U.S. patent application number 14/502170 was filed with the patent office on 2015-01-22 for antiparkinsonian action of phenylisopropylamines.
The applicant listed for this patent is Duke University. Invention is credited to Marc G. Caron, Raul R. Gainetdinov, Tatyana D. Sotnikova.
Application Number | 20150025063 14/502170 |
Document ID | / |
Family ID | 37709149 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150025063 |
Kind Code |
A1 |
Caron; Marc G. ; et
al. |
January 22, 2015 |
Antiparkinsonian Action of Phenylisopropylamines
Abstract
A method of treating a subject for Parkinson's disease comprises
administering said subject a phenylisopropylamine in an amount
effective to treat said Parkinson's disease. In some embodiments
the method is used to treat at least a motor symptom of Parkinson's
disease; in some embodiments the method is used to treat at least a
non-motor symptom of Parkinson's disease.
Inventors: |
Caron; Marc G.;
(Hillsborough, NC) ; Sotnikova; Tatyana D.;
(Chapel Hill, NC) ; Gainetdinov; Raul R.; (Chapel
Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
37709149 |
Appl. No.: |
14/502170 |
Filed: |
September 30, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11460046 |
Jul 26, 2006 |
8877802 |
|
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14502170 |
|
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60703137 |
Jul 28, 2005 |
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Current U.S.
Class: |
514/217.01 ;
514/565; 514/567 |
Current CPC
Class: |
A61K 31/198 20130101;
A61K 45/06 20130101; A61K 31/137 20130101; A61K 31/55 20130101;
A61K 31/36 20130101 |
Class at
Publication: |
514/217.01 ;
514/567; 514/565 |
International
Class: |
A61K 31/137 20060101
A61K031/137; A61K 45/06 20060101 A61K045/06; A61K 31/55 20060101
A61K031/55; A61K 31/198 20060101 A61K031/198 |
Goverment Interests
[0002] This invention was made with government support under grant
nos. NS-19576 and MH-40159 from the National Institutes of Health.
The US Government has certain rights to this invention.
Claims
1-23. (canceled)
24. A pharmaceutical composition comprising an active compound as
described herein in combination with an additional antiparkinson's
agent, with the active compound included in an amount effective to
reduce the dosage of said antiparkinson's agent, reduce at least
one undesired side effect of said antiparkinson's agent, or
synergistically enhance the efficacy of said antiparkinson's agent,
wherein said active agent is a phenylisopropylamine or a
pharmaceutically acceptable salt or prodrug thereof.
25. The composition of claim 24, wherein said active compound is a
compound of Formula I: ##STR00005## wherein: R.sup.n1, R.sup.n2 and
R.sup.a are each independently selected from the group consisting
of H, hydroxy, and loweralkyl; R.sup.1 is selected from the group
consisting of H, hydroxy and oxo (.dbd.O); and R.sup.2, R.sup.3,
R.sup.4, R.sup.5 and R.sup.6 are each independently selected from
the group consisting of H, halo, loweralkyl, haloalkyl,
loweralkoxy, haloloweralkoxy; loweralkylthio, haloloweralkylthio,
and nitro; or an adjacent pair of R.sup.2 and R.sup.3, R.sup.3 and
R.sup.4, or R.sup.4 and R.sup.5 may together form a group of the
formula --OCH.sub.2O--; or a pharmaceutically acceptable salt or
prodrug thereof.
26. The composition of claim 24, wherein said antiparkinson's agent
is selected from the group consisting of amantadine, apomorphine,
bromocriptine, levodopa, pergolide, ropinirole, selegiline,
trihexyphenidyl, atropine, scopolamine, glycopyrrolate,
pharmaceutically acceptable acid addition salts and prodrugs
thereof, and combinations thereof.
27. The composition of claim 24, wherein said composition is an
oral dosage composition.
28. A tablet or capsule comprising a composition of claim 27.
29. The tablet or capsule of claim 28, wherein said active compound
is included in an amount of from 1 to 50 milligrams.
30. The tablet or capsule of claim 28, and wherein said additional
antiparkinson's agent is included in an amount of from 0.1 to 200
milligrams.
31. The tablet or capsule of claim 28, wherein said additional
antiparkinson's agent is levodopa, and wherein said composition
further comprises carbidopa and optionally a COMT inhibitor.
32. The tablet or capsule of claim 28, wherein said additional
antiparkinson's agent is a dopamine agonist.
33. The tablet or capsule of claim 28, wherein said additional
antiparkinson's agent is an anticholinergic.
34. The tablet or capsule of claim 28, wherein said additional
antiparkinson's agent is a monoamine oxidase inhibitor.
35-40. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
patent application Ser. No. 60/703,137, filed Jul. 28, 2005, the
disclosure of which is incorporated by reference herein in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention concerns methods of treating
Parkinson's disease with phenylisopropylamines.
BACKGROUND OF THE INVENTION
[0004] The phenylethylamine derivative dopamine (DA) is critically
involved in a wide variety of vital functions such as locomotion,
feeding, emotion, and reward [1-3]. Major DA systems in the brain
originate from brainstem DA neurons located in the substantia nigra
pars compacta (SNc) and the ventral tegmental area (VTA). SNc
neurons project mainly to the caudate/putamen or dorsal striatum
(nigrostriatal system), whereas VTA neurons send their axons to the
ventral striatum including the nucleus accumbens, as well as
certain other limbic (mesolimbic system) and cortical areas
(mesocortical system). Small DA-containing cell groups located
primarily in the hypothalamus comprise the tuberoinfundibular DA
system [4-6]. DA is synthesized from tyrosine by the rate-limiting
enzyme tyrosine hydroxylase (TH), to produce L-DOPA which is
quickly decarboxylated by L-aromatic acid decarboxylase (L-AADC) to
DA [1,3]. Intraneuronal DA is accumulated into synaptic vesicles by
the vesicular monoamine transporter-2 (VMAT2) [7,8]. DA released
into the extracellular space exerts its physiological functions via
activation of G protein-coupled D1-like and D2-like DA receptors
[9]. Finally, DA in the extracellular space is subject to dilution
by diffusion and metabolic degradation; however the major route of
DA clearance from the extracellular space in the striatum/nucleus
accumbens is the rapid recycling of the neurotransmitter back into
dopaminergic terminals by the Na.sup.+/Cl.sup.--dependent plasma
membrane dopamine transporter (DAT) [10,11]. Recycled DA in the
dopaminergic terminals is then stored in the large intracellullar
storage pool available for subsequent re-release [12,13].
[0005] It is well established that DA neurotransmission in both
dorsal and ventral striatum is essential for normal locomotor
functions, and progressive degeneration of DA neurons in these
areas is a known cause of Parkinson's disease (PD). In most cases,
PD becomes clinically apparent when the loss of dopaminergic
neurons reaches 60%-70%, which leads to functional dysregulation of
the related neuronal circuitry [14-17]. Major motor manifestations
of DA deficiency in PD include, but are not limited to, resting
tremor (tremor occurring in the absence of voluntary movement),
rigidity (tonically increased muscle tone), bradykinesia/akinesia
(slowness/difficulty in initiating movement), gait disturbance and
postural instability, facial masking, and decreased eyeblinking
[18].
[0006] Presently, there is no known cure for PD [19,20], however
its symptoms can be controlled by therapeutic interventions [21].
DA replacement therapy by administration of the DA precursor,
L-DOPA, has been used for many years and remains the gold standard
for treatment of PD [22,23]. However, the efficacy of this
treatment wanes with time, and fluctuations in motor performance as
well as psychotic reactions and dyskinesias often develop. DA
agonists, as well as several other classes of drugs directly or
indirectly affecting DA function (monoamine oxidase [MAO]
inhibitors, COMT [catechol-o-methyl transferase] inhibitors, and
amantadine), have some beneficial effects in PD patients, but they
are mostly used either at early stages of PD or are applied as
adjunct medications to enhance the benefits of L-DOPA [21,24,25].
Due to these limitations of existing therapeutic approaches, the
development of better anti-Parkinsonian drugs remains a major
objective of PD research.
[0007] Several lines of evidence suggest that development of novel
non-dopaminergic approaches aimed at bypassing impaired
dopaminergic transmission would be beneficial in PD, particularly
at later stages [16,26-28], however it is still unclear if these
treatments would just potentiate action of residual DA or act
completely independently of DA. A number of animal models of DA
deficiency, based on pharmacologic, neurotoxic, or genetic
approaches, have been developed to understand basic pathological
processes leading to PD and/or to search for novel principles of
therapy [29-36]. However, in rodents, the prolonged absence of DA
is not compatible with life [3,7,8], and animals with chronic
severe DA depletion are generally not available for routine
experimentation.
[0008] We have developed mice lacking the functional DAT (DAT-KO
mice) [11] that display remarkable alterations in the
compartmentalization of DA [12,13,37]. Lack of the DAT-mediated
inward transport in these mice results in an elevated extracellular
DA and at least 95% decreased intracellular DA stores. Unlike
normal animals, these mice demonstrate remarkable dependence of the
remaining DA on ongoing synthesis, and pharmacologic blockade of DA
synthesis in DAT-KO mice provides an effective approach to
eliminate DA acutely [12,13].
[0009] Substituted phenylethylamine derivatives, amphetamines, that
are structurally similar to DA and the endogenous trace amine
beta-phenylethylamine, represent a well-known group of compounds
that potently affect psychomotor functions. Amphetamines are known
to interact with plasma membrane monoamine transporters, including
DAT, norepinephrine (NE) transporter (NET), and serotonin
transporter. This complex interaction results in
transporter-dependent efflux of monoamines into extracellular space
from intraneuronal stores [10,38,39]. It is commonly believed that
DAT-mediated efflux of DA is primarily responsible for the
psychostimulant and locomotor actions of these drugs [38,40,41].
Intriguingly, recent studies have identified novel
transporter-independent targets of amphetamines. It has been shown
that amphetamines, as well as .beta.-phenylethylamine, some
monoamine metabolites, and several drugs affecting monoaminergic
transmission, can directly activate specific G protein--coupled
trace amine (trace amine 1 [TA1]) receptors [42] with currently
unknown functional consequences [43,44]. Using DA-depleted DAT-KO
mice we observed potent DA-independent antiparkinsonian action of
several amphetamine derivatives (17 tested phenylisopropylamines
were effective as described below).
[0010] The following additional references are noted herein: [0011]
Parkes J D et al., Amphetamines in the treatment of Parkinson's
disease, J Neurol Neurosurg Psychiatry 38(3): 232-7 (1975). [0012]
Goetz C G et al., Bupropion in Parkinson's disease, Neurology
34(8):1092-4 (1984). [0013] Karoum, F. et al., Metabolism of (-)
deprenyl to amphetamine and methamphetamine may be responsible for
deprenyl's therapeutic benefit: a biochemical assessment.
Neurology. 32(5):503-9 (1982). [0014] Schmidt, W. J. et al.,
Ecstasy counteracts catalepsy in rats, an anti-parkinsonian effect?
Neurosci Lett. 330(3): 251-4 (2002) [0015] M. L. Wadenberg,
Serotonergic mechanisms in neuroleptic-induced catalepsy in the
rat. Neurosci Biobehav. Rev. 20 325-339 (1996). [0016] Banjaw M Y
et al., Anticataleptic activity of cathinone and MDMA (Ecstasy)
upon acute and subchronic administration in rat. Synapse
49(4):232-8 (2003) [0017] Fuller, R. W., Fenfluramine and
Parkinson's disease, Arch Neurol. 34(11):720 (1977) [0018] Beasley
B L et al., Fenfluramine hydrochloride treatment of parkinsonism,
Arch Neurol. 34(4):255-6 (1977)(negative study) [0019] Dawirs, R.,
Use of NeuroactiveSubstances for the Treatment of Parkinson's
Disease and Pharmaceutical Combination, US Pat. Application
2004/0147613.
SUMMARY OF THE INVENTION
[0020] A first aspect of the present invention is a method of
treating a subject for Parkinson's disease, comprising
administering said subject an active compound as described herein
in an amount effective to treat said Parkinson's disease. In some
embodiments, the Parkinson's disease is early onset Parkinson's
disease (e.g., the patient is less than 40 years old).
[0021] A second aspect of the invention is a method of treating
dysphagia in a Parkinson's disease subject, comprising
administering said subject an active compound as described herein
in an amount effective to treat said dysphagia.
[0022] A third aspect of the invention is a method of treating
incontinence in a Parkinson's disease subject, comprising
administering said subject an active compound as described herein
in an amount effective to treat said incontinence.
[0023] A further aspect of the invention is a method of treating
anxiety in a Parkinson's disease subject, comprising administering
said subject an active compound as described herein in an amount
effective to treat said anxiety.
[0024] A further aspect of the invention is a method of treating
depression in a Parkinson's disease subject, comprising
administering said subject an active compound as described herein
in an amount effective to treat said depression.
[0025] A further aspect of the invention is a method of treating
sexual dysfunction in a Parkinson's disease subject, comprising
administering said subject an active compound as described herein
in an amount effective to treat said sexual dysfunction.
[0026] A further aspect of the invention is a method of treating
fatigue in a Parkinson's disease subject, comprising administering
said subject an active compound as described herein in an amount
effective to treat said fatigue.
[0027] A further aspect of the invention is a method of treating
pain associated with Parkinson's disease in a Parkinson's disease
subject, comprising administering said subject an active compound
as described herein in an amount effective to treat said pain.
[0028] A further aspect of the invention is, in a method of
treating a subject for Parkinson's disease with an antiparkinson's
agent, the improvement comprising administering said subject an
active compound as described herein in an amount effective to
reduce the dosage of said antiparkinson's agent, reduce at least
one undesired side effect (such as dyskinesias) of said
antiparkinson's agent, and/or synergistically enhance the efficacy
of said antiparkinson's agent.
[0029] A further aspect of the present invention is a
pharmaceutical composition comprising an active agent as described
herein in combination with an additional antiparkinson's agent
(e.g., levodopa, with or without carbidopa), with the active agent
as described herein included in an amount effective to reduce the
dosage of said antiparkinson's agent, reduce at least one undesired
side effect (such as dyskinesias) of said antiparkinson's agent,
and/or synergistically enhance the efficacy of said antiparkinson's
agent.
[0030] A further aspect of the invention is the use of an active
agent as described herein for the preparation of a medicament for
carrying out a method as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1. .alpha.MT induces Severe DA Depletion in the
Striatum of DAT-KO Mice.
[0032] (A) Tissue levels of DA in the striatum of saline-treated
control WT and DAT-KO mice (n=7 per group). Striatal levels of DA
were significantly lower in DAT-KO versus WT mice (p<0.05,
Student's t-test).
[0033] (B) Dynamics of the effect of .alpha.MT (250 mg/kg IP) on
striatal tissue DA in WT and DAT-KO mice (n=5-8 per group). DA
levels were significantly lower versus control values at all the
time points after .alpha.MT treatment in DAT-KO mice and 2-24 hours
after treatment in WT mice (p<0.05, one-way ANOVA followed by
Dunnet's multiple comparison test). The magnitude of the effect was
significantly different between genotypes from 1 to 16 h after
.alpha.MT injection (p<0.05, two-tailed Mann-Whitney U
test).
[0034] (C) Tissue levels of NE in the frontal cortex of
saline-treated WT and DATKO mice (n=7 per group).
[0035] (D) Dynamics of the effect of .alpha.MT (250 mg/kg IP) on
tissue levels of NE in the frontal cortex of WT and DAT-KO mice
(n=5-8 per group). NE levels were significantly lower versus
control values at time points 2-16 after .alpha.MT treatment in
DAT-KO mice and at 4-16 hours after treatment in WT mice
(p<0.05, one-way ANOVA followed by Dunnet's multiple comparison
test). The magnitude of the effect was not different between
genotypes at any time point after .alpha.MT injection (p<0.05,
two-tailed Mann-Whitney U test).
[0036] (E) Effect of .alpha.MT on extracellular DA levels in the
striatum of WT mice, measured using in vivo microdialysis. Data are
presented as a percentage of the average level of DA measured in at
least three samples collected before the drug administration.
(Saline, n=5: .alpha.MT, n=7). .alpha.MT significantly decreased DA
levels 60-120 min after treatment (p<0.05, two-tailed
Mann-Whitney U test versus respective time points in saline-treated
controls).
[0037] (F) Effect of .alpha.MT on extracellular levels of DA in the
striatum of DAT-KO mice, measured by using in vivo microdialysis in
freely moving mice. Data are presented as a percentage of the
average level of DA measured in at least three samples collected
before drug administration. (Saline, WT: n=5: DAT-KO: n=4;
.alpha.MT, WT: n=7; DAT-KO: n=6). .alpha.MT significantly decreased
DA levels 20-120 min after treatment (p<0.05, two-tailed
Mann-Whitney U test versus respective time points in saline treated
controls). Analysis of area under curve values for 120-min periods
after drug administration revealed significant difference between
DAT-KO and WT groups (p<0.05, two-tailed Mann-Whitney U test).
Note also that the basal extracellular levels of DA in DAT-KO mice
were significantly higher than in WT mice (predrug concentrations
of DA in dialysates were: WT, 76.+-.17 fmol/20 microliters; DAT-KO,
340.+-.63 fmol/20 microliters).
[0038] FIG. 2. .alpha.MT-Induced Impairment in Motor Control in
DAT-KO Mice. Dynamics of locomotor activity following systemic
administration of .alpha.MT (250 mg/kg IP) and saline (30 min after
placement in the locomotor activity chamber) in WT (A) and DAT-KO
(B) mice (n=6-8 per group). Analysis of total distance traveled for
210 min after drug administration revealed significant effect of
.alpha.MT treatment (p<0.05; Student's t-test) in DAT-KO but not
WT mice (WT-saline, 516.+-.50 cm/210 min; WT-.alpha.MT, 505.+-.98
cm/210 min; DAT-KO-saline, 18,489.+-.4,795 cm/210 min;
DATKO-.alpha.MT, 448.+-.75 cm/210 min). .alpha.MT (injected at time
Q) induced profound alterations in the akinesia (C), catalepsy (D),
grasping (E), bracing (F) induced tremor (G), and ptosis (H) tests,
but did not affect the righting reflex (I) in DAT-KO mice.
Behavioral tests were performed as described in Materials and
Methods. At all the time points, DAT-KO mice were significantly
different versus respective values (data not shown) of
saline-treated DAT-KO controls (p<0.05; Student's t-test n=6 per
group) in these tests with exception of 15-min time point for
ptosis (H) and all time points for righting reflex test (I). In WT
mice only the akinesia test (C) revealed minor, yet significant,
effect (1.5-4 h after .alpha.MT treatment) versus values (data not
shown) of the respective saline treated WT controls (p<0.05;
Student's t-test; n=6 per group). No significant alterations in any
other test at any time point examined (D-I) was noted in
.alpha.MT-treated versus saline treated (data not shown) WT mice.
Locomotor activity is restored in DAT-KO mice 16-24 h after
.alpha.MT (250 mg/kg IP) treatment (J).
[0039] FIG. 3. L-DOPA and Nonselective DA Agonists Are Effective in
Restoring Locomotion in DDD Mice. DAT-KO mice were placed in the
locomotor activity chamber and 30 min later were treated with
.alpha.MT (250 mg/kg IP) and 1 h after .alpha.MT were challenged
with single or multiple doses of a drug (interval between
treatments is 1 h). L-DOPA itself (A) or in combination with
carbidopa (B-D) effectively restored locomotion in DDD mice, as
revealed by the significant effect of L-DOPA at doses 100 and 200
mg/kg IP, or combinations of L-DOPA/carbidopa at doses 20/20,
50/20, and 50/50 mg/kg, IP (analysis of total distance traveled for
1 h after each dose of the drug; p<0.05, two-tailed Mann-Whitney
U test versus respective values in saline-treated DDD mice; data
not shown). Nonselective DA receptor agonists, apomorphine (E) at
doses 2 and 3 mg/kg SC, and pergolide (F) at doses 5, 10, and 20
mg/kg IP, induced locomotion in DDD mice (analysis of total
distance traveled for 1 h after each dose of the drug; p<0.05,
two-tailed Mann-Whitney U test, versus respective values in
saline-treated DDD mice; data not shown). D2 DA receptor agonists
bromocriptine (G), quinpirole (H), and D1 DA receptor agonist
(+)-SKF81297 (I) were not effective, but the combinations of D1 and
D2 DA agonists (+)-SKF81297 plus quinpirole at doses 5/1 and 10/5
mg/kg IP, induced significant locomotion in DDD mice (analysis of
total distance traveled for 1 h after each treatment; p<0.05,
two-tailed Mann-Whitney U test versus respective values in
saline-treated DDD mice; data not shown). Experiments were
performed in 6-12 mice per group.
[0040] FIG. 4. Amphetamine Derivatives at High Doses Are Effective
in Reversing Abnormal Motor Behaviors of DDD Mice.
DAT-KO mice were placed in the locomotor activity chamber and 30
min later were treated with .alpha.MT (250 mg/kg IP), and 1 h after
.alpha.MT were challenged with single or multiple doses of drugs
(in cumulative dosing experiments, the interval between treatments
was 1 h). Grasping (A), catalepsy (B), and akinesia (C) tests were
performed as described in Materials and Methods 1 h after each dose
(the only exception is (+)-MDMA at 80 mg/kg IP where measurements
were performed 2 h after the drug administration). An asterisk
indicates p<0.05 versus respective values of saline-treated DDD
mice (one-way ANOVA followed by Dunnet's multiple comparison test).
Experiments were performed in 6-16 mice per group. d-AMPH indicates
damphetamine; d-METH, d-methamphetamine; and 4-chloro-AMPH,
4-chloro-amphetamine.
[0041] FIG. 5. (+)-MDMA Induces Forward Locomotion in DDD Mice.
(A-C) DAT-KO mice were placed in the locomotor activity chamber and
30 min later were treated with .alpha.MT (250 mg/kg IP) and 1 h
after .alpha.MT were challenged with single (B and C) or multiple
doses (A) of a drug (interval between treatments is 1 h) (n
1/410-16 per group). Repeated treatment with (+)-MDMA (30 and 60
mg/kg IP) induces forward locomotion in DDD mice (A). Analysis of
total distance traveled for 1 h after 60 mg/kg IP of (+)-MDMA
reveals significant effect of treatment versus respective period in
saline-treated controls (two-tailed Mann-Whitney U test, data not
shown). Dynamics (B) and dose-response (C) of locomotor effect of
(+)-MDMA in DDD mice are shown. Pretreatment with D1 and D2 DA
antagonists (SCH23390, 0.1 mg/kg SC plus raclopride, 2 mg/kg IP) 30
min before 100 mg/kg IP (+)-MDMA) did not affect locomotor action
of (+)-MDMA (C). (D) (+)-MDMA (100 mg/kg IP) fails to affect DA
dynamics in the striatum of DDD mice as measured by in vivo
microdialysis. Data are presented as a percentage of the average
level of DA measured in at least three samples collected before
.alpha.MT administration (n=4). Analysis of area under curve values
for 120-min periods after (+)-MDMA administration revealed no
significant difference in comparison with respective values in
control group (FIG. 1; p>0.05, two-tailed Mann-Whitney U test).
(E and F) (+)-MDMA (E) as well as d-amphetamine and
d-methamphetamine (F) at moderate doses potentiate
locomotor-stimulating effect of subthreshold dose of
L-DOPA/carbidopa (10/10 mg/kg IP). DAT-KO mice were treated with
.alpha.MT as described above (A-C) and 45 min after .alpha.MT were
injected with amphetamines. L-DOPA/carbidopa was injected 15 min
after amphetamines, and distance traveled for 2 h was measured
(n=6-15 per group). Note, that no forward locomotion was observed
after these doses of (+)-MDMA, d-amphetamine and d-methamphetamine
without L-DOPA/carbidopa, whereas L-DOPA/carbidopa (presented as
drug dose 0) induced only a modest but significant (p<0.05)
increase in locomotion over saline-treated controls (data not
shown). Single asterisk indicates p<0.05; double asterisks
indicate p<0.01; and triple asterisks indicate p<0.001 versus
saline-treated controls (C) or L-DOPA/carbidopa-treated (10/10
mg/kg IP) group (E and F) (two-tailed Mann-Whitney U test). d-AMPH,
d-amphetamine; METH, methamphetamine.
[0042] FIG. 6. Nomifensine, but not GBR12909, is effective in
reversing abnormal motor behaviors of DDD mice. DAT-KO mice were
placed in the locomotor activity chamber and 30 min later were
treated with .alpha.MT (250 mg/kg IP). Mice were challenged 1 h
later with two doses (10 and 30 mg/kg IP) of each drug or saline
with a 1 h interval between treatments (n=9-15 per group). Grasping
(A) and akinesia (B) tests were performed as described in Materials
and Methods 1 h after each dose. A single asterisk indicates
p<0.05, double asterisks indicate p<0.01, and triple
asterisks indicate p<0.001 versus respective values of
saline-treated DDD mice (one-way ANOVA followed by Dunnet's
multiple comparison test). Note that no significant differences
between GBR12909-treated mice and saline-treated controls in both
tests were found, whereas nomifensine-treated mice were
significantly different from GBR12909-treated mice (p<0.05) in
both experimental paradigms and doses tested.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0043] "Antiparkinson's agent" as used herein includes, but is not
limited to: levodopa (L-DOPA; with or without carbidopa), dopamine
agonists (such as apomorphine, bromocriptine, pergolide,
pramipexole, ropinirole, etc.) anticholinergics such as atropine,
scopolamine, glycopyrrolate, trihexyphenidyl, benztropine mesylate,
procyclidine, etc.), monoamine oxidase (MAO-B) inhibitors such as
selegiline, COMT inhibitors (preferably taken with levodopa) such
as entacapone and tolcapone and other medications such as
amantadine, etc., and including pharmaceutically acceptable salts
and prodrugs thereof, and combinations of any of the foregoing.
See, e.g., U.S. Pat. No. 6,833,478.
[0044] "Parkinson disease" or "Parkinson's disease" as used has its
conventional meaning and generally refers to a disease
characterized by the chronic, progressive loss of neurons in the
region of the brain known as the substantia nigra, at any point.
Parkinson's disease (including early onset and late onset
Parkinson's disease) is characterized by both motor symptoms and
non-motor symptoms. In some embodiments of the present invention
subjects are early stage Parkinson disease subjects (e.g., subjects
in stages I or II of the Hoehn and Yahr Staging scale, or subjects
with a score less than 120 or 100 on the Unified Parkinson Disease
Rating Scale (UPDRS); in some embodiments of the present invention
subjects are late stage Parkinson disease subjects (e.g., subjects
in stages IV or V of the Hoehn and Yahr Staging scale, and or
subjects with a score greater than 100 or 120 on the UPDRS)
(including late stage early onset Parkinson's disease and late
stage late onset Parkinson's disease). In some embodiments the
patients to be treated have acquired tolerance to, or have acquired
undesired side effects in response to, other antiparkinson's agents
such as L-DOPA.
[0045] "Motor symptom" of Parkinson's disease as used herein refers
to symptoms such as tremor, rigidity, difficulty of maintaining
balance or gait, and/or general slowness of movement (also called
"bradykinesia"). In some embodiments the active compounds of the
present invention are administered in an amount effective to treat
motor symptoms (at least one motor symptom) of Parkinson's
disease.
[0046] "Non-motor symptom" of Parkinson's disease as used herein
refers to one or more symptoms such as cognitive dysfunction,
autonomic dysfunction, sleep disorders, neurobehavioral
abnormalities, depression, constipation, pain, fatigue, etc. In
some embodiments the active compounds of the present invention are
administered in an amount effective to treat non-motor symptoms (at
least one non-motor symptom) of Parkinson's disease.
[0047] "Levodopa nonresponsive subject" as used herein refers to a
Parkinson's disease patient who has one or more symptom (e.g., a
motor symptom) that is no longer effectively managed by dopamine
replacement therapy (levodopa administration, with or without
carbidopa and/or a COMT inhibitor).
[0048] The term "treat" as used herein refers to any type of
treatment that imparts a benefit to a patient afflicted with a
disease, including improvement in the condition of the patient
(e.g., in one or more symptoms), delay in the progression of the
disease, reduction of tolerance or increase in efficacy of another
antiparkinson's agent, reduction in dose and corresponding
reduction in undesired side effects of another antiparkinson's
agent, etc.
[0049] The term "pharmaceutically acceptable" as used herein means
that the compound or composition is suitable for administration to
a subject to achieve the treatments described herein, without
unduly deleterious side effects in light of the severity of the
disease and necessity of the treatment.
[0050] As used herein; the administration of two or more compounds
"in combination" means that the two compounds are administered
closely enough in time that the presence of one alters the
biological effects of the other. The two compounds may be
administered simultaneously (i.e., concurrently) or sequentially.
Simultaneous administration may be carried out by mixing the
compounds prior to administration, or by administering the
compounds at the same point in time but at different anatomic sites
or using different routes of administration.
[0051] The present invention is primarily concerned with the
treatment of human subjects, but the invention may also be carried
out on animal subjects, particularly mammalian subjects such as
mice, rats, dogs, cats, livestock and horses for veterinary
purposes, and for drug screening and drug development purposes.
[0052] "Alkyl," as used herein, refers to a straight or branched
chain hydrocarbon containing from 1 to 10 carbon atoms, or 1 to 4
carbon atoms for loweralkyl. Representative examples of alkyl
include, but are not limited to, methyl, ethyl, n-propyl,
iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl,
isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,
2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the
like. "Loweralkyl" as used herein, is a subset of alkyl and refers
to a straight or branched chain hydrocarbon group containing from 1
to 4 carbon atoms. Representative examples of lower alkyl include,
but are not limited to, methyl, ethyl, n-propyl, iso-propyl,
n-butyl, iso-butyl, tert-butyl, and the like.
[0053] "Alkoxy," as used herein, refers to an alkyl group, as
defined herein, appended to the parent molecular moiety through an
oxy group, as defined herein. Representative examples of alkoxy
include, but are not limited to, methoxy, ethoxy, propoxy,
2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the
like.
[0054] "Alkylthio" as used herein refers to an alkyl group, as
defined herein, appended to the parent molecular moiety through a
thio moiety, as defined herein. Representative examples of
alkylthio include, but are not limited, methylthio, ethylthio,
tert-butylthio, hexylthio, and the like.
[0055] "Halo" as used herein refers to --Cl, --Br, --I or --F.
[0056] "Haloalkyl," as used herein, refers to at least one halogen,
as defined herein, appended to the parent molecular moiety through
an alkyl group, as defined herein. Representative examples of
haloalkyl include, but are not limited to, chloromethyl,
2-fluoroethyl, trifluoromethyl, pentafluoroethyl,
2-chloro-3-fluoropentyl, and the like.
[0057] "Haloalkoxy," as used herein, refers to an alkoxy group, as
defined herein, having at least one halo group (e.g., one, two,
three) substituted thereon. Representative examples of haloalkoxy
include, but are not limited to, trifluoromethyl, 2-chloroethoxy,
difluoromethoxy, 1,2-difluoroethoxy, 2,2,2-trifluoroethoxy,
trifluoromethoxy, and the like.
[0058] "Haloalkthio," as used herein, refers to an alkthio group,
as defined herein, having at least one halo group (e.g., one, two,
three) substituted thereon.
[0059] "Hydroxy," as used herein, refers to an --OH group.
[0060] "Nitro," as used herein, refers to a --NO.sub.2 group.
[0061] "Oxo," as used herein, refers to a .dbd.O moiety.
[0062] The disclosures of all US Patent references cited herein are
to be incorporated by reference herein in their entirety.
1. Active Compounds.
[0063] Active compounds of the present invention include
phenylisopropylamines. Such compounds are known and described in,
for example, Alexander T. Shulgin, Psychotomimetic Drugs:
Structure-Activity Relationships, Chapter 6 in Handbook of
Psychopharmacology, Volume 11: Stimulants (Edited by Leslie L.
Iversen Susan D. Iversen and Solomon H. Snyder), Plenum Press, New
York 1978).
[0064] In some embodiments the active compounds are those described
in U.S. Pat. No. 3,547,999.
[0065] In some embodiments active compounds useful for carrying out
the present invention include compounds of Formula I:
##STR00001##
wherein:
[0066] R.sup.n1, R.sup.n2 and R.sup.a are each independently
selected from the group consisting of H, hydroxy, and
loweralkyl;
[0067] R.sup.1 is selected from the group consisting of H, hydroxy
and oxo (.dbd.O); and
[0068] R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
independently selected from the group consisting of H, halo,
loweralkyl, haloalkyl, loweralkoxy, haloloweralkoxy;
loweralkylthio, haloloweralkylthio, and nitro;
[0069] or an adjacent pair of R.sup.2 and R.sup.3, R.sup.3 and
R.sup.4, or R.sup.4 and R.sup.5 may together form a group of the
formula --OCH.sub.2O--;
[0070] or a pharmaceutically acceptable salt or prodrug
thereof.
[0071] (a) In some embodiments, active compounds of the present
invention are preferably compounds of Formula Ia:
##STR00002##
wherein:
[0072] R.sup.n2 and R.sup.a are each independently selected from
the group consisting of H, hydroxy, and loweralkyl;
[0073] R.sup.1 is selected from the group consisting of H, hydroxy
and oxo (.dbd.O); and
[0074] R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
independently selected from the group consisting of H, halo,
loweralkyl, haloalkyl, loweralkoxy, haloloweralkoxy;
loweralkylthio, haloloweralkylthio, and nitro;
[0075] or an adjacent pair of R.sup.2 and R.sup.3, R.sup.3 and
R.sup.4, or R.sup.4 and R.sup.5 may together form a group of the
formula --OCH.sub.2O--;
[0076] or a pharmaceutically acceptable salt or prodrug
thereof.
[0077] (b) In some embodiments, active compounds of the present
invention are preferably compounds of Formula Ib:
##STR00003##
wherein:
[0078] R.sup.n2 and R.sup.a are each independently selected from
the group consisting of H, hydroxy, and loweralkyl;
[0079] R.sup.1 is selected from the group consisting of H, hydroxy
and oxo (.dbd.O); and
[0080] R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
independently selected from the group consisting of H, halo,
loweralkyl, haloalkyl, loweralkoxy, haloloweralkoxy;
loweralkylthio, haloloweralkylthio, and nitro;
[0081] or an adjacent pair of R.sup.2 and R.sup.3, R.sup.3 and
R.sup.4, or R.sup.4 and R.sup.5 may together form a group of the
formula --OCH.sub.2O--;
[0082] or a pharmaceutically acceptable salt or prodrug
thereof.
[0083] (c) In some embodiments, active compounds of the present
invention are preferably compounds of Formula Ic:
##STR00004##
wherein:
[0084] R.sup.n1 and R.sup.n2 are each independently selected from
the group consisting of H, hydroxy, and loweralkyl;
[0085] R.sup.1 is selected from the group consisting of H, hydroxy
and oxo (.dbd.O); and
[0086] R.sup.2, R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each
independently selected from the group consisting of H, halo,
loweralkyl, haloalkyl, loweralkoxy, haloloweralkoxy;
loweralkylthio, haloloweralkylthio, and nitro;
[0087] or an adjacent pair of R.sup.2 and R.sup.3, R.sup.3 and
R.sup.4, or R.sup.4 and R.sup.5 may together form a group of the
formula --OCH.sub.2O--;
[0088] or a pharmaceutically acceptable salt or prodrug
thereof.
[0089] (d) In some embodiments, active compounds of the present
invention are preferably compounds of Formula I, subject to the
proviso that at least one of R.sup.2, R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 is halo.
[0090] The active compounds including compounds of Formula I
contain an asymmetric carbon atom and thus normally occur as a
racemic mixture of the dextro- and levorotatory optical isomers.
Both dextro- and levorotatory isomers of these compounds, as well
as the racemic mixtures, are useful in the compositions and methods
described herein.
[0091] Specific examples of active compounds useful for carrying
out at least some embodiments of the present invention include, but
are not limited to:
(1) Methoxylated Phenylisopropylamines, such as: [0092]
4-Methoxyphenylisopropylamine; [0093]
3,4-Dimethoxyphenylisopropylamine; [0094]
2,4-Dimethoxyphenylisopropylamine; [0095]
2,5-Dimethoxyphenylisopropylamine; [0096]
3,4,5-Trimethoxyphenylisopropylamine; [0097]
2,4,5-Trimethoxyphenylisopropylamine; [0098]
2,3,4-Trimethoxyphenylisopropylamine; [0099]
2,3,5-Trimethoxyphenylisopropylamine; [0100]
2,3,6-Trimethoxyphenylisopropylamine; [0101]
2,4,6-Trimethoxyphenylisopropylamine; and [0102]
2,3,4,5-Tetramethoxyphenylisopropylamine; (2)
Methylenedioxyphenylisopropylamines, such as: [0103]
3,4-Methylenedioxyphenylisopropylamine; [0104]
N-Methyl-3,4-methylenedioxyphenylisopropylamine; [0105]
N-Ethyl-3,4-methylenedioxyphenylisopropylamine; [0106]
3-Methoxy-4,5-methylenedioxyphenylisopropylamine; [0107]
3-Methoxy-4,5-ethylenedioxyphenylisopropylamine; [0108]
2-Methoxy-4,5-methylenedioxyphenylisopropylamine; [0109]
2-Methoxy-3,4-methylenedioxyphenylisopropylamine; [0110]
4-Methoxy-2,3-methylenedioxyphenylisopropylamine; [0111]
6-Methoxy-2,3-methylenedioxy-phenylisopropylamine; [0112]
6-methoxy-2,3-methylenedioxyphenylisopropylamine; and [0113]
2,3-Dimethoxy-4,5-methylenedioxyphenylisopropylamine; (3)
Alkoxyphenylisopropylamines, such as: [0114]
4-Benzyloxy-3,5-dimethoxyphenylisopropylamine; [0115]
4-Ethoxy-2,5-dimethoxyphenylisopropylamine; [0116]
2-Ethoxy-4,5-dimethoxyphenylisopropylamine; [0117]
5-Ethoxy-2,4-dimethoxyphenylisopropylamine; and [0118]
4-(n)-Propoxy-2,5-dimethoxyphenylisopropylamine; (4)
Alkylphenylisopropylamines, such as: [0119]
4-Methylphenylisopropylamine; [0120] 2-Methylphenylisopropylamine;
[0121] 3-Methylphenylisopropylamine; [0122]
3,4-Dimethylphenylisopropylamine; [0123]
2,5-Dimethylphenylisopropylamine; [0124]
2,5-Dimethoxy-4-methylphenylisopropylamine; [0125]
2,6-Dimethoxy-4-methylisopropylamine; [0126]
2,5-Dimethoxy-4-ethylphenylisopropylamine; [0127]
2,5-Dimethoxy-4-propylphenylisopropylamine; [0128]
2,5-Dimethoxy-4-butylphenylisopropylamine; [0129]
2,5-Dimethoxy-4-amylphenylisopropylamine; and [0130]
6-(2-Aminopropyl)-2,2-dimethyl-5-methoxy-2,3-dihydrofuran; (5)
Halo- or Sulfur-Substituted Phenylisopropylamines, such as: [0131]
4-Chlorophenylisopropylamine; [0132]
4-Chloro-N-methylphenylisopropylamine; [0133]
4-Bromo-N-methylphenylisopropylamine; [0134]
4-Bromo-2,5-dimethoxyphenylisopropylamine; [0135]
2-Bromo-4,5-methylenedioxyphenylisopropylamine; and: [0136]
4-Bromo-3,5-dimethoxyphenylisopropylamine; and:
(6) Brominated Alkoxylated Phenylisopropylamines;
[0136] [0137] 4-Iodo-2,5-dimethoxyphenylisopropylamine; [0138]
4-Thiomethyl-2,5-dimethoxyphenylisopropylamine; and [0139]
4-Thioethyl-2,5-dimethoxyphenylisopropylamine. Additional examples
of active compounds useful for carrying out at least some
embodiments of the present invention include but are not limited to
the following [0140] 4-Methylthio-2,5-dimethoxyamphetamine; [0141]
4-Ethylthio-2,5-dimethoxyamphetamine; [0142]
4-Isopropylthio-2,5-dimethoxyamphetamine; [0143]
4-Phenylthio-2,5-dimethoxyamphetamine; [0144]
4-Propylthio-2,5-dimethoxyamphetamine; [0145]
4-allyloxy-3,5-dimethoxyamphetamine; [0146]
2,5-dimethoxy-4-(beta-methallylthio)amphetamine; [0147]
2,5-dimethoxy-4-allylthioamphetamine; [0148]
2,5-dimethoxy-4-cyclohexylthioamphetamine; [0149]
2,5-dimethoxy-4-(2-fluoroethylthio)amphetamine; [0150]
3,5-dimethoxy-4-bromoamphetamine; [0151]
2,5-Bismethylthio-4-methylamphetamine; [0152]
2,5-Dimethoxy-4,N-dimethylamphetamine; [0153]
N-cyclopropyl-2,5-dimethoxy-4-methylamphetamine; [0154]
4-Bromo-3,5-dimethoxyamphetamine; [0155]
2-Bromo-4,5-methylenedioxyamphetamine; [0156]
4-Benzyloxy-3,5-dimethoxyamphetamine; [0157]
4-Ethoxy-3,5-dimethoxyamphetamine; [0158] 2,4-Dimethoxyamphetamine;
[0159] 3,4,5-trimethylamphetamine; [0160]
2,4-dimethoxy-N,N-dimethylamphetamine; [0161]
2,4-dimethoxy-N,N-dimethyl-5-iodoamphetamine; [0162]
2,4-dimethoxy-N,N-dimethyl-5-fluoroamphetamine; [0163]
N,N-diethyl-2,4-dimethoxyamphetamine; [0164]
N,N-dimethyl-2-ethoxy-4-methoxyamphetamine; [0165]
2-(n)-butyloxy-N,N-dimethyl-4-methoxy-amphetamine; [0166]
2-(n)-decyloxy-N,N-dimethylamphetamine; [0167]
2,4-diethoxy-N,N-dimethylamphetamine; [0168]
N,N-dimethyl-2,4-di-(i)-propoxyamphetamine; [0169]
5-bromo-2,4-dimethoxyamphetamine; [0170] 2,5-Dimethoxyamphetamine;
[0171] 3,4-dimethylamphetamine; [0172]
2,5-dimethoxy-N,N-dimethylamphetamine; [0173]
4-fluoro-2,5-dimethoxy-N,N-dimethylamphetamine; [0174]
2,5,N,N-tetramethylamphetamine; [0175] 3,4-Dimethoxyamphetamine;
[0176] 3-bromo-2,6-dimethoxy-N,N-dimethylamphetamine [0177]
3-iodo-2,6-dimethoxy-N,N-dimethyl amphetamine [0178]
3,5-dimethoxy-N,N-dimethylamphetamine [0179]
2,5-Dimethoxy-3,4-methylenedioxyamphetamine; [0180]
2,5-dimethoxy-N-methyl-3,4-methylenedioxyamphetamine; [0181]
2,3-Dimethoxy-4,5-methylenedioxyamphetamine; [0182]
4-Amyl-2,5-dimethoxyamphetamine; [0183]
4-Bromo-2,5-dimethoxyamphetamine; [0184]
4-Butyl-2,5-dimethoxyamphetamine; [0185]
2,5-dimethoxy-4-(2-methylpropyl)-amphetamine; [0186]
2,5-dimethoxy-4-(1-methylpropyl)amphetamine; [0187]
2,5-dimethoxy-4-(1,1-dimethylethyl)amphetamine; [0188]
2,5-dimethoxy-4-cyclo-propylmethylamphetamine; [0189]
4-Chloro-2,5-dimethoxyamphetamine; [0190] 2,5-dimethoxy-4-acetamido
amphetamine; [0191] 4-(2-Fluoroethyl)-2,5-dimethoxyamphetamine;
[0192] 4-Ethyl-2,5-dimethoxyamphetamine; [0193]
4-Iodo-2,5-dimethoxyamphetamine; [0194]
4-Methyl-2,5-dimethoxyamphetamine; [0195] 2,4-dimethoxy-5-methyl
amphetamine; [0196] 4,5-dimethoxy-2-methylamphetamine; [0197]
4-Methyl-2,6-dimethoxyamphetamine; [0198]
4-Nitro-2,5-dimethoxyamphetamine; [0199]
4-Propyl-2,5-dimethoxyamphetamine; [0200]
2,5-dimethoxy-4-(1-hydroxypropyl)-amphetamine; [0201]
2,5-dimethoxy-4-ethoxyamphetamine; [0202]
3,5-dimethoxy-4-ethoxyamphetamine; [0203]
2,4,5-Triethoxyamphetamine; [0204]
2,4-Diethoxy-5-methoxyamphetamine; [0205]
2,5-Diethoxy-4-methoxyamphetamine; [0206]
2-Ethoxy-4,5-dimethoxyamphetamine; [0207]
Benzofuran-2-methyl-5-methoxy-6-(2-aminopropane); [0208]
6-(2-aminopropyl)-5-methoxy-2-methyl-2,3-dihydrobenzofuran; [0209]
7-(2-aminopropyl)-5-methoxy-2-methyl-2,3-dihydrobenzofuran; [0210]
Benzofuran-2,2-dimethyl-5-methoxy-6-(2-aminopropane); [0211]
6-(2-aminopropyl)-2,2-dimethyl-5-methoxy-2,3-dihydrobenzofuran;
[0212]
7-(2-aminopropyl)-2,2-dimethyl-5-methoxy-2,3-dihydrobenzofuran;
[0213]
6-(2-aminopropyl)-5-methoxy-2,3,3-trimethyl-2,3-dihydrobenzofuran;
[0214]
6-(2-aminopropyl)-2,3-dimethyl-5-methoxy-2,3-dihydrobenzofuran;
[0215] 6-(2-aminopropyl)-2-ethyl-5-methoxy-2,3-dihydrobenzofuran;
[0216] 7-(2-aminopropyl) 6-methoxy-1,2,3,4-tetrahydrobenzopyran;
[0217] N-Hydroxy-N-methyl-3,4-methylenedioxyamphetamine; [0218]
3,4-Trimethylene-2,5-dimethoxyamphetamine; [0219]
3,4-Tetramethylene-2,5-dimethoxyamphetamine; [0220]
3,4-Norbomyl-2,5-dimethoxyamphetamine; [0221]
3,4-Dimethyl-2,5-dimethoxyamphetamine; [0222]
1,4-Dimethoxynaphthyl-2-isopropylamine; [0223]
2,5-Dimethoxy-N,N-dimethyl-4-iodoamphetamine; [0224]
5-Ethoxy-2-methoxy-4-methylamphetamine; [0225]
4-Methoxyamphetamine; [0226]
2,N-Dimethyl-4,5-methylenedioxyamphetamine; [0227]
2,N-dimethyl-3,4-methylenedioxyamphetamine; [0228]
3,4-Methylenedioxyamphetamine; [0229]
2,3-methylenedioxyamphetamine; [0230]
N-Allyl-3,4-methylenedioxyamphetamine; [0231]
N-Butyl-3,4-methylenedioxyamphetamine; [0232] N-butylamphetamine;
[0233] N-ethylamphetamine; [0234] N-methylamphetamine; [0235]
N-Benzyl-3,4-methylenedioxyamphetamine; [0236]
3,4-methylenedioxy-N-(i)-butylamphetamine; [0237]
3,4-methylenedioxy-N-(t)-butylamphetamine; [0238]
3,4-methylenedioxy-N-amylamphetamine; [0239]
3,4-methylenedioxy-N-(n)-hexylamphetamine; [0240]
3,4-methylenedioxy-N-(4-heptyl)-amphetamine; [0241]
3,4-methylenedioxy-N-(n)-octylamphetamine; [0242]
3,4-methylenedioxy-N,N-diethylamphetamine; [0243]
3,4-methylenedioxy-N-(t)-butylaminoamphetamine; [0244]
N-Cyclopropylmethyl-3,4-methylenedioxyamphetamine; [0245]
N,N-Dimethyl-3,4-methylenedioxyamphetamine; [0246]
N-Ethyl-3,4-methylenedioxyamphetamine; [0247]
N-(2-Hydroxyethyl)-3,4-methylenedioxyamphetamine; [0248]
N-Isopropyl-3,4-methylenedioxyamphetamine; [0249]
N-Methyl-3,4-methylenedioxyamphetamine; [0250]
N-Methyl-3,4-ethylenedioxyamphetamine; [0251]
N-Methoxy-3,4-methylenedioxyamphetamine; [0252]
N-(2-Methoxyethyl)-3,4-methylenedioxyamphetamine; [0253]
N-Hydroxy-3,4-methylenedioxyamphetamine; [0254]
N-Propargyl-3,4-methylenedioxyamphetamine; [0255]
N-Propyl-3,4-methylenedioxyamphetamine; [0256]
3,4-Ethylenedioxy-5-methoxyamphetamine; [0257]
2-Methoxy-4,5-diethoxyamphetamine; [0258]
2,5-Dimethoxy-4-ethoxyamphetamine; [0259]
3-methoxy-4-ethoxy-amphetamine; [0260]
3-methoxy-4-allyloxy-amphetamine; [0261]
3-methoxy-4-methylamphetamine; [0262]
5-Bromo-2,4-dimethoxyamphetamine; [0263]
2-bromo-4,5-dimethoxyamphetamine; [0264]
5-bromo-2,3-dimethoxyamphetamine; [0265]
6-bromo-2,3-dimethoxyamphetamine; [0266]
3-bromo-2,6-dimethoxyamphetamine; [0267]
2-bromo-3,5-dimethoxyamphetamine; [0268]
2,6-dibromo-4,5-dimethoxyamphetamine; [0269]
4-bromo-2,5-dimethoxyamphetamine; [0270]
5-Methylthio-2,4-dimethoxyamphetamine; [0271]
N-Methyl-2,5-dimethoxyamphetamine; [0272]
2-methoxy-N-methylamphetamine; [0273]
5-hydroxy-2-methoxy-N-methylamphetamine; [0274]
N-methyl-3,4,5-trimethoxyamphetamine; [0275]
N-methyl-2,4,5-trimethoxyamphetamine; [0276]
N-methyl-2,4,6-trimethoxyamphetamine; [0277]
4-Bromo-2,5-dimethoxy-N-methylamphetamine; [0278]
N-Methyl-4-methoxyamphetamine; [0279]
4-methoxy-N-methylamphetamine; [0280]
4-methoxy-N,N-dimethylamphetamine; [0281]
2-methoxy-N,N-dimethylamphetamine; [0282]
N-Methyl-2-methoxy-4,5-methylenedioxyamphetamine; [0283]
2,5-dimethoxy-N-methyl-3,4-methylenedioxyamphetamine; [0284]
3-Methoxy-4,5-methylenedioxyamphetamine; [0285]
2-Methoxy-4,5-methylenedioxyamphetamine; [0286]
2-ethoxy-4,5-methylenedioxyamphetamine; [0287]
2-Methoxy-3,4-methylenedioxyamphetamine; [0288]
4-Methoxy-2,3-methylenedioxyamphetamine; [0289]
6-methoxy-2,3-methylenedioxyamphetamine; [0290]
2,4-Dimethoxy-5-ethoxyamphetamine; [0291]
2,5-Dimethoxy-4-propoxyamphetamine; [0292]
4-(n)-butoxy-2,5-dimethoxyamphetamine; [0293]
4-(n)-amyl-2,5-dimethoxyamphetamine; [0294]
2-Methylthio-4,5-dimethoxyamphetamine; [0295]
3,5-dimethoxy-4-(n)-propoxy-amphetamine; [0296]
2,3,4,5-Tetramethoxyamphetamine; [0297]
3,4-dimethoxy-2-methylthioamphetamine; [0298]
2,4-dimethoxy-3-methylthioamphetamine; [0299]
2,3-dimethoxy-4-thioamphetamine; [0300]
3,4-dimethoxy-5-methylthioamphetamine; [0301]
3,4,5-Trimethoxyamphetamine; [0302] 2,4,5-Trimethoxyamphetamine;
[0303] 2,3,4-Trimethoxyamphetamine; [0304]
2,3,5-Trimethoxyamphetamine; [0305] 2,3,6-Trimethoxyamphetamine;
[0306] 2,4,6-Trimethoxyamphetamine; [0307]
2-Methylthio-3,4-methylenedioxyamphetamine; [0308]
3-methoxy-5,4-methylenethiooxyamphetamine [0309]
2-methoxy-5,4-methylenethiooxyamphetamine [0310]
4,5-Thiomethyleneoxy-2-methoxyamphetamine; [0311]
4-Ethyl-5-methoxy-2-methylthioamphetamine; [0312]
4-Ethyl-2-methoxy-5-methylthioamphetamine; [0313]
5-Methoxy-4-methyl-2-methylthioamphetamine; [0314]
2-Methoxy-4-methyl-5-methylthioamphetamine; [0315]
2-Methoxy-4-methyl-5-methylsulfinylamphetamine; [0316]
3,5-dimethoxy-4-methylthioamphetamine; [0317]
3,5-dimethoxy-4-(n)-butylthioamphetamine; [0318] Phentermine;
[0319] 3,4-methylenedioxyphentermine; [0320] Fenfluramine; [0321]
1-amphetamine; and [0322] bupropion. Along with pharmaceutically
acceptable salts and prodrugs of any of the foregoing.
[0323] Salts.
[0324] Active compounds of the invention include pharmaceutically
acceptable salts of the foregoing. Pharmaceutically acceptable
salts are salts that retain the desired biological activity of the
parent compound and do not impart undesired toxicological effects.
Examples of such salts are (a) acid addition salts formed with
inorganic acids, for example hydrochloric acid, hydrobromic acid,
sulfuric acid, phosphoric acid, nitric acid and the like; and salts
formed with organic acids such as, for example, acetic acid, oxalic
acid, tartaric acid, succinic acid, maleic acid, fumaric acid,
gluconic acid, citric acid, malic acid, ascorbic acid, benzoic
acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,
naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic
acid, naphthalenedisulfonic acid, polygalacturonic acid, and the
like; (b) salts formed from elemental anions such as chlorine,
bromine, and iodine, and (c) salts derived from bases, such as
ammonium salts, alkali metal salts such as those of sodium and
potassium, alkaline earth metal salts such as those of calcium and
magnesium, and salts with organic bases such as dicyclohexylamine
and N-methyl-D-glucamine.
[0325] Prodrugs.
[0326] Active compounds of the present invention include prodrugs
of the foregoing. "Prodrugs" as used herein refers to those
prodrugs of the compounds of the present invention which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response and the like, commensurate
with a reasonable risk/benefit ratio, and effective for their
intended use, as well as the zwitterionic forms, where possible, of
the compounds of the invention. The term "prodrug" refers to
compounds that are rapidly transformed in vivo to yield the parent
compound of the above formulae, for example, by hydrolysis in
blood. A thorough discussion is provided in T. Higuchi and V.
Stella, Prodrugs as Novel delivery Systems, Vol. 14 of the A.C.S.
Symposium Series and in Edward B. Roche, ed., Bioreversible
Carriers in Drug Design, American Pharmaceutical Association and
Pergamon Press, 1987, both of which are incorporated by reference
herein. See also U.S. Pat. No. 6,680,299 Examples include a prodrug
that is metabolized in vivo by a subject to an active drug having
an activity of active compounds as described herein, wherein the
prodrug is an ester of an alcohol or carboxylic acid group, if such
a group is present in the compound; an acetal or ketal of an
alcohol group, if such a group is present in the compound; an
N-Mannich base or an imine of an amine group, if such a group is
present in the compound; or a Schiff base, oxime, acetal, enol
ester, oxazolidine, or thiazolidine of a carbonyl group, if such a
group is present in the compound, such as described in U.S. Pat.
No. 6,680,324 and U.S. Pat. No. 6,680,322.
2. Pharmaceutical Formulations, Administration and Dosage.
[0327] The active compounds described above may be formulated for
administration in a pharmaceutical carrier in accordance with known
techniques. See, e.g., Remington, The Science And Practice of
Pharmacy (9.sup.th Ed. 1995). In the manufacture of a
pharmaceutical formulation according to the invention, the active
compound (including the physiologically acceptable salts thereof)
is typically admixed with, inter alia, an acceptable carrier. The
carrier must, of course, be acceptable in the sense of being
compatible with any other ingredients in the formulation and must
not be deleterious to the patient. The carrier may be a solid or a
liquid, or both, and is preferably formulated with the compound as
a unit-dose formulation, for example, a tablet, which may contain
from 0.01 or 0.5% to 95% or 99% by weight of the active compound.
One or more active compounds may be incorporated in the
formulations of the invention, which may be prepared by any of the
well known techniques of pharmacy comprising admixing the
components, optionally including one or more accessory
ingredients.
[0328] The formulations of the invention include those suitable for
oral, rectal, topical, buccal (e.g., sub-lingual), vaginal,
parenteral (e.g., subcutaneous, intramuscular, intradermal, or
intravenous), topical (i.e., both skin and mucosal surfaces,
including airway surfaces) and transdermal administration, although
the most suitable route in any given case will depend on the nature
and severity of the condition being treated and on the nature of
the particular active compound which is being used.
[0329] Formulations suitable for oral administration may be
presented in discrete units, such as capsules, cachets, lozenges,
or tablets, each containing a predetermined amount of the active
compound; as a powder or granules; as a solution or a suspension in
an aqueous or non-aqueous liquid; or as an oil-in-water or
water-in-oil emulsion. Such formulations may be prepared by any
suitable method of pharmacy which includes the step of bringing
into association the active compound and a suitable carrier (which
may contain one or more accessory ingredients as noted above). In
general, the formulations of the invention are prepared by
uniformly and intimately admixing the active compound with a liquid
or finely divided solid carrier, or both, and then, if necessary,
shaping the resulting mixture. For example, a tablet may be
prepared by compressing or molding a powder or granules containing
the active compound, optionally with one or more accessory
ingredients. Compressed tablets may be prepared by compressing, in
a suitable machine, the compound in a free-flowing form, such as a
powder or granules optionally mixed with a binder, lubricant, inert
diluent, and/or surface active/dispersing agent(s). Molded tablets
may be made by molding, in a suitable machine, the powdered
compound moistened with an inert liquid binder.
[0330] Formulations suitable for buccal (sub-lingual)
administration include lozenges comprising the active compound in a
flavoured base, usually sucrose and acacia or tragacanth; and
pastilles comprising the compound in an inert base such as gelatin
and glycerin or sucrose and acacia.
[0331] Formulations of the present invention suitable for
parenteral administration comprise sterile aqueous and non-aqueous
injection solutions of the active compound, which preparations are
preferably isotonic with the blood of the intended recipient. These
preparations may contain anti-oxidants, buffers, bacteriostats and
solutes which render the formulation isotonic with the blood of the
intended recipient. Aqueous and non-aqueous sterile suspensions may
include suspending agents and thickening agents. The formulations
may be presented in unit\dose or multi-dose containers, for example
sealed ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline or water-for-injection
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described. For example, in one
aspect of the present invention, there is provided an injectable,
stable, sterile composition comprising a compound of Formula (I),
or a salt thereof, in a unit dosage form in a sealed container. The
compound or salt is provided in the form of a lyophilizate which is
capable of being reconstituted with a suitable pharmaceutically
acceptable carrier to form a liquid composition suitable for
injection thereof into a subject. The unit dosage form typically
comprises from about 10 mg to about 10 grams of the compound or
salt. When the compound or salt is substantially water-insoluble, a
sufficient amount of emulsifying agent which is physiologically
acceptable may be employed in sufficient quantity to emulsify the
compound or salt in an aqueous carrier. One such useful emulsifying
agent is phosphatidyl choline.
[0332] Formulations suitable for rectal administration are
preferably presented as unit dose suppositories. These may be
prepared by admixing the active compound with one or more
conventional solid carriers, for example, cocoa butter, and then
shaping the resulting mixture.
[0333] Formulations suitable for topical application to the skin
preferably take the form of an ointment, cream, lotion, paste, gel,
spray, aerosol, or oil. Carriers which may be used include
petroleum jelly, lanoline, polyethylene glycols, alcohols,
transdermal enhancers, and combinations of two or more thereof.
[0334] Formulations suitable for transdermal administration may be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time,
Formulations suitable for transdermal administration may also be
delivered by iontophoresis (see, for example, Pharmaceutical
Research 3 (6):318 (1986)) and typically take the form of an
optionally buffered aqueous solution of the active compound.
Suitable formulations comprise citrate or bis\tris buffer (pH 6) or
ethanol/water and contain from 0.1 to 0.2M active ingredient.
[0335] Further, the present invention provides liposomal
formulations of the compounds disclosed herein and salts thereof.
The technology for forming liposomal suspensions is well known in
the art. When the compound or salt thereof is an aqueous-soluble
salt, using conventional liposome technology, the same may be
incorporated into lipid vesicles. In such an instance, due to the
water solubility of the compound or salt, the compound or salt will
be substantially entrained within the hydrophilic center or core of
the liposomes. The lipid layer employed may be of any conventional
composition and may either contain cholesterol or may be
cholesterol-free. When the compound or salt of interest is
water-insoluble, again employing conventional liposome formation
technology, the salt may be substantially entrained within the
hydrophobic lipid bilayer which forms the structure of the
liposome. In either instance, the liposomes which are produced may
be reduced in size, as through the use of standard sonication and
homogenization techniques.
[0336] Of course, the liposomal formulations containing the
compounds disclosed herein or salts thereof, may be lyophilized to
produce a lyophilizate which may be reconstituted with a
pharmaceutically acceptable carrier, such as water, to regenerate a
liposomal suspension.
[0337] Other pharmaceutical compositions may be prepared from the
water-insoluble compounds disclosed herein, or salts thereof, such
as aqueous base emulsions. In such an instance, the composition
will contain a sufficient amount of pharmaceutically acceptable
emulsifying agent to emulsify the desired amount of the compound or
salt thereof. Particularly useful emulsifying agents include
phosphatidyl cholines, and lecithin.
[0338] In addition to active agents or their salts, the
pharmaceutical compositions may contain other additives, such as
pH-adjusting additives. In particular, useful pH-adjusting agents
include acids, such as hydrochloric acid, bases or buffers, such as
sodium lactate, sodium acetate, sodium phosphate, sodium citrate,
sodium borate, or sodium gluconate. Further, the compositions may
contain microbial preservatives. Useful microbial preservatives
include methylparaben, propylparaben, and benzyl alcohol. The
microbial preservative is typically employed when the formulation
is placed in a vial designed for multidose use. Of course, as
indicated, the pharmaceutical compositions of the present invention
may be lyophilized using techniques well known in the art.
[0339] The therapeutically effective dosage of any specific
compound, the use of which is in the scope of present invention,
will vary somewhat from compound to compound, and patient to
patient, and will depend upon the condition of the patient and the
route of delivery. As a general proposition, a dosage from about 1
or 2 to about 50, 100, 200 or 500 milligrams per day, administered
once or over two, three or four separate administrations, can be
used to carry out the present invention,
[0340] Compositions containing an active agent of the invention in
combination with an additional antiparkinson's agent are prepared
in like manner as described above and techniques that will be
apparent to those skilled in the art. Such compositions may be
prepared in any suitable unit dosage form including injectable
forms and oral dosage forms such as tablets and capsules, as
described above. The amount of active agent will depend upon the
subject to be treated and the route of administration and can be
determined in accordance with known techniques, but in some
embodiments is from 0.5 or 1 to 50, 100 or 200 milligrams per
dosage form. The amount of the one (or more) additional
antiparkinson's agent will depend upon the particular agent, but
generally will be from 0.1 or 1 to 200 or 400 milligrams per unit
dosage form. See, e.g., U.S. Pat. No. 6,797,732 (levodopa,
carbidopa, and COMT inhibitor oral pharmaceutical).
4. Screening Techniques.
[0341] The present invention provides a method of screening a
compound for antiparkinson's activity. In general, the method
comprises: (a) administering a test compound (e.g., orally or by
parenteral injection) to a dopamine-depleted dopamine transporter
deficient mouse (such as described in U.S. Pat. No. 6,218,595 to
Giros et al.), and then (b) detecting the presence or absence of
antiparkinson's activity in said mouse. The methods can generally
be carried out as described in U.S. Pat. No. 6,218,595 to Giros et
al,
[0342] In general, the recombinant mouse comprises cells (e.g.,
including brain tissue cells) containing a pair of genomic dopamine
transporter alleles, wherein at least one of said alleles is
incapable of expressing endogenous dopamine transporter protein,
and wherein there is at least about a 30% reduction in dopamine
uptake. The mouse may be a homozygote and both of said alleles are
incapable of expressing endogenous dopamine transporter protein.;
the mouse may be a heterozygote, wherein one of said alleles
expresses endogenous dopamine transporter protein. In some
embodiments the mouse is an adult.
[0343] The mouse is preferably depleted of dopamine sufficient to
develop at least one Parkinson's symptom in said mouse. Depletion
of dopamine sufficient to develop at least on Parkinson's symptom
can be carried out by any suitable technique, such as by
administering tyrosine hydroxylase inhibitors such as
alpha-methyl-para-tyrosine (c MT) to deplete dopamine (e.g., by
parenteral injection) in an amount sufficient to deplete dopamine
in the mouse and develop the at least one symptoms. Typical doses
thereof are, for example, 50-500 mg/kg. Another approach to
depletion is to block the second step in dopamine synthesis by
administering L-aromatic acid decarboxylase inhibitors such as
3-hydroxybenzylhydrazine (NSD-1015) (e.g., by parenteral injection
of 50-300 mg/kg).
[0344] Parkinson's disease in the mouse is characterized by at
least one symptom thereof, such as rigidity, akinesia, body tremor,
and ptosis (droopy eyelids). These behaviors or symptoms, and
beneficial treatment thereof by a test compound being screened, are
readily detectible by any suitable technique. For example, akinesia
can be assessed by evaluating horizontal locomotor activity and by
an "akinesia" test as described herein, rigidity assessed by a
catalepsy test, a "grasping" test, and/or a "bracing" test, while
tremor and ptosis can be visually determined.
[0345] Dopamine depleted mice as described herein are further
useful per se for identifying (e.g., by histological techniques
such as immunohistochemistry) neuronal pathways and
neurotransmitter systems involved in motor functions in conditions
of severe dopamine deficiency such as Parkinson's disease.
[0346] The present invention is explained in greater detail in the
following non-limiting Examples. The following abbreviations are
used herein: 5-HT, serotonin; .alpha.MT,
alpha-methyl-para-tyrosine; DA, dopamine; DAT, dopamine
transporter; DAT-KO mice, dopamine transporter knockout mice; DD
mice, dopamine-deficient mice; DDD mice, dopamine-deficient DAT-KO
mice; IP, intraperitoneal; L-AADC, L-aromatic acid decarboxylase;
MAO, monoamine oxidase; NE, norepinephrine; NET, norepinephrine
transporter; PD, Parkinson's disease; SC, subcutaneous; SNc,
Substantia Nigra Pars Compacta; TA1 receptor, trace amine 1
receptor; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine
transporter-2; VTA, ventral tegmental area; WT, wild-type.
EXPERIMENTAL
[0347] We report here that the pharmacologic inhibition of the
rate-limiting enzyme of DA synthesis, TH, almost immediately
depletes brain DA to undetectable levels in DAT-KO mice and induces
a transient recapitulation of essentially all PD symptoms for up to
16 h. DA-deficient DAT-KO mice (DDD mice) thus represent an acute
PD model that is useful for studying the efficacy of compounds that
potentially can restore control of locomotion in the absence of any
contribution of the dopaminergic system. By using this approach, we
found that several amphetamine derivatives can counteract the
behavioral manifestations of severe DA deficiency, suggesting that,
in addition to well-known DA-mediated effects, amphetamine-like
compounds can also affect motor functions in a DA- and
DAT-independent manner.
Materials and Methods
[0348] Animals.
[0349] DAT-KO mice were generated as previously described [11].
Animal care was in accordance with the Guide for Care and Use of
Laboratory Animals (National Institutes of Health publication
#865-23, Bethesda, Md., United States) with an approved protocol
from the Duke University Institutional Animal Care and Use
Committee. C57BL/6J3129Sv/J hybrid WT and DAT-KO mice, 3-5 mo old,
of both sexes were used. None of animals used in these studies had
the neurodegenerative phenotype sporadically observed in DAT-KO
mice [60].
[0350] Drugs.
[0351] Drugs or saline (0.9% NaCl) were administered
intraperitoneally (IP) or subcutaneously (SC) in a volume of 10
ml/kg. The drugs were either from Sigma (St. Louis, Mosouri, United
States) or supplied by the National Institute of Drug Abuse (NIDA).
Drugs provided by the NIDA Drug Supply Program included:
(.+-.)-MDMA, (+)-MDMA, (.+-.)-6-0H-MDA, (.+-.)-MDA, (.+-.)-MDE,
(+)-MDE, (-)-MDE, and AET (alpha-ethyl-tryptamine acetate).
[0352] Neurochemical assessments. Striatal tissue contents of DA
and frontal cortical tissue levels of NE were assessed using
HPLC-EC (high performance liquid chromatography with
electrochemical detection) as described [8]. In vivo microdialysis
measurements of striatal extracellular DA levels in freely moving
mice were performed at least 24 h after implantation of a
microdialysis probe as described previously [50]. Dialysate samples
were assayed for DA using HPLC-EC.
[0353] Behavioral Methods.
[0354] Locomotor activity of littermate WT and DATKO mice was
measured in an Omnitech CCDigiscan (Accuscan Instruments, Columbus,
Ohio United States) activity monitor under bright illumination
[83]. All behavioral experiments were performed between 10:00 AM
and 5:00 PM. Activity was measured at 5-min intervals. To evaluate
the effects of drugs on motor behaviors, mice were placed into
activity monitor chambers (20.times.20 cm) for 30 min and then
treated with .alpha.MT (250 mg/kg IP). A drug or combination of
drugs were injected 1 h after .alpha.MT administration, and various
parameters of locomotor activity were monitored for up to 3 h. In
cumulative dosing experiments, animals were treated with increasing
doses of drugs after a 1-h interval. For the akinesia test, the
mouse is held by the tail so that it is standing on forelimbs only
and moving on its own. The number of steps taken with both
forelimbs was recorded during a 30-s trial [57]. The presence of
catalepsy was determined and measured by placing the animal's
forepaws on a horizontal wooden bar (0.7 cm in diameter), 4 cm
above the tabletop. The time until the mouse removed both forepaws
from the bar was recorded, with a maximum cut-off time of 3 min
[53]. In the grasping test of muscular rigidity, the mouse is
suspended by its forelimbs on a metal rod (diameter: 0.25 cm)
positioned approximately 20 cm above the table. The time the animal
remains on the rod (maximum 1 min) was noted [58]. To assess
rigidity in a bracing task, the number of steps taken with each
forelimb when the mouse is pushed sideways over a distance of 50 cm
was recorded [57]. Tremor was scored visually in mice using the
rating scale [54]: 0, no tremor; 1, occasional isolated twitches;
2, moderate or intermittent tremor associated with short periods of
calm; and 3, pronounced continuous tremor. Ptosis was scored as
described [89]: 4, eyes completely closed; 2, half-open eyes; and
0, wide-open eyes; with 1 and 3 indicating intermediate values. The
righting reflex was evaluated by turning the mouse onto its back
five times. Normal mice immediately turn themselves over, to right
themselves onto all four feet. Righting reflex was scored as
follows: 0, no impairment; 1, on side one to two times; 2, on side
three to four times; 3, on side five times; 4, on back one to two
times; 5, on back three to four times; 6, on back five times; 7,
sluggish when placed on back; and 8, righting response absent when
on back and tail pinched [55].
[0355] Data Analysis.
[0356] The data are presented as mean.+-.SEM and analyzed using a
two-tailed Student's t-test and one-way analysis of variance
(ANOVA) followed by Dunnet's multiple comparison test or a
two-tailed Mann-Whitney U test when appropriate.
Results
[0357] A Pharmacologic Approach for Provoking Selective DA
Deficiency in DAT-KO Mice.
[0358] The ability of .alpha.-methyl-p-tyrosine (.alpha.MT), a
potent irreversible inhibitor of TH [29,45,46], to impede
production of brain DA suggests a simple, but straightforward,
strategy for producing an acute PD mouse model. However, numerous
studies have documented that treatment of normal animals with
.alpha.MT results only in a relatively slow and partial depletion
of DA in brain tissues that is not sufficient for generation of
PD-like symptoms [29,45,46]. This limited depletion is based upon
how DA is stored. It is believed that the large intraneuronal DA
storage pool that normally exists in striatal DA terminals provides
sufficient DA to release and recycle back into releasing terminals
up to the time when newly synthesized TH starts to regain its
functional role [29,45,46]. Thus, in a normal animal, complete
depletion of striatal DA is unachievable by TH inhibition alone,
and additional depletion of vesicular DA by VMAT2 inhibitors, such
as reserpine is required [33,47-49]. Protocols designed for
wild-type [WT] mice that use a dual inhibitor strategy (VMAT2 plus
TH inhibitor) deplete DA to 1%-2% of control levels [33,47-50], but
the levels of other monoamine eurotransmitters that are substrates
for VMAT2 are also severely affected. This nonselective targeting
of monoaminergic signaling generally results in very complicated
phenotypes that are not necessarily reflective of classic PD.
[0359] In the absence of any pharmacologic treatment, the
intraneuronal vesicular stores of DA in the striatum of DAT-KO mice
are already profoundly depleted by at least 20-fold [12]. This
selective depletion of DA in dopaminergic terminals of DAT-KO mice,
as well as analogous depletion observed in mice lacking NET [51] or
serotonin transporter [52] with NE and serotonin (5-HT),
respectively, reflects the critical role of transporter-mediated
recycling in the maintenance of intracellular storage pools [13].
With loss of the major intracellular storage pool of DA in DAT-KO
mice, both the intracellular and extracellular levels of DA in the
striatum become critically dependent upon ongoing DA synthesis.
Therefore in DAT-KO mice, acute TH inhibition alone by .alpha.MT is
sufficient to induce profound depletion of DA [12,13,37].
[0360] To explore this phenomenon in detail, we first measured the
time-course of striatal DA depletion in DAT-KO and control mice
following treatment with .alpha.MT (FIG. 1). In agreement with
previous studies [13], we observed that in untreated DAT-KO mice,
striatal tissue levels of DA were about 20-fold lower than in WT
controls (FIG. 1A). The systemic administration of .alpha.MT (250
mg/kg IP) to DAT-KO mice produced rapid (15 min) and virtually
complete (down to 5% of control levels in DAT-KO mice that is
equivalent to less than 0.2% of WT control levels) depletion of
striatal DA. In contrast, in WT mice the same treatment resulted in
a relatively slow (4 h) depletion of only 60% of striatal tissue DA
(FIG. 1B). The duration of the depletion in DAT-KO mice was
extensive, lasting up to 16 h, until a recovery of DA, related to
the de novo synthesis of TH, occurs [29,45]. Notably, the rate of
recovery of striatal DA levels was approximately the same in WT and
DAT-KO mice.
[0361] Because DA itself serves as a precursor for neuronal
production of NE in NE neurons, the inhibition of TH should also
impact NE production. To test the impact of TH inhibition on the NE
system, the frontal cortex tissue NE concentrations were measured
in WT and DAT-KO mice. As opposed to the DAT, NET expression is not
altered in DATKO mice so that the storage pool, which is by far the
predominant reservoir of NE in NE-enriched regions such as the
frontal cortex, should not be significantly altered in these
mutants. Accordingly, the levels of NE in the frontal cortex tissue
of untreated DAT-KO mice did not vary from that of WT mice (FIG.
1C). Furthermore, .alpha.MT (250 mg/kg IP) treatment induced
similar NE depletion in WT and DAT-KO mice by about 60% in 8 h
after treatment. Importantly, the rates of partial NE depletion and
recovery were almost identical between WT and DAT-KO mice (FIG.
1D). Thus, TH inhibition in DAT-KO mice induces rapid severe
depletion of DA, but only partially and slowly affects NE,
indicating selectivity of this marked depletion to neurons
expressing the DAT.
[0362] In order to demonstrate that targeting of TH by .alpha.MT
depletes the functional extracellular pool of DA in living animals,
we measured extracellular levels of striatal DA in freely moving
mice by in vivo microdialysis. In agreement with total tissue DA
data, .alpha.MT treatment essentially eliminated extracellular DA
levels in DAT-KO mice (FIG. 1F), whereas only a partial decrease
was observed in WT mice. (FIG. 1E). Thus, both intracellular and
extracellular DA levels in the striatum of DAT-KO mice are
critically dependent upon ongoing synthesis.
[0363] DA Depletion in DAT-KO Mice Results in a Loss of Motor
Control.
[0364] It is well known that DA plays a pivotal role in the control
of various aspects of locomotor behaviors. Severe depletion of DA
in .alpha.MT-treated DAT-KO mice results in a very specific
akinetic phenotype (not shown). The DA-depleted DAT-KO mice (DDD
mice) become akinetic almost immediately after treatment, in
contrast to the essentially normal motor function displayed by
.alpha.MT-treated WT mice. Moreover, DDD mice develop extreme
rigidity, body tremor, and ptosis (droopy eyelids). These behaviors
are evident on several tests (FIG. 2). Akinesia was assessed by
evaluating horizontal locomotor activity (FIGS. 2A and 2B) and by
an "akinesia" test (FIG. 2C); rigidity assessed by a catalepsy test
(FIG. 2D), a "grasping" test (FIG. 2E), and a "bracing" test (FIG.
2F); whereas tremor (FIG. 2G) and ptosis (FIG. 211) were visually
determined [3,53-58]. These behaviors were analyzed in WT and
DAT-KO mice for 4 h after of .alpha.MT treatment when depletion of
DA is most severe in DAT-KO mice but with relatively minor effect
on NE levels (see FIG. 1). In all these measures DDD mice differed
significantly from their WT littermates or saline-treated controls.
Importantly, these abnormal behaviors in DDD mice, with the
exception of ptosis, became maximal during the 30- to 60-min period
following .alpha.MT exposure, thus correlating with the rate of DA
depletion. Ptosis developed substantially later (FIG. 2H),
suggesting an additional contribution of NE depletion to the full
magnitude of this response [59]. Importantly, the righting reflex
of DDD mice was normal at all time periods analyzed (FIG. 2I),
indicating that this akinesia is not related to global sedation but
rather to deficient movement control. It should be noted also that
this global phenotype, which might be viewed as "freezing," can be
on some occasions temporarily disrupted by an acoustic startle or
other stressful stimulus. However, after manifesting a few
movements, the animals return to an akinetic state (data not
shown). Strikingly, DDD mice, when placed in water, were able to
swim with periods of floating and active swimming for at least a
3-min period (not shown), indicating that under certain conditions,
movement can occur essentially without DA. Finally, in agreement
with neurochemical data (see FIG. 1B), the recovery from this
profound akinetic phenotype in DDD mice occurs approximately 16-24
h following treatment (FIG. 2J). The full recovery of animals
allows repeated treatment with .alpha.MT, and, in fact, DAT-KO mice
chronically treated with .alpha.MT (100 mg/kg, IP, once every 3 d)
for a period of 40 wk showed no negative consequences [60].
[0365] L-DOPA and Nonselective DA Agonists Restore Motor Activity
in DDD Mice.
[0366] The locomotor restoring effects exhibited by L-DOPA and DA
agonists in various models of DA deficiency form one of the
best-established paradigms in neuroscience [3,15,45,61]. As
expected, high doses of L-DOPA alone (FIG. 3A), or lower doses of
L-DOPA given along with carbidopa (FIG. 3B-3D) to reduce its
peripheral metabolism via L-AADC inhibition, effectively restore
locomotion in DDD mice. In fact, these treatments temporarily
restore locomotion to the levels observed in untreated DAT-KO mice
(FIG. 3A-3D), which are normally at least 10 times more active than
WT mice when placed into a novel environment [11,13]. Other
manifestations associated with DA deficiency as described in FIG. 2
were also essentially completely reversed (data not shown).
[0367] Efficacy of exogenous direct DA agonists was also tested in
this model. Although the nonselective D1/D2 DA receptor agonists
apomorphine and pergolide were somewhat effective in inducing
forward locomotion (FIGS. 3E and 3F), the activity levels of DDD
mice following these treatments were substantially lower than those
induced by L-DOPA. Strikingly, the selective D1 DA receptor agonist
(+)-SKF81297 and D2 DA receptor agonists, bromocriptine and
quinpirole, were ineffective in inducing forward locomotion when
administered separately (FIG. 3G-31). However, the combined
administration of the D1 and D2 agonists (+)-SKF81297 plus
quinpirole restored movement and induced forward locomotion (FIG.
4J), supporting the well-established cooperative interaction of D1
and D2-like DA receptors in locomotor activity [62].
[0368] Movement-Restoring Actions of Amphetamine Derivatives in DOD
Mice.
[0369] The loss of DA signaling that creates the motor symptoms of
PD occurs upstream of many nondopaminergic pathways. This suggests
that activation or inhibition of some of these downstream neuronal
circuits could potentially reverse the motor deficits independent
of restoration of upstream DA activity. We, therefore, tested
several non-dopaminergic compounds that potentially could reverse
the consequences of severe DA deficiency in DDD mice (data not
shown). Many of these compounds have been found to be effective in
restoring some aspects of movement control in one or another
experimental animal model of PD and/or in PD patients
[21,26,27,48,49]. However, in DDD mice none of the drugs were
effective in restoring the major aspects of movement control
required for forward locomotion (distance traveled). Although it is
likely that the lack of locomotor effects of these drugs in DDD
mice is related to an unprecedented level of DA depletion in these
mice, it should be emphasized that in our studies only a few doses
or combinations of drugs were tested. Furthermore, several
treatments, although not inducing forward locomotion per se, were,
nevertheless, somewhat effective in reversing other manifestations
of DA deficiency. For example, the NMDA receptor antagonist MK-801
was able to reduce rigidity and promote weak, disorganized movement
that however did not result in a significant increase in forward
locomotion (data not shown). Synthetic amino acid L-DOPS
(L-threo-3,4-dihydroxyphenylserine), which is decarboxylated to NE
by L-AADC, selectively reversed ptosis in DDD mice. Cumulative
dosing experiments revealed ptosis scores (measured 1 h after each
treatment) of 2.50.+-.0.28 after 100 mg/kg, 0 after 200 mg/kg, and
0 after 400 mg/kg IP of L-DOPS (n=4), whereas corresponding values
for saline-treated controls (n=6) were 3.3.+-.0.3, 3.7.+-.0.2, and
3.7.+-.0.2, respectively. Effects of 200 and 400 mg/kg of L-DOPS on
ptosis in DDD mice were significantly different as compared to
respective control values (p<0.05, Student's t-test) supporting
an important role of NE in this behavioral manifestation [59].
Similarly, high doses of the trace amine beta-phenylethylamine
[44,63] (with or without concomitant inhibition of MAO) did not
induce forward locomotion, but did promote weak stereotypic
reactions, such as headweaving and sniffing (data not shown).
Further investigations will be required to fully evaluate the
efficacy of these drugs in DDD mice.
[0370] Unexpectedly, this initial screening revealed a potent
effect of amphetamine derivatives on behavioral manifestations of
DDD mice. High doses of d-amphetamine, d-methamphetamine,
4-chloro-amphetamine, phentermine, (.+-.)-MDE
((.+-.)-N-ethyl-3,4-methylenedioxyamphetamine HCl), (+)-MDE
((+)-N-ethyl-3,4-methylenedioxyamphetamine HCl), (-)-MDE
((-)-N-ethyl-3,4-methylenedioxyamphetamine HCl), (.+-.)-MDA
((.+-.)-3,4-methylenedioxyamphetamine HCl), (.+-.)-6-0H-MDA
((6)-6-hydroxy-3,4-methylenedioxyamphetamine HCl), (.+-.)-MDMA
((.+-.)-3,4-methylenedioxymethamphetamine HCl), and (+)-MDMA
((+)-3,4-methylenedioxymethamphetamine HCl) were effective in
reducing manifestations of akinesia and rigidity in DDD mice as
detected in the catalepsy, grasping, and akinesia tests (FIG.
4A-4C). Similar effects were observed with other amphetamine
derivatives--L-amphetamine, bupropion, DOI, isomers of fenfluramine
(see Table 1). However none of these drugs (with the exception of
(+)-MDMA, see below) was effective in restoring movement control
sufficiently to induce forward locomotion (data not shown).
[0371] DA-Independent Locomotor Effects of (+)-MDMA in DDD
Mice.
[0372] Among amphetamine derivatives, the most effective compound
to counteract manifestations of akinesia and rigidity in DDD mice
was (+)-MDMA (FIG. 4A-4C). Thus, we tested (+)-MDMA in locomotor
assay at even higher doses than those indicated in Table 1. As
presented in FIG. 5A-5C, (+)-MDMA at high doses was able to induce
significant forward locomotion in DDD mice as measured by distance
traveled in a locomotor activity test. This locomotor action of
(+)-MDMA was observed in both cumulative (FIG. 5A) and single dose
(FIGS. 5B and 5C) treatments. In cumulative dosing experiments, a
first treatment with 30 mg/kg of (+)-MDMA was not effective, but
the subsequent administration of 60 mg/kg induced significant
forward locomotion (FIG. 5A) as well as reversal of other
behavioral manifestations (see FIG. 4A-4C) in DDD mice. Finally,
testing of various single doses clearly showed a dose-dependence of
the locomotor effect of (+)-MDMA in DDD mice (FIG. 5C). The
locomotor stimulating effect of amphetamine and its derivatives are
classically thought to result from the massive efflux of DA from
presynaptic DA terminals via a mechanism including displacement of
DA from vesicular storage and reversal of DAT-mediated DA transport
[7,38-40]. However, in DDD mice, there is only a minimal amount of
DA remaining (<0.2%) and the lack of the DAT precludes the
possibility of amphetamine-mediated DA efflux. In fact, in vivo
microdialysis studies confirmed that (+)-MDMA, at the effective
dose necessary to induce significant locomotor activation in DDD
mice, did not produce any detectable increase in striatal
extracellular DA (FIG. 5D). Moreover, this locomotor stimulation by
(+)-MDMA was not inhibited by simultaneous blockade of D1/D2 DA
receptors when DDD mice were pretreated with a combination of the
D1 and D2 DA receptor antagonists, SCH23390 and raclopride (FIG.
5C). Similarly, this pretreatment did not prevent the effects of
amphetamine and phentermine on the akinesia and rigidity in DDD
mice in grasping and akinesia tests (not shown).
[0373] In contrast, the same D1/D2 DA receptor blockade completely
abolished the locomotor stimulating effects of L-DOPA/carbidopa
(50/50 mg/kg IP) in DDD mice (not shown). Taken together, these
data indicate that (+)-MDMA can affect movement control in a
DA-independent manner and, most importantly, provide a
proof-of-principle that pharmacologic activation of nondopaminergic
neuronal pathways may be sufficient to restore movement even in the
virtual absence of DA neurotransmission.
[0374] It should be noted that the locomotor-stimulating effect of
(+)-MDMA in DDD mice was observed only after high doses of the
drug, which may be potentially neurotoxic [64]. However, the lack
of the DAT renders dopaminergic neurons in DATKO mice significantly
less sensitive to the neurotoxic effects of amphetamines, such as
methamphetamine [65], as well as to MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) [66,67]; thereby
providing a unique opportunity to evaluate effects to large doses
of amphetamines that would be impossible in normal animals [38]. It
should be mentioned also that mice are generally less sensitive to
MDMA neurotoxicity, particularly with regards to the serotonergic
system [68]. Nevertheless, to directly evaluate the neurotoxic
potential of MDMA in DAT-KO mice, we treated DAT-KO and WT mice
with an established neurotoxic regimen of (.+-.)-MDMA
administration (4 injections of 20 mg/kg IP, every 2 h) [69] and
assessed striatal tissue DA and 5-HT levels 7 d later. As might be
expected, no significant differences in both DA and 5-HT levels
were found between (.+-.)-MDMA-treated and saline-treated DAT-KO
mice (saline-treated DAT-KO mice (n=6): DA, 0.53.+-.0.03 ng/mg
tissue; 5-HT, 0.36.+-.0.03 ng/mg tissue; (.+-.)-MDMA-treated DAT-KO
mice (n=7): DA, 0.58.+-.0.04 ng/mg tissue; 5-HT, 0.40.+-.0.02 ng/mg
tissue), whereas the same regimen of treatment resulted in
lethality of all treated WT mice (n=7).
[0375] Furthermore, to test whether the locomotor-stimulating
effect of (+)-MDMA may be evident under certain conditions with
lower (nonneurotoxic) doses of the drug, we coadministered (+)-MDMA
with a minimally effective dose of L-DOPA/carbidopa (10/10 mg/kg,
IP.). As shown in FIG. 5E, a potent synergistic effect of
L-DOPA/carbidopa and (+)-MDMA was observed. Furthermore, similar
effects were observed with lower doses of d-amphetamine and
d-methamphetamine (FIG. 5F). Thus, a DA-independent locomotor
effect of amphetamines can be markedly enhanced with additional
dopaminergic stimulation. It is also important to note that in a
similar experiment, MAO inhibitor deprenyl (5, 10, or 20 mg/kg IP)
failed to potentiate the effects of LDOPA/carbidopa (data not
shown), indicating that this effect is not related to the
well-known MAO-inhibiting action of amphetamines [38].
[0376] Nomifensine, but not GBR12909 Affects Rigidity and Akinesia
in DDD Mice.
[0377] Finally, to evaluate the potential of other TA1 receptor
ligands for their ability to affect motor control in DDD mice, we
elected to compare the effects of two potent DAT blockers that have
been shown to be markedly different with regards to their activity
at TA1 receptor. It has been recently reported that the mixed DAT
and NET inhibitor nomifensine can also potently activate TA1
receptor whereas selective DAT blocker GBR12909 completely lacks
the ability to interact with TA1 receptor [42]. In DDD mice, both
nomifensine and GBR12909 at doses tested (cumulative treatment with
10 and 30 mg/kg IP) were not effective in inducing forward
locomotion or reversing catalepsy (data not shown). Nevertheless,
nomifensine significantly reduced akinesia and rigidity in grasping
and akinesia tests (FIGS. 6A and 6B), whereas no such effects were
observed with equivalent doses of GBR12909 (FIGS. 6A and 6B).
Discussion
[0378] In this study we demonstrate that inhibition of DA synthesis
in DAT-KO mice represents a straightforward approach for developing
an acute model of severe DA deficiency exhibiting a characteristic
behavioral phenotype that can be utilized for testing perspective
anti-PD treatments. Furthermore, these observations provide
functional evidence for an important role of DAT-mediated recycling
mechanism in the maintenance of intraneuronal DA. Finally, the
novel DAT- and DA-independent locomotor action of amphetamines
identified in these mice directly demonstrates the possibility of
movement in a DA-independent manner.
[0379] Role of DAT-Mediated DA Recycling in the Maintenance of
Intraneuronal DA Storage.
[0380] DAT is commonly known as a major regulator of the duration
and intensity of extracellular DA signaling. However the important
role of DAT in the control and maintenance of the intraneuronal DA
storage pool frequently remains overlooked. It is generally assumed
that the intraneuronal storage of DA is replenished primarily from
newly synthesized DA with some contribution from recycled DA.
However, several lines of evidence support a predominant role of
DATmediated recycling of DA for the maintenance of the large
storage pool in DA terminals. First, mice lacking the DAT display
dramatically decreased (20-fold) striatal tissue DA content,
reflecting predominantly intraneuronal DA concentrations. Second,
as we demonstrate in the present study, the remaining DA in all
compartments is extremely sensitive to TH inhibition. Furthermore,
pharmacologic studies have shown that significant DA depletion may
occur after administration of DAT inhibitors, particularly after
chronic drug treatment [13]. Importantly, in the frontal cortex,
where DAT levels are normally low in comparison to the striatum,
tissue DA concentration is also low and can be more significantly
affected than in the striatum by .alpha.MT [70]. It is likely that
the newly synthesized DA does not contribute directly to the large
storage pool of DA in nigrostriatal terminals, but rather
contributes to it indirectly via released and recycled DA. Thus, a
cooperative function of both DA synthesis and transporter-mediated
recycling processes is necessary for the maintenance of normal
presynaptic monoamine concentrations.
[0381] A Novel Acute Mouse Model of Severe DA Deficiency,
DA-Depleted DAT-KO (DDD) Mice.
[0382] By using a combination of genetic and pharmacologic
approaches we have developed a novel acute mouse model of severe DA
deficiency, DDD mice. The lack of an active recycling mechanism in
DAT-KO mice results in a profound depletion of intraneuronal
concentrations of DA leaving the remaining DA entirely dependent on
ongoing synthesis. As a result, inhibition of DA synthesis
essentially eliminates striatal DA in these mice leading to the
extreme behavioral manifestations. In fact, DDD mice demonstrate a
unique set of behaviors that reproduces symptoms of PD with high
fidelity. Thus, the lack of DA combined with the striking and
highly reproducible behavioral phenotype in these mice can be used
as an excellent tool to evaluate the potential of drugs that can
affect locomotion in a DA-independent manner.
[0383] Furthermore, by adapting the dose of .alpha.MT to produce
various degrees of DA depletion, these mice can also be employed to
find novel approaches to restore movement under conditions of
partially impaired DA transmission that might be more relevant to
most PD cases. Several rodent models have been developed to
understand pathological processes leading to PD and/or to screen
for novel therapeutic strategies [29,30,34-36,71]. These models
either recapitulate the loss of DA through pharmacologic or genetic
manipulation, or recapitulate the neurodegenerative process through
administration of selective neurotoxins and, recently, through
mutations of specific proteins. However, in many of these models
only incomplete and highly variable levels of DA depletion are
achieved often precluding an accurate recapitulation of the
neurological manifestations of PD. This poor behavioral expression
of PD-related behaviors generally results in high level of
false-positive results in drug screening tests in general, and
particularly in those attempted to identify non-DA therapies
[72].
[0384] Among several genetic mouse models of DA deficiency
available today [73,74], the most effective was developed by
inactivation of TH in DA neurons (DA-deficient [DD mice])
[3,75-80]. DD mice have provided important insights into the role
of DA in movement control, feeding, and reward. This mutation
results in severely impaired movement and feeding, which become
apparent at 10 d and leads to death by 30 d. To maintain viable
mice with the ability to move and feed requires daily treatment
with L-DOPA, which results in an oscillation of striatal DA from
about 1% to 10% over 24 h [77,81]. Many behavioral manifestations
observed in DDD mice in this study, such as rigidity and akinesia,
were observed previously in DD mice [3,76,79]. Importantly, both of
these models showed temporal locomotor reactivity to stress and
demonstrate normal righting reflex and ability to swim, indicating
that certain movements may occur in a DA-independent manner.
[0385] Despite these similarities, some important differences were
noted between these two genetic models of severe DA dysfunction. In
DD mutant mice, a lack of TH resulting in permanently decreased DA
signaling, as well as daily treatments with L-DOPA render these
mice extremely supersensitive to DA stimulations [81], whereas
excessive DA signaling in DAT-KO mice results in compensatory
downregulation (but non-uniform) of DA receptors [11,13]. This may
explain why certain behavioral manifestations of DA deficiency such
as rigidity and akinesia may be more robust in DDD mice, whereas
tremor was not observed in DD mutants [3,76]. Furthermore, efficacy
of L-DOPA and DA agonists are remarkably higher in DD in comparison
to DDD mice [3,76,81]. Additionally, several other drugs, such as
caffeine and N-methyl-D-aspartate receptor antagonist MK-801, that
are able to induce locomotion in DD mutants [75,80] are not
effective in DDD mice (not shown). In fact, downregulation of DA
receptor responsiveness combined with the extreme level of DA
depletion in DDD mice may favor these mice as a very conservative
approach for evaluating drugs that can affect locomotion in a
DA-independent manner. Furthermore, rapid and effective elimination
of DA in DDD mice may provide a simple in vivo approach to study DA
receptor signaling [82] and/or to define neuronal circuitry
involved in locomotor control [83].
[0386] DA-Independent Locomotor Action of Amphetamines.
[0387] Intriguingly, in both DD and DDD mice d-amphetamine was
effective in restoring at least some aspects of locomotor
behaviors. In DD mice, d-amphetamine (5 mg/kg IP) induced potent
locomotor activation essentially up to the levels observed in WT
controls. At the same time, a second treatment 2 h later by the
same dose of the drug failed to induce locomotion in DD mice
suggesting that this effect is dependent upon residual (after
L-DOPA administration) DA which might be depleted by the first
treatment with the drug [76]. In DDD mice, d-amphetamine itself was
not able to induce forward locomotion at doses up to 60 mg/kg, but
it produced significant effects on other manifestations of DA
deficiency. Moreover, co-administration of relatively moderate
doses of amphetamine (15 and 20 mg/kg) with a subthreshold dose of
L-DOPA resulted in a marked locomotor activation of DDD mice. Thus,
some DA tone seems to be necessary to express the full magnitude of
locomotor activation by amphetamine, but it is evident that there
is a DA-independent component of action that contributes to the
overall effect of the drug. Further evidence for this idea relates
to the fact that many other amphetamine derivatives are also active
in reversing certain behavioral manifestations in DDD mice.
Strikingly, both single and repeated treatment with (+)-MDMA was
effective in inducing forward locomotion essentially without any
contribution of DA. It is important to note that a potent
anticataleptic effect of MDMA in haloperidol-treated rats [84] and
antiakinetic effects in 6-OH-DA-lesioned rats [85] and MPTP-treated
monkeys [64] have been recently reported. The present observations
support these findings and suggest that these actions are not
unique to MDMA but may be extended to other amphetamines. Further
characterization of these unexpected effects of amphetamines may
provide a novel framework in the search for potential
anti-Parkinsonian drugs.
[0388] Amphetamine derivatives are known mainly as indirect
enhancers of monoaminergic (DA, NE, and 5-HT) transmission via
complex interactions with the plasma membrane monoamine
transporters and the vesicular storage of these monoamines
[7,10,12,38,39]. It should be reiterated that a lack of DAT in
DAT-KO mice excludes the possibility of major effects of
amphetamines on DAT-mediated DA efflux from presynaptic DA stores
[40]. Furthermore, a blockade of D1/D2 DA receptors was ineffective
in preventing the locomotor stimulating action of (+)-MDMA. Thus,
it is virtually impossible that the observed effects of MDMA and
Other amphetamines in DDD mice are directly related to DA
transmission. Although it is possible that this effect may be due
to transporter-mediated action of amphetamines on NE or 5-HT
transmission [38,40,86], it should be noted that among several NE-
and 5-HT-related drugs tested (desipramine, clonidine, the NE
precursor DOPS, fluoxetine,
5-methoxy-N,N-dimethyltryptamine,5-methyl-N,N-dimethyltryptamine,
b-ethyltryptamine, and 5-HT1B agonist RU24969), none were effective
in DDD mice in tests of forward locomotion or akinesia and rigidity
(data not shown). Similarly, no locomotor effect of MAO-A or MAO-B
inhibitors was observed in these mice, indicating that the
locomotor effect of amphetamines may not be explained by
MAO-inhibitory action [38].
[0389] Furthermore, it should be underlined that locomotor actions
of amphetamines observed in DDD mice occur at doses that are much
higher than necessary to induce classic DA transporter mediated
effects [10,38,83].
[0390] Amphetamines share close structural similarity with an
endogenous trace amine of unknown function .beta.-phenylethylamine
[87]. Amphetamines and .beta.-phenylethylamine similarly interact
with the plasma membrane monoamine transporters to elevate
extracellular monoamine concentrations [63].
[0391] Intriguingly, recent evidence indicates that many
amphetamine derivatives, including MDMA, may also act directly as
agonists of trace amine TA1 receptors, that are known to be
activated by .beta.-phenylethylamine [42,88]. Several members of
the family of trace amine receptors have been identified, however
little is known about the pharmacology and functional role of these
receptors in mammalian physiology [43,44,63]. It is reasonable to
suggest that activation of TA1 receptors [42] or other trace amine
receptors may provide a potential mechanism for DA-independent
locomotor effect of MDMA and amphetamines in DDD mice. In line with
this hypothesis, we observed that the DAT blocker nomifensine that
can activate TA1 receptor, but not GBR12909 which is devoid this
activity [42], is able to affect motor control in DDD mice. It
should be noted, however, that in our initial exploration in DDD
mice, we did not observe clear locomotor effects for any trace
amine tested; but only a few doses, routes of administration, and
combinations with enzyme inhibitors were investigated. Further
detailed investigations will be needed to clarify the mechanism of
locomotor action of amphetamines in DDD mice.
[0392] The effects of additional isopropylamine derivatives in
DA-depleted DAT-KO (DDD) mice are also given in Table 1 below.
CONCLUSIONS
[0393] In summary, these results provide additional functional
evidence for the critical role of DAT in the maintenance of DA
storage in presynaptic terminals. Rapid and effective abolishment
of DA by inhibition of DA synthesis in DAT-KO mice provides a novel
approach to develop severe DA deficiency that might be used to
identify neuronal mechanisms involved in motor control in the
absence of DA. Amphetamines are capable of affecting neuronal
systems involved in motor control through mechanisms independent of
DAT, in particular, and DA in general.
TABLE-US-00001 TABLE 1 Effects of additional isopropylamine
derivatives in DA-depleted DAT-KO (DDD) mice. Dose, mg/kg, i.p.
Grasping test (sec) Catalepsy (sec) Akinesia (steps) Drugs (number
of mice) 1 h 2 h 3 h 1 h 2 h 3 h 1 h 2 h 3 h Saline (6) 52 .+-. 6
60 .+-. 0 60 .+-. 0 180 .+-. 0 180 .+-. 0 180 .+-. 0 4 .+-. 2 5.5
.+-. 2.sup. 2 .+-. 1 L-Amphetamine 40 and 40 (6) 25 .+-. 8* 9 .+-.
3* 180 .+-. 0 99 .+-. 36 21 .+-. 14 29 .+-. 5* Bupropion 30 and 60
(8) 25 .+-. 6* 16 .+-. 3* 92 .+-. 29* 123 .+-. 28 16 .+-. 7 29 .+-.
11 (.+-.)-DOI 5, 20 and 50 (6) 28 .+-. 8* 29 .+-. 6* 29 .+-. 9* 180
.+-. 0 180 .+-. 0 180 .+-. 0 0 .+-. 0 4 .+-. 1 12 .+-. 2* 80 (6) 10
.+-. 4* 19 .+-. 5* 180 .+-. 0 180 .+-. 0 14 .+-. 5 20 .+-. 8
(+)-Fenfluramine 30 and 60 (14) 19 .+-. 4* 9 .+-. 4* 176 .+-. 4 113
.+-. 23 7 .+-. 2 21 .+-. 3* (-)-Fenfluramine 30 and 60 (8) 9 .+-.
4* 4 .+-. 1* 157 .+-. 14 104 .+-. 31 36 .+-. 5* 28 .+-. 5*
(.+-.)-Fenfluramine 30 and 60 (8) 11 .+-. 4* 8 .+-. 4* 144 .+-. 25
138 .+-. 28 44 .+-. 8* 32 .+-. 6* Mice were treated with .alpha.MT
(250 mg/kg, i.p.) and 1 h after drugs or saline were injected. In
cumulative dosing experiments, animals were treated with different
doses of a drug every 1 h and were tested 1 h after administration.
*p < 0.05 vs corresponding time point in saline-treated controls
(two-tailed Student's t-test).
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[0482] The foregoing is illustrative of the present invention, and
is not to be construed as limiting thereof. The invention is
defined by the following claims, with equivalents of the claims to
be included therein.
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