-
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
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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.
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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.
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iv
Contribution of the authors of each Chapter to the work
presented in the Thesis
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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
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,
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
Statistical Analysis:
Design: PH, JMB, SHF; Execution: PH; Review: PH, JMB, SHF;
Critique: PH, JMB, SHF
Manuscript Preparation
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
Statistical Analysis:
Design: PH, SHF, AA, JMB; Execution: PH, AA; Review: PH, SHF,
AA, JMB Critique: PH, SHF, AA, JMB
Manuscript Preparation
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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
Statistical Analysis:
Design: PH, THJ, SHF, JMB; Execution: PH; Review: PH, THJ, SHF,
JMB; Critique: PH, SHF, JMB
Manuscript Preparation
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
Statistical Analysis:
Design: PH, THJ, SHF, MJP, JMB; Execution: PH, KDL; Review: PH,
THJ, SHF, MJP, JMB; Critique: PH,
SHF, MJP, JMB
Manuscript Preparation
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
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
Statistical Analysis:
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vii
Design: PH, THJ, SHF, MJP, JMB; Execution: PH, KDL; Review: PH,
THJ, SHF, MJP, JMB; Critique: PH,
SHF, MJP, JMB
Manuscript Preparation
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
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
Statistical Analysis:
Design: PH, THJ, SHF, MJP, JMB; Execution: PH, KDL; Review: PH,
THJ, SHF, MJP, JMB; Critique: PH,
SHF, MJP, JMB
Manuscript Preparation
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
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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
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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
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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
Materials and methods
............................................................................................................................................
55
Animals
..............................................................................................................................................................
55
High performance liquid chromatography analysis
...........................................................................................
55
Dopamine transporter autoradiography
..............................................................................................................
56
5-HT2A receptor autoradiography
.......................................................................................................................
56
Autoradiographic binding analysis
....................................................................................................................
57
Statistical 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
Technical considerations
....................................................................................................................................
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
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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
Materials and methods
............................................................................................................................................
75
Tissue collection
................................................................................................................................................
75
[3H]-Ketanserin autoradiographic binding
.........................................................................................................
76
Statistical analysis
..............................................................................................................................................
76
Results
....................................................................................................................................................................
77
Patient characteristics
.........................................................................................................................................
77
[3H]-Ketanserin binding in human post mortem brains
.....................................................................................
77
Discussion
...............................................................................................................................................................
78
Technical considerations
....................................................................................................................................
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
Materials and methods
............................................................................................................................................
96
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
Statistical analysis
..............................................................................................................................................
99
Results
..................................................................................................................................................................
100
L-745,870 does not impair L-DOPA anti-parkinsonian action
........................................................................
100
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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
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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
Materials and methods
..........................................................................................................................................
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
Statistical analysis
............................................................................................................................................
149
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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
Materials and methods
..........................................................................................................................................
172
UWA-0101 synthesis
.......................................................................................................................................
172
Behavioural assessment of UWA-0101 in the MPTP-lesioned common
marmoset ........................................ 172
Induction of parkinsonism and dyskinesia in the common marmoset
......................................................... 172
Administration of UWA-0101 in combination with L-DOPA to the
parkinsonian marmoset .................... 172
Behavioural analysis
...................................................................................................................................
173
Statistical analysis
............................................................................................................................................
173
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
Concluding remarks
..............................................................................................................................................
177
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
Materials and methods
..........................................................................................................................................
190
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
Induction of parkinsonism and dyskinesia in the common marmoset
......................................................... 192
Administration of UWA-0121/0122, in combination with L-DOPA, to
parkinsonian marmosets ............. 192
Motor activity
..............................................................................................................................................
192
Behavioural analysis
...................................................................................................................................
193
Statistical analysis
............................................................................................................................................
193
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
Concluding remarks
..............................................................................................................................................
226
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
Concluding remarks
..............................................................................................................................................
238
-
xvii
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
-
xviii
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
-
xix
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
-
xx
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
Concluding remarks
..............................................................................................................................................
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
-
xxi
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 ..