of L-745,870 on L-DOPA-induced dyskinesia and L-DOPA anti-parkinsonian efficacy ..................... 104

Concluding remarks .............................................................................................................................................. 105

Figure legends ...................................................................................................................................................... 106

Figure IV.1: motor activity ................................................................................................................................... 109

Figure IV.2: parkinsonism and ON-time duration ................................................................................................ 110

Figure IV.3: dyskinesia and quality of ON-time .................................................................................................. 111

Figure IV.4: quality of ON-time – summary ........................................................................................................ 112

Figure IV.5: L-745,870 pharmacokinetic time course the MPTP-lesioned macaque ........................................... 113

Figure IV.6: effect of L-745,870 administration on L-DOPA pharmacokinetics in the MPTP-lesioned macaque

.............................................................................................................................................................................. 114

Table IV.1: L-745,870 pharmacokinetic parameters in the MPTP-lesioned macaque ......................................... 115

Table IV.2: L-745,870 levels in the brain and cerebrospinal fluid in the MPTP-lesioned macaque at peak

behavioural effect ................................................................................................................................................. 116

Chapter V: The D4 receptor antagonist L-745,870 reduces the expression and de novo development, of

abnormal involuntary movements in the 6-OHDA-lesioned rat ......................................................................... 117

Chapter summary .................................................................................................................................................. 118

Abstract ................................................................................................................................................................ 121

Introduction .......................................................................................................................................................... 122

Materials and methods .......................................................................................................................................... 122

6-OHDA-lesioned rat model of Parkinson’s disease ....................................................................................... 122

Cylinder test ..................................................................................................................................................... 123

Drug treatment ................................................................................................................................................. 123

Acute study ................................................................................................................................................. 123

De novo study .............................................................................................................................................. 123

Behavioural testing .......................................................................................................................................... 123

Rotational study .......................................................................................................................................... 123

AIMs ........................................................................................................................................................... 124

Rotarod ........................................................................................................................................................ 124

Dopamine transporter autoradiography ............................................................................................................ 124

Statistical analysis ............................................................................................................................................ 125

Results .................................................................................................................................................................. 125

Extent of dopaminergic lesion ......................................................................................................................... 125

L-745,870 reduces the duration and amplitude of L-DOPA-induced ALO AIMs and reduces the rotational

activity ............................................................................................................................................................. 126

xiii

Simultaneous administration of L-DOPA and L-745,870 during the priming process does not prevent but

reduces the development of rotational activity ................................................................................................. 126

Simultaneous administration of L-DOPA and L-745,870 during the priming process does not prevent but

reduces the development of ALO AIMs .......................................................................................................... 127

The addition of L-745,870 to L-DOPA has a negative impact on rotarod performance .................................. 127

Discussion ............................................................................................................................................................. 128

L-745,870 reduces previously-established AIMs and rotational behaviour, but at the expense of impairing the

anti-parkinsonian efficacy of L-DOPA ............................................................................................................ 128

L-745,870 reduces the development of AIMs and rotational behaviour when administered de novo with L-

DOPA ............................................................................................................................................................... 129

Concluding remarks – therapeutic implications ............................................................................................... 129

Figure legends ...................................................................................................................................................... 131

Figure V.1: effect of 6-hydroxydopamine (6-OHDA) lesion on the cylinder test and dopamine transporter (DAT)

autoradiography .................................................................................................................................................... 135

Figure V.2: effect of acute challenges of L-745,870 on axial, limbs and orolingual (ALO) abnormal involuntary

movements (AIMs) and rotational behaviour ....................................................................................................... 136

Figure V.3: effect of chronic treatment with L-745,870 on the development of rotational behaviour ................. 137

Figure V.4: effect of chronic treatment with L-745,870 on the development of axial, limb and orolingual (ALO)

abnormal involuntary movements (AIMs) ........................................................................................................... 138

Figure V.5: effect of L-DOPA/ vehicle and L-DOPA/ L-745,870 on rotarod performance ................................. 139

Chapter VI: Characterisation of 3,4-methylenedioxymethamphetamine (MDMA) enantiomers in vitro and in

the MPTP-lesioned primate: R-MDMA reduces severity of dyskinesia whereas S-MDMA extends duration of

ON-time .................................................................................................................................................................... 140

Chapter summary .................................................................................................................................................. 141

Abstract ................................................................................................................................................................ 144

Introduction .......................................................................................................................................................... 145


R- and S-MDMA synthesis .............................................................................................................................. 145

In vitro pharmacology ...................................................................................................................................... 146

Tissue preparation ....................................................................................................................................... 146

Radioligands and drugs ............................................................................................................................... 146

Receptor and transporter binding assays ..................................................................................................... 146

Determination of R- and S-MDMA affinity at selected receptor/ transporters ........................................... 147

Behavioural assessment of R- and S-MDMA in the MPTP-lesioned non-human primate .............................. 147

Induction of parkinsonism and dyskinesia in the common marmoset ......................................................... 147

Administration of R- and S-MDMA, in combination with L-DOPA, to parkinsonian marmosets ............. 147

Behavioural analysis ................................................................................................................................... 148


xiv

Results .................................................................................................................................................................. 149

Pharmacological profile of R- and S-MDMA .................................................................................................. 149

R-MDMA decreases the severity of L-DOPA-induced dyskinesia without reducing the anti-parkinsonian

action of L-DOPA ............................................................................................................................................ 149

S-MDMA extends duration of anti-parkinsonian action of L-DOPA, but worsens the severity of dyskinesia 150

Effects of R- and S-MDMA on L-DOPA-induced psychosis-like behaviour .................................................. 150

Discussion ............................................................................................................................................................. 151

Technical considerations .................................................................................................................................. 151

R- and S-MDMA binding profile ................................................................................................................ 151

5-HT2A receptors and dyskinesia ...................................................................................................................... 151

Serotonergic and dopaminergic transporters inhibition and ON-time extension ............................................. 152

MDMA enantiomers and dopaminergic psychosis-like behaviours ................................................................. 153

Concluding remarks ......................................................................................................................................... 153

Figures and legends .............................................................................................................................................. 154

Figure VI.1: synthesis of MDMA enantiomers via a novel enantiospecific process ............................................ 158

Figure VI.2: time course of dyskinesia and parkinsonism .................................................................................... 159

Figure VI.3: peak dose dyskinesia ........................................................................................................................ 160

Figure VI.4: peak dose parkinsonism ................................................................................................................... 161

Figure VI.5: ON-time and quality of ON-time ..................................................................................................... 162

Figure VI.6: psychosis-like behaviour .................................................................................................................. 163

Table VI.1: Experimental parameters for receptor/ transporters binding assays .................................................. 164

Table VI.2: MDMA enantiomers binding profiles ............................................................................................... 165

Chapter VII: The monoamine re-uptake inhibitor UWA-0101 acutely improves motor fluctuations in the

parkinsonian marmoset .......................................................................................................................................... 166

Chapter summary .................................................................................................................................................. 167

Abstract ................................................................................................................................................................ 170

Introduction .......................................................................................................................................................... 171


UWA-0101 synthesis ....................................................................................................................................... 172

Behavioural assessment of UWA-0101 in the MPTP-lesioned common marmoset ........................................ 172


Administration of UWA-0101 in combination with L-DOPA to the parkinsonian marmoset .................... 172



Results .................................................................................................................................................................. 174

Effects of UWA-0101 on L-DOPA anti-parkinsonian action in the MPTP-lesioned common marmoset ....... 174

Effects of UWA-0101 on L-DOPA-induced dyskinesia in the MPTP-lesioned common marmoset ............... 174

xv

Effects of UWA-0101 on L-DOPA-induced psychosis-like behaviours in the MPTP-lesioned common

marmoset .......................................................................................................................................................... 175

Discussion ............................................................................................................................................................. 175

UWA-0101 extends duration of ON-time without exacerbating dyskinesia severity ...................................... 175

UWA-0101 exacerbates the severity of psychosis-like behaviour ................................................................... 176


Figure legends ...................................................................................................................................................... 178

Figure VII.1: ON-time and quality of ON-time .................................................................................................... 180

Figure VII.2: dyskinesia ....................................................................................................................................... 181

Figure VII.3: psychosis-like behaviour ................................................................................................................ 182

Figure VII.4: quality of ON-time – summary ....................................................................................................... 183

Chapter VIII: UWA-0121, a mixed dopamine and serotonin re-uptake inhibitor, acutely enhances L-DOPA

anti-parkinsonian action without worsening dyskinesia or psychosis-like behaviours in the MPTP-lesioned

common marmoset .................................................................................................................................................. 184

Chapter summary .................................................................................................................................................. 185

Abstract ................................................................................................................................................................ 188

Introduction .......................................................................................................................................................... 189


UWA-0121 and -0122 syntheses ..................................................................................................................... 190

In vitro pharmacology ...................................................................................................................................... 190

Tissue preparation ....................................................................................................................................... 190

Radioligands and drugs ............................................................................................................................... 190

Receptor and transporter binding assays ..................................................................................................... 191

Monoamine re-uptake assays ...................................................................................................................... 191

Determination of UWA-0121/0122 affinity at selected receptor/ transporters ........................................... 191

Behavioural assessment of UWA-0121/0122 in the MPTP-lesioned non-human primate .............................. 192


Administration of UWA-0121/0122, in combination with L-DOPA, to parkinsonian marmosets ............. 192

Motor activity .............................................................................................................................................. 192



Results .................................................................................................................................................................. 194

Pharmacological profile of UWA-0121/0122 .................................................................................................. 194

UWA-0121, but not UWA-0122, increases motor activity induced by L-DOPA ............................................ 194

UWA-0121 extends duration of L-DOPA anti-parkinsonian action, and provides “good quality” extra ON-time

......................................................................................................................................................................... 195

UWA-0121 does not exacerbate the severity of L-DOPA-induced dyskinesia................................................ 195

UWA-0121 increases the duration of ON-time without psychosis-like behaviours ........................................ 196

xvi

Discussion ............................................................................................................................................................. 197

Figure legends ...................................................................................................................................................... 199

Figure VIII.1: synthesis of UWA-0121 and UWA-0122 ...................................................................................... 204

Figure VIII.2: motor activity ................................................................................................................................ 205

Figure VIII.3: ON-time and quality of ON-time .................................................................................................. 206

Figure VIII.4: dyskinesia ...................................................................................................................................... 207

Figure VIII.5: psychosis-like behaviour ............................................................................................................... 208

Figure VIII.6: ON-time with psychosis-like behaviour ........................................................................................ 209

Figure VIII.7: quality of ON-time – summary ...................................................................................................... 210

Table VIII.1: Experimental conditions for receptor/ transporters binding assays ................................................ 211

Table VIII.2: Experimental conditions for monoamine re-uptake assays ............................................................. 212

Table VIII.3: UWA-0121/0122 binding profiles .................................................................................................. 213

Table VIII.4: UWA-0121/0122 monoamine re-uptake profiles ........................................................................... 214

General Discussion: Importance of the work presented in this Thesis to the field of Parkinson’s disease ..... 215

General Discussion I: Anatomically-selective 5-HT1A and 5-HT2A therapies for Parkinson’s disease – an

approach to reducing dyskinesia without exacerbating parkinsonism? ............................................................. 219

Abstract ................................................................................................................................................................ 221

Introduction .......................................................................................................................................................... 222

The role of 5-HT1A receptors in parkinsonism and dyskinesia ............................................................................. 224

The role of 5-HT2A receptors in parkinsonism and dyskinesia ............................................................................. 224

Anatomically selective targeting of 5-HT1A and 5-HT2A transmission ................................................................. 225


Figure legend ........................................................................................................................................................ 227

Table 1: 5-HT1A agonists in parkinsonism and dyskinesia ................................................................................... 228

Table 2: 5-HT2A antagonists in parkinsonism and dyskinesia .............................................................................. 229

Figure 5-HT1A and 5-HT2A receptor levels in the normal, untreated parkinsonian, and L-DOPA-treated

parkinsonian states ................................................................................................................................................ 230

General Discussion II: Dopamine re-uptake inhibitors in Parkinson’s disease – how selective should we make

them and how should we use them? ....................................................................................................................... 231

Abstract ................................................................................................................................................................ 233

Introduction .......................................................................................................................................................... 234

Selective dopamine transporter inhibitors ............................................................................................................ 234

Dual dopamine and noradrenaline transporter inhibitors ...................................................................................... 235

Dual dopamine and serotonin transporter inhibitors ............................................................................................. 236

Triple dopamine, noradrenaline, and serotonin transporter inhibitors .................................................................. 237

Dopamine transporter inhibitors in PD: where next?............................................................................................ 237


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Figure DII.1 legend ............................................................................................................................................... 240

Figure DII.1: the dopamine transporter (DAT) and DAT inhibitors .................................................................... 241

Table DII.1: summary of the trials performed with dopamine transporter inhibitors in the parkinsonian non-

human primate and idiopathic Parkinson’s disease .............................................................................................. 242

Table DII.2: dopamine transporter inhibitors and their effect on parkinsonism, ON-time, and dyskinesia

according to their pharmacoselectivity ................................................................................................................. 245

Future directions ..................................................................................................................................................... 247

Appendix I: The MPTP-lesioned non-human primate model of Parkinson’s disease ....................................... 248

Introduction .......................................................................................................................................................... 249

MPTP ............................................................................................................................................................... 249

MPTP handling and administration ................................................................................................................. 250

The MPTP-lesioned non-human primate ......................................................................................................... 251

Limitations the MPTP-lesioned non-human primate model of Parkinson’s disease ........................................ 252

Appendix II: the 6-hydroxydopamine-lesioned rat model of Parkinson’s disease............................................. 255

Introduction .......................................................................................................................................................... 256

6-hydroxydopamine ......................................................................................................................................... 256

Bilateral 6-OHDA lesion ................................................................................................................................. 257

Unilateral 6-OHDA lesion ............................................................................................................................... 257

Behavioural testing in the unilaterally 6-OHDA-lesioned rat .......................................................................... 258

Rotational behaviour ................................................................................................................................... 259

Limitations of the 6-OHDA-lesioned rat model of Parkinson’s disease .......................................................... 260

Haloperidol-induced catalepsy in the rat .......................................................................................................... 262

The reserpine-treated rat .................................................................................................................................. 263

Appendix III: receptor binding .............................................................................................................................. 265

Introduction .......................................................................................................................................................... 266

Key concepts .................................................................................................................................................... 266

The law of mass action ................................................................................................................................ 266

The equilibrium dissociation constant (Kd) ................................................................................................ 266

The fractional occupancy ............................................................................................................................ 267

Limitations of the law of mass action ......................................................................................................... 267

Allosteric regulation .................................................................................................................................... 268

Radioligand binding ......................................................................................................................................... 268

Total, specific and non-specific binding ..................................................................................................... 268

Detection of radioactivity ............................................................................................................................ 270

Saturation binding ....................................................................................................................................... 271

Competition binding .................................................................................................................................... 272

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Single dose screening .................................................................................................................................. 273

Kinetic binding experiments ............................................................................................................................ 274

Methodological considerations ........................................................................................................................ 274

Limitations of receptor binding ........................................................................................................................ 275

Monoamine re-uptake assays ........................................................................................................................... 276

Figure legends ...................................................................................................................................................... 278

Figure AIII.1: fractional occupancy...................................................................................................................... 280

Figure AIII.2: relation between total, non-specific and specific binding .............................................................. 281

Figure AIII.3: representation of binding data ....................................................................................................... 282

Figure AIII.4: nonlinear and linear representation of receptor binding data ........................................................ 283

Figure AIII.5: dose-response curve ...................................................................................................................... 284

Appendix IV: stereotaxic surgery in the rat .......................................................................................................... 285

Stereotaxic surgery in the rat ................................................................................................................................ 286

Stereotaxic surgery: background and protocol ................................................................................................. 286

Delivery of 6-OHDA in the brain using stereotaxic surgery ............................................................................ 287

Advantages and limitations of stereotaxic surgery ........................................................................................... 288

Figure legend ........................................................................................................................................................ 289

Figure AIV.1: the rat skull .................................................................................................................................... 290

Appendix V: determining protein concentration using the folin phenol reagent .............................................. 291

Determining protein concentration using the folin phenol reagent ....................................................................... 292

Appendix VI: high-performance liquid chromatography and liquid chromatography/ mass spectrometry .. 294

Introduction .......................................................................................................................................................... 295

High-performance liquid chromatography ....................................................................................................... 295

General principles ....................................................................................................................................... 295

HPLC instrumentation ................................................................................................................................ 296

Figure legends ...................................................................................................................................................... 299

Figure AVI.1: chromatogram showing peaks corresponding to the catecholamines and their metabolites .......... 300

Figure AVI.2: detection of L-745,870 in macaque plasma by LC/ MS analysis .................................................. 301

Table AVI.1: characteristics of good mobile and stationary phases for HPLC analysis ...................................... 302

Table AVI.2: characteristics of a good detector for HPLC analysis ..................................................................... 303

Appendix VII: monoamine re-uptake inhibitors in Parkinson’s disease ............................................................ 304

Abstract ................................................................................................................................................................ 306

Introduction .......................................................................................................................................................... 307

Methods ................................................................................................................................................................ 307

Monoamine transporters ....................................................................................................................................... 308

Dopamine transporter and Parkinson’s disease ................................................................................................ 308

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Serotonin transporter and Parkinson’s disease ................................................................................................. 309

Noradrenaline transporter and Parkinson’s disease .......................................................................................... 309

Interactions between the monoamine systems and relevance to Parkinson’s disease ...................................... 309

SERT inhibitors .................................................................................................................................................... 310

Overview .......................................................................................................................................................... 310

Citalopram and escitalopram ............................................................................................................................ 311

Citalopram ................................................................................................................................................... 311

Escitalopram ................................................................................................................................................ 313

Clomipramine .................................................................................................................................................. 313

Duloxetine ........................................................................................................................................................ 313

Fenfluramine .................................................................................................................................................... 314

Fluoxetine ........................................................................................................................................................ 314

Fluvoxamine .................................................................................................................................................... 317

Imipramine ....................................................................................................................................................... 317

Paroxetine ........................................................................................................................................................ 319

R-MDMA ......................................................................................................................................................... 320

Sertraline .......................................................................................................................................................... 320

Trazodone ........................................................................................................................................................ 321

UWA-0122 ....................................................................................................................................................... 321

Venlafaxine ...................................................................................................................................................... 322

SERT inhibitors: summary ............................................................................................................................... 322

SERT = NET inhibitors ........................................................................................................................................ 323

Amitriptyline .................................................................................................................................................... 323

Milnacipran ...................................................................................................................................................... 324

SERT = NET inhibitors: summary ................................................................................................................... 324

NET inhibitors ...................................................................................................................................................... 325

Amoxapine ....................................................................................................................................................... 325

Amphetamine, methamphetamine, propylhexedrine ........................................................................................ 325

Atomoxetine ..................................................................................................................................................... 327

Desipramine ..................................................................................................................................................... 328

Levoamphetamine ............................................................................................................................................ 329

Maprotiline ....................................................................................................................................................... 329

Mazindol .......................................................................................................................................................... 329

Mianserin ......................................................................................................................................................... 330

Mirtazapine ...................................................................................................................................................... 330

Nisoxetine ........................................................................................................................................................ 331

Nortriptyline ..................................................................................................................................................... 331

Reboxetine ....................................................................................................................................................... 332

NET inhibitors: summary ................................................................................................................................. 332

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DAT inhibitors ...................................................................................................................................................... 333

Amineptine ....................................................................................................................................................... 333

Modafinil ......................................................................................................................................................... 333

SEP-228,791 and SEP-226,330 ........................................................................................................................ 335

Vanoxerine ....................................................................................................................................................... 336

DAT inhibitors: summary ................................................................................................................................ 336

DAT = NET inhibitors .......................................................................................................................................... 337

Benztropine ...................................................................................................................................................... 337

Brasofensine ..................................................................................................................................................... 337

Bupropion ........................................................................................................................................................ 338

Cocaine ............................................................................................................................................................ 339

Dextroamphetamine ......................................................................................................................................... 339

Methamphetamine ............................................................................................................................................ 339

Methylphenidate .............................................................................................................................................. 339

Nomifensine ..................................................................................................................................................... 342

DAT = NET inhibitors: summary .................................................................................................................... 343

DAT = SERT inhibitors ........................................................................................................................................ 343

UWA-0101, UWA-0121, UWA-0122 ............................................................................................................. 344

DAT = SERT inhibitors: summary .................................................................................................................. 344

DAT = NET = SERT inhibitors ............................................................................................................................ 345

BTS 74,398 ...................................................................................................................................................... 345

MDMA, R-MDMA, S-MDMA ........................................................................................................................ 346

Nefazodone ...................................................................................................................................................... 347

S-MDMA ......................................................................................................................................................... 347

Tesofensine ...................................................................................................................................................... 348

DAT = NET = SERT inhibitors: summary ...................................................................................................... 349

SERT enhancer ..................................................................................................................................................... 350

Tianeptine ........................................................................................................................................................ 350

SERT enhancer: summary ............................................................................................................................... 350


Figure legends ...................................................................................................................................................... 352

Figure AVII.1: chemical formulae of selective SERT inhibitors ......................................................................... 353

Figure AVII.2: chemical formulae of dual SERT = NET inhibitors ..................................................................... 354

Figure AVII.3: chemical formulae of selective NET inhibitors ........................................................................... 355

Figure AVII.4: chemical formulae of selective DAT inhibitors ........................................................................... 356

Figure AVII.5: chemical formulae of dual DAT = NET inhibitors ...................................................................... 357

Figure AVII.6: chemical formulae of dual DAT = SERT inhibitors .................................................................... 358

Figure AVII.7: chemical formulae of non-selective DAT = NET = SERT inhibitors .......................................... 359

Figure AVII.8: chemical formula of the SERT enhancer tianeptine .................................................................... 360

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Table AVII.1: keywords used to perform the literature search ............................................................................. 361

Table AVII.2: Affinity of the monoamine re-uptake inhibitors studied in idiopathic PD and animal models of PD

.............................................................................................................................................................................. 362

Table AVII.3: selectivity profile of the monoamine re-uptake inhibitors studied in idiopathic PD and animal

models of PD ........................................................................................................................................................ 365

Table AVII.4: summary of the effects of SERT inhibitors in idiopathic PD and animal models of PD .............. 366

Table AVII.5: summary of the effects of SERT = NET inhibitors in idiopathic PD and animal models of PD ... 370

Table AVII.6: summary of the effects of NET inhibitors in idiopathic PD and animal models of PD ................ 371

Table AVII.7: summary of the effects of DAT inhibitors in idiopathic PD and animal models of PD ................ 374

Table AVII.8: summary of the effects of DAT = NET inhibitors in idiopathic PD and animal models of PD .... 376

Table AVII.9: summary of the effects of DAT = SERT inhibitors in idiopathic PD and animal models of PD .. 378

Table AVII.10: summary of the effects of DAT = NET = SERT inhibitors in idiopathic PD and animal models of

PD ......................................................................................................................................................................... 379

Table AVII.11: summary of the effects of SERT enhancer in idiopathic PD and animal models of PD ............. 381

Appendix VIII: the serotonergic system in Parkinson’s disease ......................................................................... 382

Abstract ................................................................................................................................................................ 384

Introduction .......................................................................................................................................................... 385

Methods ................................................................................................................................................................ 385

Serotonin............................................................................................................................................................... 386

Serotonin, 5-HIAA, and tryptophan hydroxylase in Parkinson’s disease ..

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