Biphenyl-indanone A, a positive allosteric modulator of the metabotropic glutamate receptor subtype 2, has antipsychotic- and anxiolytic-like effects in mice

Article abstract of DOI:10.1124/jpet.106.102046

Previous studies indicate that agonists of the group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, may provide a novel approach for the treatment of anxiety disorders and schizophrenia. However, the relative contributions of the mGluR2

Full text of DOI:10.1124/jpet.106.102046

0022-3565/06/3181-173–185$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
JPET 318:173–185, 2006
Vol. 318, No. 1
102046/3121168
Printed in U.S.A.
Biphenyl-indanone A, a Positive Allosteric Modulator of the
Metabotropic Glutamate Receptor Subtype 2, Has
Antipsychotic- and Anxiolytic-Like Effects in Mice
Ruggero Galici,1 Carrie K. Jones, Kamondanai Hemstapat, Yi Nong,
Nicholas G. Echemendia, Lilly C. Williams, Tomas de Paulis, and P. Jeffrey Conn
Program in Translational Neuropharmacology, Department of Pharmacology, Vanderbilt University Medical Center,
Nashville, Tennessee
Received January 30, 2006; accepted March 24, 2006
ABSTRACT
Previous studies indicate that agonists of the group II metabo- the mGluR2/3 agonist-induced inhibition of excitatory synaptic
tropic glutamate receptors (mGluRs), mGluR2 and mGluR3, transmission at the medial perforant path-dentate gyrus syn-
may provide a novel approach for the treatment of anxiety apse. BINA was also efficacious in several models predictive of
disorders and schizophrenia. However, the relative contribu- antipsychotic- and anxiolytic-like activity in mice. The behav-
tions of the mGluR2 and mGluR3 subtypes to the effects of the ioral effects of BINA were blocked by the mGluR2/3 antagonist
group II mGluR agonists remain unclear. In the present study, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-
we describe an alternate synthesis and further pharmacological yl) propanoic acid (LY341495), suggesting that the in vivo effects
characterization of a recently reported positive allosteric mod- of BINA are mediated by increased activation of mGluR2. Collec-
ulator of mGluR2 termed biphenyl-indanone A (BINA). In re- tively, these results indicate that BINA is a selective mGluR2
combinant systems, BINA produced a robust and selective positive allosteric modulator and provide further support for the
potentiation of the response of mGluR2 to glutamate with no growing evidence that selective allosteric potentiators of mGluR2
effect on the glutamate response of other mGluR subtypes. In mimic many of the in vivo actions of mGluR2/3 agonists that may
hippocampal brain slices, BINA (1 ␮M) significantly potentiated predict therapeutic utility of these compounds.
Metabotropic glutamate receptors (mGluRs) are classified nists (Conn and Pin, 1997). Group II mGluR subtypes, mGluR2
into three major groups based on sequence homologies, coupling
to second messenger systems, and selectivities for various ago-
and mGluR3, couple to Gi/o and associated effector pathways,
such as the inhibition of adenylyl cyclase and the regulation of
ion channels. Group II mGluRs reduce transmission at gluta-
matergic synapses in multiple brain regions, where excessive
glutamatergic neurotransmission has been implicated in the
underlying pathophysiology of anxiety disorders and schizo-
phrenia (Walker and Davis, 2002; Moghaddam and Jackson,
2003). Based on these findings, it has been postulated that
selective agonists of group II mGluRs may provide anxiolytic
and/or antipsychotic effects through a reduction in glutamater-
gic neurotransmission within these brain regions.
This work was supported by grants from the National Institute of Neuro-
logical Disorders and Stroke and the National Institute of Mental Health.
Vanderbilt is a site in the National Institutes of Health-supported Molecular
Libraries Screening Centers Network.
R.G. and C.K.J. contributed equally to this work.
A portion of these studies was presented at the 5th International Meeting on
Metabotropic Glutamate Receptors; 2005 September 18–23; Taormina, Italy.
1Current affiliation: Johnson and Johnson, San Diego, CA.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.102046.
ABBREVIATIONS: mGluR, metabotropic glutamate receptor; LY379268, (Ϫ)-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid; LY354740,
(1S,2S,5R,6S)-2-oxabicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate; LY487379, N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsul-
fonyl)pyrid-3-ylmethylamine; BINA, biphenyl-indanone A, 3Ј-[[(2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy]methyl]biphenyl-
4-carboxylic acid; 2, 2Ј-cyclopentyl-2,3-dimethyl-4-methoxyacetophenone, cyclopentylacetophenone; 3, 1-(4-methoxy-2,3-dimethylphenyl)-
2-cyclopentyl-2-propenone; 4, 2-cyclopentyl-5-methoxy-6,7-dimethylindan-1-one, methoxyinandone; 5, 2-cyclopentyl-5-hydroxy-6,7-dimethyl-
indan-1-one, indanol; 8, ethyl 3Ј-methylbiphenyl-4-carboxylate, biphenyl; THF, tetrahydrofuran; 9, ethyl 3Ј-bromomethylbiphenyl-4-carboxylate;
10, ethyl 3Ј-(((2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yl)oxy)methyl)biphenyl-4-carboxylate; r, rat; DMEM, Dulbecco’s modified Ea-
gle’s medium; FBS, fetal bovine serum; HEK, human embryonic kidney; CHO, Chinese hamster ovary; h, human; GTP␥S, guanosine 5Ј-[␥-thio]triphos-
phate; L-AP4, L-(ϩ)-2-amino-4-phosphonobutyric acid; DMSO, dimethyl sulfoxide; ACSF, artificial cerebrospinal fluid; MPP, medial perforant path;
fEPSP, field excitatory postsynaptic potentials; DCG-IV, (2S,2ЈR,3ЈR)-2-(2Ј,3Ј-dicarboxycyclopropyl)glycine; PCP, phencyclidine; PPI, prepulse inhibition;
LY341495, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; ANOVA, analysis of variance; DG, dentate gyrus.
173

174
Galici et al.
There have been considerable advances in the development and anxiolytic-like activity of group II mGluR agonists.
of selective agonists of group II mGluRs. Systemic adminis- These studies provide strong support for the growing evi-
tration of the mGluR2/3 agonists LY379268, LY354740, and dence that mGluR2 is responsible for many of the known in
their analogs have behavioral effects in several rodent mod- vivo effects of mGluR2/3 agonists and that allosteric poten-
els predictive of anxiolytic and antipsychotic activity (for tiators of mGluR2 may provide an exciting alternative to
reviews, see Schoepp and Marek, 2002; Schoepp et al., 2003; group II mGluR agonists for development of therapeutic
Marek, 2004). Moreover, the selective group II mGluR ago- agents.
nist LY354740 has been shown to have anxiolytic activity in
several clinical studies (Grillon et al., 2003; Schoepp et al.,
2003; Swanson et al., 2005). However, only one structural
class of selective group II mGluR agonists has been devel-
Materials and Methods
Synthesis of BINA
The improved synthesis of BINA was completed via an alternate
route (Fig. 1) from the method used by Bonnefous et al. (2005).
Cyclopentylacetyl chloride (1, 11.3 g) and 2,3-dimethylanisole (8.6 g)
in methylene chloride were treated with aluminum trichloride (10.5
g) for 16 h at 20°C to give 16.1 g (98%) of cyclopentylacetophenone
(2). Treatment of 2 with paraformaldehyde (6.8 g) and dimethyl-
amine hydrochloride (20 g) in acetic acid (1.5 ml) for 18 h at 95°C
according to Woltersdorf et al. (1977) gave 6.0 g (84%) of intermedi-
ate 3. Cyclization of 3 in 18 M sulfuric acid (35 ml) for 3 h at 20°C
gave 4.9 g (82%) of methoxyindanone (4). Demethylation of 4 with 0.5
oped, and these compounds activate both mGluR2 and
mGluR3. To date, the relative contributions of mGluR2 and
mGluR3 to the actions of the group II mGluR agonists re-
main unknown. Moreover, the development of tolerance to
the effects of the direct-acting group II mGluR agonists has
been observed in several rodent behavioral models (Cartmell
et al., 2000; Galici et al., 2005; Jones et al., 2005). Thus, it is
important to develop novel approaches for the subtype-selec-
tive activation of the group II mGluRs.
One alternative approach to direct-acting group II mGluR M boron tribromide in methylene chloride for 4 h at 20°C gave 2.3 g
(51%) of the indanol (5) after crystallization from methanol.
3-Toluoylzinc bromide (6), prepared from 3-bromotoluene (3.2 g), 1.6
M butyl lithium (12 ml), and 0.5 M zinc chloride (38 ml) in anhydrous
tetrahydrofuran (THF) and 4-ethoxycarbonylphenyl-bis(triphenyl-
phosphine)palladium (7), prepared from ethyl 4-iodobenzoate (4.4 g)
and bis(triphenylphosphine)palladium dichloride (0.7 g) in THF,
were combined and stirred for 16 h at 20°C according to Klein et al.
(1998), yielding 2.8 g (73%) of biphenyl (8) after gravity column
chromatography on silica in hexane-ethyl acetate (10:1). Free radi-
cal-induced bromination of 8 with N-bromosuccinimide (2.1 g) and
agonists is the use of subtype-selective positive allosteric
modulators of mGluR2 or mGluR3. Johnson et al. (2003)
reported the initial discovery of LY487379 as a selective
positive allosteric modulator of mGluR2. The pharmacologi-
cal characterizations of several compounds within the
LY487379 structural class have now been reported (Johnson
et al., 2003, 2005; Schaffhauser et al., 2003). These com-
pounds are highly selective for mGluR2 relative to other
mGluR subtypes. They do not activate mGluR2 directly but
act at an allosteric site on the receptor, which induces a 2,2Ј-azobisisobutyronitrile (0.2 g) as a free radical initiator in carbon
tetrachloride for 16 h at 80°C according to Gillig et al. (2004) yielded
3.2 g (86%) of biphenylmethyl bromide (9). Coupling of intermediates
5 (2.4 g) and 9 (3.2 g) with potassium carbonate in dimethylform-
amide for 14 h at 75°C gave 3.6 g (75%) of the ester 10 after flash
column chromatography on silica in hexane-ethyl acetate (10:1).
Hydrolysis of 10 with 2 N sodium hydroxide (10 ml) in THF-metha-
nol (1:1, 100 ml) for 2 h at 60°C, followed by extraction with 1 N
sodium hydroxide from diethyl ether and neutralization of the aque-
ous layer with 12 N hydrochloric acid yielded 2.0 g (63%) of BINA.
Melting point: 238–241°C. 1H nuclear magnetic resonance (300 MHz
leftward shift of the glutamate concentration-response curve
(Johnson et al., 2003, 2005; Schaffhauser et al., 2003). Inter-
estingly, these mGluR2 potentiators mimic some of the be-
havioral effects of mGluR2/3 agonists in animal models used
to predict anxiolytic and antipsychotic activity (Johnson et
al., 2003, 2005; Galici et al., 2005). However, members of this
first series of mGluR2 allosteric potentiators have modest
potencies in several behavioral models (Johnson et al., 2003;
Galici et al., 2005) and a short duration of action (Galici et al.,
2005), which prevents a more systematic characterization of in CDCl3): 8.21 (d, 2H, 3,5-CH), 7.73 (d, 2H, 2,6-CH), 7.71 (m, 1H,
2Ј-CH), 7.64 (dd, 1H, 6Ј-CH), 7.53 (m, 2H, 4Ј,5Ј-CH), 6.81 (s, 1H,
4Љ-CH), 5.22 (s, 2H, OCH2), 3.07 (dd, 1H, 2ٞ-CH), 2.73 (m, 2H,
3ٞ-CH2), 2.64 (s, 3H, 7Ј“-CH3), 2.33 (m, 1H, 2٣-CH), 2.23 (s, 3H,
6ٞ-CH3), 1.94 (m, 1H, 5٣-CH), 1.59 (m, 5H, 3٣,4٣-CH2 and 1٣-CH),
1.42 (m, 1H, 2٣-CH), 1.09 (m, 1H, 5٣-CH). Elemental analysis
(C30H30O4) C: calculated 79.27%, found 78.52%; H: calculated 6.65%,
found 6.73%.
their pharmacologic effects in vivo.
More recently, a novel series of selective allosteric poten-
tiators of mGluR2 have been discovered (Bonnefous et al.,
2005; Pinkerton et al., 2005). These compounds are chemi-
cally and structurally unrelated to LY487379 or to the group
II agonist LY354740. Preliminary studies suggest that some
of these newer compounds are centrally penetrant and block
ketamine-induced hyperlocomotor activity in rats, an animal
model predictive of antipsychotic activity (Rodriguez et al.,
2004; Bonnefous et al., 2005; Govek et al., 2005). However,
Cell Culture and Transfections
Baby hamster kidney cells stably expressing the rmGluR1a were
generously provided by Dr. Betty Haldeman at Zymogenetics Inc.
the pharmacological properties of these compounds have not (Seattle, WA). Cells were grown in Dulbecco’s modified Eagle’s me-
dium (DMEM) containing 5% heat-inactivated fetal bovine serum
(FBS), 2 mM GlutaMax I, 100 units of penicillin, 100 ␮g of strepto-
mycin, and 0.25 ␮g of amphotericin B (antibiotic-antimycotic), 1 mM
sodium pyruvate, 20 mM HEPES, and 250 nM methotrexate. Human
embryonic kidney (HEK) 293A cells stably expressing the rmGluR5a
were grown in DMEM containing 10% heat-inactivated FBS, 2 mM
GlutaMax I, antibiotic-antimycotic, 0.1 mM nonessential amino ac-
ids, 20 mM HEPES, and 500 ␮g/ml G418 sulfate. rmGluR2 and the
promiscuous G proteins (Gqi5) were cotransfected into HEK293A
cells using Lipofectamine 2000 and were grown in the same manner
been rigorously evaluated, and no individual compounds
have emerged as optimal tools for in vivo and in vitro studies.
Interestingly, Rodriguez et al. (2004) presented a preliminary
report suggesting that a member of this series, 3Ј-[[(2-
cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-
yl)oxy]methyl]biphenyl-4-carboxylic acid, termed biphenyl-
indanone A (BINA) may provide a useful tool for in vivo studies.
We now report that BINA is a highly selective allosteric
potentiator of mGluR2 with robust, long-lasting effects in
animal models that have been used to predict antipsychotic- as rmGluR5a except that the G418 sulfate was omitted.

BINA, a Positive Allosteric Modulator of mGluR2
175
Fig. 1. Synthesis of BINA. BINA was prepared in
nine steps from 3-bromotoluene, 2,3-dimethyl-
anisole, ethyl 4-iodobenzoate, and cyclopentylac-
etic acid in 16% overall yield.
Chinese hamster ovary (CHO) cells stably expressing the was resuspended and homogenized for 5 s in ice-cold buffer contain-
hmGluR2 were transiently transfected with Gqi5, and CHO cells ing 20 mM HEPES and 0.1 mM EDTA, pH 7.4. The final pellet was
stably expressing the hmGluR4/Gqi5 were grown in DMEM contain- resuspended and homogenized using a glass homogenizer. Aliquots
ing 10% heat-inactivated, dialyzed FBS, 2 mM GlutaMax I, antibi- were stored at Ϫ80°C until use. Protein concentrations were measured
otic-antimycotic, 1 mM sodium pyruvate, 20 mM HEPES, 5 nM using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) using
methotrexate, and 20 ␮g/ml L-proline. CHO cells stably expressing serum albumin (Pierce Chemical, Rockford, IL) as the standard.
the rmGluR3 were grown in DMEM containing 10% heat-inactivated
After thawing, the membranes were diluted and homogenized
FBS, 2 mM GlutaMax I, 100 units of penicillin, 100 ␮g of strepto- using a glass homogenizer in ice-cold assay buffer containing 50 mM
mycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids,
and 20 mM HEPES.
Tris-HCl (pH 7.4), 5 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 10
␮g/ml saponin, and 1 ␮M GDP. Assay mixtures contained 10 ␮g of
All recombinant cell lines were plated at a seeding density of 7 to membrane protein, test compound, glutamate, 0.1 nM [35S]GTP␥S,
8 ϫ 105 cells/well, in clear-bottomed, poly-D-lysine-coated 96-well and assay buffer to yield a total volume of 100 ␮l. Nonspecific binding
plates (Becton Dickinson Labware, Bedford, MA). Cells were then was determined in the presence of 10 ␮M unlabeled GTP␥S. The
incubated in glutamate-glutamine-free medium overnight at 37°C in assay mixtures were incubated at 30°C for 60 min. The binding
5% CO2, with the exception of baby hamster kidney cells stably equilibrium was terminated by rapid filtration through Unifilter-96
expressing mGluR1a, which were maintained in regular medium.
GF/B filter plates (presoaked with ice-cold assay buffer), and the
filters plates were washed three times with ice-cold assay buffer
using a 96-well Brandel harvester (Brandel, Gaithersburg, MD).
Filter plates were dried and filled with 30 ␮l of MicroScint-20, the
radioactivity was determined by TopCount NXT Microplate Scintil-
lation and Luminescence Counter (PerkinElmer Life and Analytical
Sciences, Downers Grove, IL).
[
35S]GTP␥S Binding Assay
Membranes were prepared from CHO cells stably expressing ei-
ther hmGluR2 or rmGluR3. In brief, cells were washed once with
ice-cold 1ϫ phosphate-buffered saline, pH 7.4. Cells were then har-
vested with a cell scraper and resuspended in ice-cold buffer (20 mM
HEPES and 10 mM EDTA, pH 7.4) and were homogenized using a
Polytron homogenizer for 2 s. The homogenate was centrifuged at
4°C for 20 min at 40,000g. The pellet was resuspended with the same
Calcium Mobilization Assay
Cells were loaded with calcium indicator dye (Calcium 3 Assay
buffer and was homogenized for 10 s. After centrifugation, the pellet Kit) from Molecular Devices (Sunnyvale, CA) for 1 h at 37°C. Dye

176
Galici et al.
was removed and replaced with the appropriate volume of assay
care covered in Principles of Laboratory Animal Care (National In-
buffer containing 1ϫ Hanks’ balanced salt solution (Invitrogen, stitutes of Health publication 85-23, revised 1985) and were ap-
Carlsbad, CA), 20 mM HEPES, and 2.5 mM probenecid, pH 7.4. Cells proved by the Institutional Animal Care and Use Committee.
were then preincubated with either vehicle or various concentrations
Apparatus. Amphetamine- and phencyclidine (PCP)-induced hy-
of BINA (5ϫ stock) for 5 min followed by the 1-min stimulation of an perlocomotor activity studies were conducted in open field chambers
EC20 of agonists (i.e., glutamate or L-AP4). BINA was dissolved in (27 ϫ 27 ϫ 20 cm) (Med Associates, Inc., St. Albans, VT) equipped
100% DMSO and then serially diluted into assay buffer containing with 16 horizontal (x- and y-axes) and 16 vertical (z-axis) infrared
0.1% bovine serum albumin for a 5ϫ stock in 0.5% DMSO. The stock
was then added to the assay for a final DMSO concentration of 0.1%.
Glutamate and L-AP4 were prepared to a 10ϫ stock solution in assay
buffer before addition to the assay plate. Calcium mobilization was
measured using the FLEXstation-II (Molecular Devices).
photobeams located 1 and 5.5 cm above the floor of the chamber,
respectively. Changes in locomotor activity were measured as the
number of photobeam breaks (counts) and were recorded with a
Pentium I computer equipped with a mouse activity monitoring
system software (Med Associates).
Prepulse inhibition (PPI) studies were conducted in sound-atten-
uating acoustic startle cubicles (Med Associates) equipped with two
speakers, a mouse holder, and a transducer system (a platform with
load cells) through which startle responses were recorded. Chambers
were connected to an amplifier and to a Pentium IV computer
equipped with Startle Reflex software (Med Associates).
For the stress-induced hyperthermia studies, changes in core body
temperature were measured rectally using a digital thermometer
with an accuracy of 0.1°C using a Physitemp (model BAT-12) digital
thermometer (Physitemp Instruments, Inc. Clifton, NJ).
Hippocampal Slice Electrophysiology
Extracellular field recordings were performed using hippocampal
brain slices prepared from male Sprague-Dawley rats, 3 to 4 weeks
of age, as previously described with minor modifications (Macek et
al., 1996, Marino et al., 1998). After decapitation, brains were rap-
idly removed and submerged in an ice-cold sucrose replacement
solution containing: 200 mM sucrose, 2.5 mM KCl, 1.2 mM
NaH2PO4, 1.3 mM MgCl2, 8 mM MgSO4, 20 mM glucose, and 46 mM
NaHCO3 and equilibrated with 95% O2-5% CO2. Coronal hippocam-
pal slices (400 ␮m) were cut using a microtome (Leica Microsystems,
Nussloch GmbH, Nußloch, Germany) and transferred to a holding
chamber containing artificial cerebrospinal fluid (ACSF) with 124
mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 2 mM
CaCl2, 20 mM glucose, and 26 mM NaHCO3, equilibrated with 95%
O2-5% CO2 and maintained at room temperature. For all experi-
ments, both sucrose replacement buffer and holding chamber ACSF
buffer were supplemented with 5 ␮M glutathione, 500 ␮M pyruvate,
and 250 ␮M kynurenic acid to increase slice viability.
After 1 h of recovery in the holding chamber, hippocampal slices
were transferred to the slice recording chamber and maintained fully
submerged with continuous perfusion of ACSF (2–3 ml/min). By
using a microscope with Hoffman modulation contrast, the middle
third of the molecular layer of the dentate gyrus was identified for
placement of the recording and stimulation electrodes. Recording
electrodes were pulled from borosilicate glass on a Narashige vertical
patch pipette puller and filled with 2.5 mM NaCl (0.3–1.0 M⍀ resis-
tance). A bipolar tungsten microelectrode (FHC Inc., Bowdoinham,
ME) was used for stimulation of the medial perforant path (MPP)
fibers; stimuli (100 ␮s in duration) were delivered at 0.05 Hz using a
Grass S48 stimulator and a Grass isolator. The field excitatory
postsynaptic potentials (fEPSPs) were recorded using an Axon Multi-
Clamp 700B amplifier (Molecular Devices) in current clamp mode, data
were digitized using DigiData 1322A, filtered (2 kHz), and acquired
using the pClampex v.9.2 program. An input-output curve was per-
formed for each experiment, and the stimulus intensity was adjusted to
produce a fEPSP ϳ50 to 70% of the maximal amplitude. The fEPSP was
analyzed using pClampfit to calculate the slope of the fEPSP. The first
six consecutive slope values were averaged to define the baseline fEPSP
slope value for a given experiment. The subsequent fEPSP slopes were
normalized to the baseline fEPSP slope value and expressed as a per-
centage. Six averaged consecutive fEPSP slopes after 12 min of appli-
cation of DCG-IV were averaged and defined as the value for applying
DCG-IV. All normalized fEPSP slopes for each concentration of DCG-IV
were then expressed as the mean Ϯ S.E.M.
Elevated plus maze studies were conducted using an automated
elevated plus maze constructed from black Plexiglas using a design
validated by Lister (1987). The maze (Hamilton-Kinder, Poway, CA)
consisted of two open arms (37.5 ϫ 5 ϫ 0.3 cm) and two closed arms
(37.5 ϫ 5 ϫ 15 cm), which extended from the central platform (5 ϫ 5
cm). The maze was elevated 63 cm above the floor. Each arm was
outfitted with four infrared photobeam sensors. Automated software
recorded the number of entries and time spent in the open and closed
arms of the plus maze by analyzing photobeam breaks using Motor
Monitor software (Hamilton-Kinder). A modified zone map was used.
The central area was extended to include the first 2.5 cm of each arm;
thus the arm area was defined as the most distal (35 cm). An arm
entry was recorded when the mouse entered the distal part of the
arm based on Lister (1987).
Procedure. Amphetamine- and PCP-induced hyperlocomotor ac-
tivity. Experiments were conducted every 3 to 4 days in the same
mice using a within-subject counterbalanced design. Specifically, for
each session, half of the mice received an acute injection of vehicle
(10 ml/kg i.p.) and the remaining half received a dose of BINA (10
and 32 mg/kg i.p.). Animals were placed in the open field for 60 min
(habituation period) and, thereafter, mice received amphetamine
(3.2 mg/kg s.c.) or PCP (5.6 mg/kg s.c.), and locomotor activity was
measured for an additional 120 min. These pretreatment times for
amphetamine and PCP were chosen based on previous open field
studies (Galici et al., 2005). The duration of action of BINA (32 mg/kg
i.p.) was evaluated in the same mice used for the acute studies by
administering BINA at various pretreatment times, specifically 0, 1,
4, 8, or 16 h before PCP administration.
To evaluate the selectivity of the observed behavioral effects of
BINA, pretreatment with a dose of BINA (32 mg/kg i.p.) or vehicle for
40 min, was followed by a dose of the orthosteric mGluR2/3 antago-
nist LY341495 (3 mg/kg, i.p.) before the end of the 60-min habitua-
tion period. Thereafter, mice received PCP (5.6 mg/kg s.c.), and
locomotor activity was evaluated for an additional 120 min.
In a separate experiment, the effects of BINA (10 and 32 mg/kg
i.p.) on locomotor activity when administered alone were assessed
using a between-subject design in nonhabituated naive mice. In this
experiment, BINA was given 60 min before the habituation period,
and locomotor activity was studied for 180 min. For all of the loco-
motor activity studies, changes in locomotor activity were expressed
as the average number of counts or photobeam breaks Ϯ S.E.M.
PCP-induced disruption of PPI of startle reflex. The effects of
Behavioral Studies
Subjects. All behavioral studies were conducted using C57BL6/J
male mice (Charles River Laboratories, Inc., Wilmington, MA) 8 to
10 weeks of age. Subjects were housed in groups of four to five per
cage in a large colony room under a 12-h light/dark cycle (lights on at
6:00 a.m.) with food and water provided ad libitum. Test sessions BINA (32 and 100 mg/kg i.p.) on PCP-induced (5.6 mg/kg s.c.) dis-
were performed between 6:00 a.m. and 6:00 p.m. All dose groups ruption of PPI was studied using a within-subject design. Eight
consisted of 6 to 12 mice. All experiments were conducted in accor- different treatment conditions were assigned to 16 naive mice in
dance with the National Institutes of Health regulations of animal each session. Vehicle and BINA were administered 60 min before

BINA, a Positive Allosteric Modulator of mGluR2
177
whereas water and PCP were given 15 min before the beginning of
the session. These pretreatment times were chosen according to
published open field studies in the literature (Geyer and Ellenbroek,
2003). Each session started with a 5-min acclimatization period,
during which a 65-dB background noise was continuously present,
and included a total of 54 trials. Six different trial types were ran-
domly assigned and delivered (every 15–20 s on average) for 9 times
throughout the session: 40-ms broadband 120-dB burst (pulse only),
65-dB background noise (noise only), and 20-ms prepulse of 70, 76,
82, and 88 dB followed by 100 ms of a 120-dB pulse. Sessions were
conducted every 3 to 4 days.
Statistical Analysis
For the calcium mobilization assay, all data were normalized to
percentage glutamate or L-AP4 maximum defined by 10 ␮M gluta-
mate or L-AP4 for each cell line, respectively. For the [35S]GTP␥S
binding assay, all data were normalized to percent glutamate max-
imum defined as 10 ␮M glutamate. Data analysis was performed
using GraphPad Prism 3.0 software (GraphPad Software, Inc., San
Diego, CA). Concentration-response curves were generated from the
mean data of at least three independent experiments. Error bars
represent Ϯ S.E.M. For the electrophysiology experiments, data
were analyzed using Clampfit version 9.2 (Molecular Devices). All
results are expressed as means Ϯ S.E.M., and statistical significance
was determined using Student’s t test. For the locomotor activity
time course and antagonist studies, data were analyzed using a
repeated measures ANOVA with treatment and time (i.e., 120 min
postinjection) as within-group factors followed by analyses of simple
main effects and, when appropriate, post hoc analysis with least
significant difference test. To quantify the potency and duration of
action of BINA, locomotor activity data were calculated as the aver-
age counts/5 min that occurred in the first 60 min postinjection (i.e.,
after habituation time) and were expressed as a percentage of control
(i.e., average of two determinations of PCP effects by itself). ED50
values were calculated by interpolation (only two data points were
available) for each mouse and were expressed as mean Ϯ S.E.M.
Startle amplitude data were expressed as the mean value Ϯ S.E.M.
during presentation of the background noise only (i.e., 65 dB) and the
pulse alone (i.e., 120 dB) and were analyzed with one-way repeated
measures ANOVA. Levels of PPI were determined by the formula
(100 Ϫ [(prepulse pulse/pulse alone) ء 100]) and were expressed as
percentage PPI Ϯ S.E.M. Data were analyzed using repeated mea-
sures ANOVA with the prepulse intensity and treatment as within-
group factors, followed by analyses of simple main effects and, when
appropriate, post hoc analysis with the least significant difference
test. In the stress-induced hyperthermia and elevated plus maze
tests, data were expressed as means Ϯ S.E.M. Treatment groups
were compared with the appropriate control groups using a one-way
ANOVA and Dunnett’s t test. Statistical analyses were performed
using JMP version 4.04 statistical software (SAS Institute, Cary,
NC). For all behavioral data, a probability of p Յ 0.05 was taken as
the level of statistical significance.
Stress-induced hyperthermia. For the stress-induced hyperther-
mia experiments, a between-subject design was used. Mice were
group housed until the day before the experiment and then singly
housed overnight. On the test day, mice were pretreated with vehicle
or a dose of either BINA (10 or 32 mg/kg i.p.) or the benzodiazepine
chlordiazepoxide (2.5, 5, or 10 mg/kg i.p.) for 1 h before the beginning
of the experiment. To evaluate the selectivity of BINA, mice were
pretreated with a dose of LY341495 (3 mg/kg i.p.) 30 min before a
dose of 32 mg/kg BINA i.p. or vehicle and then were tested 60 min
later. With the use of a modified method from Van der Heyden et al.
(1997) mice were brought into the testing room one at a time, and
baseline temperature was taken by inserting a thermistor probe,
dipped into mineral oil, 2 cm into the rectum for 20 s to obtain core
body temperature (T1). After a 15-min interval, core body tempera-
ture for each mouse was measured a second time (T2). Stress-induced
hyperthermia was calculated as the change in core body temperature
between the first and second temperature readings (⌬T ϭ T2 Ϫ T1).
Elevated plus maze. For the elevated plus maze experiments, a
between-subject design was used. On the test day, mice were pre-
treated with vehicle or a dose of either BINA (10 or 32 mg/kg i.p.) or
chlordiazepoxide (2.5, 5, or 10 mg/kg i.p.) and then acclimated in the
testing room for 1 h. A 5-min test session began by placing each
mouse on the central platform facing the open arm of the elevated
plus maze. After each session, the maze was cleaned with 70%
ethanol before the next mouse was tested. Data are expressed as
either the time spent in the open arms as a percentage of the total
testing time, the total number of open arm entries, or the total
number of photobeam breaks or ambulations. To evaluate the selec-
tivity of BINA, mice were pretreated with a dose of LY341495 (3
mg/kg i.p.) 30 min before a dose of 32 mg/kg BINA i.p. or vehicle and
then tested 60 min later.
Results
Drugs
Synthesis of BINA. BINA was synthesized through a
All reagents and solvents for the synthesis of BINA were obtained nine-step reaction pathway as outlined in Fig. 1 using an
from Sigma-Aldrich (St. Louis, MO). All cell culture reagents were
obtained from Invitrogen. G418 sulfate was obtained from Mediatech
(Herndon, VA). Methotrexate was obtained from Calbiochem (La
Jolla, CA). [35S]GTP␥S (1250 Ci/mmol), Unifilter-96 GF/B plates,
and MicroScint-20 were obtained from PerkinElmer Life Sciences
(Boston, MA). Unlabeled GTP␥S was obtained from Sigma-Aldrich
(St. Louis, MO). Saponin was obtained from Fluka (Buchs, Switzer-
land). EDTA was obtained from Mallinckrodt Baker, Inc. (Phillips-
burg, NJ). L-Glutamic acid, l-AP4, and LY341495 were obtained from
Tocris Cookson Inc. (Ellisville, MO). Probenecid, L-proline, PCP hy-
alternate synthetic route from that by Bonnefous et al.
(2005). The spectral data of BINA and its precursors were
consistent with the structure of BINA.
BINA Induces a Selective Allosteric Potentiation of
mGluR2 in Recombinant Systems. Activation of mGluR2
was assessed by measuring glutamate-induced increases in
intracellular calcium in CHO cells expressing hmGluR2 co-
expressed with the G protein (Gqi5). Consistent with the
effects of other allosteric potentiators of mGluR2, increasing
drochloride, chlordiazepoxide hydrochloride, and amphetamine hy- concentrations of BINA induced parallel leftward shifts of
drochloride were obtained from Sigma-Aldrich.
the glutamate concentration-response curves in the hm-
GluR2 CHO cells with no increase in the maximal response
to glutamate (Fig. 2A). The EC50 value of glutamate in the
presence of a maximally effective concentration of BINA was
108.0 Ϯ 15.2 nM. Thus, BINA induced a maximal change in
the glutamate EC50 of approximately 11-fold (Fig. 2A).
To assess the potency of BINA, we determined the effects of
increasing concentrations of BINA on the response to an
EC20 concentration (200–300 nM) of glutamate in CHO cells
expressing hmGluR2/Gqi5 (Fig. 2B). BINA produced a maxi-
For the electrophysiology experiments, BINA was dissolved in
DMSO and then diluted to the appropriate concentration using
ACSF. LY341495 and DCG-IV were dissolved in double-deionized
water with 1 N sodium hydroxide. For behavioral experiments, BINA
was dissolved in 10% Tween 80 and 1% lactic acid (Sigma-Aldrich),
and the pH was adjusted with 1 N sodium hydroxide (Mallinckrodt,
St. Louis, MO). PCP, amphetamine, chlordiazepoxide, and LY341495
were dissolved in double-deionized water. Doses refer to the form of
the drug listed. All drugs were administered i.p. in a volume of 10
ml/kg.

178
Galici et al.
Fig. 2. A, concentration-response curves for glutamate in the absence or presence of increasing concentrations of BINA. B and C, concentration-
response curves of BINA in the presence of a submaximal concentration (EC20) of glutamate in CHO cells stably expressing hmGluR2 (B) and rmGluR2
(C). Cells were preincubated with vehicle or BINA for 5 min before the addition of a submaximal concentration (EC20) of glutamate or increasing
concentrations of glutamate. Calcium mobilization was measured using FLEXstation-II. The fluorescence responses were normalized as a percentage
of the maximal response to glutamate (10 ␮M) and are the means Ϯ S.E.M. of three independent experiments performed in triplicate.
mal potentiation of approximately 3.5-fold, with an EC50 in the presence of 300 nM BINA. Interestingly, in the ab-
value of 33.2 Ϯ 4.9 nM (Fig. 2B). Similar to the potentiation sence of glutamate, BINA also increased [35S]GTP␥S binding
observed in hmGluR2, BINA also produced a concentration- in membranes expressing hmGluR2. In contrast, BINA had
dependent potentiation of the glutamate response of no effect on glutamate-induced [35S]GTP␥S binding in
HEK293A cells expressing rmGluR2/Gqi5 with a maximal rmGluR3 (Fig. 3B). Moreover, BINA had no effect on gluta-
potentiation of approximately 6-fold and an EC50 value of mate-induced activation of the other mGluR subtypes tested,
98.0 Ϯ 8.2 nM (Fig. 2C).
including rmGluR1a (Fig. 4A), rmGluR5a (Fig. 4B), or
To determine the selectivity of BINA for mGluR2 relative hmGluR4 (Fig. 4C) using a calcium mobilization assay. Fur-
to that of other mGluR receptor subtypes, we assessed the thermore, application of BINA alone did not elicit a calcium
effects of this compound on glutamate-induced activation of response in any of the other cell lines tested. These present
mGluR3 as well as members of the other mGluR subgroups. data suggest that BINA is a potent and highly selective
In particular, the ability of BINA to shift glutamate-induced allosteric potentiator of mGluR2.
[
35S]GTP␥S binding in membranes expressing either
BINA Potentiates Agonist-Induced Inhibition of Ex-
hmGluR2 or rmGluR3 was measured (Fig. 3, A and B). In- citatory Synaptic Transmission at the Medial Per-
creasing concentrations of BINA produced a leftward shift in forant Path-Dentate Gyrus Synapse in Rat Hippocam-
the glutamate-induced [35S]GTP␥S binding in the hmGluR2 pal Slices. Previous studies have demonstrated that group
with a maximal potentiation of approximately 10-fold, result- II mGluRs, located presynaptically within the MPP, inhibit
ing in an EC50 value for glutamate of 0.50 Ϯ 0.0 ␮M (Fig. 3A) transmission at the MPP-dentate gyrus (DG) synapse by
Fig. 3. A, and B, concentration-re-
sponse curves for glutamate-induced
[
35S]GTP␥S binding to membranes
prepared from recombinant cell lines
expressing either hmGluR2 (A) and
rmGluR3 (B) in the absence or pres-
ence of 30 or 300 nM BINA.
[
A
35S]GTP␥S binding assay was incu-
bated at 30°C for 60 min, in which
the assay mixtures contained 10 ␮g
of membrane protein, BINA, gluta-
mate, and 0.1 nM [35S]GTP␥S. Data
were normalized as a percentage of
the maximal response to 1 mM or
31.6 ␮M glutamate for hmGluR2 and
rmGluR3, respectively. The means of
three individual experiments per-
formed in triplicate are shown, and
error bars represent S.E.M.

BINA, a Positive Allosteric Modulator of mGluR2
179
Fig. 4. A–C, concentration-response curves for glutamate in the absence or presence of 300 nM or 1 ␮M BINA in cell lines expressing rmGluR1a (A),
rmGluR5a (B), or hmGluR4 (C). Calcium mobilization was measured using FLEXstation-II, and the signal amplitude was normalized as a percentage
of the maximal response to glutamate or L-AP4 (10 ␮M). The means of three individual experiments performed in triplicate are shown, and error bars
represent S.E.M.
reducing glutamate release from presynaptic terminals locomotor activity, significant at a dose of 32 mg/kg i.p. (Fig.
(Macek et al., 1996, 1998). Furthermore, Schaffhauser et al.
(2003) reported that the mGluR2 positive allosteric modula-
tor LY487379 potentiates the effects of group II mGluR
agonists at this synapse. These previous findings at the
MPP-DG synapses provide an excellent response for deter-
mining whether BINA potentiates activation of native
mGluR2 at an identified synapse. Thus, extracellular field
recordings were performed in the middle third of the molec-
ular layer of the DG to determine whether BINA had effects
on synaptic transmission at the MPP-DG synapse. As re-
ported previously (Macek et al., 1996), bath application of the
group II mGluR agonist DCG-IV reduced MPP excitatory
synaptic responses in a concentration-dependent manner
(Fig. 5A), with an inhibition of 70.3% when the concentration
of DCG-IV was 3 ␮M (n ϭ 18). The observed effects of
DCG-IV were blocked using the group II mGluR antagonist
LY341495 (1 ␮M) (Macek et al., 1998) (data not shown). The
effect of BINA was evaluated using a concentration of
DCG-IV (100 nM) that induced a submaximal inhibition of
the fEPSP slope (18.9 Ϯ 2.6% inhibition; n ϭ 8) (Fig. 5, B and
C). Consistent with the effects of BINA observed in the re-
combinant systems, BINA (1 ␮M) significantly potentiated
the effect of DCG-IV on inhibition of the fEPSP slope (33.1 Ϯ
3.9% inhibition; n ϭ 8) (Fig. 5, B and C).
BINA Blocks PCP-Induced Hyperlocomotor Activity.
The finding that BINA is a highly selective allosteric poten-
tiator of mGluR2 with activity in native systems is encour-
aging in light of previous studies suggesting that this com-
pound crosses the blood-brain barrier (Rodriguez et al., 2004;
Bonnefous et al., 2005). We next performed a series of studies
to determine whether BINA could mimic the effects of group
II mGluR agonists in animal models used to predict potential
antipsychotic-like and anxiolytic activity. The effects of BINA
on PCP-induced hyperlocomotor activity were determined
using C57BL6 mice (Fig. 6, A–D). A two-way repeated mea-
sures ANOVA indicated that there was a significant effect of
dose [F(4,20) ϭ 4.9, p Ͻ 0.05]. Post hoc analysis indicated
that PCP (5.6 mg/kg s.c.) significantly increased locomotor
activity compared with the vehicle control group (Fig. 6A).
6A). The ED50 value of BINA for blocking the effects of PCP
was 16.8 Ϯ 2.3 mg/kg (Fig. 6B).
The effects of BINA on the PCP-induced hyperlocomotor
activity were reversed with pretreatment of the mGluR2/3
antagonist LY341495 (3 mg/kg i.p.), indicating that the ef-
fects of BINA were mediated through activation of mGluR2
(Fig. 6A). LY341495 (3 mg/kg i.p.) administered alone had no
effects on the PCP-induced hyperlocomotor activity. Two-way
repeated measures ANOVA also indicated that there was a
time effect (Fig. 4A) [F(35,175) ϭ 17.6, p Ͻ 0.05] and a
treatment ϫ time interaction (Fig. 6A) [F(140,700) ϭ 2.01,
p Ͻ 0.05]. BINA produced a modest decrease in locomotor
activity when administered alone, significant at 32 mg/kg i.p.
[F(2,26) ϭ 4.8, p Ͻ 0.05] (Fig. 6C). There was also a signifi-
cant time effect (Fig. 6C) [F(35,910) ϭ 34.6, p Ͻ 0.05] and a
treatment ϫ time interaction (Fig. 6C) [F(70,910) ϭ 2.7].
We next determined the time course for the effects of a
maximally effective dose of BINA (32 mg/kg i.p.) on PCP-
induced hyperlocomotor activity to determine the duration of
action of this compound. BINA had no significant effect when
it was coadministered with PCP but had a robust effect at a
1-h pretreatment time. This finding suggests that this com-
pound may not fully reach the site of action within the 1st
min of administration, but is fully active by 1 h (Fig. 6D).
Importantly, BINA had a relatively long duration of action
and produced a significant blockade of PCP-induced hyper-
locomotor activity that persisted for at least 8 h after admin-
istration (Fig. 6D) [F(6,36) ϭ 8.7, p Ͻ 0.05]. However, BINA
had no significant effect when pretreated 16 h before PCP.
BINA Has No Effect on Amphetamine-Induced Hy-
perlocomotor Activity. We next determined the effects of
BINA on amphetamine-induced hyperlocomotor activity, an-
other response that has been shown to be sensitive to the
effects of group II mGluR agonists (Cartmell et al., 2000) and
the mGluR2 allosteric potentiator LY487379 (Galici et al.,
2005). Interestingly, BINA had no effect on amphetamine-
induced hyperlocomotor activity after coadministration (data
not shown) or a 60-min pretreatment time (Fig. 7). Thus,
BINA produced a robust blockade of the PCP-induced hyper- BINA does not share all of the in vivo activities of group II

180
Galici et al.
induced disruption of PPI, which was significant across all
prepulse intensities tested after 100 mg/kg s.c. The effect of
BINA was also significant with the 82- and 88-dB prepulse
intensities after 32 mg/kg s.c. (Fig. 8). Two-way repeated
measures ANOVA indicated that there was a treatment ef-
fect (Fig. 8) [F(7,77) ϭ 11.98, p Ͻ 0.05]. In addition, there was
a prepulse intensity effect (Fig. 8) [F(3,33) ϭ 45.8, p Ͻ 0.05],
but no treatment ϫ prepulse intensity interaction. One-way
ANOVA also indicated that PCP significantly increased the
startle amplitude when only the background noise (i.e., 65
dB) was presented (Table 1) [F(7,77) ϭ 7.74, p Ͻ 0.05).
However, there was no effect on startle amplitude when the
startle pulse alone (120 dB) was presented under any of the
treatment conditions (Table 1).
BINA Decreases Stress-Induced Hyperthermia in
Mice. In addition to effects in animal models used to predict
antipsychotic efficacy, group II mGluR agonists are also ef-
fective in multiple animal models of anxiolytic drug activity
(for review, see Swanson et al., 2005). More importantly,
mGluR2/3 agonists have been shown to have anxiolytic
activity in multiple clinical studies (Grillon et al., 2003;
Schoepp et al., 2003; Swanson et al., 2005). Thus, it is critical
to determine whether allosteric potentiators of mGluR2 have
activity similar to that of mGluR2/3 agonists in these animal
models. We determined the effect of BINA on stress-induced
hyperthermia,
a response that is highly sensitive to
mGluR2/3 agonists (Spooren et al., 2002; Swanson et al.,
2005). BINA produced an inhibition of stress-induced hyper-
thermia, as evidenced by a main effect for dose [F(3,56) ϭ
34.1; p Ͻ 0.05], significant after doses of 32 mg/kg i.p. by a
Dunnett’s comparison with the vehicle group (Fig. 9A). The
effects of BINA (32 mg/kg i.p.) on stress-induced hyperther-
mia were blocked by pretreatment with a dose of LY341495
(3 mg/kg i.p.), indicating that the effects were mediated by
activation of mGluR2 (Fig. 9A). Larger doses of BINA pro-
duced substantial decreases in basal body temperature,
which precluded further evaluation in the stress-induced
hyperthermia model. For comparison, the effects of the ben-
zodiazepine chlordiazepoxide were also evaluated in the
stress-induced hyperthermia model. Chlordiazepoxide pro-
duced a dose-dependent inhibition of the stress-induced hy-
perthermia as evidenced by a main effect for dose [F(3,82) ϭ
25.6; p Ͻ 0.05], significant after 5 and 10 mg/kg i.p. by a
Dunnett’s comparison with the vehicle group (Fig. 9B).
BINA Produces Anxiolytic-Like Effects in the Ele-
vated Plus Maze Test. Anxiolytic effects of BINA were also
evaluated in the elevated plus maze, another animal model
that is sensitive to anxiolytic agents. In the elevated plus
maze test, BINA increased the percentage of time spent in
the open arms of the maze as evidenced by a main effect for
dose [F(3,39) ϭ 3.4; p Ͻ 0.05], significant after a dose of 32
mg/kg i.p. by a Dunnett’s comparison with the vehicle group
Fig. 5. BINA potentiates the effect of DCG-IV on fEPSPs at MPP-DG
synapses in rat hippocampal slices. A, concentration-response curves for
the inhibitory effect of DCG-IV on fEPSP slopes by activation of group II
mGluR at MPP-DG synapses (n ϭ 50 slices). B, representative traces
show the recorded fEPSPs before and after 12 min of application of DCG-
IV alone (100 nM, left); or DCG-IV (100 nM) in the presence of BINA (1
␮M, right). Each trace is an average of six consecutive evoked responses
at MPP-DG synapses. C, bar graph illustrates normalized fEPSP slopes
at MPP-DG synapses after a 12-min application of DCG-IV alone (100
nM, n ϭ 8 slices) or DCG-IV (100 nM) in the presence of BINA (1 ␮M, n ϭ
8 slices). All values are expressed as means Ϯ S.E.M., Calibration bar, 0.2
mV/5 ms. ء, p Ͻ 0.01; Student’s t test.
mGluR agonists or LY487379 in models used to assess the (Fig. 10A). In addition, BINA also increased the number of
activity of potential antipsychotic agents. entries into the open arms of the maze after doses of 10 or 32
BINA Blocks PCP-Induced Disruption of PPI. An- mg/kg i.p. by a Dunnett’s comparison with the vehicle group
other animal model that is often used to assess the potential (Fig. 10C). When administered alone, BINA had no effect on
antipsychotic activity of novel compounds is the reversal of total ambulations (Fig. 10E). The increased entries and time
PCP-induced disruption of PPI of the acoustic startle re- spent in the open arms of the elevated plus maze after ad-
sponse (Geyer and Ellenbroek, 2003). Consistent with previ- ministration of BINA (32 mg/kg i.p.) were blocked with pre-
ous studies, PCP (5.6 mg/kg s.c.) produced a robust decrease treatment of LY341495 (3 mg/kg i.p.), indicating that these
in PPI across all of the prepulse intensities tested (Fig. 8). effects were mediated through the activation of mGluR2 (Fig.
Pretreatment with BINA produced a robust blockade of PCP- 10, A and C). For comparison, the effects of the chlordiazep-

BINA, a Positive Allosteric Modulator of mGluR2
181
Fig. 6. Effects of BINA on PCP-in-
duced hyperlocomotor activity and
spontaneous locomotor activity in
mice. A, BINA blocked the effects of
PCP-induced hyperlocomotor activity.
B, potency of BINA in blocking PCP-
induced hyperlocomotor activity. Av-
erage counts/60 min, expressed as the
mean percentage Ϯ S.E.M of the av-
erage of two determinations of PCP
effects (i.e., percentage control), are
plotted as a function of BINA dose
expressed in milligrams per kilogram.
C, BINA administered alone (32
mg/kg i.p.) decreased spontaneous lo-
comotor activity. Data are expressed
as means Ϯ S.E.M. D, time course
effects of BINA on PCP-induced hy-
perlocomotor activity in mice. BINA
was administered with (i.e., at time
0 h) and 1, 4, 8, and 16 h before PCP
treatment. Vertical lines represent
Ϯ S.E.M. and are absent when less
than the size of the point. (n ϭ 7–8
per dose) Ordinate, average counts/5
min. Abscissa, time in minutes. ء, p Ͻ
0.05 indicates statistical difference
from vehicle (V). ءء, p Ͻ 0.05 indicates
statistical difference from PCP (5.6
mg/kg).
Discussion
We report an alternate synthesis and further pharmaco-
logical characterization of the previously reported positive
allosteric modulator of mGluR2, termed here biphenyl-in-
danone A (BINA). Derived from a series of substituted in-
danones (Bonnefous et al., 2005; Pinkerton et al., 2005),
BINA is structurally and chemically distinct from both group
II mGluR agonists and the mGluR2 positive allosteric mod-
ulator LY487379. In recombinant systems, BINA is a potent
and highly selective potentiator of the response of mGluR2 to
glutamate with no effect on the glutamate response of the
other mGluR receptor subtypes tested. At native mGluR2,
BINA functions as a positive allosteric modulator in a man-
ner similar to its actions on recombinant mGluR2. Most
encouraging, BINA produces robust and long-lasting effects
in mouse behavioral models of antipsychotic- and anxiolytic-
like activity that mimic previously observed effects using
group II mGluR agonists. Taken together, our findings dem-
Fig. 7. BINA had no effect on amphetamine-induced hyperlocomotor
activity in mice. Data are expressed as means
counts/60 min are plotted as a function of time in hours (n ϭ 7–8 per
condition). Vertical lines represent Ϯ S.E.M. and are absent when less
than the size of the point. Ordinate, average counts/5 min. Abscissa, time
in minutes.
Ϯ
S.E.M. Average onstrate that BINA is a selective tool for dissecting the role of
mGuR2 in the observed effects of group II mGluR agonists in
vivo.
The development of subtype-selective allosteric modulators
for the activation of mGluRs offers many potential advan-
tages over direct-acting agonists, including greater subtype-
selectivity maintenance of activity dependent receptor acti-
vation. The discovery of LY487379 and related analogs as
selective positive allosteric modulators of mGluR2 (Johnson
et al., 2003, 2005; Schaffhauser et al., 2003) represented an
important breakthrough in the field of mGluR research.
However, modest potency and a short duration of action limit
the utility of these compounds for in vivo studies (Johnson et
al., 2003; Galici et al., 2005). The present findings confirm
and extend previously reported studies showing that BINA, a
member of a novel series of mGluR2 potentiators (Bonnefous
et al., 2005; Pinkerton et al., 2005), is highly selective and
oxide were also evaluated in the elevated plus maze test.
Chlordiazepoxide increased the percentage of time spent in
the open arms of the maze as evidenced by a main effect for
dose [F(3,39) ϭ 4.8; p Ͻ 0.05], significant after doses of 10
mg/kg i.p. by a Dunnett’s comparison with the vehicle group
(Fig. 10B). In the dose range tested, chlordiazepoxide also
produced an increase in the number of entries into the open
arms of the maze, significant after doses of 5 and 10 mg/kg
i.p. by a Dunnett’s comparison with the vehicle group (Fig.
10D). After administration alone, chlordiazepoxide produced
a modest increase in total ambulations, significant with a
dose of 10 mg/kg (Fig. 10F).

182
Galici et al.
Fig. 8. BINA blocked PCP-induced disruption of PPI in
mice. Data are expressed as means Ϯ S.E.M. Percentage
PPI is plotted as a function of prepulse intensity (decibels)
used. ء, p Ͻ 0.05 indicates statistical difference from vehi-
cle (V). ءء, p Ͻ 0.05 indicates statistical difference from
PCP (PCP; 5.6 mg/kg). Vertical lines represent Ϯ S.E.M.
Ordinate, percentage prepulse inhibition. Abscissa, pre-
pulse intensity amplitude (decibels) (n ϭ 7–8 per dose
group).
TABLE 1
Effects of BINA on startle amplitude of the acoustic startle reflex
Data are expressed as means Ϯ S.E.M. when the background noise stimulus (65 dB) or the pulse stimulus (120 dB) was presented alone.
Startle Amplitude
Treatment Dose
65
120
dB
Vehicle Ϯ water
200.4 Ϯ 28.6
211.9 Ϯ 31.1
100.1 Ϯ 10.5
418.5 Ϯ 80.0*
259.9 Ϯ 37.9
117.3 Ϯ 17.1
151.9 Ϯ 13.7
249.4 Ϯ 17.5
765.3 Ϯ 70.2
817.3 Ϯ 101.7
766.9 Ϯ 73.4
666.4 Ϯ 59.0
721.2 Ϯ 49.9
654.8 Ϯ 106.5
590.0 Ϯ 74.2
548.4 Ϯ 67.5
BINA (32 mg/kg i.p.) Ϯ water
BINA(100 mg/kg i.p.) Ϯ water
Vehicle Ϯ PCP (5.6 mg/kg i.p.)
BINA (32 mg/kg i.p.) Ϯ PCP (5.6 mg/kg i.p.)
BINA (100 mg/kg i.p.) Ϯ PCP (5.6 mg/kg i.p.)
BINA (100 mg/kg i.p.) Ϯ LY341495 (3 mg/kg i.p.) Ϯ PCP (5.6 mg/kg i.p.)
Vehicle Ϯ LY341495 (3 mg/kg i.p.) Ϯ PCP (5.6 mg/kg i.p.)
* P Ͻ 0.05 compared with control (i.e., vehicle Ϯ water).
Fig. 9. BINA inhibited stress-induced
hyperthermia in mice. The dose-re-
lated effects of BINA and the benzodi-
azepine chlordiazepoxide (CDP) were
evaluated on stress-induced hyper-
thermia. The dose of the mGluR2/3
antagonist LY341495 used was 3 mg/
kg. Data are expressed as means Ϯ
S.E.M. Bar graphs above Veh repre-
sent the effects of vehicle alone. Ver-
tical lines represent Ϯ S.E.M. (n ϭ
10–12 mice per dose group). Abscissa,
dose of drug in milligrams per kilo-
gram. Ordinate, ⌬T (degrees Centi-
grade). ء, p Ͻ 0.05 versus vehicle,
Dunnett’s t test.
has robust in vivo activity, thereby providing an important mGluR2 agonist activity in this assay. It is unlikely that this
tool for investigating the positive allosteric modulation of is due to potentiation of the response to low concentrations of
mGluR2.
glutamate present in the medium because the [35S]GTP␥S
In addition to inducing a leftward shift of the glutamate binding assay is performed in a washed membrane prepara-
concentration response curve, BINA induced a modest acti- tion in which glutamate should not be present. This direct
vation of mGluR2 in the absence of glutamate when activation of mGluR2 by BINA is reminiscent of the previ-
[
35S]GTP␥S binding was measured in membranes from cells ously reported effects of 3-cyano-N-(1,3-diphenyl-1H-pyrazol-
expressing mGluR2. Thus, BINA has modest intrinsic 5-yl)benzamide, a positive allosteric modulator of mGluR5

BINA, a Positive Allosteric Modulator of mGluR2
183
Fig. 10. BINA produced anxiolytic ef-
fects in the elevated plus maze test in
mice. The dose-related effects of BINA
and the benzodiazepine chlordiazep-
oxide were evaluated on time spent in
open arms as a percentage of total
testing time (A and B), total number
of entries into the open arms (C and
D), or total ambulations (E and F).
Bar graphs above ϮLY341495 (A, C,
and E) represent the effects of the
mGlu2/3 antagonist LY341495 (3 mg/
kg) given in combination with a 32
mg/kg dose of BINA. Data are ex-
pressed as means
Ϯ S.E.M. Bar
graphs above Veh represent the ef-
fects of vehicle alone. Vertical lines
represent Ϯ S.E.M. Abscissa, dose of
drug in milligrams per kilogram (n ϭ
10–12 mice per dose group). Ordinate,
percentage of time in the open arms,
total number of entries into the open
arm, or total ambulations (total num-
ber of beam breaks/5 min). ء, p Ͻ 0.05
versus vehicle, Dunnett’s t test.
that also exhibits modest intrinsic agonist activity at within the MPP-DG synapse is one region of the CNS
mGluR5 (Kinney et al., 2005). Furthermore, previous studies thought to be associated with the pathogenesis of anxiety and
have yielded direct allosteric agonists for mGluR7 (Mit- fear conditioning (Walker and Davis, 2002; Bergink et al.,
sukawa et al., 2005) and the M1 muscarinic receptor (Spal- 2004). Thus, our data provide important evidence to further
ding et al., 2002; Sur et al., 2003). However, it is not yet clear support the role for mGluR2 in the physiological responses
whether this direct activation of mGluR2 by BINA is relevant mediated by group II mGluR activation in systems thought to
in vivo or is an artifact of this expression system. Impor- be relevant for anxiolytic and antipsychotic action of these
tantly, BINA did not appear to have direct agonist activity in compounds.
the calcium mobilization assay or at native mGluR2 at the
In the present study, BINA also mimicked the effects of
MPP synapse. However, this does not necessarily imply that group II mGluR agonists and mGluR2 allosteric potentiators
BINA is incapable of direct activation of this receptor at other in animal models used to predict potential antipsychotic-like
synapses or in other neuronal populations.
and anxiolytic-like activity. Previous studies have shown
In addition to providing insights into the pharmacological that the N-methyl-D-aspartate receptor antagonist PCP and
properties of the positive allosteric modulation of mGluR2 in the indirect acting dopamine agonist amphetamine produce
recombinant systems, BINA also served as a valuable ligand behavioral changes in animals, such as increases in locomo-
for the study of physiological and behavioral responses me- tor activity and disruption of prepulse inhibition, which are
diated by native mGluR2. BINA potentiated mGluR2 agonist reversed by currently available antipsychotic therapies (for
effects on excitatory synaptic responses at the MPP-DG syn- review, see Geyer and Ellenbroek, 2003). Systemic adminis-
apse. Potential hyperactivty of glutamatergic transmission tration of BINA produced a dose-related inhibition of PCP-

184
Galici et al.
induced hyperlocomotor activity and disruption of PPI. For elevated plus maze after pretreatment with BINA were sim-
the most part, the effects of BINA in these models were ilar to effects observed previously with group II mGluR ago-
similar to those previously shown for group II mGluR ago- nists (Helton et al., 1998; Linden et al., 2004). BINA also
nists or the mGluR2-specific allosteric potentiator LY487379 prevented the development of stress-induced hyperthermia
(Cartmell et al., 1999; Spooren et al., 2000; Galici et al., as observed using both group II mGluR agonists and N-[4Ј-
2005). Consistent with mediation by mGluR2, each of the cyanobiphenyl-3-yl)-N-(3-pyridinylmethyl)ethanesulfonamide
effects of BINA were blocked by the mGluR2/3 antagonist hydrochloride, a structural analog of LY487379 (Johnson et
LY341495. However, unlike the short-acting LY487379 al., 2005; Rorick-Kehn et al., 2006). In both preclinical anx-
(Galici et al., 2005), the effects of BINA were long-lasting iety models, the effects of BINA were blocked by LY341495,
over a duration of at least 8 h, indicating that this compound confirming a role for the activation of mGluR2. Thus, our
is suitable for a further pharmacologic evaluation in vivo (i.e., findings extend previous studies suggesting a role for
chronic studies).
mGluR2 in anxiety disorders and support the fundamentally
Although the effects of BINA and the structurally distinct novel approach to treatment of anxiety disorders using allo-
mGluR2 potentiator LY487379 in animal models of antipsy- steric potentiators of mGluR2.
chotic-like activity were comparable, there were some impor-
In summary, BINA constitutes a highly selective positive
tant differences. For instance, BINA did not reverse amphet- allosteric modulator of mGluR2 with a long duration of action
amine-induced hyperlocomotor activity whereas, group II and robust efficacy in several preclinical models used to
mGluR agonists (Cartmell et al., 1999, 2000) and LY487379 predict anxiolytic and antipsychotic-like activity. Our find-
(Galici et al., 2005) inhibit amphetamine-induced hyperloco- ings confirm and extend a role for the mGluR2 receptor in
motor activity. Furthermore, in contrast to LY487379 (Galici many of the documented effects of mGluR2/3 agonists in vivo
et al., 2005), BINA blocked the disruptive effects of PCP on and suggest that positive allosteric modulators of mGluR2
PPI. Although the reasons for these important differences provide an important alternative to group II mGluR agonists
between the effects of BINA and LY487379 are unclear, for the treatment of anxiety disorders and schizophrenia.
previous studies revealed that different orthosteric agonists
can differentially activate different signaling pathways of a Acknowledgments
single receptor, a phenomenon referred to as agonist receptor
trafficking (Brink et al., 2000; Gazi et al., 2003). Based on
this finding, it has been suggested that structurally distinct
agonists can have different in vivo effects by actions at a
single receptor. Interestingly, we recently showed that the
same principle applies to allosteric potentiators and that
different classes of allosteric potentiators of mGluR5 can
have different effects on coupling of this receptor to activa-
tion of calcium transients versus extracellular signal-regu-
lated kinase 2 phosphorylation (Zhang et al., 2005). It is
conceivable that potentiation of mGluR2 by different classes
of allosteric modulators could have distinct effects on cou-
pling of this receptor to different cellular responses. If so, this
could lead to different in vivo effects of different classes of
allosteric potentiators. It is unlikely that the effects of BINA
and LY487379 are due to off-target activity because they are
both blocked by the group II mGluR antagonist LY341495.
Thus, the present data provide the first evidence that two
classes of allosteric potentiators of an mGluR have subtly
different effects in vivo. The combined actions of BINA re-
ported here provide further preclinical evidence that mGluR2
may play a key role in the antipsychotic-like effects of
mGluR2/3 agonists. Although it is tempting to speculate that
selective positive allosteric potentiators of mGluR2 may pro-
vide a novel therapeutic approach for the treatment of schizo-
phrenia, particularly as an important alternative to group II
mGluR agonists (Johnson et al., 2005), further characteriza-
tion of BINA in other preclinical models predictive of anti-
psychotic activity is warranted.
We thank William Nobis for excellent technical assistance.
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Address correspondence to: Dr. P. Jeffrey Conn, Professor, Director, Pro-
gram in Translational Neuropharmacology, Department of Pharmacology,
Vanderbilt University Medical Center, 23rd Ave. S. at Pierce, 417-D Preston
Research Building, Nashville, TN 37232-6600. E-mail: jeff.conn@vanderbilt.
edu