Title Page The mGlu4 positive allosteric modulator VU0364770 produces efficacy alone and in ...

65 Pages·2011·2.25 MB·English

by  Michael Bubser

JJPPEETT FFaasstt FFoorrwwaarrdd.. PPuubblliisshheedd oonn NNoovveemmbbeerr 1166,, 22001111 aass DDOOII:: 1100..11112244//jjppeett..111111..118877444433
JPET FaTTshht iissF aaorrttriiccwlleea hhraadss .nn ooPtt ubbeebeelnni sccoohppeyyeeddd iiotteendd aaNnnddo ffvoorremmmaattttbeedde..r TT 1hhee6 ff,ii nn2aa0ll vv1ee1rrss iiaoosnn mmDaaOyy ddI:iiff1ffee0rr. ff1rroo1mm2 4tthh/iijssp vveeerrtss.ii1oo1nn..1.187443
JPET #187443

Title Page

The mGlu positive allosteric modulator VU0364770 produces efficacy alone and
4
in combination with L-DOPA or an adenosine A antagonist in preclinical rodent
2A
models of Parkinson’s disease

Carrie K. Jones, Michael Bubser, Analisa D. Thompson, Jonathan W. Dickerson
,
Nathalie Turle-Lorenzo, Marianne Amalric, Anna L. Blobaum, Thomas M. Bridges, Ryan
D. Morrison, Satyawan Jadhav, Darren W. Engers, Kimberly Italiano, Jacob Bode, J. DD
oo
Scott Daniels, Craig W. Lindsley, Corey R. Hopkins, P. Jeffrey Conn, Colleen M. ww
nn
lolo
Niswender aa
dd
ee
dd
fro fro
mm
jp jp
ee
Vanderbilt Center for Neuroscience Drug Discovery (C.K.J., M.B., A.D.T., J.W.D., t.ast.as
pp
ee
A.L.B., T.M.B., R.D.M., S.J., D.W.E., J.S.D., C.W.L., C.R.H., P.J.C., C.M.N.), tjoutjou
rnrn
aa
Departments of Pharmacology (C.K.J., M.B., A.D.T., J.W.D., A.L.B., T.M.B., R.D.M., lsls
.o.o
rgrg
S.J., D.W.E., J.S.D., C.W.L., C.R.H., P.J.C., C.M.N.) and Chemistry (C.W.L., C.R.H.), a a
t At A
SS
Vanderbilt Center for Accelerated Probe Development (MLPCN) (D.W.E., C.W.L., PP
EE
TT
J J
C.R.H.), Vanderbilt University, Nashville, TN 37212, Laboratoire de Neurobiologie de la oo
uu
rnrn
aa
Cognition CNRS UMR6155 Université de Provence. Case C, 3 Place Victor Hugo, lsls
o o
nn
13331 Marseille cdx 3 (N.T-L., M.A.), Millipore Corp, Billerica, MA USA (K.I., J.B.) M M
aa
rcrc
. h 1h 1
33
, 2, 2
00
22
33

1

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

Running Title Page

Running Title: mGlu4 PAM in preclinical models of Parkinson’s disease

Corresponding Author information:
Colleen M. Niswender
D
12478C MRB IV o
w
n
lo
a
d
Vanderbilt Center for Neuroscience Drug Discovery e
d
fro
m
Department of Pharmacology jp
e
t.a
s
p
Vanderbilt University, Nashville, TN 37212.Phone: [phone] etjo
u
rn
a
Fax: [phone] ls
.o
rg
a
[email protected] t A
S
P
E
T
J
Number of text pages: 49 o
u
rn
a
ls
Number of tables: 6 on
M
a
rc
Number of figures: 10 h 1
3
, 2
0
2
Number of references: 40 3
Number of words in Abstract: 250
Number of words in Introduction: 742
Number of words in Discussion: 1498
Number of Supplemental Figures: 1
2
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

List of nonstandard abbreviations: BG, basal ganglia; FLA, forelimb asymmetry; mGlu,
metabotropic glutamate receptor; PAM, positive allosteric modulator; PD, Parkinson’s
disease;VU0155041,(±)-cis-2-(3,5-Dicholorphenylcarbamoyl)cyclohexanecarboxylic;
VU0364770, N-(3-chlorophenyl) picolinamide; VU0364772, N-(6-(trifluoromethyl)pyridin-
2-yl)picolinamide; GPCR, G protein coupled receptor; GIRK, G-protein-regulated
Inwardly Rectifying K (potassium) channel; HEK, human embryonic kidney; CHO,
Chinese Hamster Ovary, PHCCC, N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-
carboxamide; DMSO, Dimethylsulfoxide; 6-OHDA, 6-hydroxydopamine; A2A receptor,
adenosine 2A receptor; L-DOPA, L-3,4-dihydroxyphenylalanine; CYP 450, cytochrome
D
P ; HBSS, Hanks Balanced Salt Solution; HEPES, 4-(2-hydroxyethyl)-1- o
450 w
n
lo
piperazineethanesulfonic acid; preladenant (SCH-420,814), 2-(2-Furanyl)-7-[2-[4-[4-(2- a
d
e
d
methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5- fro
m
c]pyrimidine-5-amine, ACPT-I, (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic jp
e
t.a
acid. sp
e
tjo
u
rn
a
ls
.o
rg
a
t A
S
P
E
T
J
o
u
rn
a
ls
on
M
a
rc
h 1
3
, 2
0
2
3
3
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

Abstract
Parkinson's disease (PD) is a debilitating neurodegenerative disorder associated
with severe motor impairments caused by the loss of dopaminergic innervation of the
striatum. Previous studies have demonstrated that positive allosteric modulators
(PAMs) of metabotropic glutamate receptor 4 (mGlu ), including PHCCC, can produce
4
antiparkinsonian-like effects in preclinical models of PD. However, these early mGlu
4
PAMs exhibited unsuitable physiochemical properties for systemic dosing, requiring
intracerebroventricular administration and limiting their broader utility as in vivo tools to
further understand the role of mGlu in modulation of basal ganglia function relevant to
4 D
o
w
PD. In the present study, we describe the pharmacologic characterization of a n
lo
a
d
systemically active mGlu PAM, VU0364770, in several rodent PD models. VU0364770 e
4 d
fro
showed efficacy alone or when administered in combination with L-DOPA or an m
jp
e
adenosine 2A (A2A) receptor antagonist, currently in clinical development, termed t.a
s
p
e
preladenant. When administered alone, VU0364770 exhibited efficacy in reversing tjo
u
rn
haloperidol-induced catalepsy, forelimb asymmetry-induced by unilateral 6- a
ls
.o
hydroxydopamine (6-OHDA) lesions of the median forebrain bundle and attentional rg
a
t A
deficits induced by bilateral 6-OHDA nigrostriatal lesions in rats. In addition, S
P
E
T
VU0364770 enhanced the efficacy of preladenant to reverse haloperidol-induced J
o
u
catalepsy when given in combination. The effects of VU0364770 to reverse forelimb rna
ls
o
asymmetry were also potentiated when the compound was coadministered with an n M
a
inactive dose of L-DOPA, suggesting that mGlu PAMs may provide L-DOPA sparing rc
4 h
1
3
activity. The present findings provide exciting support for the potential role of selective , 2
0
2
3
mGlu PAMs as a novel approach for the symptomatic treatment of PD and as a
4
possible augmentation strategy with either L-DOPA or A antagonists.
2A

4
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

Introduction
Parkinson's disease (PD) is a chronic neurodegenerative disorder that affects
approximately 1% of the population worldwide over the age of 55 and is associated with
motor symptoms including tremor, muscle rigidity, bradykinesia and gait/postural
imbalances (Jankovic, 2008). The primary neuropathology of PD involves the
progressive degeneration of dopamine neurons in the substantia nigra pars compacta
(SNc), which provide the major dopaminergic innervation to the striatum and other basal
ganglia nuclei (Hassler, 1938). While dopamine replacement therapies, such as L-
DOPA, remain the gold standard for the symptomatic treatment of PD, these treatments
D
o
ultimately fail to provide reliable efficacy with disease progression and are associated w
n
lo
a
with numerous dose-limiting side effects, including on/off oscillations, dyskinesias, and d
e
d
cognitive impairments (Stacy et al., 2008). fro
m
jp
e
Over the last decade, an enhanced understanding of basal ganglia (BG) circuitry t.as
p
e
has allowed the investigation of mechanisms that alter BG function in ways that do not tjo
u
rn
a
rely on dopamine replacement. Within the BG, normal motor movement is regulated by ls
.o
rg
two primary output nuclei, the substantia nigra pars reticulata (SNr) and the internal a
t A
S
globus pallidus (GPi; entopeduncular nucleus in non-primates) (Conn et al., 2005; P
E
T
Niswender et al., 2010). Output from the SNr/GPi is maintained by a delicate balance Jo
u
rn
between two distinct striatal GABAergic projection pathways: the direct pathway, which als
o
n
projects directly to the SNr/GPi; and the indirect pathway, which projects from the M
a
rc
striatum to the external globus pallidus (GPe). The GPe then sends additional h 1
3
GABAergic projections to the subthalamic nucleus (STN), which in turn provides , 20
2
3
excitatory input to the SNr/GPi (Conn et al., 2005). Loss of striatal dopaminergic
innervation from the SNc results in overactivation of the indirect pathway (excessive
inhibitory tone at the level of the GPe and subsequent disinhibition of the STN), leading
to increased excitation of the SNr/GPi nuclei and the motor impairments observed in PD
(Conn et al., 2005). Recent surgical strategies have focused on correcting this
overactivity within the indirect pathway; deep brain stimulation of the STN, for example,
has shown efficacy in correcting PD motor symptoms, suggesting that pharmacological
5
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

manipulation of synaptic transmission in the indirect pathway may provide an important
approach for nondopaminergic PD treatment.
Two important targets for potential pharmacologic modulation of the indirect
pathway are the adenosine A receptor and the metabotropic glutamate receptor 4
2A
(mGlu ) (Johnson et al., 2009; Morelli et al., 2009). Antagonism of adenosine A
4 2A
receptors, expressed on medium spiny GABAergic neurons that project to the GPe,
results in decreased inhibition of the GPe and antiparkinsonian-like effects in animal PD
models (Hettinger et al., 2001; Hodgson et al., 2010). In a recent phase II, double-blind,
clinical trial, positive effects were reported on the motor impairments in Parkinson's
D
o
w
disease patients using the A receptor antagonist preladenant (Hauser et al., 2011). n
2A lo
a
d
With regard to mGlu , activation of this receptor, expressed presynaptically on e
4 d
fro
GABAergic neurons within the striato-pallidal synapse, also reduces excessive inhibitory m
jp
e
tone within the GPe and reverses motor deficits in rodent PD models (Bradley et al., t.a
s
p
e
1999; Matsui et al., 2003; Valenti et al., 2003; Lopez et al., 2007; Sibille et al., 2007; tjo
u
rn
Niswender et al., 2008; Beurrier et al., 2009). Unfortunately, development of selective a
ls
.o
orthosteric mGlu agonists has been hindered by the high conservation of the glutamate rg
4 a
t A
binding site across the mGlu subtypes. S
P
E
T
J
o
Using an alternative strategy, our group and others have recently identified highly u
rn
a
selective mGlu4 ligands that potentiate receptor activity via action at sites that are less ls o
n
M
highly conserved and topographically distinct relative to the glutamate binding site,
a
rc
h
termed positive allosteric modulators (PAMs). For example, VU0155041 and PHCCC 1
3
, 2
represent two chemically and structurally distinct classes of mGlu4 PAMs that bind to 023
allosteric sites on mGlu and potentiate the effects of glutamate (Maj et al., 2003;
4
Marino et al., 2003; Niswender et al., 2008). These early mGlu PAMs provided
4
important proof of concept data suggesting antiparkinsonian-like efficacy in preclinical
PD models, but had limited utility as in vivo tools due to unsuitable physiochemical
properties (e.g. limited solubility and central penetration) for systemic dosing studies.
Recently, we reported the discovery of a novel series of biaryl amides with mGlu PAM
4
activity that exhibited submicromolar potency at rat and human mGlu as well as
4
improved physiochemical properties for systemic dosing (Engers et al., 2009). Here, we
6
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

describe the pharmacologic characterization of a systemically active mGlu PAM,
4
VU0364770, derived from this biaryl amide chemical series. Using multiple rodent PD
models, we show that VU0364770 exhibits efficacy when administered alone as well as
in combination with L-DOPA or the adenosine A receptor antagonist, preladenant.
2A

Methods
Animals
Adult male Sprague-Dawley rats (Harlan, Indianapolis) weighing 250-300 g used
for the haloperidol and forelimb asymmetry studies were group housed with food and D
o
w
water available ad libitum. Rats for the haloperidol studies were kept under a 12 h nlo
a
d
light/dark cycle with lights on from 6 AM to 6 PM and were tested during the light phase ed
fro
unless stated otherwise. Rats for the forelimb asymmetry studies were housed under a m
jp
e
reversed 12h/12h light-dark cycle with lights on between 4PM and 4AM and were tested t.a
s
p
e
during the first half of the dark cycle. For the Reaction time task studies, male Wistar tjo
u
rn
rats (Charles River, l’Arbresle, France), weighing 175–185 g at the beginning of the als
.o
experiment, were used and fed 15-17 g/d of laboratory chow delivered 3 h after the arg
t A
testing period, so as to maintain 85% of their free feeding body weight with water S
P
E
T
provided ad libitum. All experimental procedures were approved by the Vanderbilt J
o
u
rn
University Animal Care and Use committee and followed the guidelines set forth by the a
ls
o
n
Guide for the Care and Use of Laboratory Animals and the requirements of the M
a
European Communities council (directive n.86/609/EEC, November 24th, 1986). rch
1
3
, 2
0
Drugs 2
3
Apomorphine hydrochloride, benserazide hydrochloride, clorgyline, R-(-)-
deprenyl hydrochloride, haloperidol, L-DOPA methyl ester, and 6-hydroxydopamine
hydrobromide were obtained from Sigma (St. Louis, MO). L-glutamate was obtained
from Tocris Biosciences (Ellisville, MO). Preladenant, VU0364770, VU0364772, and the
A antagonist from Neurocrine Biosciences (Slee et al., 2008) were synthesized in-
2A
house in the laboratory of Dr. Hopkins. Clorgyline, R-(-)-deprenyl hydrochloride,
VU0364770, VU0364772, and preladenant were suspended in an aqueous solution of
10% Tween 80. Benserazide and L-DOPA methyl ester were dissolved in 0.9% saline,
7
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

while haloperidol and the Neurocrine A2A antagonist were dissolved in 0.85% lactic
acid (Sigma) and 0.5% Cremophor EL (Sigma), respectively. Except for apomorphine
and L-DOPA, which are unstable at neutral pH, all drug formulations were adjusted to a
pH of approximately 7 using 1N sodium hydroxide. Drugs were administered in a
volume of 1 to 2 ml/kg (i.p. and s.c.) or 10 ml/kg (p.o.).
The chemical synthesis for the biaryl amides VU0364770 and VU0364772 was
completed by an in-house synthesis using the following methods:
D
o
w
n
lo
a
d
e
d
fro
m
jp
e
t.a
s
p
e
tjo
u
rn
N-(3-chlorophenyl)picolinamide: To a solution of 3-chloroaniline (29.5 mL, 0.28 als
.o
mol) in DMF (300 mL) at 0 °C was added DMAP (50 mg) and DIEA (118 mL, 0.84 mol). arg
t A
Picolinoyl chloride hydrochloride (50 g, 0.28 mol) was added portion wise over 30 min S
P
E
T
and after an additional 15 min, the ice bath was removed. After 16h at rt, the reaction J
o
u
rn
was added to EtOAc: NaHCO (sat’d) (1:1; 1000 mL). The organic layer was separated a
3 ls
o
n
and washed with water (5 x 200 mL), brine (200 mL), dried (MgSO4), filtered and M
a
concentrated under reduced pressure. After recrystallization of the crude solid from rch
1
3
EtOH, the desired product was obtained as an off-white solid (63.5g, 84% yield): R = , 2
f 0
2
3
0.47 (33% EtOAc/hexanes); Mp = 81-83C; Analytical LCMS: single peak (214 nm),
3.417 min; 1H NMR (400 MHz, CDCl ):  10.06 (br s, 1H), 8.62 (d, J = 4.4 Hz, 1H), 8.30
3
(d, J = 8.0 Hz, 1H), 7.95-7.91 (m, 2H), 7.63 (dd, J = 8.0, 1.2 Hz, 1H), 7.51 (dd, J = 7.6,
4.8 Hz, 1H), 7.31 (dd, 8.4, 8.0 Hz, 1H), 7.13 (dd, J = 8.0, 1.2 Hz, 1H); HRMS, calc’d for
C H N OCl (M+H+), 233.0482; found 233.0483.
12 10 2
8
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

N-(6-(trifluoromethyl)pyridin-2-yl)picolinamide: To a solution of 6-
(trifluoromethyl)pyridin-2-amine (5.0 g, 30.8 mmol) in DMF (60 mL) at 0 °C was added
DMAP (10 mg) and DIEA (13.0 mL, 92.4 mmol). Picolinoyl chloride hydrochloride (5.8 D
o
w
g, 32.4 mmol) was added portion wise over 10 min and after an additional 15 min, the nlo
a
d
e
ice bath was removed. After 12h at rt, the reaction was added to EtOAc: NaHCO d
3 fro
(sat’d) (1:1; 100 mL). The organic layer was separated and washed with water (3 x 20 m
jp
e
mL), brine (20 mL), dried (MgSO ), filtered and concentrated under reduced pressure. t.a
4 s
p
e
After recrystallization, the desired product was obtained as a white solid (4.7 g, 57%): Rf tjou
rn
a
= 0.46 (33% EtOAc/hexanes); Mp = 141-142C; Analytical LCMS: single peak (214 nm), ls
.o
rg
3.546 min; 1H NMR (400 MHz, CDCl ):  10.68 (br s, 1H), 8.68-8.64 (m, 2H), 8.30 (d, J a
3 t A
S
= 7.6 Hz, 1H), 7.94 (dd, J = 7.6, 7.6 Hz, 1H), 7.93 (dd, J = 8.0, 7.6 Hz, 1H), 7.53 (ddd, J P
E
T
= 7.6, 4.8, 1.2 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H); HRMS, calc’d for C H N OF (M+H+), Jo
12 9 3 3 u
rn
268.0698; found 268.0695. als
o
n
M
HCl Salt Formation for in vivo studies: To a solution of the amide in DCM arc
h
1
(0.2M) at 0 C was added 4M HCl in 1,4-dioxane (5 eq.) dropwise. After 15 min, the ice 3
, 2
0
2
bath was removed. The solvent was removed after additional 30 min at rt to provide a 3
pure HCl salt of the appropriate amide.
In Vitro Studies
Cell culture. Human mGlu /G /CHO cells were grown in 90% Dulbecco's
4 qi5
Modified Eagle Media (DMEM), 10% dialyzed fetal bovine serum (FBS), 100 units/ml
penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 20 μg/ml
proline, 2 mM glutamine, 400 μg/ml G418 sufate (Mediatech, Inc., Herndon, VA) and 5
nM methotrexate (Calbiochem, EMD Chemicals, Gibbstown, NJ). Rat mGlu /HEK/GIRK
4
9
JPET Fast Forward. Published on November 16, 2011 as DOI: 10.1124/jpet.111.187443
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #187443

cells, as well as HEK/GIRK lines expressing rat mGlu , mGlu , mGlu , mGlu , and
2 3 7 8
human mGlu were cultured in 45% DMEM, 45% F-12, 10% FBS, 20 mM HEPES, 2
6
mM l-glutamine, 100 units/ml penicillin/streptomycin, 1X nonessential amino acids, 1
mM sodium pyruvate, 700 μg/mL G418, and 0.6 μg/mL puromycin. Rat mGlu and
1
mGlu HEK cell lines were grown in DMEM, 10% FBS, 20 mM HEPES, 2 mM l-
5
glutamine, 1X nonessential amino acids, 1 mM sodium pyruvate, 500 μg/mL G418. All
cell culture reagents were from Invitrogen (Carlsbad, CA) unless otherwise noted.
Calcium Mobilization Assays. Human mGlu /G /CHO cells (30,000 cells/20
4 qi5
μl/well) were plated in black-walled, clear-bottomed, TC treated, 384 well plates
D
o
w
(Greiner Bio-One, Monroe, North Carolina) in DMEM containing 10% dialyzed FBS, 20 n
lo
a
d
mM HEPES, 100 units/ml penicillin/streptomycin, and 1 mM sodium pyruvate (Plating e
d
fro
Medium). The cells were grown overnight at 37°C in the presence of 5% CO . The next m
2 jp
e
day, the medium was removed and replaced with 20 μL of 1 μM Fluo-4, AM (Invitrogen, t.a
s
p
e
Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% tjo
u
rn
(w/v) pluronic acid F-127 and diluted in Assay Buffer (Hank's balanced salt solution, 20 a
ls
.o
mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) for 45 minutes at rg
a
t A
37°C. Dye was removed and replaced with 20 μL of Assay Buffer. For concentration- S
P
E
T
response curve experiments, compounds were serially diluted 1:3 into 10 point J
o
u
concentration response curves in DMSO, transferred to daughter plates using an Echo rna
ls
o
acoustic plate reformatter (Labcyte, Sunnyvale, CA) Echo, and diluted in Assay Buffer n M
a
to a 2X final concentration. Ca2+ flux was measured using the Functional Drug rc
h
1
3
Screening System 6000 (FDSS6000, Hamamatsu, Japan). After establishment of a , 2
0
2
3
fluorescence baseline for 4 seconds (4 images at 1 Hz; excitation, 470 ± 20 nm;
emission, 540 ± 30 nm), 20 µl of test compounds were added to the cells, and the
response was measured. 142 seconds later, 10 µl (5X) of an EC concentration of
20
glutamate was added to the cells, and the response of the cells was measured; after an
additional 120 seconds, 12 µl (5X) of an EC concentration of agonist was added and
80
readings taken for an additional 40 seconds. Calcium fluorescence was recorded as fold
over basal fluorescence and raw data were normalized to the maximal response to
glutamate. Potency (EC ) and maximum response (% Glu Max) for compounds were
50
determined using a four parameter logistical equation in GraphPad Prism (La Jolla, CA).
10