JPET#136580 1 Title Page ADX47273: A Novel Metabotropic

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Title Page

ADX47273: A Novel Metabotropic Glutamate Receptor 5 Selective Positive

Allosteric Modulator with Preclinical Antipsychotic-Like and Pro-cognitive

Activities

Feng Liu, Steve Grauer, Cody Kelley, Rachel Navarra, Radka Graf, Guoming Zhang,

Peter J. Atkinson, Michael Popiolek, Caitlin Wantuch, Xavier Khawaja, Deborah Smith,

Michael Olsen, Evguenia Kouranova, Margaret Lai, Farhana Pruthi, Claudine

Pulicicchio, Mark Day, Adam Gilbert, Mark H. Pausch, Nicholas J. Brandon, Chad E.

Beyer, Tom A. Comery, Sheree Logue, Sharon Rosenzweig-Lipson and Karen L.

Marquis

Wyeth Neuroscience Discovery Research, CN 8000, Princeton, NJ 08543 (F.L., S.G., C.

K., R.N., R.G., G.Z., P.J.A., M.P., C.W., X.K., D.S., M.O., E.K., M.L., F.P., C.P.,

M.H.P., N.J.B., C.E.B., T.A.C., S.L., S.R-L., K.L.M.)

Wyeth Translational Medicine, Collegeville, PA (M.D.)

Exploratory Medicinal Chemistry, Chemical and Screening Sciences, Wyeth Research,

Pearl River, NJ 10965 (A.G.)

JPET Fast Forward. Published on August 27, 2008 as DOI:10.1124/jpet.108.136580

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on August 27, 2008 as DOI: 10.1124/jpet.108.136580

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Running Title Page Running Title ADX47273 Preclinical Antipsychotic and Pro-cognitive Effects To whom correspondence should be addressed:

Feng Liu, PhD

Discovery Neuroscience

Wyeth Discovery

865 Ridge Road

Monmouth Junction, NJ 08852

USA

phone – (732) 274-4017

fax – (732) 274-4020

E-mail: [email protected]

Page Number 48

Abstract 250 words

Introduction 754 words

Discussion 1462 words

Number of Table 0

Number of Figures 10

References 40


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Abbreviation List

ADX (Addex)

PAM (positive allosteric modulator)

CAR (condition avoidance response)

AIC (apomorphine induced climbing)

NOR (novel object recognition)

5-CSRT (5-choice serial reaction time)

MED (minimum effective dose)

PCP (phencyclidine)

FLIPR (fluorometric imaging plate reader)

GPT (glutamate pyruvate transaminase)

HEK (human embryonic kidney)

DMEM (Dulbecco/Vogt modified Eagle's minimal)

FSB (fetal bovine serum)

GFP (green fluorescence protein)

LED (light emitting diode)

ITI (inter-trial interval)

SD (stimulus duration)

HPLC (high performance liquid chromatography)

MPEP (2-methyl-6-(phenylethynyl)pyridine)

MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine)

DFB (3,3’-difluorobenzaldazine)

CPPHA (N-100-2-hydrobenzamide)


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CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide)

GABA (γ-aminobutyric acid)

GPCR (G-protein coupled Receptor)

NMDA (N-methyl-D-aspartic acid)

ERK (extracellular signal-regulated kinase)

CREB (cAMP response element-binding)

MK801 ((5R,10S)-(-)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cylc ohepten-5,10-imine)

L-AP4 (L-(+)-2-Amino-4-phosphonobutyric acid)

DHPG ((S)-3,5-Dihydroxyphenylglycine)

ADX47273 (S-(4-Fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol—5-yl]-

piperidin-1-yl}-methanone)


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Abstract

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5

(mGlu5) enhance N-methyl-d-aspartate (NMDA) receptor function and may represent a

novel approach for the treatment of schizophrenia. ADX47273, a recently identified a

potent and selective mGlu5 PAM, increased (9-fold) the response to threshold

concentration of glutamate (50 nM) in fluorometric Ca2+ assays (EC50 = 170 nM) in HEK

293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently

shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 μM) and

competed for binding of 3H-MPEP (Ki = 4.3 μM), but not 3H-quisqualate. In vivo,

ADX47273 increased extracellular signal-regulated protein kinase (ERK) and cyclic-

AMP responsive element-binding protein (CREB) phosphorylation in hippocampus and

prefrontal cortex, both of which are critical for glutamate-mediated signal transduction

mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced

rat conditioned avoidance responding (MED = 30 mg/kg, IP) and decreased mouse

apomorphine-induced climbing (MED = 100 mg/kg, IP), with little effect on stereotypy

or catalepsy. Furthermore, ADX47273 blocked PCP, apomorphine and amphetamine-

induced locomotor activities (MED = 100 mg/kg, IP) in mice, and decreased extracellular

levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In

cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg, IP)

and reduced impulsivity in 5-choice serial reaction time (5-CSRT) test (MED = 10

mg/kg, IP) in rats. Taken together, these effects are consistent with the hypothesis that

allosteric potentiation of mGluR5 may provide a novel approach for development of

antipsychotic and pro-cognitive agents.


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Introduction

The metabotropic glutamate receptor (mGlu) receptor family includes eight G-

protein-coupled receptor (GPCR) subtypes classified on the basis of structural homology,

mechanism of signaling transduction, and pharmacological properties. Stimulation of

group I receptor (subtypes 1 and 5) leads to activation of phospholipase C, increased

phosphoinositide (PI) hydrolysis and mobilization of intracellular calcium (Conn and Pin,

1997).

Positive allosteric modulators (PAM)s of mGlu receptors offer an attractive

alternative to the direct activation of mGlu receptors by orthosteric competitive agonists.

PAMs have been discovered for mGlu1, mGlu2 and mGlu5 receptors (Knoflach et al.,

2001; Johnson et al., 2003; O'Brien et al., 2003a). These molecules offer the potential to

increase the efficiency of normal glutamate transmission without the risk of inappropriate

stimulation. Furthermore, such compounds are more likely to achieve high receptor

subtype selectivity by targeting regions of the receptor that are different than those

affected by the endogenous ligand. PAMs of mGlu5 include DFB (3,3’-

difluorobenzaldazine), CPPHA (N-100-2-hydrobenzamide) (O'Brien et al., 2003a;

O'Brien et al., 2004) and CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide)

(Lindsley et al., 2004; Kinney et al., 2005a). These compounds have little or no direct

agonist activity, but act as selective PAMs of competitive agonists of human and rat

mGlu5; however, they suffer from relative weak in vitro activity, such as poor potency

and efficacy (DFB and CPPHA) (O'Brien et al., 2003a; O'Brien et al., 2004) and

solubility (CDPPB) (Kinney et al., 2005a).


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Recently S-(4-Fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol—5-yl]-

piperidin-1-yl}-methanone (ADX47273) (De Paulis et al., 2006) was identified as a novel

selective mGlu5 PAM at the 5th International Metabotropic Glutamate Receptors

Meeting (Le Poul et al., 2005). Up to 60 μM, ADX47273 showed no agonist, antagonist,

or allosteric modulator activity at other rat and/or human family III GPCRs (mGlu1-8 and

GABA-B). In addition, ADX47273 (10 μM) failed to displace radioligand binding to 56

GPCRs, transporters, enzymes and ion channels (Le Poul et al. 2005). ADX47273 is

reported to have a brain/plasma ratio of 1.6 and a 2 hr half-life in mouse (Poli et al.

2005). In general, it appears that ADX47273 has pharmacological properties, such as

potency and efficacy as well as blood brain barrier penetrability, which make it an

improved tool for profiling the in vivo effects of modulating mGlu5 receptor activity.

Several lines of evidence suggest that impaired NMDA receptor-mediated

neurotransmission is a major component of the pathophysiology of schizophrenia

(Lindsley et al., 2006). Activation of mGlu5 has been suggested as one of the approaches

by which NMDA receptor function can be augmented (Marino and Conn, 2002). For

example, activation of mGlu5 enhances NMDA receptor mediated currents in slices from

rat hippocampus (Doherty et al., 1997) and subthalamic nucleus (Awad et al., 2000).

Conversely, the mGlu5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP)

potentiates the effects of NMDA receptor antagonists on spontaneous burst and spike

activity of cortical neurons (Homayoun and Moghaddam, 2006) and enhances the effect

of NMDA antagonists on behavior, such as prepulse inhibition, locomotion and working

memory impairments (Campbell et al., 2004; Homayoun et al., 2004). The ability of

mGlu5 antagonists to potentiate the detrimental effects of NMDA receptor antagonists


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suggests that activation of mGlu5 may represent an approach towards ameliorating

symptoms of schizophrenia (Marino and Conn, 2002). As predicted, CDPPB exhibits

antipsychotic-like activity on amphetamine-induced hyperlocomotion and prepulse

inhibition deficits (Kinney et al., 2005b). More recently, CDPPB prevents NMDA

receptor antagonist MK801-induced excessive firing in prefrontal cortex (PFC)

(Lecourtier et al., 2007). Thus, mGlu5 PAMs may also be effective in treating the

cognitive deficits in schizophrenic patients by ameliorating the NMDA receptor

hypofunction thought to underlie these cognitive deficits.

The effects of mGlu5 PAMs on the molecular mechanisms associated with

learning and memory processes also suggests efficacy in cognitive enhancement.

Phosphorylation of extracellular signal-regulated protein kinase ERK1/2 and the

transcription factor cyclic-AMP-responsive element-binding protein (CREB) play a

major role in synaptic plasticity and cognition (Thomas and Huganir, 2004b; Carlezon et

al., 2005). Previously, we demonstrated that CPPHA potentiated the response to a

subthreshold concentration of the non-selective Group 1 agonist 3,5-dihydroxy-

phenylglycine (DHPG) on ERK and CREB phosphorylation in cortical and hippocampal

slices (Liu et al., 2006), suggesting that modulation of mGlu5 by a PAM can regulate

these major signaling molecules important in learning and memory.

The present set of studies used ADX47273 to further explore the potential of a

mGlu5 PAM as a therapeutic approach for the treatment of positive symptoms and

cognitive deficits associated with schizophrenia. ADX47273 was evaluated in models

predictive of antipsychotic efficacy (apomorphine-induced climbing/stereotypy,

conditioned avoidance responding, and hyperactivity induced by PCP, apomorphine and


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amphetamine) and cognition (novel objective recognition and the 5-choice serial reaction

time task).


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Methods

Subjects Male CF-1 mice (20–28 g; Charles River Laboratories, Inc., Wilmington, MA) were used

in the antagonism of apomorphine-induced behaviors and locomotor hyperactivity

studies. Male Sprague-Dawley rats were used for the conditioned avoidance test (350–

450 g; Charles River Laboratories, Inc). Male Long-Evans rats (350–450 g; Charles

River Laboratories, Inc) were used for the novel object recognition and 5-choice serial

reaction time (5-CSRT) tasks. All animals were group-housed (except for conditioned

avoidance response, novel object recognition and 5-CSRT subjects which were housed

singly) in an Association for Assessment and Accreditation of Laboratory Animal Care

International-accredited facility that was maintained on a 12-h light/dark cycle (lights on

at 6:00 AM). Food and water were available ad libitum, except where noted. All studies

were approved by the Institutional Animal Care and Use Committee and were performed

in accordance with the Principles of Laboratory Animal Care as adopted and promulgated

by the National Institutes of Health (publication 85-23, 1996).

Drugs

All drugs and vehicles were administrated intraperitoneally (IP). The compound

ADX47273 was synthesized by Wyeth Research. Phencyclidine (PCP) hydrochloride,

apomorphine hydrochloride, d-amphetamine sulphate and carboxymethyl-cellulose were

obtained from Sigma-Aldrich (St. Louis, MO). MPEP hydrochloride and MTEP

hydrochloride were purchased from Tocris (Bristol, UK). Drugs were dissolved in saline

(PCP and d-amphetamine) or suspended in 0.2%Tween/water (apomorphine) or 2%


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Tween + 0.5% Carboxymethyl-cellulose (ADX47273, MPEP and MTEP). Solutions

were administered at a volume of 10 ml/kg to mice and 1 ml/kg to rats unless otherwise

noted. The dose administered for each compound refers to the amount of the active

component, rather than the salt. All other materials were analytical grade and were

purchased from Aldrich Chemical Co. (Milwaukee, WI) and Sigma-Aldrich.

Procedures

Fluorometric Imaging Plate Reader (FLIPR). Ca2+ influx measurements were made

using the FLIPR384 flurometric imaging plate reader (Molecular Devices, Sunnyvale,

CA). HEK 293 cells expressing rat mGlu5 or mGlu1 receptor were plated in clear-

bottomed 384-well plates in glutamate/glutamine-free media and loaded the next day

with calcium-sensitive fluorescent dye Calcium 3, and placed in FLIPR384. HEK 293

cells expressing the cloned rat mGlu5 and mGlu1 receptor show concentration-dependent

increases in Fluo-3 fluorescence in response to glutamate with an EC50 value of 300 nM

and 2 μM, respectively. A range of concentrations of ADX47273 alone or together with

50 nM glutamate (EC10 concentration) was added to the cells, and the Ca2+ response was

measured by FLIPR384. For MPEP experiments, MPEP (10 μM) was added 30 min

prior to the FLIPR assay as a pretreatment. For primary astrocyte cultures, astrocytes

were pre-loaded for 50 min at 370C / 5 % C02 using the FLIPR calcium 3-assay kit

(containing 3 U ml-1 GPT, 3 mM pyruvate and 2.5 mM probenecid) according to the

manufacturer’s instructions (Molecular Devices). Cells were left to equilibrate at room

temperature for 15 min before basal fluorescence (t = 10 s) was determined using

FLIPR384 (Molecular Devices). Allosteric modulators were added 5 min prior to the

addition of 50 nM (EC10 concentration) glutamate. For the astrocytes, the EC20 of


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glutamate was determined as 300 nM and was subsequently used for PAM experiments.

Data were normalized to the calcium signal produced by maximal concentrations of

glutamate on each plate.

Primary Astrocyte Cultures. This protocol was adapted from (Marriot et al. 1995).

Mixed glia cultures were prepared from rat cortex using 2-day-old neonates. Cultures

were grown in DMEM containing 10 % heat-inactivated, dialyzed FBS, L-glutamine and

1 % penicillin/streptomycin (Invitrogen). After 10 days in-vitro G-5 (1x) supplement

(Invitrogen) was added to the cultures and astrocytes were purified by overnight shaking

(120 r.p.m). The following day, astrocytes were lifted and plated into poly (D-lysine)-

coated 384-well plates at a density of 10,000 cell well-1 48 h prior to experimentation.

Using this protocol the final preparation contained ∼90 % GFAP-positive astrocytes (not

shown).

Radioligand Binding Assays. [3H]-MPEP binding: [3H]-MPEP was used to evaluate

the interaction of compounds with the receptor. Membranes were prepared from HEK

293 cells expressing rat mGlu5 receptor. Aliquots of these membranes were added to

tubes containing ADX47273 (0.4% DMSO final concentration) or vehicle and [3H]-

MPEP (2 nM final concentration in 50 mM Tris/0.9% NaCl, pH 7.4). The tubes were

incubated for 60 min at room temperature with shaking, and the membrane-bound ligand

was separated from the free ligand by filtration onto glass-fiber filters presoaked with 20

mM HPEPS, 2 mM CaCl2 and MgCl2, pH 7.2. Membrane bound radioactivity was

determined by scintillation counting of the filters. Competition binding data were

analyzed and Ki determined using Prizm 4.0 (GraphPAD Software, Inc.). [3H]-

quisqualate binding: Membranes from CHO cells expressing rat mGlu5 were


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resuspended in ice-cold assay buffer (20 mM HEPES-NaOH (pH 7.2) containing 2 mM

MgCl2 and 2 mM CaCl2) with 20 nM [3H]quisqualate either in the absence or presence

of 10 μM ADX47273 for 1 h at room temperature. Nonspecific binding was defined in

the presence of 1 mM glutamate. At the end of the incubation, the suspension was filtered

onto Whatman GF/C glass fiber filters and washed rapidly three times with 1 ml of cold

binding buffer. The radioactivity trapped on the filters was measured by liquid

scintillation in a Tri-Carb model 2500 TR counter (Canberra Packard).

Functional Calcium Mobilization Assay for mGlu2. Recombinant CHO cell lines co-

expressing human mGlu2 and GqGi3 were plated at a seeding density of 0.5 x 105

cells/well in clear-bottomed, non-poly-D-lysine-coated 96-well plates. Cells were

incubated in glutamate/glutamine-free medium overnight at 37°C in an atmosphere of

95% O2/5%CO2. Cells were loaded with calcium indicator dye (Calcium 3 Assay Kit)

containing 3U/ml of glutamic pyruvic transaminase, 3mM sodium pyruvate, and 2.8mM

probenecid at 37°C for 1 h. At this stage, cells were used for the calcium mobilization

assay. ADX47273 was dissolved to a stock solution of 10mM in 100% DMSO and then

half-log diluted into H2O. The stock solution was added to the assay plate to a final

DMSO concentration of 0.4%. Agonist activity was determined by adding ADX47273

(0.1 nM-10 μM) to the well. Antagonist activity was assessed by pre-treating cells with

ADX47273 (0.1 nM -10 μM) for 5 min followed by a submaximal concentration (30 μM)

of glutamate (EC80). PAM activity was evaluated by performing a L-glutamate

concentration response curve (3 nM -100 μM) in the absence or presence of 10 μM

ADX47273. Calcium mobilization was measured using the FLEXstation II (Molecular

Devices).


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Functional Cyclase Inhibition assay for mGlu4. CHO cells expressing human mGlu4

were plated in poly-D-lysine-coated 96-well plates 1 day before assay. Cells were washed

with Hank’s Balanced Salt Solution for 10 min at room temperature. Compounds were

half-log diluted in 4% DMSO. The assay procedure according to the DiscoverX

HitHunter cAMP XP Assay Kit was followed. To measure agonist activity, cells were

incubated with forskolin (10 μM), ADX47273 (0.1 nM -10 μM), and cAMP XP antibody

supplemented with 500 µM 3-isobutyl-1-methylxanthine, 3U/ml of glutamic pyruvic

transaminase, and 3mM sodium pyruvate for 30 min at 37°C. Antagonist activity was

evaluated by treating cells with ADX47273 (0.1 nM-10 μM), 10 μM forskolin, and 1 μM

L-AP4 (EC80). PAM activity was assessed by performing a L-AP4 concentration-

response curve (0.1 nM -10 μM) in the absence or presence of 10 μM ADX47273.

cAMP was measured using the luminescent reader of the Packard TopCount.

Immunoblot analysis. Drugs were administered IP to six male Long-Evans rats per dose

level. Thirty minutes later, animals were sacrificed (decapitation) and hippocampus and

prefrontal cortex were dissected and homogenized by sonication in 1% SDS/50 mM NaF

buffer. Equivalent amounts of protein from each individual sample were resolved in 4-

12% SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel and transferred to

nitrocellulose membranes. The membranes were blocked for 1 hr in Tris-buffered saline

(TBS) containing Tween-20 and then incubated with the phospho-specific antibody of

interest [phospho-ERK antibody, 1:1000; phospho-CREB Ser-133) antibody, 1:1000,

(Cell Signaling, Cambridge, MA)] overnight at 4oC followed by incubation with

horseradish peroxidase-linked goat anti-rabbit IgG (1:10000) and developed using

enhanced chemiluminescence (ECL) (Promega). The blots then were incubated in


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stripping buffer (62 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM β-mercaptoethanol).

The stripped blots were incubated with antibody-directed against total levels for the

respective protein overnight at 4oC (ERK, antibody, 1:1000; CREB antibody, 1:1000,

(Cell Signaling, Cambridge, MA)). Densitometric analysis of phospho-immunoreactivity

and total immunoreactivity for each protein was conducted using the Bio-Rad GS-710

Calibrated Imaging Densitometer and quantified using Quantity One version 4.1.0.

Phosphorylated immunoreactivity was normalized to total immunoreactivity. Data were

statistically analyzed by student t-test (unpaired) using Microsoft Excel.

Conditioned Avoidance Responding. Rats were maintained on a food-restricted

schedule (15 g of standard rodent feed each day after training/testing). Four shuttlebox

test chambers (Med Associates, St. Albans, VT) were used (divided into two

compartments by an archway). Each chamber floor half was composed of 13 3/16” -inch

diameter stainless steel grid rods placed on ½”-inch centers wired for the presentation of

a scrambled electric foot shock (0.5 mA). In addition, each side of the chamber was

equipped with a stimulus light and tone (Sonalert) and two infrared beam

source/detectors used to locate the rat within the chamber. Rats trained to avoid the foot

shock were placed in the experimental chambers for a 4-min habituation period followed

by 50 trials presented on a 15-s variable interval schedule (range = 7.5–22.5 s). Each trial

consisted of a 10-s warning tone and stimulus light (conditioned stimulus) followed by a

10-s shock (unconditioned stimulus), presented through the grid floor on the side where

the rat was located, in the presence of the tone and light. If during the initial 10 s of the

trial, an animal crossed through the archway which divides the shuttlebox thereby

breaking the infrared beam location 13 cm from the center archway, the tone and light


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were terminated, and the response was considered an avoidance response. If an animal

crossed through the archway which divides the shuttlebox after a foot shock was initiated,

the tone, light, and shock were terminated, and the response was considered an escape

response. If a response was made during an intertrial interval, the response was punished

with a 0.5-s shock (0.5 mA). A Med Associates computer with MedState Notation

software controlled the test session and counted the number of trials in which the animal

avoided shock, escaped shock, and did not respond. On test days, ADX47273 was

administered IP 30 min before testing. In the reversal experiments, MPEP (10 mg/kg, IP)

or MTEP (1 mg/kg, IP) was administered 45 min prior to testing, while ADX47273 was

administered 30 min prior to testing. The dose of MPEP or MTEP was chosen based on

literature and in-house data demonstrating anxiolytic and/or antidepressant-like effects

(Palucha and Pilc, 2007). The same eight animals, part of a colony of trained subjects

used for antipsychotic screening, received each treatment with at least three days

intervening. Demonstration of baseline performance (greater than 90% avoidance

responses) was a criteria for subsequent testing. The order of treatments was ADX47273

at 0, 10, 30 and then 100 mg/kg followed by MPEP (10 mg/kg pretreatment) plus

ADX47273 (100 mg/kg). In a second group of 8 subjects, the effects of ADX47273 (100

mg/kg IP) were replicated followed by an evaluation of the effect of MTEP (1 mg/kg IP)

pretreatment. Yet separate groups of subjects were used to test MPEP (10 mg/kg, IP) or

MTEP (1 mg/kg IP) alone (N=8). Avoidance response and response failure data were

analyzed by repeated-measures analyses of variance with post hoc least significant

difference tests (p < 0.05).


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Antagonism of Apomorphine-Induced Climbing and Stereotypy. Drugs were

administered IP to 6-18 mice per dose level. A control group, run simultaneously with

drug-treated groups, received saline at equal volumes. Thirty minutes later, experimental

and control animals were challenged with 1 mg/kg apomorphine subcutaneously (SC).

Five minutes after the apomorphine injection, the sniffing-licking gnawing (0 = absent

and 1 = present) syndrome (stereotyped behavior) and climbing behavior (0 = all four

feet on ground, 1 = two feet up on wire cage, and 2 = all four feet on wire cage) induced

by apomorphine were scored and recorded for each animal. Readings were repeated

every 5 min during a 30-min test session. Scores for each animal were totaled over the

30-min test session for each syndrome (stereotyped behavior and climbing). Mean

climbing and stereotypy scores were then expressed as a percentage of control values

observed in vehicle-treated mice that received apomorphine. One-way analysis of

variance (ANOVA) followed by the least significance difference test was used to

determine the minimal effective dose (MED). In the reversal experiments, MPEP (10

mg/kg, IP) or MTEP (10 mg/kg, IP) was administered 45 min followed by ADX47273

(300 mg/kg, IP) 30 min prior to apomorphine. Data in the reversal experiments were

analyzed with a two-way analysis of variance followed by a least significant difference

test (p < 0.05).

PCP, apomorphine and amphetamine-induced locomotor hyperactivity. Mice (10

mice per treatment group) were weighed and placed in the locomotor chambers and

allowed to habituate for 90 minutes. After habituation, the mice were dosed with vehicle

or ADX47273 and activity was measured for another 30 minutes at which time the mice

were dosed with PCP (3 mg/kg, IP), apomorphine (1 mg/kg, SC), amphetamine (1 mg/kg,


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SC) or vehicle. Locomotor activity was measured for an additional 30 minutes.

Locomotor activity data was recorded under room light using Accuscan infrared beam

activity monitors with enclosed Plexiglas chambers (8 in. x 8 in.). Accuscan Versamax

and Versadat software (Columbus, OH) was used to convert the infrared beam-breaks

into centimeters traveled in each 10-minute bin. Data were analyzed with two-way

repeated measures ANOVAs, followed by a least significant difference post-hoc test,

comparing the treatment groups across time during the 30 min pretreatment period and

during the 30 min following stimulant challenge (p < 0.05). Separate analyses were

conducted for PCP, apomorphine, amphetamine and vehicle challenged groups.

Cataleptogenic Potential in Mice. ADX47273 (0, 10, 30, 100 or 300 mg/kg, IP) was

administered to six mice per treatment group. Every 30 min for 2 h post-dosing, the

animal's forelegs were draped over a thin horizontal rod 1 inches high. The amount of

time (in seconds) for which the animal maintained this awkward position was recorded

(60 s maximum). Maintenance of this position was considered catalepsy. Mean seconds

spent in the catalepsy position for each dose at each time point was calculated. The time

point at which the peak catalepsy was exhibited was analyzed with a one-way analysis of

variance with a post hoc least significant difference test (p < 0.05) and expressed

graphically.

48-hour-delay novel object recognition. The test arena consisted of a circular field

(diameter ~70cm, 30cm high) constructed of plastic and containing bedding. The novel

object recognition task was divided into 3 sessions: habituation, trial 1, and trial 2. Rats

were placed in the arena with two identical assemblies of Lego® blocks and were allowed

to explore freely for a total of 10 minutes to allow habituation on Day 1. On Day 2, 24 hr


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following habituation, rats were treated 30 minute prior to Trial 1. Rats were then placed

in the arena with a different set of two identical Lego® objects and allowed to explore

freely for a total of 5 minutes. Time spent sniffing each object was recorded by an

observer and totaled for an overall object exploration time for each rat. On Day 4, 48 hrs

following Trial 1 and without further drug treatment, rats were placed back in the arena

with one novel and one familiar object and allowed to explore freely for a total of 5

minutes. Time (seconds) spent sniffing each of the objects was recorded. Seconds spent

on each object were analyzed by repeated measures ANOVA with main effects of trial

and treatment, followed by a post hoc least significant difference test comparing novel

versus familiar object exploration time for each treatment (p<0.05).

5-choice serial reaction time (5-CSRT) task. The test apparatus consisted of ten 25 x

25 cm operant chambers (Med Associates, USA). The rear wall of each chamber was

concavely curved and contained 5 accessible apertures, each 2.5 cm square, and 4 cm

deep and set 2 cm above floor level. A standard 3-watt LED located at the rear of each

aperture provided illumination of each hole. The ten chambers were individually housed

within sound-attenuating cabinets and were ventilated by low-level noise fans, which also

served to mask extraneous background noise. Each chamber was illuminated by a 3W

house-light mounted near the ceiling in the center of the front wall alongside a small

general-purpose loud speaker. The program controlling the software was developed by

Conclusive Solutions (UK).

Prior to drug treatments, rats were trained to discriminate a brief visual stimulus

presented randomly in one of the 5 spatial locations. At the beginning of each test

session, the house light was illuminated and free delivery of a single food pellet to the


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magazine was made. Trial initiation was triggered when the rat opened the magazine to

collect this pellet. After a fixed 5 sec inter-trial interval (ITI), the light at the rear of one

of the 5 openings was illuminated for 500-msec stimulus duration (SD). A nose-poke in

this opening during illumination and for a limited hold period of 5 sec afterwards was

reinforced by the delivery of a food pellet and a correct response was recorded. A

response in a non-illuminated opening during the signal period including the 5 sec

immediately afterward (incorrect response) and failures to respond with the limited hold

period (missed trial) were followed by a “time-out” period of darkness for 5 sec where no

food pellet was delivered. Premature responses, those nose-pokes into apertures prior to

illumination, were also followed by the time out period and reset the ITI. The

consequence of a premature response is that the ITI further delayed the aperture

illumination. Once animals had been trained to a baseline performance of 75% correct

responding on the standard procedure (500-msec light SD / 5 second ITI) they began

testing.

In order to increase impulsivity and decrease attention during test sessions,

animals were exposed to light stimuli (500-msec SD) presented on a variable ITI

schedule (ITI lengths of 10, 7, 5, and 4 sec). Equal numbers of each of the 4 ITIs were

randomly presented during each 100 trial test session. All subjects received drug

treatments and testing on the variable ITI schedule on Tuesday and Friday of each week

giving a minimum of 2 days washout period. Interspersed with those treatment days

were standard training days (500-msec SD / 5 second ITI) reinstating baseline

performance of >75% correct responding. A within subjects design was employed such

that all animals received all treatments in a fully counterbalanced regimen.


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The various performance measures were analyzed with a mixed linear model,

because a standard linear model does not allow correlation of observations or non-

constant variability across different levels of certain factors. The fixed factors included

in the model were dose, ITI, and interactions of dose and ITI. The correlation of

observations taken from the same animal was modeled by covariance structure

″compound symmetry″, which assumed any two different observations from the same

animal were correlated to the same degree. After fitting the data, the residuals — the

observed values minus the predicted values — were plotted against the predicted values.

A tool to accommodate possible unequal variability in different levels of dose or ITI is to

group covariance structure, e.g., grouping by ITI would use different sets of covariance

parameters for different levels of ITI. The selection of grouping was aided by values of

Akaike’s Information Criterion (Akaike et al. 1974).

In Vivo Microdialysis. Animal Housing and Surgery: Male Sprague-Dawley rats

weighed between 280 and 325 g at the time of surgery. Following induction of

anesthesia with 3% isoflurane, rats were secured in a stereotaxic frame with ear and

incisor bars (David Kopf, Tujunga, CA). A microdialysis probe guide cannula (CMA/12,

CMA Microdialysis, Stockholm, Sweden) was implanted into the striatum

(anteroposterior +0.2 mm, lateral –3.0 mm and ventral –3.2 mm) or nucleus accumbens

(anteroposterior +2.2 mm, lateral –1.2 mm, and ventral –6.0 mm). Coordinates were

taken according to (Paxinos et al., 1985) reference points taken from bregma and vertical

from dura using a flat skull position. Guide cannulae were fixed to the skull with two

stainless-steel screws (Small Parts, Roanoke, VA) and dental acrylic (Plastics One,


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Roanoke, VA). Following surgery, animals were individually housed in Plexiglas cages

(45 cm sq.) for approximately 24 hrs where they had access to food and water ad libitum.

Acute ADX47273 Treatment: Concentric-style microdialysis probes (CMA/12; 20

kD cut-off) were purchased from CMA/Microdialysis and consisted of a 2-mm active

membrane (OD 0.5 mm) and a 14-mm stainless steel shaft (OD 0.64mm). Probes were

perfused with artificial CSF (aCSF; 125mM NaCl, 3mM KCl, 0.75mM MgSO4 and

1.2mM CaCl2, pH 7.4) for at least 18 hrs according to the manufacturer’s specifications.

On the morning of the microdialysis experiment, probes were inserted, via the guide

cannula, into the nucleus accumbens or striatum and perfused with aCSF at a flow rate of

1ul/min. After a 3-hr stabilization period following probe insertion, dialysis samples

were collected every 30 min. Initially, three dialysate samples were taken prior to drug

injection to demonstrate a steady baseline. At the end of baseline, animals received an

acute injection of either ADX47273 (175 mg/kg, IP) or vehicle (2% Tween80 / 0.5%

Methylcellulose). After injections, dialysis samples were collected for the following 4

hrs. At the end of these experiments, animals were euthanized and probe placement was

verified histologically. Data from animals with incorrect probe placements were

discarded from the study.

Neurochemical Analysis: Microdialysis samples (30 μl) containing dopamine

was separated by HPLC (C18 ODS3 column, 150 x 3.0 mm, Metachem, Torrance, CA)

and detected using an ANTEC electrochemical detector set at a potential of 0.65V vs. a

Ag/AgCl reference electrode. Mobile phase (0.15 M NaH2PO4, 0.25 mM EDTA, 1.75

mM 1-octane sulphonic acid, 2% isopropanol and 4% methanol, pH = 4.6) was delivered

by a Jasco PU1580 HPLC pump (Jasco Ltd, Essex, U.K) at a flow rate of 0.5 ml/min.


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Neurochemical data were compared to an external standard curve and all data was

acquired using the Atlas software package (Thermo Labsystems, Beverley, MA) for the

PC. Neurotransmitter levels (fmol concentrations) collected during the baseline samples

were averaged and this value was denoted as 100%. Subsequent sample values were

expressed as a percent change from this pre-injection baseline value (% change from

baseline). Neurochemical data, excluding pre-injection values, were analyzed by two-

way analysis of variance (ANOVA) with repeated measures (time). Post-hoc analyses

were made using the Bonferroni / Dunns adjustment for multiple comparisons (p < 0.05).

All statistical calculations were performed using the Statview software application

(Abacus Concepts Inc., Berkeley, CA) for the PC.


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Results

ADX47273 potentiates glutamate response in HEK 293 cells expressing rat mGlu5

receptors and primary astrocyte cultures. In FLIPR384 assays, HEK 293 cells

expressing rat mGlu5 exhibited concentration-dependent increases in Fluo-3 fluorescence

in response to glutamate with an EC50 value 300 nM. A subthreshold concentration of

glutamate 50 nM caused approximately 10% of the maximum glutamate response (Fig.

1A). ADX47273 caused concentration-dependent increased in the response to 50 nM

glutamate in HEK 293 cells expressing rat mGlu5 without eliciting a response by itself.

The maximal increased in the response was approximately 9-fold, with an EC50 value for

potentiation of 0.17 ± 0.03 μM (n = 8) (Fig. 1A). Preincubation with 10 μM MPEP for

30 min completely blocked the effects of ADX47273 on potentiation (Fig. 1A). In

primary astrocyte cultures, ADX47273 caused concentration-dependent increased in the

response to 300 nM glutamate with EC50 value of 0.23 ± 0.07 μM (n = 12) (Fig. 1B).

Increasing concentrations of ADX47273 caused a parallel, leftward shift of the glutamate

concentration response curve. In the presence of 0.1 μM or 1 μM of ADX47273, the

EC50 for glutamate decreased 4- fold and 9-fold, respectively, in HEK 293 cells

expressing rat mGlu5 (Fig. 2A). Similarly in astrocytes, in the presence of 1 μM or 3 μM

of ADX47273, the EC50 for glutamate decreased 4- and 9-fold, respectively (Fig. 2B).

The functional cross-selectivity of ADX47273 was evaluated in rat mGlu1, human

mGlu2 (Group II receptor) and mGlu4 (Group III receptor) receptors cell lines. No

agonist, antagonist or allosteric responses were observed with ADX47273 (up to 10 μM)

in any of cell lines (data not shown).


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ADX47273 competes with [3H]-MPEP but not [3H]-quisqualate binding.

ADX47273 inhibited binding of [3H]-MPEP to membranes prepared from HEK 293 cells

expressing mGlu5 receptors (Fig. 3) [Ki value: 4.3 ± 0.5 μM (n= 5), Hill Slope = 0.833

based on one-site model, R2 = 0.96, p < 0.05). In the in vitro 3H-quisqualate binding

assay, ADX47273 did not affect the binding of the radioligand at the glutamate

recognition (orthosteric) site of the rat mGlu5 receptor at concentrations up to 10 μM

(specific 3H quisqualate binding: mean ± SEM of 1506 ± 66 vs 1546 ± 65 fmoles/mg

protein in the absence and presence of ADX47273, respectively; n = 3, not significant by

Student's t-test).

ADX47273 increases ERK and CREB phosphorylation in hippocampus and cortex.

Rats were administered ADX47273 (1 and 10 mg/kg, IP) and sacrificed 30 minutes later.

The homogenates from prefrontal cortex and hippocampus of these subjects were

analyzed by immunobloting with anti-phospho-ERK and anti-phospho-CREB antibodies.

ADX47273 (10 mg/kg, IP) treatment increased ERK and CREB phosphorylation in both

prefrontal cortex and hippocampus (Fig. 4). Pretreatment with MPEP (10 mg/kg, IP 60

min prior to sacrifice) had no significant effects on its own and blocked ADX47273

induced increases in ERK and CREB phosphorylation (Fig. 4).

ADX47273 produces an antipsychotic-like decrease in conditioned avoidance

responding. ADX47273 (10-100 mg/kg, IP) produced dose-dependent decreases in

avoidance responding in rats (Fig. 5A) (p < 0.0001) and increased escapes, at doses that

did not produce any response failures. These effects of ADX47273 were significantly

attenuated by pretreatment with either the mGlu5 receptor antagonist MPEP (10 mg/kg,

IP) or MTEP (1 mg/kg IP) 15 min prior to ADX47273 (Fig. 5B, C) (p < 0.05). MPEP


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alone (10 mg/kg, IP) given to a separate group of animals reduced avoidance responding

and increased escape responding by approximately 30% relative to vehicle treatment

while MTEP alone (1 mg/kg IP) did not significantly affect avoidance responding (data

not shown).

ADX47273 blocks apomorphine-induced climbing. Under vehicle treatment, the

absolute values (mean ± SEM) for apomorphine-induced climbing were 9.67 ± 0.42 and

for apomorphine-induced stereotypy were 6.0 ± 0. ADX47273 (10–300 mg/kg, IP)

produced a dose-dependent decrease in apomorphine-induced climbing (Fig. 6A) (F4,49 =

25.663, p < 0.001; MED = 100 mg/kg) at doses that had negligible effects on

apomorphine-induced stereotypy (F4,49 = 4.729, p < 0.001; MED = 300 mg/kg), a profile

similar to that produced by the atypical antipsychotic clozapine (Marquis et al., 2007).

These effects of ADX47273 in blocking apomorphine-induced climbing were

significantly attenuated by pretreatment with MPEP (10 mg/kg, IP) (F3,20 = 4.767, p <

0.05) or MTEP (10 mg/kg, IP) ) (F3,20 = 61.7, p < 0.05) 15 min prior to ADX47273 (Fig.

6B, C, respectively). MPEP (1-30 mg/kg, IP) alone had no significant effect on

apomorphine-induced climbing (F4,25 = 0.822, p > 0.05) but did produce a small (8 to

11% relative to control) but statistically significant decrease in stereotypy (F4,25 = 3.750,

p < 0.05) (data not shown). MTEP (10 mg/kg, IP) did not affect either apomorphine-

induced climbing or stereotypy (Fig. 6C).

ADX47273 blocks PCP, apomorphine or amphetamine-induced locomotor activity.

ADX47273 (10-100 mg/kg, IP) was tested for its effects on PCP, apomorphine or

amphetamine-induced locomotor activity in habituated mice (Fig. 7). When locomotor

activity during the pretreatment period was analyzed, there was no significant main effect


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of ADX47273 pretreatment (F3,31 = 0.73, F3,32 = 0.74, F3,29 = 2.84, in subjects earmarked

for PCP (Fig. 7A), apomorphine (Fig. 7B) and amphetamine (Fig. 7C) challenge,

respectively, all p >0.05). No treatment by time interaction was detected in subjects

earmarked for PCP (F6,62 = 0.65, p>0.05) or apomorphine (F6,64 = 0.47, p >0.05) while

those subjects earmarked for amphetamine challenge (F6,58 = 2.72, p <0.05) tended to

show increased activity at 10 mg/kg and decreased activity at 100 mg/kg compared to

vehicle in the initial 10 min period following ADX47273. These effects were not

observed in the other cohorts earmarked for stimulant challenge, or a separate group of

subjects pretreated with ADX47273 (100 mg/kg, IP) followed by vehicle challenge

(Treatment main effect F1,16= 1.49, p >0.05; Treatment by time interaction F2,32= 1.18, p

>0.05) (Fig. 7D).

Analysis of locomotor activity after challenge with psychostimulant revealed a

significant main effect of ADX47273 (F3,31 = 1.97, F3,32 = 1.69, F3,29 = 12.94, p <0.05 for

PCP (Fig. 7A), apomorphine (Fig. 7B) and amphetamine (Fig. 7C) challenged subjects,

respectively) as well as treatment by time interactions (F6,62 = 2.91, F6,64 = 2.6, p > 0.05,

F6,58 = 4.73, p <0.05 for PCP, apomorphine and amphetamine challenged subjects,

respectively). Post-hoc analysis revealed that pretreatment with ADX47273 at 100

mg/kg decreased locomotor activity compared to vehicle pretreatment at 20 min after

PCP, 30 min after apomorphine and at all time points after amphetamine challenge. In a

separate group of vehicle-challenged mice which were habituated to the locomotor

apparatus, ADX47273 (100 mg/kg, IP) failed to affect motor activity significantly

different from vehicle pretreatment over this same time frame (F1,16 = 1.22, p >0.05) (Fig.

7D). The effect on unhabituated motor activity was not tested.


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ADX47273 induced catalepsy. While there was a significant effect of treatment (F4,25 =

5.41, p < 0.05), there was no significant time (F3,75 = 1.5) or treatment by time interaction

(F12,75 = 1.59). At 300 mg/kg, mice maintained the cataleptic position for 37 ± 15.7% of

the maximum 60 second response at 90 min post treatment. Catalepsy was detected at

this dose at 30 and 60 min post treatment, but was of a lesser magnitude (approximately

25% of maximum). (Data not shown).

ADX47273 decreases dopamine levels in the nucleus accumbens, but not in the

dorsal striatum. Basal levels of extracellular dopamine in the nucleus accumbens were

on average 927 and 1212 fmol/10 μL sample in vehicle and ADX47273 treated subjects,

respectively. In the striatum, dopamine basal levels were 4823 and 4086 fmol/10 μL

sample in vehicle and ADX47273 treated subjects, respectively. Acute administration of

ADX47273 (175 mg/kg, IP) lowered dopamine levels in the rat nucleus accumbens (Fig.

8A), but not in the dorsal striatum relative to vehicle-treated animals (Fig. 8B). The

maximal decrease in dopamine in the nucleus accumbens was 66.02 ± 8.35%. A two-

way ANOVA with repeated measures (time) revealed a significant decrease in dopamine

at 175 mg/kg (F1,14 = 23.91, p < 0.05) as well as a significant time effect (F7,98 = 3.79 , p

< 0.05). Post-hoc analyses revealed that ADX47273 effect on extracellular dopamine

levels in the nucleus accumbens was significantly different from vehicle treatment as

early as 60 min post treatment and were sustained for the remainder of the experiment.

No significant effect occurred in striatum of rats dosed with ADX47273 (175 mg/kg, IP)

(Treatment Effect: F1,11.9 = 0.07, Time Effect: F6,58.3 = 0.58, Treatment x Time: F6,58.3 =

1.47, p > 0.05).


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ADX47273 improves recall after a 48-hr delay in novel object recognition.

ADX47273 (0.1-50 mg/kg, IP) was evaluated for cognition enhancement in novel object

recognition paradigm, which is a task for testing memory performance based on the rat's

natural differential exploration of new and familiar objects (Ennaceur and Delacour,

1988; Ennaceur and Meliani, 1992). During the learning trial, there was no difference in

the behavior of the animals in the different groups (vehicle- versus ADX47273-treated)

and the time spent exploring the objects was very similar (between 60 and 80 s, for a 5-

min trial) among all groups (F5,64= 1.110, p > 0.05) (Fig. 9A). In the test trial,

ADX47273 dose-dependently improved recall with the 1-50 mg/kg groups exploring the

novel object more than the familiar while the vehicle and 0.1 mg/kg groups explored the

object equally (F5,64 = 4.45, p < 0.05). (Fig. 9B).

ADX47273 reduces impulsivity in 5-CSRT. To investigate the effects ADX47273 on

attention and impulsivity, we employed the 5-choice serial reaction time (5-CSRT) test

(Robbins, 2002). Varying the length of inter-trial interval (ITI) reduced percent correct

responses (Fig. 10A) (F3,143 = 4.5, p < 0.005). A post-hoc analysis revealed that there

was a decrease in percent correct responses at the 10 sec ITI to 78% from the 86% correct

responses made at the baseline ITI of 5 sec (p < 0.05). ADX47273 did not produce any

treatment effects on percent correct responses either as a main effect of dose (F2,143 =

0.120, p >0.05) or as a dose x ITI interaction (F2,143 = 0.82, p >0.05). Varying the ITI

also produced a significant main effect on the rate of premature responses (F3,14.2 = 68.04,

p < 0.0001) (Fig. 10B). According to post-hoc analysis, significantly more premature

responses were made during the 10 sec ITI as compared to all other ITIs and decreased as

ITI length was decreased. Treatment with ADX47273 resulted in a main effect of drug


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treatment (F2,29.3 = 4.63, p < 0.05) with post-hoc analysis revealing a decrease in the

number of premature responses at both the 10 and 30 mg/kg doses (p < 0.05). There was

also a significant dose x ITI interaction on premature responding (F6,31.9 = 2.67, p < 0.05)

with a post-hoc analysis indicating that the 10 mg/kg dose significantly decreased the

number of premature responses made at the 10 and 7 sec ITIs (p < 0.05). The decrease in

premature responding was not due to a global decrease in motor activity because

ADX47273 did not increase the number of missed trials (F2,143 = 0.69, p > 0.05) or the

latency to collect the reward (F2,143 = 2.12, p > 0.05) (data not shown). Thus in the 5-

CSRT task, ADX47273 had no effect on the modest decrease in percent correct

responding produced by instituting a variable ITI, but was effective in decreasing the

premature responding elicited by the 10 sec ITI within the variable ITI session.


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Discussion

Reversal of NMDA receptor hypofunction has been suggested as a possible

strategy for the treatment of schizophrenia (Lindsley et al., 2006). One mechanism for

enhancing NMDA activity is through activation of mGlu5 receptors (Gasparini et al.,

2002; Marino and Conn, 2002). In this report, the mGlu5 PAM ADX47273 (Le Poul et

al. 2005) is used to demonstrate that modulation of mGlu5 can produce antipsychotic-like

and pro-cognitive activities in rodent models thereby extending other reports of

antipsychotic-like effects of mGlu5 PAMs (Kinney et al., 2005b). In addition, the

enhancement of recognition in the NOR assay is the first direct demonstration of the

impact of an mGlu5 PAM on cognition. This effect was predicted by the ability of an

mGlu5 PAM to enhance NMDA receptor activation (O'Brien et al., 2003b; Kinney et al.,

2005b) and to effect biochemical end points relevant to cognition (Thomas and Huganir,

2004a; Carlezon et al., 2005; Liu et al., 2006). These results underscore the value of

mGlu5 PAMs as a novel approach to treating the positive symptoms and cognitive

deficits of schizophrenia.

ADX47273 behaves as a positive allosteric modulator in vitro as evidenced by its

ability to potentiate the effect of a subthreshold concentration of glutatmate in the Ca2+

mobilization assay with an EC50 = 170 nM. As this effect can be blocked in the presence

of mGlu5 receptor antagonist MPEP, it appears due to interactions of ADX47273 at the

allosteric modulatory site of mGlu5. In addition, ADX47273 (at concentrations up to 10

μM) did not affect the binding of 3H-quisqualate at the extracellular glutamate

recognition (orthosteric) site of rat mGluR5, but was shown to inhibit 3H-MPEP binding

by as much as 20% at 10 μM. These results suggest that ADX47273 and MPEP share

slightly overlapping sites of interaction within the mGlu5 allosteric pocket that is distinct


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from the 3H-quisqualate orthosteric site (i.e., an allosteric mechanism). This in vitro

pharmacological profile is consistent with a previous report (Le Poul et al. 2005).

ADX47273 was evaluated in the conditioned avoidance model, a standard

screening model for antipsychotic efficacy (Arnt, 1982). In this model, ADX47273 dose-

dependently decreased avoidance responding, without increasing the number of no

responses trials, a profile similar to the typical antipsychotic haloperidol and the atypical

antipsychotic clozapine (Marquis et al., 2007). The antipsychotic-like effect of

ADX47273 is apparently mediated by the mGlu5 as both mGlu5 receptor antagonists

MPEP and MTEP attenuated the effect of ADX47273, despite the observation that MPEP

(but not MTEP) itself affected avoidance responding.

Additionally, ADX47273 selectively reduced apomorphine-induced climbing

behavior, a second model predictive of antipsychotic efficacy that is mediated by the

mesolimbic dopaminergic pathway (Costall et al., 1980). Both MPEP and MTEP

significantly attenuated the effect of ADX47273 on apomorphine-induced climbing,

suggesting a role for mGlu5 in this effect of ADX47273. In addition, ADX47273

reduced amphetamine-induced hyperactivity and reduced the extracellular concentration

of dopamine in the nucleus accumbens, the projection target of the mesolimbic

dopaminergic pathway. The effect of ADX47273 on direct (apomorphine) and indirect

(amphetamine) dopamine agonist induced behaviors may be, in part, explained by the

ability of ADX47273 to decrease extracellular dopamine levels in the nucleus

accumbens. Another mGlu5 receptor PAM CDPPB was reported to reduce

amphetamine-induced locomotor activity and reverse amphetamine-induced deficits in

prepulse inhibition in rats (Kinney et al., 2005b). These data suggest a possible


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neurochemical explanation for the antipsychotic-like behavioral results of ADX47273 in

that reduction of dopamine neurotransmission by blockade of D2 dopamine receptors in

mesolimbic brain regions is argued to be a mechanism whereby current antipsychotics

exert their efficacy.

In the same dose range, ADX47273 had minimal effects on apomorphine-induced

stereotypy, an endpoint reportedly mediated by the nigrostriatal dopaminergic pathway

(Costall et al., 1975) and useful in predicting Parkinson’s-like extrapyramidal motor

system (EPS) side-effects of current typical antipsychotics (Marquis et al., 2007).

Additionally, ADX47273 did not significantly affect striatal dopamine levels further

supporting a mesolimbic selective effect of the compound. When assessed in the

catalepsy assay, ADX47273 did induce a modest level of catalepsy at the dose 3 times the

MED for its effect on apomorphine-induced climbing. These effects of ADX47273 in the

apomorphine-induced climbing/stereotypy, catalepsy and microdialysis models are

suggestive of an atypical antipsychotic-like profile for ADX47273, potentially mediated

via selective reduction of mesolimbic relative to nigrostriatal dopamine, as has been

reported with other non-dopaminergic mechanisms with preclinical antipsychotic-like

activity (Marquis et al., 2007).

It is worth noting that the mGlu5 receptor PAM ADX47273 also blocks PCP-

induced locomotor activity. This result provides direct evidence that activation of mGlu5

receptor can reverse NMDA receptor hypofunction and adds to the emerging evidence

that alterations in dopamine-glutamate interactions may contribute to the

pathophysiology of schizophrenia (de Bartolomeis et al., 2005). Position emission

tomography studies in human showed that the NMDA receptor antagonist PCP induced


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alterations in striatal dopamine (Kegeles et al., 2002). Preclinical studies also suggest a

dopamine-glutamate interaction based on the converging effect of different drugs, such as

amphetamine and PCP on dopamine modulating factors (dopamine and cAMP-regulated

phosphoprotein, DARPP-32) (Svenningsson et al., 2003). Furthermore, antipsychotic

compounds are shown to regulate postsynaptic density proteins (e.g. Homer proteins)

(Polese et al., 2002), which regulate the function of glutamate receptors.

Previously it has been reported that the mGlu5 antagonist MPEP enhanced the

detrimental effects of the NMDA antagonist MK-801 on cognition in assays dependent

on medial prefrontal cortex (mPFC) (Homayoun et al., 2004). More recently, it has been

shown that the mGlu5 PAM CDPPB prevented MK801-induced excessive firing and

reduced spontaneous bursting in the mPFC (Lecourtier et al., 2007). These observations

suggest that mGlu5 receptors play a role in regulating NMDA receptor-dependent

functions and that mGlu5 PAMs may be effective in ameliorating cognitive deficits in

schizophrenia. We have previously shown that CPPHA, another mGlu5 PAM can

increase phosphorylation of ERK and CREB in hippocampal slices (Liu et al., 2006), two

signaling molecules implicated in learning and memory. In the current study, we found

that ADX47273 also increases ERK and CREB phosphorylation in the hippocampus as

well as the prefrontal cortex, suggesting the potential for mGlu5 PAMs to improve

cognition, an area of major unmet medical need in schizophrenia.

To evaluate the potential cognitive enhancing activity of mGlu5 PAMs, we used

two models, novel object recognition task and 5-choice serial reaction time (5-CSRT)

task. In the object recognition memory task, it has been reported that the activation of

group I and group II metabotropic glutamate receptors in the perirhinal cortex is


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necessary for the acquisition, but not consolidation or retrieval, of long-term familiarity

discrimination (Barker et al., 2006). We found that in rats ADX47273 can enhance

object recognition suggesting that positive allosteric modulation of mGlu5 alone is

sufficient to improve performance in this particular behavior paradigm. Our data are

consistent with what has been reported on the effect of ADX47273 on object recognition

performance in mice (Epping-Jordan et al., 2005).

The continuous performance test has been widely used to measure attention

performance in humans (Robbins, 2002) and is sensitive in detecting attention deficits

across several disorders (Nieuwenstein et al., 2001). Schizophrenic patients show

impairments in the task compared to controls (Moeller et al., 2001). The 5-choice serial

reaction time (5-CSRT) test, a preclinical analogue of continuous performance test

(CPT), monitors attentional function using measures of percent correct responding and

response latency to the visual stimuli, as well as impulse control by measuring level of

premature responding (Robbins, 2002). It has been reported that impairment of

glutamatergic transmission following treatment with the NMDA antagonist PCP can

induce deficits in attentional functioning and response control in 5-CSRT task in mice.

PCP decreased percent correct responding in DBA, but not C57Bl6, mice and increased

premature responding in both strains, effects normalized in part by treatment with agents

that affect glutamatergic neurotransmission such as the mGlu2/3 agonist LY 379268

(Greco et al., 2005).

In 5-CSRT, ADX47273 inhibited premature responding (impulsivity), but did not

improve percent correct responding (attention). These effects occurred in the absence of

significant effects on correct response or reward retrieval latencies. In contrast, MPEP is


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reported to decrease premature responding similar to ADX47273, but with a concomitant

increase in response latency suggesting a disruption in motivation produced by MPEP in

this model (Semenova and Markou, 2007). The ability of ADX47273 to specifically

decrease premature responding suggests a mGlu5 PAM may effective in treating the

impulsivity observed in schizophrenia (Moeller et al., 2001).

In summary, ADX47273 acts as a selective positive modulator of mGlu5 and

produces behavioral effects suggestive of an antipsychotic-like profile. The effects on

CREB and ERK phosphorylation paired with effects in novel object recognition and 5-

CSRT tasks suggest that ADX47273 may effectively treat the cognitive symptoms of

schizophrenia as well. Taken together, these results suggest that a positive allosteric

modulator of mGlu5 may be a novel approach to treating symptoms of schizophrenia and

broaden the therapeutic response beyond the treatment of psychosis.


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References

Akaike, H. (1974), A New Look at the Statistical Model Identification, IEEE

Transaction on Automatic Control, vol, AC-19, 716-723.

Arnt J (1982) Pharmacological specificity of conditioned avoidance response

inhibition in rats: inhibition by neuroleptics and correlation to dopamine receptor

blockade. Acta Pharmacol Toxicol (Copenh) 51:321-329.

Awad H, Hubert GW, Smith Y, Levey AI and Conn PJ (2000) Activation of

metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA

receptor currents in neurons of the subthalamic nucleus. Journal of Neuroscience

20:7871-7879.

Barker GR, Bashir ZI, Brown MW and Warburton EC (2006) A temporally

distinct role for group I and group II metabotropic glutamate receptors in object

recognition memory. Learn Mem 13:178-186.

Campbell UC, Lalwani K, Hernandez L, Kinney GG, Conn PJ and Bristow LJ

(2004) The mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) potentiates

PCP-induced cognitive deficits in rats. Psychopharmacology (Berl) 175:310-318.

Carlezon WA, Jr., Duman RS and Nestler EJ (2005) The many faces of CREB.

Trends in Neurosciences 28:436-445.

Conn PJ and Pin JP (1997) Pharmacology and functions of metabotropic

glutamate receptors. Annu Rev Pharmacol Toxicol 37:205-237.

de Bartolomeis A, Fiore G and Iasevoli F (2005) Dopamine-glutamate interaction

and antipsychotics mechanism of action: implication for new pharmacological strategies

in psychosis. Curr Pharm Des 11:3561-3594.


at ASPE



ownloaded from


JPET#136580

38

De Paulis T, Hemstapat K, Chen Y, Zhang Y, Saleh S, Alagille D, Baldwin

Ronald M, Tamagnan GD, Conn PJ (2006) Substituent effects of N-(1,3-diphenyl-1H-

pyrazol-5-yl)benzamides on positive allosteric modulation of the metabotropic glutamate-

5 receptor in rat cortical astrocytes. J Med Chem 49: 3332-3344.

Doherty AJ, Palmer MJ, Henley JM, Collingridge GL and Jane DE (1997) (RS)-

2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5, but no mGlu1, receptors

expressed in CHO cells and potentiates NMDA responses in the hippocampus.

Neuropharmacology 36:265-267.

Ennaceur A and Delacour J (1988) A new one-trial test for neurobiological

studies of memory in rats. 1: Behavioral data. Behav Brain Res 31:47-59.

Ennaceur A and Meliani K (1992) Effects of physostigmine and scopolamine on

rats' performances in object-recognition and radial-maze tests. Psychopharmacology

(Berl) 109:321-330.

Epping-Jordan M.P., Nayak, S., Derouet, F., Dominguez, H., Bessis A.S., Le

Poul, E., Ludwig, B., Mutel, V., Poli S.M. and Rocher J.P. (2005) In vivo

Characterization of mgluR5 Positive Allosteric Modulators as Novel Treatments For

Schizophrenia And Cognitive Dysfunction. 5th International Metabotropic Glutamate

Receptors Meeting, Tormina, Italy.

Gasparini F, Kuhn R and Pin JP (2002) Allosteric modulators of group I

metabotropic glutamate receptors: novel subtype-selective ligands and therapeutic

perspectives. Curr Opin Pharmacol 2:43-49.


at ASPE



ownloaded from


JPET#136580

39

Greco B, Invernizzi RW and Carli M (2005) Phencyclidine-induced impairment

in attention and response control depends on the background genotype of mice: reversal

by the mGLU(2/3) receptor agonist LY379268. Psychopharmacology (Berl) 179:68-76.

Homayoun H and Moghaddam B (2006) Bursting of prefrontal cortex neurons in

awake rats is regulated by metabotropic glutamate 5 (mGlu5) receptors: rate-dependent

influence and interaction with NMDA receptors. Cereb Cortex 16:93-105.

Homayoun H, Stefani MR, Adams BW, Tamagan GD and Moghaddam B (2004)

Functional Interaction Between NMDA and mGlu5 Receptors: Effects on Working

Memory, Instrumental Learning, Motor Behaviors, and Dopamine Release.

Neuropsychopharmacology 29:1259-1269.

Johnson MP, Baez M, Jagdmann GE, Jr., Britton TC, Large TH, Callagaro DO,

Tizzano JP, Monn JA and Schoepp DD (2003) Discovery of allosteric potentiators for the

metabotropic glutamate 2 receptor: synthesis and subtype selectivity of N-(4-(2-

methoxyphenoxy)phenyl)-N-(2,2,2- trifluoroethylsulfonyl)pyrid-3-ylmethylamine. J Med

Chem 46:3189-3192.

Kegeles LS, Martinez D, Kochan LD, Hwang DR, Huang Y, Mawlawi O,

Suckow RF, Van Heertum RL and Laruelle M (2002) NMDA antagonist effects on

striatal dopamine release: positron emission tomography studies in humans. Synapse

43:19-29.

Kinney GG, O'Brien JA, Lemaire W, Burno M, Bickel DJ, Clements MK, Chen

TB, Wisnoski DD, Lindsley CW, Tiller PR, Smith S, Jacobson MA, Sur C, Duggan ME,

Pettibone DJ, Conn PJ and Williams DL, Jr. (2005a) A novel selective positive allosteric


at ASPE



ownloaded from


JPET#136580

40

modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and

antipsychotic-like effects in rat behavioral models. J Pharmacol Exp Ther 313:199-206.

Knoflach F, Mutel V, Jolidon S, Kew JN, Malherbe P, Vieira E, Wichmann J and

Kemp JA (2001) Positive allosteric modulators of metabotropic glutamate 1 receptor:

characterization, mechanism of action, and binding site. Proc Natl Acad Sci U S A

98:13402-13407.

Lecourtier L, Homayoun H, Tamagnan G and Moghaddam B (2007) Positive

Allosteric Modulation of Metabotropic Glutamate 5 (mGlu5) Receptors Reverses N-

Methyl-D-Aspartate Antagonist-Induced Alteration of Neuronal Firing in Prefrontal

Cortex. Biol Psychiatry 62:739-46.

Le Poul, E., Bessis A.S. , Lutgens R., Bonnet B., Rocher J.P., Epping-Jordan M

and Mutel V. (2005) In vitro Pharmacological Characterisation of Selective MgluR5

Positive Allosteric Modulators. 5th International Metabotropic Glutamate Receptors

Meeting, Tormina, Italy.

Lindsley CW, Shipe WD, Wolkenberg SE, Theberge CR, Williams DL, Jr., Sur C

and Kinney GG (2006) Progress towards validating the NMDA receptor hypofunction

hypothesis of schizophrenia. Curr Top Med Chem 6:771-785.

Lindsley CW, Wisnoski DD, Leister WH, O'Brien J A, Lemaire W, Williams DL,

Jr., Burno M, Sur C, Kinney GG, Pettibone DJ, Tiller PR, Smith S, Duggan ME, Hartman

GD, Conn PJ and Huff JR (2004) Discovery of positive allosteric modulators for the

metabotropic glutamate receptor subtype 5 from a series of N-(1,3-diphenyl-1H- pyrazol-

5-yl)benzamides that potentiate receptor function in vivo. J Med Chem 47:5825-5828.


at ASPE



ownloaded from


JPET#136580

41

Liu F, Zhang G, Hornby G, Vasylyev D, Bowlby M, Park K, Gilbert A, Marquis

K and Andree TH (2006) The effect of mGlu5 receptor positive allosteric modulators on

signaling molecules in brain slices. Eur J Pharmacol 536:262-268.

Marino MJ and Conn PJ (2002) Direct and indirect modulation of the N-methyl

D-aspartate receptor. Curr Drug Targets CNS Neurol Disord 1:1-16.

Marquis KL, Sabb AL, Logue SF, Brennan JA, Piesla MJ, Comery TA, Grauer

SM, Ashby CR, Jr., Nguyen HQ, Dawson LA, Barrett JE, Stack G, Meltzer HY, Harrison

BL and Rosenzweig-Lipson S (2007) WAY-163909 [(7bR,10aR)-1,2,3,4,8,9,10,10a-

octahydro-7bH-cyclopenta-[b][1,4]diazepino[ 6,7,1hi]indole]: A novel 5-

hydroxytryptamine 2C receptor-selective agonist with preclinical antipsychotic-like

activity. J Pharmacol Exp Ther 320:486-496.

Moeller FG, Barratt ES, Dougherty DM, Schmitz JM and Swann AC (2001)

Psychiatric aspects of impulsivity. Am J Psychiatry 158:1783-1793.

Nieuwenstein MR, Aleman A and de Haan EH (2001) Relationship between

symptom dimensions and neurocognitive functioning in schizophrenia: a meta-analysis of

WCST and CPT studies. Wisconsin Card Sorting Test. Continuous Performance Test. J

Psychiatr Res 35:119-125.

O'Brien JA, Lemaire W, Chen TB, Chang RS, Jacobson MA, Ha SN, Lindsley

CW, Schaffhauser HJ, Sur C, Pettibone DJ, Conn PJ and Williams DL, Jr. (2003a) A

family of highly selective allosteric modulators of the metabotropic glutamate receptor

subtype 5. Mol Pharmacol 64:731-740.

O'Brien JA, Lemaire W, Wittmann M, Jacobson MA, Ha SN, Wisnoski DD,

Lindsley CW, Schaffhauser HJ, Rowe B, Sur C, Duggan ME, Pettibone DJ, Conn PJ and


at ASPE



ownloaded from


JPET#136580

42

Williams DL, Jr. (2004) A novel selective allosteric modulator potentiates the activity of

native metabotropic glutamate receptor subtype 5 in rat forebrain. J Pharmacol Exp Ther

309:568-577.

Palucha A and Pilc A (2007) Metabotropic glutamate receptor ligands as possible

anxiolytic and antidepressant drugs. Pharmacol Ther 115:116-147.

Paxinos G, Watson C, Pennisi M and Topple A (1985) Bregma, lambda and the

interaural midpoint in stereotaxic surgery with rats of different sex, strain and weight. J

Neurosci Methods 13:139-143.

Paxinos G and Watson C (1986) The rat brain in stereotaxic coordinates.

Academic Press, New York.

Polese D, de Serpis AA, Ambesi-Impiombato A, Muscettola G and de

Bartolomeis A (2002) Homer 1a gene expression modulation by antipsychotic drugs:

involvement of the glutamate metabotropic system and effects of D-cycloserine.

Neuropsychopharmacology 27:906-913.

Poli S. M., Barvier S., Bessif A., Bessis A.S., Epping-Jordan M.P., Le Poul E.,

Ludwig B.L., Mingard B., Mutel V., Nayak S. Rocher J.P. (2005) In vivo

Characterization of mGluR5 Positive Allosteric Modulators as Novel Antipsychotic

Agents: Pharmacokinetic/Pharmacodynamic Correlation. 5th International Metabotropic

Glutamate Receptors Meeting, Tormina, Italy.

Robbins TW (2002) The 5-choice serial reaction time task: behavioural

pharmacology and functional neurochemistry. Psychopharmacology (Berl) 163:362-380.


at ASPE



ownloaded from


JPET#136580

43

Semenova S and Markou A (2007) The effects of the mGluR5 antagonist MPEP

and the mGluR2/3 antagonist LY341495 on rats' performance in the 5-choice serial

reaction time task. Neuropharmacology 52:863-872.

Svenningsson P, Tzavara ET, Carruthers R, Rachleff I, Wattler S, Nehls M,

McKinzie DL, Fienberg AA, Nomikos GG and Greengard P (2003) Diverse

psychotomimetics act through a common signaling pathway. Science 302:1412-1415.

Thomas GM and Huganir RL (2004a) MAPK cascade signalling and synaptic

plasticity. Nature Reviews Neuroscience 5:173-183.


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Legends for Figures

Fig. 1. ADX47273 potentiates activation of mGlu5 receptor by glutamate. ADX47273

has partial agonist-like activity at concentrations above 1 μM. (A) Rat mGlu5 receptor

HEK 293 cells were plated in clear-bottomed 384-well plates in glutamate/glutamine-free

medium and loaded with calcium-sensitive fluorescent dye Fluo-3. A range of

concentrations of ADX47273 was added to the cells with or without 50 nM of glutamate

and the Ca2+ response was measured by FLIPR384. For the blocking experiment, 10 μM

of MPEP was added 30 min before adding ADX47273. Concentration-response curves

were generated from mean data of three experiments. Error bars are S.E.M. The fold

potentiation was calculated from the maxima and minima determined by non-linear curve

fitting of the meaned data. (B) ADX47273 potentiates mGlu5 receptor-mediated calcium

signals in primary astrocyte cultures. Shown are calcium signals (shown as % of

maximal glutamate response), measured using FLIPR384 in response to increasing

concentrations of ADX47273 in the presence or absence of 300 nM glutamate (EC20).

Each data point represents the mean ± S.E.M from 12 independent experiments carried

out in quadruplicate.

Fig. 2. Potentiation of mGlu5 receptor activity by ADX47273 is manifested as an

increased sensitivity to agonist. (A) Rat mGlu5 receptor HEK 293 cells and (B) primary

astrocyte cultures, were plated in clear bottomed 384-well plates in glutamate/glutamine-

free medium, loaded the next with calcium sensitive fluorescent dye Fluo-3, and place in

FLIPR384. Several fixed concentrations of glutamate was added to the cells with or

without a range of concentrations of ADX47273. The basal activity of the glutamate


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concentration-response curves seems to increase at the highest concentration of

ADX47273. Concentration response curves were generated from mean data of three

experiments. Error bars are S.E.M. EC50 values were determined by non-linear curve

fitting of the meaned data.

Fig. 3. ADX47273 inhibits [3H] MPEP binding to rat mGlu5 receptor HEK cell

membranes. Membranes prepared from rat mGlu5 receptor HEK cells were incubated

with the radiolabeled MPEP for 60 min at room temperature in the presence of varying

concentrations of ADX47273. Samples were then filtered onto glass fiber filters.

A representative competition experiment is shown. Data are mean ± SD. Ki was

generated from the 5 experiments. Hill Slope = 0.833 based on one-site model, R2 =0.96,

p < 0.05.

Fig. 4. ADX47273 dose-dependently increased ERK and CREB phosphorylation in rat

hippocampus and prefrontal cortex. Vehicle, or MPEP (10 mg/kg, IP) was administered

30 min before ADX47273 (IP) to Long-Evans rats. Thirty minutes later ADX47273,

hippocampus (A) and prefrontal cortex (B) were dissected out and subjected to

immunoblotting with anti-phospho-ERK and anti-phospho-CREB antibodies. Phospho-

ERK and -CREB immunoactivity were normalized to total ERK and total CREB

immunoactivity, and followed by normalizing to untreated control immunoactivity. Data

were statistically analyzed by student t-test (unpaired). *Denotes statistical significance

compared with control (p < 0.05, n = 6).


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Fig. 5. ADX47273 was evaluated in the conditioned avoidance response in Sprague

Dawley rats. (A)ADX47273 was administered IP 30 min before testing. (B) vehicle, or

MPEP (10 mg/kg, IP) or (C) MTEP (1 mg/kg, IP) was administered 15 min before

ADX47273 (100 mg/kg, IP), which was administered IP 30 min before testing. Data are

expressed as average (means means ± S.E.M.) avoidance response and average number

of escape failures observed over 50 trials (n = 8 rats per group). The results following 100

mg/kg ADX47273 in (A) are also displayed in (B) and are replicated in a separate cohort

in (C). *Denotes statistical significance compared with vehicle-treated control (p < 0.05).

#Denotes statistical significance compared with ADX47273 treated rats (p < 0.05).

Fig. 6. ADX47273 evaluated in the apomorphine-induced climbing and stereotypy

assay in CF-1 mice. (A) ADX47273 was administrated IP 30 min before administration

of apomorphine (1 mg/kg, s.c.). For the blocking experiments, (B) MPEP (10 mg/kg, IP)

or (C) MTEP (10 mg/kg, IP) was administrated 15 min before ADX47273 (300 mg/kg,

IP) which was administered 30 min before administration of apomorphine. Data are

expressed as the percentage of climbing and stereotypy observed in the vehicle-treated

group. Data are the mean percentage of control ± S.E.M (n = 6 mice per group).

*Denotes statistical significance compared with vehicle-treated control (p < 0.05).

#Denotes statistical significance compared with ADX47273 treated mice (p < 0.05).

Fig. 7. ADX47273 evaluated in the PCP, apomorphine or amphetamine induced

locomotor activity in mice. After habituation for 90 minutes, the mice were administered

with vehicle or ADX47273 and locomotor activity was measured for another 30 minutes


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at which time the mice were dosed with either (A) PCP (3 mg/kg IP), (B) apomorphine (1

mg/kg SC), (C) amphetamine (1 mg/kg SC) or (D) vehicle. Locomotor activity was

measured for an additional 30 minutes. Data are expressed as the distance traveled. Data

are the mean ± S.E.M (n =10 mice per group). *Denotes statistical significance

compared to vehicle treated control (p < 0.05).

Fig. 8. The effect of ADX47273 on extracellular levels of dopamine in awake, freely

moving Sprague-Dawley rats. ADX47273 (175 mg/kg, IP) was administered in a single

bolus via SC cannulae at T = 0 min (marked by arrow) in nucleus accumbens (A) or

striatum (B). Data are expressed as a percentage of baseline levels and represent means ±

S.E.M (n = 7-9 animals per group). *Denotes significant effect from vehicle treatment.

Fig. 9. ADX47273 was evaluated in the novel object recognition task in rats.

ADX47273 was administered IP 30 minutes prior to Trial 1. No overall difference in

total object exploration time was observed in Trial 1 (A). Treatment with ADX47273

(0.1-50 mg/kg, IP) enhanced recognition of the novel object, compared to the familiar (*

p < 0.002), when subjects were evaluated in trail 2, 48 hr after Trial 1 (B).

Fig. 10. ADX47273 was evaluated in the 5-CSRT in rats. ADX47273 was administered

IP 30 min before testing. The inter-trial interval was varied from 4 to 10 sec. Correct and

premature responses were evaluated to measure effects on attention (A) and impulsivity

(B). Results are expressed as mean ± S.E.M. Data were analyzed with a mixed linear


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model to allow correlation of observations or non-constant variability across dose, ITI,

and interactions of dose and ITI. *denotes p < 0.05 from vehicle at each ITI.


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JPET#136580 1 Title Page ADX47273: A Novel Metabotropic