Biotransformation of a novel positive allosteric modulator of metabotropic glutamate receptor subtype 5 contributes to seizure-like adverse events in rats involving a receptor agonism-dependent mechanism

Article abstract of DOI:10.1124/dmd.113.052084

Activation of metabotropic glutamate receptor subtype 5 (mGlu5) represents a novel strategy for therapeutic intervention into multiple central nervous system disorders, including schizophrenia. Recently, a number of positive allosteric modulators (PAMs) of mGlu5 were discovered to exhibit in vivo efficacy in rodent models of psychosis, including PAMs possessing varying degrees of agonist activity (ago-PAMs), as well as PAMs devoid of agonist activity. However, previous studies revealed that ago-PAMs can induce seizure activity and behavioral convulsions, whereas pure mGlu5 PAMs do not induce these adverse effects. We recently identified a potent and selective mGlu5 PAM, VU0403602, that was efficacious in reversing amphetamine-induced hyperlocomotion in rats. The compound also induced time-dependent seizure activity that was blocked by coadministration of the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine. Consistent with potential adverse effects induced by ago-PAMs, we found that VU0403602 had significant allosteric agonist activity. Interestingly, inhibition of VU0403602 metabolism in vivo by a pan cytochrome P450 (P450) inactivator completely protected rats from induction of seizures. P450-mediated biotransformation of VU0403602 was discovered to produce another potent ago-PAM metabolite-ligand (M1) of mGlu5. Electrophysiological studies in rat hippocampal slices confirmed agonist activity of both M1 and VU0403602 and revealed that M1 can induce epileptiform activity in a manner consistent with its proconvulsant behavioral effects. Furthermore, unbound brain exposure of M1 was similar to that of the parent compound, VU0403602. These findings indicate that biotransformation of mGlu5 PAMs to active metabolite-ligands may contribute to the epileptogenesis observed after in vivo administration of this class of allosteric receptor modulators. Copyright

Full text of DOI:10.1124/dmd.113.052084

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2013/07/02/dmd.113.052084.DC1.html
1521-009X/41/9/1703–1714$25.00
DRUG METABOLISM AND DISPOSITION
http://dx.doi.org/10.1124/dmd.113.052084
Drug Metab Dispos 41:1703–1714, September 2013
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Biotransformation of a Novel Positive Allosteric Modulator of
Metabotropic Glutamate Receptor Subtype 5 Contributes to Seizure-
Like Adverse Events in Rats Involving a Receptor Agonism-
Dependent Mechanisms
Thomas M. Bridges, Jerri M. Rook, Meredith J. Noetzel, Ryan D. Morrison, Ya Zhou,
Rocco D. Gogliotti, Paige N. Vinson, Zixiu Xiang, Carrie K. Jones, Colleen M. Niswender,
Craig W. Lindsley, Shaun R. Stauffer, P. Jeffrey Conn, and J. Scott Daniels
Departments of Pharmacology (T.M.B., R.D.M., Y.Z., R.D.G., J.S.D., J.M.R., M.J.N., P.N.V., Z.X., C.M.N., P.J.C., C.K.J., C.W.L.,
S.R.S.) and Chemistry (C.W.L.),Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center,
Nashville, Tennessee
Received March 21, 2013; accepted July 2, 2013
ABSTRACT
Activation of metabotropic glutamate receptor subtype 5 (mGlu5)
effects induced by ago-PAMs, we found that VU0403602 had
represents a novel strategy for therapeutic intervention into mul- significant allosteric agonist activity. Interestingly, inhibition of
tiple central nervous system disorders, including schizophrenia.
Recently, a number of positive allosteric modulators (PAMs) of
VU0403602 metabolism in vivo by a pan cytochrome P450 (P450)
inactivator completely protected rats from induction of seizures.
mGlu5 were discovered to exhibit in vivo efficacy in rodent models P450-mediated biotransformation of VU0403602 was discovered to
of psychosis, including PAMs possessing varying degrees of produce another potent ago-PAM metabolite-ligand (M1) of mGlu5.
agonist activity (ago-PAMs), as well as PAMs devoid of agonist Electrophysiological studies in rat hippocampal slices confirmed
activity. However, previous studies revealed that ago-PAMs can agonist activity of both M1 and VU0403602 and revealed that M1
induce seizure activity and behavioral convulsions, whereas pure
mGlu5 PAMs do not induce these adverse effects. We recently
identified a potent and selective mGlu5 PAM, VU0403602, that was
efficacious in reversing amphetamine-induced hyperlocomotion in
can induce epileptiform activity in a manner consistent with its
proconvulsant behavioral effects. Furthermore, unbound brain
exposure of M1 was similar to that of the parent compound,
VU0403602. These findings indicate that biotransformation of mGlu5
rats. The compound also induced time-dependent seizure activity PAMs to active metabolite-ligands may contribute to the epilepto-
that was blocked by coadministration of the mGlu5 antagonist, genesis observed after in vivo administration of this class of
2-methyl-6-(phenylethynyl) pyridine. Consistent with potential adverse allosteric receptor modulators.
Introduction
(Olney et al., 1999; Tsai and Coyle, 2002; Conn et al., 2009; Marek
et al., 2010; Gregory et al., 2011; Vinson and Conn, 2012). To this end,
we have successfully identified potent and selective positive allosteric
modulators (PAMs) of mGlu5 that are orally active in rodent models of
psychosis and in various cognitive paradigms (Hammond et al., 2010;
Rodriguez et al., 2010; Williams et al., 2011). Recently, we described
several PAMs possessing intrinsic agonist activity that stimulate
glutamate-independent signaling (Noetzel et al., 2012). Some of these
agonist-PAM (ago-PAM) compounds induce mGlu5-dependent limbic
seizures similar to the adverse events (AEs) reported in rodents re-
ceiving intracerebral administration of the mGlu5 agonist, dihydroxy-
phenylglycine (DHPG) (Tizzano et al., 1995a,b; Rook et al., 2012).
Moreover, results from electrophysiological experiments confirm an
Initiated largely by the N-methyl-D-aspartate receptor hypofunction
hypothesis, we and others have identified metabotropic glutamate
receptor subtype 5 (mGlu5) as a potential means for therapeutic inter-
vention in central nervous system (CNS) disorders such as schizophrenia
This work was supported by the National Institutes of Health [Grants R01-
MH062646, R01-MH074953, and U01-MH087965].
Portions of this work were previously presented: Bridges T, Morrison R, Daniels
J (2012) North American Meetings of the International Society for the Study of
Xenobiotics, October 2012, Dallas, TX.
dx.doi.org/10.1124/dmd.113.052084.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: 1-ABT, 1-aminobenzotriazole; ACSF, artificial cerebrospinal fluid; AE, adverse event; ago-PAM, positive allosteric modulator with
agonist activity; AUC, area under the curve; CLint, intrinsic clearance; CNS, central nervous system; CRC, concentration-response curve; DHPG,
dihydroxyphenylglycine; DME, drug-metabolizing enzyme; DMEM, Dulbecco’s modified Eagle’s medium; DMPK, drug metabolism and pharmacokinetics;
DMSO, dimethylsulfoxide; FBS, fetal bovine serum; fEPSP, field excitatory postsynaptic potential; GIRK, G-protein–coupled inwardly rectifying potassium
channel; HEK293A, human embryonic kidney cell line 293A; HPLC, high-performance liquid chromatography; 5-HT, human serotonin; LTD, long-term
depression; mGlu5, metabotropic glutamate receptor subtype 5; MPEP, 2-methyl-6-(phenylethynyl) pyridine; NAM, negative allosteric modulation; P450,
cytochrome P450; PAM, positive allosteric modulator; PO, by mouth; RCF, relative centrifugal force; RED, rapid equilibrium dialysis; S9, subcellular
fraction containing microsomes and cytosol; SAM, silent allosteric modulation; SAR, structure-activity relationship; TFA, trifluoroacetic acid.
1703

1704
Bridges et al.
induction of epileptiform activity after treatment of rat hippocampal seizures, further implicating the role of a metabolite-ligand in the
slices with mGlu5 agonists; this effect was attenuated by an mGlu5 onset of AEs. In vivo pharmacokinetic studies revealed that both
antagonist (Lee et al., 2002; Zhao et al., 2004; Wong et al., 2005). parent VU0403602 and the metabolite permeated the CNS of rats
Collectively, our observations are consistent with recent assertions that and that the exposure of the metabolite was significantly reduced
group I mGlu5 activation can be a critical initiation step contributing to in rats that had been pretreated with ABT. An in vitro and in vivo
propagation of epileptogenic discharges in the CNS (Wong et al., 2005). appraisal of the metabolism of VU0403602 identified a principal
We previously reported on a group of highly selective mGlu5 oxidative metabolite that displayed potent ago-PAM activity.
allosteric modulators displaying favorable pharmacokinetic properties Additional studies involving systemic administration of the metab-
and PAM or ago-PAM activity as determined by Ca2+ mobilization in olite to rats resulted in a rapid onset of seizures, the severity of which
recombinant cells and in cortical astrocytes (Rook et al., 2012) (Fig. was similar to that observed after the administration of VU0403602.
1). Importantly, electrophysiological experiments indicated that ago- Subsequent in vitro electrophysiological experiments indicated that
PAMs such as VU0424465 induced prolonged long-term depression the metabolite-ligand induced prolonged LTD and epileptogenic
(LTD) and increased both the amplitude and frequency of spontaneous discharges in rat hippocampal slices, similar to those observed with
population spikes determined by extracellular field potential record- the mGlu5 agonist DHPG (Tizzano et al., 1995a,b; Lee et al., 2002;
ings in rat hippocampal slices (Rook et al., 2012). However, PAMs Zhao et al., 2004; Wong et al., 2005). Together, these findings support
that were devoid of intrinsic agonist activity (VU0360172 and the hypothesis that the mGlu5 ago-PAM VU0403602 undergoes P450-
VU0361747) did not induce epileptiform activity (Rook et al., 2012). mediated biotransformation to an active metabolite, which itself
Finally, rats receiving a systemic administration of the ago-PAM, possesses potent agonist-PAM activity and subsequently contributes
VU0424465, experienced pronounced seizures, which were com- to proconvulsant behavioral effects in vivo.
pletely prevented by pretreatment with the selective mGlu5 antagonist,
2-methyl-6-(phenylethynyl) pyridine (MPEP) (Rook et al., 2012).
We have observed that several mGlu5 PAM chemical series contain
Materials and Methods
Reagents
VU0403602 (N-cyclobutyl-5-((3-fluorophenyl)ethynyl)picolinamide) and
compounds possessing diverse modes of pharmacology, including
positive allosteric modulation, negative allosteric modulation (NAM),
and silent allosteric modulation (SAM) (Sharma et al., 2009; Lamb
et al., 2011; Lindsley, 2011; Williams et al., 2011; Wood et al., 2011).
In many cases, single-atom modifications to compounds have resulted
in a molecular switch that effectively converts PAMs to NAMs or ago-
PAMs. This propensity for pharmacological “mode-switching” repre-
sents a barrier to the optimization of mGlu5 allosteric modulators
owing to the range of drug-metabolizing enzymes (DMEs) that are
capable of introducing subtle single atom oxidations to PAMs.
Biotransformation of a PAM could result in the formation of active
metabolites capable of 1) silencing the effect (i.e., SAM) of the parent
compound, 2) activating the receptor in the absence of glutamate (ago-
PAMs), or 3) reversing the allosteric modulation (i.e., NAM) of the
parent PAM in vivo.
Herein, we describe the in vitro and in vivo pharmacology and drug
metabolism and pharmacokinetic (DMPK) profile of a selective mGlu5
ago-PAM, VU0403602, which induced pronounced dose- and time-
dependent seizures in rats, an effect that was prevented by pretreat-
ment with MPEP. Together, these data indicate an mGlu5-mediated
mechanism to the neurotoxicity observed in vivo. Interestingly, pre-
treatment of rats with the pan cytochrome P450 (P450) inactivator
1-aminobenzotriazole (ABT) effectively prevented VU0403602-induced
metabolites M1 and M2 (5-((3-fluorophenyl)ethynyl)-N-(3-hydroxycyclobutyl)
picolinamide and 5-((3-fluorophenyl)ethynyl)picolinic acid, respectively)
were prepared by the Medicinal Chemistry Laboratories of the Vanderbilt
Center for Neuroscience Drug Discovery. Potassium phosphate, ammonium
formate, formic acid, NADPH, magnesium chloride (MgCl2), adenosine 39-
phosphate 59-phosphosulfate lithium salt hydrate, uridine 59-diphosphoglu-
curonic acid trisodium salt, and 1-aminobenzotriazole (ABT) were purchased
from Sigma-Aldrich Chemical Company (St. Louis, MO). Rat hepatic sub-
cellular fractions were obtained from BD Biosciences (Woburn, MA). Rat
brain S9 fractions were obtained from Celsis In Vitro Technologies (Baltimore,
MD). Rat intestinal S9 fractions were obtained from Xenotech (Lenexa, KS).
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS),
and antibiotics were purchased from Invitrogen (Carlsbad, CA). DHPG was
obtained from Ascent Scientific (Bristol, UK). [3H]mPEPy was purchased
from PerkinElmer (Waltham, MA). 5MPEP and MPEP were synthesized as
described previously.
Chemical Synthesis
General. All NMR spectra were recorded on a Bruker 400-mHz instrument
(Bruker, Billerica, MA). 1H chemical shifts are reported in d values in parts per
million downfield from TMS (tetramethylsilane) as the internal standard in
d3-MeOH. Data are reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), integration, coupling
constant (Hz). Low-resolution mass spectra were obtained on an Agilent 1200
series 6130 mass spectrometer (Agilent, Santa Clara, CA). High-resolution mass
spectra were recorded on a Waters Q-TOF API-US (Waters, Millford, MA).
Analytical thin-layer chromatography was performed on Analtech silica gel GF
250-mm plates (Analtech, Newark, DE). Analytical high-performance liquid
chromatography (HPLC) was performed on an HP1100 with UV detection at 214
and 254 nm along with ELSD (evaporative light scattering detector) detection,
liquid chromatographic-tandem mass spectrometry analysis (LC/MS/MS)
(J-Sphere80-C18, 3.0 Â 50 mm, 4.1-minute gradient, 5%[0.05% TFA(trifluoro-
acetic acid)/CH3CN]:95%[0.05% TFA/H2O] to 100%[0.05%TFA/CH3CN].
Preparative reverse phase HPLC purification was performed on a custom
HP1100 automated purification system with collection triggered by mass
detection or using a Gilson Inc. (Middleton, WI) preparative UV-based system
using a Phenomenex Luna C18 column (50 Â 30 mm I.D. [internal diameter],
5 mm; Phenomenex, Torrance, CA) with an acetonitrile (unmodified)-water
(0.1% TFA) custom gradient. Normal-phase silica gel preparative purification
was performed using an automated Combi-flash companion from ISCO (Lincoln,
NE). Solvents for extraction, washing, and chromatography were HPLC grade.
Fig. 1. Structures of mGlu5 allosteric modulators.

Biotransformation of mGlu5 PAM to Active Agonist Metabolite
1705
All reagents were purchased from Sigma Aldrich Chemical Co. and were used %ECmax response was plotted versus the log of the molar concentration of test
without purification. All polymer-supported reagents were purchased from compound. The data were fit to a four-parameter logistic equation to determine
Argonaut Technologies (Redwood City, CA) and Biotage (Uppsala, Sweden). the minimum response, maximum response (percentage response achieved
Individual compound synthetic methods are contained in the Supplemental relative to the maximal response obtained with glutamate, % Glumax), the log
Material.
concentration giving the half-maximal response (log EC50), and the slope factor
of the curve.
Cell Culture and Mutagenesis
mGlu1 Selectivity Screening
Human embryonic kidney cell line (HEK293A) cells lines stably expressing
rat mGlu5 were maintained in complete DMEM supplemented with 10% FBS,
2 mM L-glutamine, 20 mM HEPES, 0.1 mM nonessential amino acids, 1 mM
sodium pyruvate, antibiotic-antimycotic, and G418 (500 mg/ml; Mediatech,
Manassas, VA) at 37°C in a humidified incubator containing 5% CO2, 95% O2.
Point mutations in rat mGlu5-pCl:Neo were generated using a site-directed
mutagenesis kit (Quikchange II; Agilent), and mutant plasmids were fully
sequenced to verify the desired mutations. HEK293A cells were transfected
with the mutant plasmids using Fugene6 (Promega, Madison, WI), and the cells
were maintained under 1 mg/ml G418 selection for 2 weeks to obtain the stable
cell lines expressing these mutants. Stable polyclonal mutant rat mGlu5 cells
and HEK293A cells stably expressing rat mGlu1 were maintained in the same
media as the mGlu5 wild-type cells. HEK293A cells stably expressing
G-protein–coupled inwardly rectifying potassium channels (HEK293A-GIRK)
and the individual group II and group III mGlus were maintained in growth
media containing 45% DMEM, 45% F-12, 10% FBS, 20 mM HEPES, 2 mM
L-glutamine, antibiotic/antimycotic, nonessential amino acids, 700 mg/ml
G418, and 0.6 mg/ml puromycin.
HEK293A cells stably expressing rat mGlu1 were plated in black-walled,
clear-bottomed, poly-D-lysine coated 384-well plates (Greiner Bio-One, Monroe,
NC) in assay medium at a density of 20,000 cells/well for 24 hours before the
assay. Assays were performed at the Vanderbilt University High-Throughput
Screening Center as described previously (Hammond et al., 2010; Rodriguez
et al., 2010). Calcium flux was measured using the FDSS 6000; vehicle or
a fixed concentration of test compound (10 mM) was added, followed by
a CRC to glutamate 2.5 minutes later. The change in relative fluorescence
over basal was calculated before normalization to the maximal response to
glutamate.
Group II and Group III mGlu Selectivity Screening
Test compound activity at the rat group II and III mGlus was assessed using
thallium flux through GIRK channels as previously described in detail
(Niswender et al., 2008; Hammond et al., 2010). Briefly, HEK293A-GIRK
cells expressing mGlu subtypes (2, 3, 4, 6, 7, or 8) were plated into 384-well,
black-walled, clear-bottom poly-D-lysine coated plates at a density of 15,000
cells/well in assay medium the day before the assay. On the day of the assay,
the medium was aspirated and replaced with assay buffer (Hanks’ balanced salt
solution, 20 mM HEPES, pH 7.4) supplemented with 0.16 mM Fluozin2-AM
(Invitrogen). For these assays, vehicle or fixed concentration of test compound
(10 mM) was added, followed by a CRC to glutamate (or L-AP4 in the case of
mGlu7) diluted in thallium buffer (125 mM NaHCO3, 1 mM MgSO4, 1.8 mM
CaSO4, 5 mM glucose, 12 mM thallium sulfate, 10 mM HEPES), and fluo-
rescence was measured using an FDSS 6000. Data were analyzed as described
previously (Niswender et al., 2008). The presence of activity at each receptor
was determined by comparing the fold shift imparted by the compound (EC50
of glutamate under control conditions divided by EC50 in the presence of
compound), as well as the effect on the maximum response of glutamate. A
compound was determined to be a PAM if it resulted in a fold shift of 2.0 or
greater, a NAM if it decreased the maximum response from 100% to 75% or
less, and an antagonist if it resulted in a fold shift of 0.5 or less and the
maximum response remained above 75%.
Fluorescence-Based Calcium Assays in Rat mGlu5 Cells
Measurement of mGlu5-mediated intracellular Ca2+ mobilization was per-
formed using the Ca2+-sensitive dye Fluo-4 and a Flexstation II (Molecular
Devices, Sunnyvale, CA), as described previously (Noetzel et al., 2012). HEK293A
cells expressing either mGlu5 wild-type or mGlu5 mutants were incubated
with test compound for 60 seconds before stimulation with glutamate. Base-
line fluorescence was subtracted from peak fluorescence before normalization
to the maximal peak response elicited by glutamate alone (10–100 mM). Data
were transformed and fitted using GraphPad Prism 5.0 (GraphPad Software,
Inc., San Diego, CA).
Fluorescence-Based Calcium Assays in Rat Astrocytes
Rat cortical astrocytes were plated in 384-well poly-D-lysine coated, black-
walled, clear-bottomed plates (BD Falcon, San Jose, CA) in a 20-ml volume of
astrocyte growth medium at a density of approximately 15,000–20,000 cells/
well and supplemented the following day with G5 diluted 1:100 in astrocyte
growth medium. On the day of the experiment, concentration-response curves
(CRCs) of test compounds were prepared. Stock compounds were made in
dimethylsulfoxide (DMSO) at a concentration of 10 mM. CRCs were made at
a 2Â concentration in assay buffer (Hanks’ balanced salt solution, 20 mM
HEPES, 2.5 mM probenecid, pH 7.4) to achieve final concentrations in the
assay that ranged from 0.37 nM to 30 mM in an 11-point curve and a final
DMSO concentration of 0.3%. Each curve occurred in triplicate in each plate
along with vehicle-matched controls. CRCs were generated using a Labcyte
Echo 555 employed with Labcyte Dose Response Software package (Labcyte,
Sunnyvale, CA). Calcium flux was measured using the Functional Drug
Screening System 6000 (FDSS 6000, Hamamatsu, Japan); compound alone was
added at 3 seconds to detect any agonist activity, followed by a EC20 of
glutamate at 143 seconds, and finally a EC80 glutamate at 240 seconds. A
glutamate concentration (60 mM) resulting in a maximal response was also added
in the third addition to wells not receiving compound or previous glutamate
additions. The resulting ECmax response was used to normalize test responses
during data analysis. FDSS data were analyzed using a Microsoft Excel analysis
template using Excel formulas for data reduction and IDBS XLfit (Guildford,
UK) for curve fitting. Each point was normalized to the initial value for that well
(static ratio). For each window (agonist, potentiator, antagonist), the peak static
ratio response was determined and corrected by subtraction of the minimum
response at the beginning of the data collection. Each corrected response was
then expressed as a percentage of the average baseline corrected static ratio of the
ECmax. For the agonist, potentiator, and antagonist windows, each corresponding
Ancillary Pharmacology Screening
VU0403602 was sent to Ricerca Biosciences (Painesville, OH) for testing in
their LeadProfiling Screen, a panel of ancillary pharmacology binding assays
measuring the percentage of displacement of an orthosteric radioligand at 66
distinct molecular targets. Two independent determinations were made using
10 mM test compound concentration. Significant activity was defined as .50%
inhibition of binding at any target. Additionally, a functional human serotonin
5-HT2B assay was performed using rat stomach fundus (n = 2) and a 30 mM test
compound concentration.
Radioligand Binding Assays
The allosteric antagonist MPEP analog [3H]methoxyPEPy (Cosford et al.,
2003) was used to evaluate the interaction of the test compounds with the
allosteric MPEP site on mGlu5. Membranes were prepared from HEK293A
cells stably expressing rat mGlu5. Compounds were diluted into assay buffer
(50 mM Tris and 0.9% NaCl, pH 7.4) to a 2Â stock, and 125 ml of test
compound was added to each well of a 96-well assay plate; 100-ml aliquots of
membranes diluted in assay buffer (50 mg/well) were added to each well.
Twenty-five microliters of [3H]methoxyPEPy (5 nM final concentration in
assay buffer) was added, and the reaction was incubated at room temperature
for 60 minutes with shaking. After the incubation period, the membrane-bound
ligand was separated from free ligand by filtration through glass fiber 96-well
filter plates (Unifilter-96, GF/B; PerkinElmer Life and Analytical Sciences,
Boston, MA). The contents of each well were transferred simultaneously to the

1706
Bridges et al.
filter plate and washed three times with assay buffer (Brandel cell harvester;
Brandel Inc., Gaithersburg, MD). Forty microliters of scintillation fluid was
added to each well, and the membrane-bound radioactivity was determined by
scintillation counting (TopCount; PerkinElmer Life and Analytical Sciences).
Nonspecific binding was estimated using 10 mM MPEP.
Plasma Protein Binding and Nonspecific Binding in Brain Homogenate
The extent of plasma protein binding and nonspecific binding of test com-
pounds was determined in vitro in male (Sprague-Dawley) rat plasma and brain
homogenate via rapid equilibrium dialysis (RED; ThermoFisher Scientific,
Rochester, NY). A 96 well plate containing plasma and an individual test
article (5 mM) was vortex mixed. A portion (200 ml) of the mixture was
transferred to the cis-chamber of the RED insert and dialyzed against
phosphate buffer (350 ml, 25 mM, pH 7.4) in the trans-chamber and in-
cubated (37°C) with shaking (4 hours). After the incubation, an aliquot from
each chamber was diluted (1:1; v/v) with either plasma or buffer from the cis-
or trans-chamber, respectively, and transferred to a new 96-well plate. Protein
precipitation of the matrices was executed by the addition of ice-cold
acetonitrile containing carbamazepine (50 nM) as an internal standard for
LC-MS/MS analysis. The plate was centrifuged [3000 relative centrifugal
force (RCF), 10 minutes] and the supernatants transferred to a new 96-well
plate, where they were diluted in H2O (1:1; v/v). The plate was then sealed for
LC/MS/MS analysis.
Animal Use
All animal studies and experiments were conducted in accordance with the
National Institutes of Health’s Guide for the Care and Use of Laboratory
Animals and were approved by the Institutional Animal Care and Use
Committee. Animals were housed under a 12-hour light/dark cycle with access
to food and water (ad libitum).
Electrophysiology
Extracellular Field Potential Recordings. In the LTD experiments, 4- to 6-
week-old male Sprague-Dawley rats (Charles River, Wilmington, MA) were
sacrificed and the brains quickly removed and submerged into ice-cold cutting
solution (in mM: 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 5
D-glucose, 0.6 (+)-sodium-L-ascorbate, 0.5 CaCl2, 7 MgCl2) continuously
bubbled with 95% O2/5% CO2. Then 400-mm transverse brain slices were
Hepatic Microsome Stability Assessment of VU0403602
The metabolic stability of VU0403602 was investigated in male S-D rat
made using a vibratome (Leica VT100S; Leica Microsystems, Nussloch, hepatic microsomes using substrate depletion methods (percentage of parent
Germany). Individual hippocampi were microdissected from the slice and compound remaining). In a 96-well plate, a potassium phosphate-buffered (0.1 M,
transferred to a room temperature mixture containing equal volumes of cutting pH 7.4) solution of VU0403602 (1 mM), microsomes (0.5 mg/ml), + NADPH
solution and artificial cerebrospinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl,
1.25 NaH2PO4, 25 NaHCO3, 25 glucose, 2 CaCl2, 1 MgCl2) and allowed to
(1 mM), + MgCl2 (3 mM) was incubated at 37°C under ambient oxygenation;
reactions were initiated by the addition of NADPH. At designated times (t =
equilibrate for 30 minutes. The hippocampi were then transferred to ACSF 0, 3, 7, 15, 25, and 45 minutes), an aliquot of the incubation mixture was
continuously bubbled with 95% O2/5% CO2 for a minimum of an additional removed and precipitated by the addition of two volumes of ice-cold
hour. For recordings, the slices were transferred to a submersion recording
chamber and allowed to equilibrate for 5–10 minutes at 30–32°C with a flow plates were centrifuged at 3000 RCF (4°C) for 10 minutes. The resulting
rate of 2 ml/min. Schaffer collaterals were stimulated by placing a bipolar- supernatants were diluted 1:1 (supernatant: water) into a new 96-well plates in
stimulating electrode in the stratum radiatum near the CA3-CA1 border. preparation for LC/MS/MS analysis. The compound was assayed in triplicate
acetonitrile containing carbamazepine as an internal standard (50 ng/ml). The
Recording electrodes were pulled with a Flaming/Brown micropipette puller
(Sutter Instruments, Novato, CA), filled with ACSF, and placed in the stratum
radiatum of area CA1. Field potential recordings were acquired using a
MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, CA) and pClamp
10 software (Molecular Devices). Input-output curves were generated to
determine the stimulus intensity that produced 50%–60% of the maximum
field excitatory postsynaptic potential (fEPSP) slope before each experi-
ment, which was then used as the baseline stimulation. Test compounds
were diluted to the appropriate concentrations in DMSO (0.1% final) in
ACSF and applied to the bath for 10 minutes using a perfusion system.
Chemically induced mGlu LTD was initiated by the application of DHPG in
ACSF (75 mM) for 10 minutes. Sampled data were analyzed offline using
Clampfit 10. The slopes from three sequential fEPSPs were averaged. All
fEPSP slopes were normalized to the average slope calculated during the
predrug period (percentage of baseline). Data were analyzed using GraphPad
Prism.
Epileptiform Experiments. Male Sprague-Dawley rats (24–30 days old,
Charles River) were sacrificed and the brains quickly removed and submerged
into ice-cold cutting solution (as described above) continuously bubbled with
95% O2/5% CO2. Then 400-mm transverse slices were made as described
above. Individual hippocampi were transferred to artificial cerebrospinal fluid
(ACSF, in mM: 124 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2
CaCl2, 1.2 MgCl2), maintained at room temperature, and allowed to equilibrate
for a minimum of 1 hour. For recordings, the slices were transferred to
within the same 96-well plate. The substrate depletion method previously
described was used to estimate the in vitro intrinsic clearance (CLint; ml/min per
kilogram) of VU0403602 in rat liver microsomes (Obach and Reed-Hagen,
2002).
Hepatic Biotransformation of VU0403602
The in vitro metabolism of VU0403602 was investigated in hepatic S9
fractions from S-D rats (male, pooled; BD Biosciences, San Jose, CA). A
potassium phosphate-buffered solution (0.1 M, pH 7.4) of test compound
(25 mM) and hepatic S9 fractions (5 mg/ml) was incubated (37°C) in boro-
silicate glass tubes under ambient oxygenation for 1 hour with select reactions
being fortified with NADPH (2 mM), UDGPA (2 mM), and/or adenosine
39-phosphate 59-phosphosulfate lithium salt hydrate (2 mM). Protein was
precipitated by the addition of two volumes of acetonitrile with subsequent
centrifugation (3000 RCF, 10 minutes). The supernatant was dried under
a stream of nitrogen gas and reconstituted in 85:15 (v/v) ammonium formate
(10 mM, pH 4.1, aqueous):acetonitrile in preparation for LC/MS/MS analysis.
In an identical manner, and where indicated, the metabolism of VU0403602
was investigated in Sprague-Dawley rat intestinal S9 fractions (4 mg/ml,
pooled male S-D rats, BD Biosciences).
Pharmacokinetic Studies in Rats after Intravenous Administration
Male Sprague-Dawley rats (250–300 g) were purchased from Harlan
a submersion recording chamber and allowed to equilibrate for 5–10 minutes at Laboratories (Indianapolis, IN). Catheters were surgically implanted in the
30–32°C with a flow rate of 2 ml/min. Recording electrodes were pulled with
a Flaming/Brown micropipette puller (Sutter Instruments, CA), filled with
ACSF and placed in the cell body layer of CA3. Field potential recordings
carotid artery and jugular vein. The cannulated animals were acclimated to their
environment for approximately 1 week before dosing. Parenteral administration
of test compounds to rats was achieved via a jugular vein catheter at a dose of 1
(spontaneous events) were acquired using a MultiClamp 700B amplifier or 3 mg/kg and a dose volume of 1 ml/kg [10% ethanol (EtOH)/50% PEG
(Molecular Devices) and pClamp10 software (Molecular Devices). Compounds (polyethylene glycol) 400/40% saline] or 3 ml/kg (10% EtOH/90% PEG 400)
of interest were diluted to the appropriate concentrations in DMSO (0.1% final)
in ACSF and applied to the bath for 10 minutes using a perfusion system.
for metabolite M1 and parent VU0403602, respectively. Blood collections via
the carotid artery were performed at 2, 7, 15, and 30 minutes and 1, 2, 4, 7, and
DHPG was used as a positive control (50 mM). Sampled data were analyzed 24 hours postadministration. Samples were collected into chilled, EDTA-
offline using MiniAnalysis (Synaptosoft Inc., Fort Lee, NJ) to determine the
amplitude and interevent interval of the spontaneous events and normalized to
the predrug period.
fortified tubes, centrifuged for 10 minutes (3000 RCF, 4°C), and the resulting
plasma stored at 280°C until analysis. Pharmacokinetic parameters were
obtained from noncompartmental analysis (NCA, WinNonLin, v5.3; Pharsight

Biotransformation of mGlu5 PAM to Active Agonist Metabolite
1707
Corp., Mountain View, CA) of individual animal concentration-time profiles
following the parenteral administration of a test compound.
held for 0.2 minute and was linearly increased to 90% B over 0.8 minute, with
an isocratic hold for 0.6 minute, before transitioning to 30% B over 0.1 minute.
The column was re-equilibrated (1 minute) before the next sample injection. The
total run time was 2.5 minutes, and the HPLC flow rate was 0.5 ml/min. The
source temperature was set at 500°C, and mass spectral analyses were performed
using multiple-reaction monitoring of transitions specific for the test articles and
metabolites and using a Turbo-Ionspray source in positive ionization mode
(5.0-kV spray voltage). All data were analyzed using AB Sciex Analyst 1.5.1
software. The lower limits of quantitation for VU0403602, VU0453103 (M1),
and VU0451326 (M2), were determined at 1 ng/ml in plasma and 0.5 ng/g in
brain homogenates.
Samples from in vitro DMPK assays (CLint, plasma protein and brain
homogenate binding), were analyzed on a TSQ Quantum Ultra (Thermo Fisher
Scientific, Waltham, MA) using electrospray ionization (ESI). The mass
spectrometer was coupled to an Accella HPLC pump system (Thermo Fisher
Scientific) and a CTC PAL autosampler. Analytes were separated by gradient
elution employing two Acquity BEH C18 columns (2.1 Â 50 mm, 1.7 m;
Waters Corp., Milford, MA) that were heated at 50°C. HPLC mobile phase A
was a 95:5:0.1 mixture of water/acetonitrile:formic acid, respectively; mobile
phase B was a 95:5:0.1 mixture of acetonitrile/water/formic acid, respectively.
Determination of Systemic and CNS Exposure of VU0403602
Male Sprague-Dawley rats (250–300 g) were purchased from Harlan
Laboratories and acclimated to their surroundings for approximately 1 week
before dosing. Administration (i.p.) of test compounds to rats was performed at
various doses (3–30 mg/kg). When warranted, rats received pretreatment with
ABT (PO, 50 mg/kg) or coadministration of test article and MPEP (i.p., 5 or
10 mg/kg) (Balani et al., 2002). Plasma and whole brains were collected at
varied time points using nonserial sampling methods from multiple animals.
Plasma samples were collected into chilled, EDTA-fortified tubes and centri-
fuged for 10 minutes (3000 RCF, 4°C), and the resulting plasma stored at 280°C
until analysis. Brain samples were rinsed with cold phosphate-buffered saline,
snap frozen (dry ice), and stored at 280°C until LC/MS/MS analysis. Plasma and
brain time-concentration area-under-the-curves (AUCs) were calculated by the
trapezoidal method employing Prism software (GraphPad).
Behavioral Manifestations of Seizure Activity
To study mGlu5 PAM-induced behavioral manifestation of seizure activity, The following was the gradient program that was used in these separations:
rats received various doses (i.p., 30 mg/kg) of the test compound with or
without the mGlu5 antagonist MPEP (5 and 10 mg/kg). Compounds were
formulated in 10% Tween 80 and administered at a volume of 3 ml/kg. Animals
were monitored continuously for 2 hours. After this procedure, rats were
euthanized and brains removed and processed for compound exposure levels.
Rats were scored for behavioral manifestations of seizure activity in periods of
5 minutes, once every 5 minutes for the first 15 minutes, then once every 15
pump 1 ran the gradient: 95:5 (A:B) at 800 ml/min hold 0 to 0.5 minute, linear
ramp to 5:95 (A:B) 0.5 to 1.0min, 5:95 (A:B) hold 1.0 to 1.9 minutes, return to
95:5 (A:B) at 1.9 minutes. While pump 1 ran the gradient method, pump 2
equilibrated the previously used column under isocratic conditions (95:5; A:B).
The total run time was 2.0 minutes. All compounds were optimized and
analyzed using QuickQuan (Thermo Fisher Scientific) software.
Samples from in vitro metabolism experiments and authentic standards of
minutes up to 1 hour, and every 30 minutes up to 2 hours. Compound-induced test compound/metabolites were analyzed on an Agilent 1100 HPLC system
behavioral manifestations of seizures were scored using a five-grade modified employing a Supelco Discovery C18 column (2.1 Â 150 mm, 5 mm; Sigma-
Racine scoring system (Rook et al., 2012). A score of 0 represents no
behavioral alteration; score 1, immobility, mouth and facial movements, facial
clonus; score 2, head nodding, forelimb and/or tail extension, rigid posture;
Aldrich Chemical Company). Solvent A was 10 mM (pH 4.1) ammonium
formate and solvent B was acetonitrile. The initial mobile phase was 15% and
by linear gradient transitioned to 80% over 20 minutes. The flow rate was 0.400
score 3, forelimb clonus, repetitive movements; score 4, rearing, forelimb ml/min. The HPLC eluent was first introduced into an Agilent 1100 DAD
clonus with rearing, rearing and falling; and score 5, continuous rearing and
falling, jumping, severe tonic-clonic seizures.
(single wavelength selected, 254 nM), followed by electrospray ionization
introduction into a Finnigan LCQ Deca XPPLUS ion trap mass spectrometer
(Thermo Fisher Scientific) operated in either the positive or negative ionization
mode. Ionization was assisted with sheath and auxiliary gas (ultrapure nitrogen)
set at 60 and 40 psi, respectively. The electrospray voltage was set at 5 kV with
the heated ion transfer capillary set at 300°C and 30 V. Relative collision
energies of 25%–35% were used when the ion trap mass spectrometer was
operated in the MS/MS or MSn mode.
Reversal of Amphetamine-Induced Hyperlocomotion
Studies were conducted using male Sprague-Dawley rats purchased from
Harlan Laboratories weighing 275–300 g. Dose groups consisted of 5 to 12 rats
per group. VU0403602 was dissolved in 10% Tween 80 and double-deionized
water, and the pH was adjusted to approximately 7.0 using 1 N NaOH.
VU0403602 was administered i.p. at a dose of 3, 10, or 30 mg/kg in a 3 ml/kg
volume. Studies were performed as previously described (Noetzel et al., 2012).
Briefly, rats were placed in the open-field chambers and allowed to habituate
for 30 minutes, followed by pretreatment (i.p.) with vehicle or test compound.
After an additional 30 minutes, rats received a saline vehicle or 1 mg/kg
amphetamine via s.c. injection. Locomotor activity was measured for an
additional 60 minutes. Ambulation or locomotor activity was measured as the
number of total photobeam breaks per 5-minute interval using Motor Monitor
System software (Kinder Scientific, Poway, CA). Main effects of test
compound treatment on the amphetamine-induced locomotor activity area
under the time-course curve were evaluated using one-way analysis of variance.
Comparisons of treatment group effects relative to the vehicle + amphetamine
group were completed across the time interval from t = 60 to 120 minutes using
Dunnett’s post hoc tests with a P value , 0.05 considered significant.
Results
VU0403602 Displays a Mixed mGlu5 Agonist-PAM Pharmacology
Profile In Vitro
We previously reported a series of small-molecule selective mGlu5
PAMs that were developed based on a biaryl acetylene scaffold;
a number of highly potent compounds were identified that display in
vivo activity in rodent behavioral models of antipsychotic efficacy
(Williams et al., 2011; Rook et al., 2012). We recently identified
a cyclobutyl picolinamide analog, VU0403602, that displayed potent
mGlu5 agonist (31.1 nM, 49% Glumax) and PAM (4 nM, 100%
Glumax) activity in vitro (Fig. 2), inducing a progressive concentration-
dependent left-shift (9-fold maximal shift at 1 mM, data not shown) of
the glutamate CRC in a functional calcium mobilization assay in
mGlu5-expressing HEK293A cells (Gregory et al., 2013). Importantly,
VU0403602 is absent of activity at mGlu1 (Ca2+ assay), group II
(GIRK thallium flux assay), and group III receptors (GIRK thallium
flux assay), indicating that the compound is a subtype-selective mGlu5
ligand (Supplemental Fig. 1). VU0403602 displayed minimal off-
target ancillary pharmacology, as radioligand binding displacement of
a broad panel (.65) of GPCRs, ion channels, transporters, and other
targets (n = 2, 10 mM test compound) indicated significant activity
(.50%) for only three targets: human norepinephrine transporter
Liquid Chromatography-UV-Mass Spectrometry Analysis of VU0403602
and Corresponding Metabolites
Plasma and brain tissue samples originating from in vivo studies were
analyzed with electrospray ionization by an AB Sciex Q-TRAP 5500 (Foster
City, CA) that was coupled to a Shimadzu LC-20AD pump (Columbia, MD)
and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC). Analytes
were separated by gradient elution using a C18 column (3 Â 50 mm, 3 mm;
Fortis Technologies Ltd, Cheshire, UK) that was thermostated at 40°C. HPLC
mobile phase A was 0.1% formic acid in water (pH unadjusted); mobile phase
B was 0.1% formic acid in acetonitrile (pH unadjusted). A 30% B gradient was

1708
Bridges et al.
cells revealed a fully competitive interaction by VU0403602, similar
to that of the unlabeled MPEP control (Supplemental Fig. 2). In
additional cell-based functional experiments using mGlu5-expressing
cells, CRCs of VU0403602 in the presence of multiple fixed con-
centrations of the noncompetitive mGlu5 antagonist, 5PMEP (occu-
pies the MPEP allosteric site), exhibited parallel right shifts, with
a corresponding Schild regression analysis indicating a competitive
mode of interaction (slope = 1.06)). These data support a fully com-
petitive interaction of VU0403602 at the prototypical MPEP binding
site of mGlu5.
In Vivo Administration of VU0403602 Induces Adverse Events in
Rats
Although VU0403602 produced a dose-dependent (3, 10, 30, mg/kg,
i.p.; ED50, 21 mg/kg) efficacy in the reduction of hyperlocomotion in
Sprague-Dawley rats (Supplemental Fig. 3) receiving amphetamine
(1 mg/kg, s.c.), the systemic administration of this mGlu5 PAM also
induced pronounced AEs marked by the onset of seizures, forelimb
asymmetry, tremor, and facial bleeding, the severity of which were
recorded using the Racine assessment method (Rook et al., 2012). The
onset of VU0403602-mediated AEs was similar to those observed in
S-D rats that received a single administration (i.p.) of the Ago-PAM
VU0424465 (agonist EC50: 171 nM, 65% Glumax, PAM EC50: 2 nM,
88% Glumax; Rook et al., 2012). When rats received a single i.p. dose
(30 mg/kg) of VU0403602 in the absence of amphetamine, subsequent
observations revealed a time-dependent manifestation of the afore-
mentioned behavioral effects (Fig. 3). To determine whether the AEs
observed in rats were mGlu5 target-mediated, we administered (i.p.)
VU0403602 (30 mg/kg) with the mGlu5 allosteric antagonist, MPEP
(5 and 10 mg/kg) and monitored the behavioral effects for 4 hours
post-treatment. The lower dose (5 mg/kg) of MPEP reduced the
severity of the VU0403602-induced behavioral AEs by approximately
50%, whereas rats receiving the 10 mg/kg dose of MPEP were
completely devoid of the behavioral manifestations (Fig. 3). These
data implicate a role for mGlu5 in the mechanism underlying the AEs
resulting from the administration of VU0403602. Interestingly,
Fig. 2. VU0403602 is a potent positive allosteric modulator of mGlu5 with direct
agonist activity in the absence of glutamate in vitro. VU0403602 causes
concentration-dependent increases in the mobilization of intracellular calcium in
recombinant cells expressing mGlu5 in the presence (top) and absence (bottom) of
a fixed submaximal (;EC20) concentration of glutamate and with a PAM EC50 of
4 nM (100% response achieved relative to the maximal response obtained with
glutamate, % Glumax) and an agonist EC50 of 31.1 nM (49.4% Glumax). Data
represent the mean 6 S.E.M. of four independent experiments performed in
duplicate.
(66% inhibition), human dopamine transporter (DAT, 67% inhibition), pretreatment of rats with the pan cytochrome P450 inactivator, 1-
and a human serotonin receptor (5-HT2B, 92% inhibition). Subsequent aminobenzotriazole (ABT; 50 mg/kg, i.p.), completely mitigated the
5-HT2B functional assays with VU0403602 (30 mM) in rat stomach onset of the behavioral AEs resulting from the systemic administration
fundus tissue revealed no significant activity (data not shown).
of VU0403602, indicating that one or more metabolites of this PAM
Competition binding experiments using the MPEP site radioligand may be contributing to the mGlu5-mediated toxicity observed in vivo
[3H]-mPEPy to label membranes from rat mGlu5-expressing HEK (Fig. 3) (Balani et al., 2002).
Fig. 3. The mGlu5 ago-PAM VU0403602 induces time-dependent increases in behavioral convulsions, which are mitigated by pretreatment with the mGlu5 antagonist
MPEP or the P450 inactivator ABT in male Sprague-Dawley rats. VU0403602 administration (i.p., 30 mg/kg, n = 9) causes full clonic-tonic seizure activity (modified Racine
score 5); the same administration of VU0403602 to rats pretreated with a lower dose of MPEP (i.p., 5 mg/kg, n = 3) or a higher dose of MPEP (i.p., 10 mg/kg, n = 3) reduced
(modified Racine score 2) or completely blocked (modified Racine score 0), respectively, the severity of seizure activity. In rats pretreated with ABT (PO, 50 mg/kg, n = 6),
administration of VU0403602 (i.p., 30 mg/kg) failed to induce any seizure activity (modified Racine score 0). Data represent mean 6 S.E.M.

Biotransformation of mGlu5 PAM to Active Agonist Metabolite
1709
In Vitro and In Vivo Disposition of VU0403602 in Sprague- in the MS/MS of M1 (m/z 275). Another fragmentation observed for
Dawley Rats Reveals Hepatic Metabolism as the Mechanism of M1 was the gas phase, water-assisted fragmentation of the amide bond
Clearance
that resulted in the formation of an ion at m/z 242 (20% relative). The
water-assisted amide fragmentation was also observed in the MS/MS
of VU0403602, producing a fragment ion at m/z 242 in addition to the
loss of the cyclobutyl moiety producing an [M + 2H]+ at m/z 241. The
proposed carboxylic acid metabolite M2, observed at 15.7 minutes
([M + H]+ at m/z 242), resulted in the gas phase decarboxylation of
M2, producing a fragment ion at m/z 196.
To determine the relative stereochemistry of oxidation of the cy-
clobutyl moiety of VU0403602 to the hydroxylated metabolite, M1,
we synthetically prepared authentic cis- and trans-hydroxylated com-
pounds from the ci-s/trans-3-hydroxy-1-amino-cyclobutane and a
requisite amide coupling; chiral SFC was used to separate the cis- and
trans-standards (Supplemental Schemes 1–3). The cis- (VU0453102)
and trans- (VU0453103) structures were confirmed based on both 1D
NMFR studies (Supplemental Figs. 5–8), including the examination of
nuclear overhauser effects between the methine protons within the
cyclobutane ring system (Supplemental Fig. 9). Subsequent LC/MS/MS
analysis confirmed the stereochemistry of M1 as that of the trans-
compound (i.e., VU0453103) based on the corresponding LC reten-
tion time and MS/MS fragmentation pattern. The structure proposed
for M2 was also confirmed via LC/MS/MS comparison with its au-
thentic standard (VU0451326).
After the initial rat behavioral findings, the DMPK profile of
VU0403602 was determined using conventional in vitro subcellular
fraction metabolism and in vivo dosing paradigms to define the
biotransformation pathways for VU0403602 and the contribution(s) of
metabolites toward precipitating AEs in rat (Balani et al., 2005). The
CLint of VU0403602 was assessed in rat hepatic microsomes and
subsequently used to predict the extent of hepatic clearance in vivo.
The CLint and hepatic clearance values (271 ml/min per kilogram and
55.6 ml/min per kilogram, respectively) for VU0403602 correlated
well with the total body plasma clearance value (CLp: 36.2 ml/min per
kilogram) observed in rats after parenteral administration (3 mg/kg)
(Supplemental Fig. 4), indicating that hepatic metabolism was likely
the predominant mechanism of clearance for VU0403602. Consistent
with this assessment, we detected negligible amounts of unchanged
VU0403602 excreted in the urine. The volume of distribution
estimated at steady state of VU0403602 (Vss, 6.73 l/kg) exceeded
that of the total body water in rats and, coupled with its moderate CLp
in vivo, resulted in a mean residence time of approximately 3 hours
(Supplemental Fig. 4). In vitro rapid equilibrium dialysis (RED)
indicated the plasma protein binding of VU0403602 to be extensive in
rat plasma, producing a corresponding fraction unbound (Fu) of , 0.01;
RED data also indicated extensive nonspecific binding of VU0403602
(Fu , 0.01) in rat brain homogenates.
The Hydroxylated Metabolite (M1) is a Metabolite-Ligand of
mGlu5 Displaying Agonist and PAM Activity
A pharmacological characterization of M1 on the mGlu5 receptor
was obtained through the use of functional calcium mobilization
assays measuring agonist, PAM, and antagonist responses at mGlu5 in
recombinant HEK293A cells. In vitro analysis revealed M1 to be a
robust ago-PAM, possessing agonist and PAM EC50 values of 400 nM
(78% Glumax) and 16.9 nM (94% Glumax), respectively (Fig. 5). Re-
lated glutamate CRC experiments demonstrated that M1 (10 mM)
induced a maximal left shift of 8.2-fold, similar to that of 9-fold for the
parent PAM, VU0403602 (1 mM, data not shown). However, unlike
the partial mGlu5 agonist activity of the parent VU0403602 (49.4%
Glumax), the hydroxylated metabolite M1 displayed a near full agonist
activity (94% Glumax). By contrast, the carboxylic acid metabolite
(M2) displayed no activity at mGlu5 up to the limit of solubility
(agonism, PAM, antagonism EC50 values .30 mM, data not shown).
In Vitro Biotransformation of VU0403602 Results in the Formation
of Two Principal Metabolites
LC/MS/MS was used to characterize the metabolites of VU0403602
produced in vitro from rat S9 fractions (intestinal, hepatic, brain),
potentially representing metabolite-ligands of mGlu5 that could con-
tribute to the AEs observed in rats. Data from these experiments
revealed two principal biotransformation pathways for VU0403602
(Scheme 1), NADPH-dependent oxidation of the cyclobutyl moiety of
VU0403602 to the hydroxylated metabolite, M1, and the NADPH-
independent amide hydrolysis of VU0403602 to the carboxylic acid
metabolite, M2. Metabolite M1 was observed at approximately 16.1
minutes as the [M + H]+ at m/z 311 (Fig. 4). The primary fragmentation
observed (100% relative) was the gas phase loss of water (218 Da; 2H2O)
to produce the ion at m/z 293, representing a 16-Da increase over the
water loss fragment observed in the MS/MS of VU0403602 (m/z 277).
A secondary fragmentation from the ion at m/z 293 was also observed
In Vivo Metabolism of VU0403602 Resulted in the Formation and
Distribution to the CNS of the Active Metabolite-Ligand M1
Based on the abundant formation in vitro and the potent mGlu5
pharmacological activity observed for M1, we suspected that the
systemic formation and CNS exposure of this hydroxylated metabo-
lite in vivo may contribute to the adverse events observed in rats.
Moreover, a role of M1 in eliciting the AEs in rats would be consistent
with the ABT results, where the absence of AEs was noted in rats that
had received the single pretreatment with the P450 inactivator, ABT.
Therefore, we administered VU0403602 to rats (i.p., 30 mg/kg; n = 2)
that were treated with either ABT (PO, 50 mg/kg) or vehicle (0.5%
methylcellulose in water) followed by brain and blood collections (1.5
hour postdose) to determine the concentrations of VU0403602 and its
metabolites (M1 and M2) in the plasma and CNS (Table 1). The
separation of plasma and brain homogenates was first demonstrated
with a protracted LC/MS/MS gradient to profile qualitatively the range
of metabolites observed in vivo; results from this LC/MS/MS analysis
indicated that VU0403602, M1, and M2 were the principal com-
ponents observed in vivo, with a negligible component observed
Scheme 1. Principal pathways of VU0403602 metabolism observed in vitro in male
Sprague-Dawley rat hepatic S9 fractions.

1710
Bridges et al.
Fig. 4. Representative reconstructed LC/MS/MS total ion current (TIC) (A) of extracts from a Rat hepatic S9 incubation (+NADPH) of VU0403602, showing the principal
metabolites M1 and M2, as well as a minor hydroxylated metabolite M3. A corresponding TIC (B) from the LC/MS/MS analysis (MS/MS, m/z 311) of brain extracts from
rats receiving a 30 mg/kg dose (i.p.) of VU0403602 reveals M1 to be the principal hydroxylated metabolite in vivo.
representing the cis-isomer of M1. Hence, the remaining quantitative concentrations may also be best applied to the pharmacological activity
bioanalysis of plasma and brain homogenate samples was executed elicited by VU0403602 and M1 in vivo. However, regardless of
with a standard high-throughput LC/MS/MS gradient as described consideration of total or unbound levels achieved, the results obtained
already herein without the resolution of the cis- and trans-hydroxylated from in vivo analysis are consistent with that generated from the in vitro
metabolites. In the absence of ABT, VU0403602 reached an average metabolism experiments; rats that had received the P450 inactivator
maximal concentration (Cmax) of 3.2 mM (5 nM unbound) with ABT produced an approximate 11-fold lower Cmax of the hydroxylated
a corresponding time to reach Cmax (Tmax) of 0.25 hour in plasma; the metabolite M1 in plasma (13-fold lower Cmax, brain) relative to the
Cmax observed in brain was 11.5 mM (6 nM, unbound) with an average vehicle control (-ABT) treated rats (Table 1). Likewise, ABT pre-
Tmax of 0.88 hour (Table 1). In plasma, M1 reached an average total treatment also reduced the area-under-the-curve (AUC0-6h) of M1 in
Cmax of 64 nM (4 nM unbound) with an average Tmax of 0.25 hour plasma by approximately 17-fold in rats receiving a single administra-
(Table 1). The brain penetration observed for M1 was substantial, tion (i.p.) of VU0403602, with a corresponding 8.4-fold reduction in the
reaching an approximate 9:1 distribution relative to the systemic plasma brain AUC0-6 of M1 (Table 1). Overall, the impact of ABT pretreatment
compartment. M1 reached an average brain Cmax of 400 nM (4 nM, on the metabolism of VU0403602 was profound, resulting in the
unbound) and a corresponding Tmax of 1.5 hours (Table 1). Because of substantial reduction of M1 exposure in vivo, both in terms of maximal
the extensive plasma protein- and nonspecific-binding of VU0403602 plasma and brain levels and systemic exposure. As expected for a
(Fu , , 0.01) and M1 (Fu, 0.01) observed in vitro, the framing of the non-P450–mediated biotransformation such as amide hydrolysis, ABT
relevant concentrations observed between the two tissue compartments pretreatment had little to no effect on the Cmax or AUC0-6h of the
(i.e., systemic circulation and the CNS) may be most appropriate when carboxylic acid metabolite, M2. Collectively, these data demonstrate
considering the total concentrations achieved. Moreover, this framing of the in vivo relevance of the mGlu5 ago-PAM metabolite-ligand, M1, in

Biotransformation of mGlu5 PAM to Active Agonist Metabolite
1711
agonists as well as allosteric ago-PAMs. Importantly, we have de-
monstrated that allosteric mGlu5 modulators displaying exclusive
PAM pharmacology are incapable of inducing LTD in the absence
of an orthosteric ligand (e.g., DHPG). As previously discussed, the
allosteric modulator VU0403602 displayed mixed ago-PAM pharma-
cology at mGlu5 in recombinant cells expressing the rat receptor. To
demonstrate the ago-PAM behavior of VU0403602 in a native system,
we investigated alterations in electrophysiological responses in rat
brain slices following in vitro exposure to VU0403602. Data gen-
erated from these experiments indicated that VU0403602 (10 mM)
induced pronounced hippocampal LTD at the SC-CA1 synapse (Fig. 6),
with a prolonged depression of the fEPSP slope (39.8 6 5.9% of
baseline) well after the compound had been washed from the slice pre-
paration (55 minutes after washout). Likewise, the hydroxylated me-
tabolite M1 (10 mM) also induced significant LTD at the SC-CA1 synapse
(Fig. 8) with a similar magnitude of depression of the fEPSP (slope 68.9
6 4.9% of baseline) and duration of action (55 minutes post wash-
out). (Fig. 6).
VU0403602 and Metabolite M1 Induce Epileptiform Activity in
the Rat Hippocampal CA3 Region In Vitro. Having demonstrated
that VU0403602 and its principal oxidative metabolite M1 were ca-
pable of inducing alterations in LTD, additional experiments inves-
tigating whether these ago-PAMs are capable of inducing epileptiform
activity in rat hippocampus, specifically in CA3 pyramidal neurons,
were performed. Similar to the agonist DHPG, the hydroxylated
metabolite M1 (10 mM) and parent VU0403602 (10 mM) induced
pronounced epileptiform activity, as evidenced by decreases in the
interevent intervals of spontaneous field population spikes (48.1 6
7.8% and 48.8 6 11% of baseline, respectively) detected by ex-
tracellular field potential recordings in the pyramidal cell body layer of
area CA3 in the hippocampal slices (Fig. 7). Similar to the positive
control DHPG (50 mM), neither VU0403602 nor M1 significantly
affected the amplitude of spontaneous events (Fig. 7). Together, re-
sults from the rat hippocampal electrophysiology experiments indicate
that the principal oxidative metabolite-ligand M1 is capable of in-
ducing LTD and epileptiform activity in native systems, a finding
consistent with mGlu5 activation and one that advances our under-
standing of the mechanism of AEs observed in rats receiving systemic
administration of the parent compound, VU0403602.
Fig. 5. M1 is a potent positive allosteric modulator of mGlu5 with direct agonist
activity in the absence of glutamate in vitro. M1 causes concentration-dependent
increases in the mobilization of intracellular calcium in recombinant cells expressing
mGlu5 in the presence (top) and absence (bottom) of a fixed submaximal (;EC20
)
concentration of glutamate and with a PAM EC50 of 16.9 nM (94% Glumax) and an
agonist EC50 of 400 nM (79% Glumax). Data represent the mean 6 S.E.M. of at least
three independent experiments performed in duplicate.
the onset of adverse behavioral events observed in rats receiving i.p.
administration of VU0403602.
In Vitro Rat Brain Slice Electrophysiology
VU0403602 and its Hydroxylated Metabolite M1 Induce Long-
Term Depression in Rat Hippocampus. We and others have demon-
strated the induction of LTD at the Schaffer-collateral-CA1 (SC-CA1)
Direct Administration of M1 Results in Behavioral AEs in Rats
The systemic administration of M1 to rats was executed to
synapse in the hippocampus as a result of mGlu5 activation by orthosteric determine whether this mGlu5 metabolite-agonist was capable of
TABLE 1
Pharmacokinetics of VU0403602 and the formation of its principal metabolites (M1, M2) in vivo in male Sprague-
Dawley rats after i.p administration (30 mg/kg) in the absence and presence of pretreatment with the cytochrome P450
inactivator 1-aminobenzotriazole (ABT)
Data represent mean values (n = 2).
Analyte
Pretreatment
Plasma Cmax
mM
Plasma Tmax
Plasma AUC0-inf
Brain Cmax
mM
Brain AUC0-inf
h
mM*h
mM*h
VU0403602
M1
—
+ABT
—
3.21 (0.005)a
11.0 (0.018)a
0.064 (0.004)c
0.032 (0.002)c
17.8
0.25
0.75
1.5
1.5
1.25
1.5
3.63 (0.006)a
9.64 (0.015)a
0.046 (0.003)c
0.003 (0.001)c
15.8
11.5 (0.006)b
12.9 (0.006)b
0.406 (0.005)d
0.031 (0.001)d
3.71
7.79 (0.004)b
9.64 (0.005)b
0.258 (0.003)d
0.031 (0.001)d
3.15
+ABT
—
M2e
+ABT
20.3
11.7
6.73
4.25
—, Not done; AUC, area under the curve.
aUnbound concentrations, f u 0.0016.
bNonspecific binding, f u , 0.005.
cUnbound concentrations, f u 0.06.
dNonspecific binding, f u 0.011.
eUnbound concentrations for M2 not determined.

1712
Bridges et al.
Fig. 6. VU0403602 and M1 (VU0453103) induce long-term depression at the
Schaffer collateral-CA1 synapse in hippocampus. A stimulus that produced a
50%–60% fEPSP slope was used as baseline stimulation for each recording.
Bath application of 75 mM DHPG for 10 minutes (solid line) induced LTD that
lasted at least 55 minutes after washout (n = 8). Similarly, bath application of either
10 mM VU0403602 or 10 mM M1 for 10 minutes (solid line) resulted in LTD that
lasted at least 55 minutes after washout of the compound (n = 8, 7, respectively).
Insets are sample traces measured predrug (black) or 55 minutes after compound
washout (gray). Data represent mean 6 S.E.M.
inducing the pronounced AEs that we observed after the administra-
tion of the parent ligand VU0403602. Previously, after VU0403602
administration to rats (i.p., 30 mg/kg), we observed a rapid Tmax of
both the parent ligand VU0403602 (Tmax, 0.25 hour) and M1 (Tmax
,
0.25 hour), as well as concurrent onset of behavioral AEs that marked
this rodent neurotoxicity. After direct administration of M1 to rats
(i.p., 30 mg/kg), we observed a similar rapid onset of AEs (Fig. 8).
Moreover, the M1-mediated AEs were strikingly similar to those
achieved by systemic administration of VU0403602 and the aforemen-
tioned ago-PAM, VU0422465, the occurrence of which were char-
acterized by seizures, forelimb asymmetry, tremor, and facial bleeding.
Considering the rapid formation of M1 in vivo, its extensive distribution
to the CNS, and the consistency between the P450-mediated formation
of M1 and the subsequent mitigation of AEs following ABT pre-
treatment, we submit that this metabolite contributes to the in vivo
toxicity observed in rats via an mGlu5 receptor-mediated mechanism.
Fig. 7. VU0403602 and M1 (VU0453103) induce epileptiform activity in CA3
neurons in hippocampus. Amplitude and interevent interval of spontaneous firing
were determined using field potential recordings in CA3 in the hippocampal
formation. (A) Application of 50 mM DHPG (n = 11), 10 mM VU0403602 (n = 8),
or 10 mM M1 (n = 7) for 10 minutes resulted in a robust decrease in the interevent
interval of spontaneous events while having no significant effect on amplitude of
spontaneous events. Data represent mean 6 S.E.M. (B) Sample traces from
individual experiments taken before and after the addition of the compound.
Discussion
In an effort to advance new approaches in the treatment of the
negative and cognitive symptoms common among schizophrenia
patients, we have identified PAMs of mGlu5 as potential novel ther-
apeutics to rescue the N-methyl-D-aspartate receptor hypofunction
believed to underpin this devastating neurologic disorder (Olney et al.,
However, similar to previous reports from our laboratories re-
1999; Tsai and Coyle, 2002; Williams et al., 2011; Rook et al., 2012). garding the systemic administration of ago-PAMs to rodents (e.g.,
Herein, we have introduced an mGlu5 PAM, VU0403602, residing VU0422465), we observed an AE profile in rats after systemic
within a biarylacetylene scaffold that, in addition to displaying robust administration of VU0403602 characterized by behavioral disturban-
potency and a corresponding ninefold shift in the glutamate response, ces such as seizure activity (Noetzel et al., 2012; Rook et al., 2012).
also displayed an intrinsic partial agonist profile in vitro (31.1 nM; The onset of behavioral disturbances was dose- and concentration-
49% Glumax). Such ago-PAMs have previously been shown to produce dependent, and the severity of the AEs increased with time as mea-
efficacy in preclinical models of psychosis, such as in the reversal of sured by Racine scoring methodology. Moreover, the neurotoxicity
amphetamine-induced hyperlocomotion rodent model (Noetzel et al., observed in rats was discovered to be target-mediated, as coadministration
2012; Rook et al., 2012). Indeed, after i.p. administration of VU0403602 of VU0403602 with an allosteric antagonist of mGlu5 (MPEP) effec-
to S-D rats, VU0403602 produced a dose-dependent reversal of hy- tively blocked the onset of AEs. Interestingly, an in vitro metabolism
perlocomotion, with a minimally active dose of 3 mg/kg.
appraisal implicated oxidative metabolism as the primary biotransformation

Biotransformation of mGlu5 PAM to Active Agonist Metabolite
1713
inactivator ABT) and at the concentrations achieved in the present
studies. It is also possible that VU0403602 and M1 may have differential
effects in varied brain regions that are known to express mGlu5.
Ultimately, these findings serve to underscore the importance of avoiding
allosteric agonist activity in the design of mGlu5 modulators and
thereby retaining dependency on activation of mGlu5 by endogenous
glutamate activity for the safe and efficacious modulation of mGlu5.
Recent academic and pharmaceutical research accounts of pro-
convulsant side effects arising from overactivation of mGlu5 may
represent an impediment to the development of mGlu5-targeted
allosteric modulators for the treatment of schizophrenia (Rook et al.,
2012; Parmentier-Batteur et al., 2013). Recent reports from our
laboratory comparing PAMs with ago-PAMs support the hypothesis
that positive allosteric modulators of mGlu5 that are devoid of intrinsic
agonist activity do not carry proconvulsant and epileptogenic side
effect liabilities; however, a recent publication describes an mGlu5
PAM devoid of agonist activity, which nevertheless carried seizure-
like adverse effects in rodents (Noetzel et al., 2012; Rook et al., 2012;
Parmentier-Batteur et al., 2013). The present report highlights the
Fig. 8. The mGlu5 ago-PAM metabolite M1 induces dose- and time-dependent
increases in behavioral adverse effects in male Sprague-Dawley rats receiving
a single administration (i.p., 30 mg/kg, n = 2), whereas the inactive metabolite M2 is
devoid of AEs (i.p., 30 mg/kg, n = 2). Data represent mean 6 S.E.M.
pathway for VU0403602, resulting in the formation of a principal possibility that in vivo biotransformation of mGlu5 PAMs may result
hydroxylated metabolite M1 (+16 Da) in Sprague-Dawley rat hepatic in the CNS exposure of a potent metabolite ligand that bears
subcellular fractions (e.g., S9). Having demonstrated the P450- noticeable switches in pharmacology and/or pharmacokinetic dispo-
mediated mechanism of M1 formation in vitro, we examined the extent sition that are distinct from its parent ligand.
of P450 metabolism in vivo and determined M1 to be the principal
Most SAR common to classes of enzyme inhibitors or receptor agonists
oxidative metabolite detected in the circulation and central nervous or antagonists targeting orthosteric sites often display an intolerance with
system (i.e., brain tissue analysis) of rats receiving VU0403602 respect to structural modifications that are installed by DMEs (e.g., P450s),
(Kalvass and Maurer, 2002; Liu et al., 2008). Moreover, the distribution resulting in the pharmacological deactivation of the chemotherapeutic
of M1 to the CNS (AUC0-‘) was discovered to be approximately ;6:1 agent. The biotransformation of allosteric receptor modulators and/or
relative to the plasma of rats receiving VU0403602. Coupled with an allosteric enzyme inhibitors/activators by DMEs represents an unexpected
unbound brain fraction (fu) of 0.011 for M1, the CNS distribution of the hurdle for rational drug design efforts due to the possibility of metabolism-
primary oxidative metabolite (M1) far exceeded that of the parent mod- mediated formation of active metabolites, ligands that either display
ulator, VU0403602 (AUC0-‘ brain-to-plasma, ;2:1; brain fu, , 0.001). pharmacology distinct from that of the parent compound or bear potent
Previously established structure-activity relationships (SAR) for intrinsic agonist activity that exceeds that of the parent compound, as is the
allosteric modulation of mGlu5 indicated a range of tolerance for apparent case with VU0403602. The SAR observed in allosteric receptor
oxidation (e.g., hydroxylation) of this scaffold with respect to resulting modulation are sensitive to subtle, single atom modifications commonly
PAM activity of analogs, including switches in primary pharmacology to catalyzed by so-called phase I oxidative enzymes (e.g., P450, flavin
NAMs and SAMs (Sharma et al., 2009; Lindsley, 2011; Wood et al., monooxygenases, aldehyde oxidase), thus oxidative metabolism could
2011). Considering mono hydroxylation of organic molecules is a common bioactivate a parent compound to a metabolite-ligand, which could
outcome of P450–mediated metabolism, we hypothesized a role of DMEs attenuate or exacerbate the desired pharmacodynamic effects, seriously
in the AEs observed in vivo following VU0403602 administration to rats. complicating proof-of-concept studies and/or the safety assessment of a
Importantly, rats that were pretreated with the pan P450 inactivator, ABT, clinical candidate. These and other related scenarios underline
failed to manifest the AEs previously demonstrated following VU0403602 the importance of continued basic science development into un-
exposure, implicating the role of an oxidative metabolite (e.g., M1) in the derstanding the biotransformation and metabolic fate of allosteric
induction of in vivo neurotoxicity. Indeed, in vitro investigations revealed modulators for the potential treatment of human disease.
a pharmacological profile for M1 characterized by a mixed ago-PAM
profile in rat mGlu5-expressing cells. Although the PAM efficacy of M1
was similar to VU0403602 (8.2- and 9.0-fold left-shift in glutamate
potency, respectively), we noted an agonist profile of higher efficacy for
the metabolite (ꢀ80% Glumax) compared with that of VU0403602
Conducted experiments: Bridges, Rook, Noetzel, Morrison, Vinson.
Authorship Contributions
Participated in research design: Bridges, Rook, Noetzel, Morrison, Jones,
Niswender, Xiang, Stauffer, Conn, Daniels.
(, 50% Glumax). Likewise, the ago-PAM character of M1 was observed
in the electrophysiological assessments, as exposure of rat hippocampal
slices to the metabolite alone induced profound LTD at the SC-CA1
synapse, as well as epileptiform activity in area of CA3 of the
hippocampus. The present findings are in agreement with previous data
from our laboratory and other recent reports demonstrating that direct
agonists and mixed ago-PAMs can over activate mGlu5 and perturb
normal neuronal activity in rats, which can include an induction of
epileptiform activity in distinct hippocampal regions (e.g., CA3) (Lee
et al., 2002; Wong et al., 2005; Rook et al., 2012). Although VU0403602
was able to induce LTD and epileptiform activity in vitro, its intrinsic
partial agonism and overall lower efficacy in vivo appears insufficient to
induce overt adverse effects on its own (i.e., in the presence of the P450
Contributed new reagents or analytic tools: Zhou, Gogliotti, Lindsley,
Stauffer.
Performed data analysis: Bridges, Rook, Morrison, Noetzel, Xiang,
Niswender, Daniels.
Wrote or contributed to the writing of the manuscript: Bridges, Rook,
Noetzel, Niswender, Lindsley, Stauffer, Conn, Daniels.
References
Balani SK, Miwa GT, Gan LS, Wu JT, and Lee FW (2005) Strategy of utilizing in vitro and in
vivo ADME tools for lead optimization and drug candidate selection. Curr Top Med Chem 5:
1033–1038.
Balani SKZ, Zhu T, Yang TJ, Liu Z, He B, and Lee FW (2002) Effective dosing regimen of 1-
aminobenzotriazole for inhibition of antipyrine clearance in rats, dogs, and monkeys. Drug
Metab Dispos 30:1059–1062.

1714
Bridges et al.
Conn PJ, Lindsley CW, and Jones CK (2009) Activation of metabotropic glutamate receptors as
a novel approach for the treatment of schizophrenia. Trends Pharmacol Sci 30:25–31.
Cosford ND, Roppe J, Tehrani L, Schweiger EJ, Seiders TJ, Chaudary A, Rao S, and Varney MA
(2003) [3H]-methoxymethyl-MTEP and [3H]-methoxy-PEPy: potent and selective radioligands
for the metabotropic glutamate subtype 5 (mGlu5) receptor. Bioorg Med Chem Lett 13:
351–354.
Gregory KJ, Dong EN, Meiler J, and Conn PJ (2011) Allosteric modulation of metabotropic
glutamate receptors: structural insights and therapeutic potential. Neuropharmacology 60:
66–81.
Parmentier-Batteur S, Hutson PH, Menzel K, Uslaner JM, Mattson BA, O’Brien JA, Magliaro
BC, Forest T, Stump CA, and Tynebor RM, et al. (2013) Mechanism based neurotoxicity of
mGlu5 positive allosteric modulators: development challenges for a promising novel anti-
psychotic target. Neuropharmacology, in press.
Rodriguez AL, Grier MD, Jones CK, Herman EJ, Kane AS, Smith RL, Williams R, Zhou Y,
Marlo JE, and Days EL, et al. (2010) Discovery of novel allosteric modulators of metabo-
tropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo
activity in rat behavioral models of anxiolytic and antipsychotic activity. Mol Pharmacol 78:
1105–1123.
Gregory KJ, Nguyen ED, Reiff SD, Squire EF, Stauffer SR, Lindsley CW, Meiler J, and Conn PJ
(2013) Probing the metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulator
(PAM) binding pocket: discovery of point mutations that engender a “molecular switch” in
PAM pharmacology. Mol Pharmacol 83:991–1006.
Rook JM, Noetzel MJ, Pouliot WA, Bridges TM, Vinson PN, Cho HP, Zhou Y, Gogliotti RD,
Manka JT, Gregory KJ, et al. (2012) Unique signaling profiles of positive allosteric modulators
of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity. Bi-
ological Psychiatry 73(6):501–9.
Hammond AS, Rodriguez AL, Townsend SD, Niswender CM, Gregory KJ, Lindsley CW,
and Conn PJ (2010) Discovery of a novel chemical class of mGlu(5) allosteric ligands with
distinct modes of pharmacology. ACS Chem Neurosci 1:702–716.
Kalvass JC and Maurer TS (2002) Influence of nonspecific brain and plasma binding on CNS
exposure: implications for rational drug discovery. Biopharm Drug Dispos 23:327–338.
Lamb JP, Engers DW, Niswender CM, Rodriguez AL, Venable DF, Conn PJ, and Lindsley CW
(2011) Discovery of molecular switches within the ADX-47273 mGlu5 PAM scaffold that
modulate modes of pharmacology to afford potent mGlu5 NAMs, PAMs and partial antago-
nists. Bioorg Med Chem Lett 21:2711–2714.
Sharma S, Kedrowski J, Rook JM, Smith RL, Jones CK, Rodriguez AL, Conn PJ, and Lindsley
CW (2009) Discovery of molecular switches that modulate modes of metabotropic glutamate
receptor subtype 5 (mGlu5) pharmacology in vitro and in vivo within a series of functionalized,
regioisomeric 2- and 5-(phenylethynyl)pyrimidines. J Med Chem 52:4103–4106.
Tizzano JP, Griffey KI, and Schoepp DD (1995a) Induction or protection of limbic seizures in
mice by mGluR subtype selective agonists. Neuropharmacology 34:1063–1067.
Tizzano JP, Griffey KI, and Schoepp DD (1995b) Receptor subtypes linked to metabotropic
glutamate receptor agonist-mediated limbic seizures in mice. Ann N Y Acad Sci 765:230–235,
discussion 248.
Lee AC, Wong RK, Chuang SC, Shin HS, and Bianchi R (2002) Role of synaptic metabotropic
glutamate receptors in epileptiform discharges in hippocampal slices. J Neurophysiol 88:
1625–1633.
Lindsley CW (2011) Molecular switches’ on mGluR allosteric ligands that modulate modes of
pharmacology and/or subtype selectivity, in 7th International Metabotropic Glutamate
Receptors Meeting, Taormina, Italy. Curr Neuropharmacol 9(1):36
Tsai G and Coyle JT (2002) Glutamatergic mechanisms in schizophrenia. Annu Rev Pharmacol
Toxicol 42:165–179.
Vinson PN and Conn PJ (2012) Metabotropic glutamate receptors as therapeutic targets for
schizophrenia. Neuropharmacology 62:1461–1472.
Williams R, Manka JT, Rodriguez AL, Vinson PN, Niswender CM, Weaver CD, Jones CK, Conn
PJ, Lindsley CW, and Stauffer SR (2011) Synthesis and SAR of centrally active mGlu5
positive allosteric modulators based on an aryl acetylenic bicyclic lactam scaffold. Bioorg Med
Chem Lett 21:1350–1353.
Liu X, Chen C, and Smith BJ (2008) Progress in brain penetration evaluation in drug discovery
and development. J Pharmacol Exp Ther 325:349–356.
Marek GJ, Behl B, Bespalov AY, Gross G, Lee Y, and Schoemaker H (2010) Glutamatergic (N-
methyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired
brain? Mol Pharmacol 77:317–326.
Wong RK, Bianchi R, Chuang SC, and Merlin LR (2005) Group I mGluR-induced epilepto-
genesis: distinct and overlapping roles of mGluR1 and mGluR5 and implications for antiepi-
leptic drug design. Epilepsy Curr 5:63–68.
Niswender CM, Johnson KA, Weaver CD, Jones CK, Xiang Z, Luo Q, Rodriguez AL, Marlo JE,
de Paulis T, and Thompson AD, et al. (2008) Discovery, characterization, and antiparkinsonian
effect of novel positive allosteric modulators of metabotropic glutamate receptor 4. Mol
Pharmacol 74:1345–1358.
Noetzel MJ, Rook JM, Vinson PN, Cho HP, Days E, Zhou Y, Rodriguez AL, Lavreysen H,
Stauffer SR, and Niswender CM, et al. (2012) Functional impact of allosteric agonist activity of
selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in reg-
ulating central nervous system function. Mol Pharmacol 81:120–133.
Wood MR, Hopkins CR, Brogan JT, Conn PJ, and Lindsley CW (2011) “Molecular switches”
on mGluR allosteric ligands that modulate modes of pharmacology. Biochemistry 50:
2403–2410.
Zhao W, Bianchi R, Wang M, and Wong RK (2004) Extracellular signal-regulated kinase 1/2 is
required for the induction of group I metabotropic glutamate receptor-mediated epileptiform
discharges. J Neurosci 24:76–84.
Obach RS and Reed-Hagen AE (2002) Measurement of Michaelis constants for cytochrome
P450-mediated biotransformation reactions using a substrate depletion approach. Drug Metab
Dispos 30:831–837.
Address correspondence to: Dr. J. Scott Daniels, Department of Pharmacology,
Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical
Center, Nashville, TN 37232-6600. E-mail: scott.daniels@vanderbilt.edu.
Olney JW, Newcomer JW, and Farber NB (1999) NMDA receptor hypofunction model of
schizophrenia. J Psychiatr Res 33:523–533.