Excess glutamatergic neurotransmission has been implicated in the pathophysiology of schizophrenia, and the activation of metabotropic glutamate 2 (mGlu2) receptor may exert antipsychotic effects by normalizing glutamate transmission. In the present study, we investigated the neurophysiologic and antipsychotic profiles of TASP0433864 [(2S)-2-[(4-tert-butylphenoxy)methyl]-5-methyl-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide], a newly synthesized positive allosteric modulator (PAM) of mGlu2 receptor. TASP0433864 exhibited PAM activity at human and rat mGlu2 receptors with EC50 values of 199 and 206 nM, respectively, without exerting agonist activity at rat mGlu2 receptor. TASP0433864 produced a leftward and upward shift in the concentration-response curve of glutamate-increased guanosine 5′-O-(3-[35S]thio)triphosphate binding to mGlu2 receptor. In contrast, TASP0433864 had negligible activities for other mGlu receptors, including mGlu3 receptor, and did not have any affinity for other receptors or transporters. In hippocampal slices, TASP0433864 potentiated an inhibitory effect of DCG-IV [(2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)glycine], a mGlu2/3 receptor agonist, on the field excitatory postsynaptic potentials in the dentate gyrus, indicating that TASP0433864 potentiates the mGlu2 receptor–mediated presynaptic inhibition of glutamate release. Moreover, TASP0433864 inhibited both MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate]- and ketamine-increased cortical γ band oscillation in the rat cortical electroencephalogram, which have been considered to reflect the excess activation of cortical pyramidal neurons. The inhibitory effect of TASP0433864 on cortical activation was also observed in the mouse 2-deoxy-glucose uptake study. In a behavioral study, TASP0433864 significantly inhibited both ketamine- and methamphetamine-increased locomotor activities in mice and rats, respectively. Collectively, these findings indicate that TASP0433864 is a selective mGlu2 receptor PAM with antipsychotic activity, and the attenuation of excess glutamatergic neurotransmission may be involved in the action of TASP0433864.
Glutamate is the primary excitatory neurotransmitter in the mammalian brain, and is responsible for most corticocortical, thalamocortical, and corticofugal neurotransmission through ionotropic (ligand-gated cation channels) and metabotropic (G protein–coupled) glutamate receptors (Nakanishi, 1992; Kew and Kemp, 2005). The metabotropic glutamate (mGlu) receptor family includes eight receptor subtypes classified into three groups based on sequence homology, signaling transduction mechanisms, and pharmacologic properties: group I (mGlu1 and -5) receptors are positively coupled to phospholipase C via Gq/11 protein, whereas both group II (mGlu2 and -3) and group III (mGlu4, -6, -7, and -8) receptors are negatively coupled to adenylate cyclase via Gi/o protein (Conn and Pin, 1997; Schoepp et al., 1999). Among them, mGlu2/3 receptors are abundantly distributed in the forebrain regions and limbic areas responsible for mental function, including the cerebral cortex, hippocampus, striatum, nucleus accumbens, thalamus, and amygdala (Neki et al., 1996; Shigemoto et al., 1997; Tamaru et al., 2001; Wright et al., 2013). Thus, mGlu2/3 receptors are thought to be implicated in several psychiatric disorders, including schizophrenia, anxiety, depression, and addiction (Nicoletti et al., 2011). In addition, because mGlu2/3 receptor perisynaptically localized on glutamatergic synaptic terminals negatively regulates glutamate release as an autoreceptor (Cartmell and Schoepp, 2000), the activation of mGlu2/3 receptors is considered an attractive strategy for the treatment of disorders associated with excessive glutamatergic neurotransmission, such as schizophrenia (Chaki, 2010; Nicoletti et al., 2011; Fell et al., 2012).
Indeed, mGlu2/3 receptor orthosteric agonists, which have similar potency for both mGlu2 and -3 receptors, have been reported to exhibit antipsychotic and anxiolytic efficacy in preclinical animal models (Monn et al., 1997; Moghaddam and Adams, 1998; Cartmell et al., 1999; Nakazato et al., 2000; Rorick-Kehn et al., 2007) as well as in patients with schizophrenia and anxiety disorders (Grillon et al., 2003; Schoepp et al., 2003; Kellner et al., 2005; Krystal et al., 2005; Patil et al., 2007; Dunayevich et al., 2008). In support of the aforementioned mechanisms, mGlu2/3 receptor agonists have been reported to inhibit excitatory neurotransmission in electrophysiologic studies (Kilbride et al., 1998; Kew et al., 2001; Benneyworth et al., 2007), to normalize excessive glutamate levels in the prefrontal cortex (PFC) in schizophrenia animal models (Moghaddam and Adams, 1998; Lorrain et al., 2003), and to reduce the aberrant PFC activity caused by N-methyl-d-aspartic acid (NMDA) receptor antagonists (Homayoun et al., 2005; Gozzi et al., 2008; Chin et al., 2011; Dedeurwaerdere et al., 2011; Jones et al., 2012). The relative contributions of mGlu2 and -3 to the mechanisms of action of mGlu2/3 receptor agonists remain unknown. However, results in studies using mice lacking either mGlu2 or -3 receptor have indicated that mGlu2 receptor, rather than mGlu3 receptor, likely contributes to the presynaptic autoreceptor function (Kew et al., 2002) and the antipsychotic activities of mGlu2/3 receptor agonists (Fell et al., 2008; Woolley et al., 2008), suggesting that mGlu2 receptor is an attractive potential therapeutic target.
Recently, the structural diversity of mGlu2 receptor positive allosteric modulators (PAMs), which provide an approach to the selective activation of mGlu2 receptor, has been noted (Fraley, 2009), and these PAMs reportedly exhibit antipsychotic and anxiety activities in preclinical models (Galici et al., 2005, 2006; Johnson et al., 2005; Benneyworth et al., 2007; Fell et al., 2011; Lavreysen et al., 2013). However, the roles of mGlu2 receptor in normalizing excessive glutamate release as well as aberrant PFC activity, which mimic the pathophysiologic states of schizophrenia, and the effects of mGlu2 receptor PAMs on these states have not been fully addressed.
In the present study, we investigated the neurophysiologic and antipsychotic properties of a newly synthesized mGlu2 PAM, TASP0433864 [(2S)-2-[(4-tert-butylphenoxy)methyl]-5-methyl-2,3-dihydroimidazo[2,1-b][1,3]oxazole-6-carboxamide; Fig. 1], with a high selectivity and good brain penetration, to clarify the involvement of glutamatergic neurotransmission in the mechanism of action of TASP0433864.
Materials and Methods
The animals were group-housed [except for the electrode-implanted rats that were used for the electroencephalogram (EEG) recordings, which were housed singly] with food and water provided ad libitum, and under a 12-hour light/dark cycle (lights on at 7:00 AM), a constant temperature of 23°C, and a relative humidity of 50%. All experimental procedures were reviewed and approved by the Taisho Pharmaceutical Co., Ltd. Animal Care Committee and met the Japanese Experimental Animal Research Association standards, as defined in the Guidelines for Animal Experiments (1987).
TASP0433864 was synthesized in Taisho Research Laboratories (Saitama, Japan). TASP0433864 was dissolved in dimethylsulfoxide (DMSO) and was diluted with each assay buffer to various concentrations for the in vitro studies, or was suspended in 0.5 w/v% methyl cellulose and administered intraperitoneally for the in vivo studies. [35S]GTPγS (guanosine 5′-O-(3-[35S]thio)triphosphate; specific radioactivity: 46.25 TBq/mmol) was purchased from PerkinElmer Life Science (Boston, MA). DCG-IV [(2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)glycine] and LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] were purchased from Tocris Bioscience (Bristol, UK). MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate] and memantine hydrochloride (memantine) were purchased from Sigma-Aldrich (St. Louis, MO). Ketamine hydrochloride (ketamine) was purchased from Sankyo Yell Pharmaceutical (Tokyo, Japan) as Ketalar for intravenous injection. Methamphetamine hydrochloride (methamphetamine) was purchased from Dainippon Sumitomo Pharma (Osaka, Japan) as Philopon. For the in vivo studies, all drugs were dissolved in saline and administered subcutaneously. Each drug, including TASP0433864, was injected in a volume of 2 or 10 ml/kg for the rats or mice, respectively.
In Vitro Assays
Cell Culture and Membrane Preparation.
Chinese hamster ovary (CHO) cell lines stably expressing human mGlu2 or -3 receptor were established in house. CHO cell lines stably expressing rat mGlu2 receptor were kindly donated by Dr. S. Nakanishi (Kyoto University, Kyoto, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% dialyzed fetal bovine serum, 2 mM l-glutamine, 1% proline, 1 mM sodium pyruvate, 1 mM succinic acid, 1 mM succinic disodium salt, penicillin (50 units/ml for rat mGlu receptor or 100 units/ml for human mGlu receptor), and streptomycin (50 μg/ml for rat mGlu receptor or 100 μg/ml for human mGlu receptor), and were maintained at 37°C in the humidified atmosphere of a 5% CO2 incubator. For the expression of the human mGlu receptor, hygromycin B (400 μg/ml for mGlu2 receptor or 300 μg/ml for mGlu3 receptor) was also added to the culture medium. Confluent cells expressing human or rat mGlu2 and mGlu3 receptor were washed in phosphate-buffered saline, scraped, and centrifuged at 190g for 5 minutes at 4°C. The pellet was homogenized with 20 mM HEPES buffer (pH 7.4), which contained 1 mM EDTA for mGlu3 receptor, then centrifuged at 48,000g for 20 minutes at 4°C. The pellet was washed twice and suspended with 20 mM HEPES buffer with/without 1 mM EDTA (pH 7.4) to obtain the crude membrane fraction, which was stored at −80°C.
[35S]GTPγS Binding Assay
The aforementioned membranes were diluted in 20 mM HEPES buffer (pH 7.4) containing 100 mM NaCl, 10 mM MgCl2, 8.4 μM GDP, 10 μg/ml saponin, and 0.1% bovine serum albumin, which also contained 1 mM EDTA for mGlu3 receptor, to yield a protein concentration of 10 μg/assay (for mGlu2 receptor) or 15 μg/assay (for mGlu3 receptor). The membranes were preincubated with various concentrations of TASP0433864 for 20 minutes at 30°C; various concentrations of glutamate and 0.15 nM [35S]GTPγS were then added, and the membranes were incubated for 60 minutes at 30°C. The reaction was terminated by rapid filtration under a vacuum through a UniFilter GF/C microplate (PerkinElmer Life Science), after which the filters were washed with 1 ml of ice-cold 20 mM HEPES buffer (pH 7.4) using a UniFilter-96 harvester (PerkinElmer Life Science). After drying the filters, 20 μl of MicroScint-O (PerkinElmer Life Science) was added, and the membrane-bound radioactivity was counted with a TopCount NXT (PerkinElmer Life Science). The specific binding of [35S]GTPγS was calculated by subtracting a nonspecific binding in the absence of glutamate. The amount of [35S]GTPγS binding was normalized with the maximal response to 1 mM (for mGlu2 receptor) or 30 μM (for mGlu3 receptor) glutamate. The EC50 values of TASP0433864 for the mGlu2 PAM activity were determined in the presence of an EC20-equivalent concentration of glutamate (1 μM for human mGlu2 receptor or 3 μM for rat mGlu2 receptor). The Emax values of TASP0433864 for the mGlu2 PAM activity were defined as the maximum specific binding of [35S]GTPγS in the presence of TASP0433864. The effect of TASP0433864 on human mGlu3 receptor was examined in the presence of an EC40-equivalent concentration of glutamate at 100 nM.
Measurement of cAMP Accumulation.
Eighteen hours before the assay, CHO cells expressing rat mGlu2 receptor were plated at a density of 20,000 cells/well in a 96-well plate in 100 μl of the aforementioned medium. After the removal of the medium, the cells were incubated in a medium without l-glutamine for 4−5 hours at 37°C, then washed using Hanks’ balanced salt solution containing 20 mM HEPES, 0.3 mM 3-isobutyl-1-methylxanthine, and 0.1% bovine serum albumin (incubation buffer, pH 7.4). TASP0433864 at each concentration was preincubated with the incubation buffer for 20 minutes at 37°C, followed by the addition of glutamate and forskolin to yield a final concentration of 3 and 10 μM, respectively. After incubation for 15 minutes at 37°C, 1.25% Triton X-100 containing incubation buffer (lysis buffer) was added to the cells to terminate the reaction, and the cells were incubated at room temperature for 20 minutes after gentle mixing. The inhibition of forskolin-stimulated cAMP accumulation by TASP0433864 was measured using the homogeneous time-resolved fluorescence technology of a cAMP assay kit (Cisbio, Bedford, MA). To measure the cAMP levels, the treated cells were incubated with cAMP-d2 conjugate and anti–cAMP-cryptate conjugate in lysis buffer at room temperature for 60 minutes. The homogeneous time-resolved fluorescence signal was detected using an EnVision plate reader (PerkinElmer) to calculate the ratio of fluorescence at 665–620 nm. After the raw data were converted to the cAMP amount (pmol/well) using a cAMP standard curve generated for each experiment, the inhibition of cAMP accumulation was normalized with the maximal response to 1 mM glutamate.
TASP0433864 was tested at a concentration of 10 μM by CEREP (Celle L’Evescault, France) and in house for its ability to inhibit radioligand binding or enzymatic activity to a battery of neurotransmitter and peptide receptors, ion channels, transporters, and enzymes.
Ex Vivo Electrophysiology
Hippocampal slices were prepared from young male Wistar rats (4 weeks old; Japan SLC, Inc., Hamamatsu, Japan) or Sprague-Dawley rats (5−7 weeks old; Charles River Laboratories, Yokohama, Japan) for the DCG-IV with LY341495 study or the DCG-IV with TASP0433864 study, respectively. The animals were sacrificed, and their brains were removed and submerged into ice-cold artificial cerebrospinal fluid (ACSF), which contains the following (in mM): 124 NaCl, 3.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2.5 CaCl2, 10 glucose, and 22 NaHCO3 (for the DCG-IV with LY341495 study) or 124 NaCl, 5 KCl, 1.25 NaH2PO4, 1.5 MgSO4, 2.5 CaCl2, 10 glucose, and 24 NaHCO3 (for the DCG-IV with TASP0433864 study), saturated with 95% O2−5% CO2. The isolated hippocampus was cut into 400-μm-thick transverse slices using a vibratome (DTK-1000; Dosaka EM, Kyoto, Japan; or VT1200S; Leica, Nussloch, Germany). Slices were transferred immediately to an incubation chamber filled with ACSF saturated with 95% O2−5% CO2 at room temperature and kept under this condition for at least an hour before the extracellular recordings.
Slices were transferred to a recording chamber continuously perfused (2−3 ml/min) with ACSF saturated with 95% O2−5% CO2 at a temperature of about 30−31°C. The field excitatory postsynaptic potentials (fEPSPs) were recorded using a standard extracellular recording technique from the molecular layer of the dentate gyrus (DG), where mGlu2 receptor is abundantly distributed (Neki et al., 1996; Shigemoto et al., 1997; Wright et al., 2013). A bipolar tungsten electrode (Unique Medical, Tokyo, Japan) was placed in the middle third of the molecular layer of the DG for stimulation of the medial perforant path (MPP) fibers; this stimulation consisted of paired-pulse stimuli (200-microsecond duration, 40-millisecond interval) delivered at 0.033 Hz. A glass micropipette (Clark Electromedical Instruments, Pangbourne, UK) filled with ACSF (pipette resistance: 2−4 MΩ) was placed in the middle third of the molecular layer of the DG to record the fEPSPs evoked in the DG. Voltage signals were amplified using a microelectrode amplifier (MEZ-8300; Nihon Kohden, Tokyo, Japan), low-pass filtered at 1 kHz, and stored on the computer hard disk for off-line analysis (PowerLab System; ADInstruments, Oxford, UK). The stimulus intensity was adjusted to produce about 50–70% of the maximal fEPSP amplitude.
Slices in which stable fEPSP could be recorded for over 10 minutes were used. Each drug was dissolved in the ACSF and added to the perfusate. DCG-IV at each concentration with/without LY341495 (0.1 or 1 μM) was bath-applied for 30 minutes to examine the concentration-dependent effect of DCG-IV. To examine the potentiating effect of TASP0433864 on mGlu2 receptor agonistic activity, the preapplication of TASP0433864 or 0.1% DMSO for 30 minutes was followed by the bath-application of DCG-IV at 50 nM with TASP0433864 for 30 minutes, subsequently washed out with ACSF and antagonized with LY341495. Four to five independent experiments were performed for each treatment group.
The first fEPSP amplitude and the paired-pulse ratio (PPR), which was defined as the amplitude ratio of the second fEPSP relative to the first fEPSP, were calculated in each fEPSP recording. These values were averaged in 1-minute bins and were normalized with the baseline response for 10 minutes before drug application. The minimal amplitude of the first fEPSP and the maximal PPR during the application of DCG-IV were used for the concentration-response curve of DCG-IV to calculate the EC50 values. In the TASP0433864 study, the first fEPSP amplitude and the PPR after the application of DCG-IV were compared between the absence and presence of TASP0433864.
Measurement of Brain Metabolic Activity Using a Nonradioisotope Method
Male C57BL/6J mice (8 weeks old; Charles River Laboratories) were used in the experiment after fasting for more than 15 hours. Vehicle or TASP0433864 was intraperitoneally administered 30 minutes before the subcutaneous administration of memantine (30 mg/kg) or saline. 2-Deoxy-d-glucose (2-DG; 3 mg/kg) was subcutaneously injected 15 minutes after the administration of memantine or saline. Animals were decapitated following cervical dislocation at 45 minutes after the injection of 2-DG. Then the brains were removed and the PFC including the anterior cingulate cortex, where 2-DG uptake is remarkably increased by NMDA receptor antagonists (Miyamoto et al., 2000), was dissected. The dissected tissues were homogenized and subsequently heated at 95°C for 15 minutes. Following centrifugation (17,360g for 15 minutes at 4°C) to remove the cell debris, the resulting supernatants were used to measure 2-DG uptake. The measurement of 2-DG uptake in the tissue was performed using the 2-deoxyglucose uptake measurement kit (Cosmo Bio, Tokyo, Japan) according to the manufacturer’s instructions, enabling the 2-DG uptake into the cells to be quantified as the amount of 2-deoxyglucose 6-phosphate (2-DG6P) without the interference of the remaining extracellular 2-DG based on the enzymatic photometric method (Saito et al., 2011).
In Vivo Electroencephalogram Recording
The rat EEG recording was performed using our previously reported methods, with some modification (Hiyoshi et al., 2014).
Implantation of Electrodes.
To implant electrodes for the EEG recording, male Sprague-Dawley rats (10−11 weeks old; Charles River Laboratories) were anesthetized with pentobarbital sodium (50 mg/kg i.p.) and fixed in a stereotaxic frame. After holes were drilled in the skull, stainless steel screw electrodes (E363/20; Plastics One, Roanoke, VA) were placed on the cerebral dura mater according to a rat brain atlas (Paxinos and Watson, 2007) as the follows: the recording electrode (placed over the occipital cortex at 6.0 mm posterior and 2.0 mm lateral from the bregma), the reference electrode (placed over the cerebellum at 11.5 mm posterior and 0 mm lateral from the bregma), and the ground electrode (placed over the contralateral occipital cortex at 6.0 mm posterior and 2.0 mm lateral from the bregma). All electrodes were socketed into an electrode pedestal (Plastics One), which was then fixed onto the skull using a combination of dental acrylic resin (ADFA; Shofu, Kyoto, Japan) and α-cyanoacrylate adhesive. The electrode-implanted rats were then singly housed and allowed to recover for more than 5 days before the EEG recording.
EEG Recording and Drug Administration.
The EEG recordings were performed during the light phase of the light/dark cycle. Rats (11−14 weeks old) were individually transferred to an acrylic chamber (30 cm wide × 30 cm deep × 35 cm high) placed within an electrically shielded sound-proof box, and were tethered to a lead wire connected to a slip-ring commutator (Hikari Denshi, Tokyo, Japan). Cortical EEG recordings and drug administrations were performed under freely moving and unrestrained conditions. The EEG signals were amplified (20,000 times) and bandpass filtered (0.5−1000 Hz) using a biophysical amplifier (AB-611J; Nihon Kohden), digitized at a sampling rate of 2.5 kHz with an analog-to-digital converter [AD16-16U(PCI)EH; Contec, Osaka, Japan], and recorded using the data acquisition program VitalRecorder (version 1.3; Kissei Comtec, Matsumoto, Japan). After a baseline EEG recording for 30 minutes in parallel with the acclimation of the animal to the measurement environment, vehicle or TASP0433864 was intraperitoneally administered, followed 30 minutes later by subcutaneous administration of MK-801 (0.1 mg/kg) or ketamine (5 mg/kg). The EEG recording was continued for 180 minutes after the administration of MK-801 or ketamine. The animals were repeatedly used for the EEG recordings a maximum of three times after a more than 7-day interval for the washout of the drug.
Data Analysis for Quantitative EEG.
The EEG data were individually analyzed off-line using the data analysis program SleepSign (version 3.0; Kissei Comtec) for quantification. After digital filtering (0.5−200 Hz bandpass), a fast-Fourier transform was performed for each 4-second epoch for a power spectrum analysis. The total power in the γ band frequency (30−80 Hz) was averaged for 1-minute bins and was normalized with the mean of 30-minute baseline recordings to plot the time course. The area under the curve for the γ power change for 90 minutes after the MK-801 injection or for 60 minutes after the ketamine injection was calculated to evaluate the effects of TASP0433864 on the MK-801– or ketamine-increased γ power, respectively.
Measurement of Locomotor Activity in Mice.
The locomotor activities of mice were recorded using an infrared motion detector with the AB system (Neuroscience Inc., Tokyo, Japan). Male CD-1 (ICR) mice (5 weeks old; Japan SLC, Inc.) were individually habituated to a test chamber (34.5 cm wide × 40.3 cm deep × 17.7 cm high) equipped with an infrared sensor for 120 minutes, and were then subcutaneously administered with saline or ketamine at a dose of 30 mg/kg. The locomotor activity was recorded in 10-minute bins for 90 minutes immediately after the administration of saline or ketamine. Vehicle or TASP0433864 was intraperitoneally administered 30 minutes before the administration of saline or ketamine.
Measurement of Locomotor Activity in Rats.
The locomotor activities of rats were measured using a SCANET apparatus (Melquest Ltd., Toyama, Japan). Male Wistar rats (7 weeks old; Charles River Laboratories) were individually habituated to a test chamber (47 cm wide × 28 cm deep × 30 cm high) placed in a sound-proof box for 60 minutes, and were then subcutaneously administered with saline or methamphetamine at a dose of 1 mg/kg. The locomotor (horizontal movement) and rearing (vertical movement) activities were recorded in 10-minute bins for 120 minutes immediately after the administration of saline or methamphetamine. Vehicle or TASP0433864 was intraperitoneally administered 30 minutes before the administration of saline or methamphetamine.
Pharmacokinetic Follow-Up in Rats.
To evaluate the exposure levels of TASP0433864 in the plasma, brain, and cerebrospinal fluid (CSF) after systemic administration, TASP0433864 was intraperitoneally administered at doses of 10 and 30 mg/kg to male Wistar rats on the same experimental day as the locomotor measurements. Animals were sacrificed and a blood sample was collected 1 hour after TASP0433864 administration; the CSF and brain were then rapidly collected and removed, respectively. The plasma samples were separated by centrifugation (860g for 10 minutes at 4°C), and all samples were stored at −30°C until assay. Each sample was extracted using protein precipitation, and was subsequently analyzed for the determination of TASP0433864 levels using a liquid chromatography/tandem mass spectrometry–qualified research method on an API4000 instrument (AB SCIEX, Foster City, CA).
Data are presented as the mean ± S.E.M. In vitro EC50 values were calculated from nonlinear regression analyses using the least-squares algorithms of a GraphPad Prism 5 analysis program (GraphPad Software, San Diego, CA). The statistical analyses were performed using SAS software (SAS Institute Japan, Tokyo, Japan). The effects of TASP0433864 on the time course plot were analyzed using a two-way repeated-measures analysis of variance (ANOVA). Data were analyzed using Student’s t test or a one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons. A value of P < 0.05 was regarded as significant.
TASP0433864 Exhibits Selective PAM Activity for Human and Rat mGlu2 Receptor.
In membranes expressing human and rat mGlu2 receptor, glutamate increased [35S]GTPγS binding in a concentration-dependent manner, with EC50 values of 5.4 and 8.2 µM, respectively. In the presence of a fixed glutamate concentration at an equivalent EC20 value (1 µM for human mGlu2 and 3 µM for rat mGlu2), TASP0433864 potentiated the glutamate-increased [35S]GTPγS binding to the membranes of CHO cells expressing human and rat mGlu2 receptor in a concentration-dependent manner, with EC50 values of 252 and 180 nM and Emax values of 91 and 135%, respectively (Fig. 2, A and B; Table 1). In contrast, TASP0433864 alone had a negligible effect on [35S]GTPγS binding to the membranes of CHO cells expressing rat mGlu2 receptor (Fig. 2B). Moreover, TASP0433864 shifted the concentration-response of the glutamate-increased [35S]GTPγS binding curve both upward and leftward in a concentration-dependent manner (Fig. 2C).
In addition, glutamate inhibited forskolin-stimulated cAMP accumulation in CHO cells expressing rat mGlu2 receptor in a concentration-dependent manner, with an EC50 value of 8.1 µM. TASP0433864 enhanced the inhibitory effect of glutamate at a concentration of 3 µM (as an equivalent EC20 value) on forskolin-stimulated cAMP accumulation in a concentration-dependent manner, with an EC50 value of 87 nM and an Emax value of 111% (Table 1).
In contrast, TASP0433864 did not affect the response to glutamate (100 nM) in [35S]GTPγS binding of membranes expressing human mGlu3 receptor (Table 1), whereas glutamate increased [35S]GTPγS binding in a concentration-dependent manner, with an EC50 value of 286 nM. The selectivity of TASP0433864 for some other subtypes of mGlu receptor was evaluated using a [35S]GTPγS binding assay (human mGlu6, mGlu8 receptor) or a Ca2+ mobilization assay (human mGlu5 receptor). No agonist, antagonist, or PAM activity was observed with TASP0433864 (up to 10 µM) in any of the subtypes that were tested, except that TASP0433864 showed agonist activity at mGlu5 receptor at a high concentration (71% at 10 μM versus 1 mM glutamate response) (data not shown). Further TASP0433864 profiling against a diverse panel of 48 central nervous system molecular targets revealed that TASP0433864 at a concentration of 10 μM showed no significant displacement of radioligand binding, except for human serotonin 5-HT2B receptor (72% inhibition at 10 μM) and human monoamine oxidase B (MAO-B; 92% inhibition at 10 μM) (Table 2). Moreover, the IC50 values of TASP0433864 for the inhibition of radioligand binding to human 5-HT2B receptor and the inhibition of enzymatic reactivity for human MAO-B were 4.0 µM (n = 1) and 0.59 µM (n = 1), respectively.
TASP0433864 Enhances DCG-IV–Induced fEPSP Depression in Rat Hippocampal Slices.
In rat hippocampal slices, the mGlu2/3 receptor orthosteric agonist DCG-IV reduced the amplitude of fEPSP and increased the PPR in the MPP-DG synapses in a concentration-dependent manner, with EC50 values of 175 and 355 nM, respectively (Fig. 3). Both of the concentration-response curves of DCG-IV for the fEPSP amplitude and the PPR shifted rightward in the presence of mGlu2/3 receptor antagonist LY341495 (Fig. 3, C and D).
In the combination study with DCG-IV at a slightly effective concentration of 50 nM, a two-way repeated-measures ANOVA revealed significant concentration-time interactions of TASP0433864 (0.3−3 µM) on the fEPSP amplitude (F237,1264 = 11.95, P < 0.001) and the PPR (F237,1264 = 12.09, P < 0.001) (Fig. 4, B and C). TASP0433864 at concentrations of 0.3–3 µM did not affect the fEPSP amplitude or the PPR by itself (Fig. 4, D and E). However, TASP0433864 concentration dependently reduced the amplitude of fEPSP (F3,16 = 33.80, P < 0.001) and increased the PPR (F3,16 = 19.12, P < 0.001) in the presence of DCG-IV at 50 nM (Fig. 4), showing significant potentiating actions at concentrations of 1 and 3 µM compared with the 0.1% DMSO–treated group (P < 0.01 or 0.001). In addition, these effects were reversed by washout or the application of LY341495 at 1 µM.
TASP0433864 Reduces Memantine-Elicited Brain Metabolic Activity in Mouse PFC.
A noncompetitive NMDA receptor antagonist, memantine (30 mg/kg), significantly increased 2-DG6P accumulation in the PFC after the administration of 2-DG to mice (P < 0.001) (Fig. 5), indicating an increase in 2-DG uptake into the area. Pretreatment with TASP0433864 (3−30 mg/kg) inhibited memantine-elicited 2-DG6P accumulation in a dose-dependent manner (F3,28 = 7.06, P < 0.01) with a significant inhibition at a dose of 30 mg/kg (P < 0.01) (Fig. 5). In addition, TASP0433864 at a dose of 30 mg/kg did not change blood glucose levels compared with the vehicle (a satellite study; data not shown).
TASP0433864 Reverses Noncompetitive NMDA Receptor Antagonist–Increased Cortical γ Power in Rat Quantitative EEG.
In the rat quantitative EEG study, both MK-801 (0.1 mg/kg) and ketamine (5 mg/kg), noncompetitive NMDA receptor antagonists, remarkably increased the γ band oscillation (GBO) power (Fig. 6, A and C). In the time-course analyses, a two-way repeated-measures ANOVA demonstrated that TASP0433864 (10−30 mg/kg) showed significant dose-time interactions (F418,3762 = 2.23, P < 0.001; F418,4389 = 2.43, P < 0.001) on the MK-801– and ketamine-increased GBO power, respectively (Fig. 6, A and C). TASP0433864 dose-dependently reduced the MK-801–induced GBO increase (F2,18 = 5.52, P < 0.05), showing a significant inhibition at 30 mg/kg compared with the vehicle (P < 0.01) (Fig. 6B). TASP0433864 also attenuated the ketamine-induced GBO increase in a dose-dependent manner (F2,21 = 18.21, P < 0.001), showing a significant inhibition at 10 and 30 mg/kg compared with the vehicle (all P < 0.001) (Fig. 6D).
TASP0433864 Inhibits Both Ketamine-Induced Hyperlocomotion in Mice and Methamphetamine-Induced Hyperlocomotion in Rats.
The administration of ketamine at a dose of 30 mg/kg significantly increased the locomotor activity in mice (P < 0.001; Fig. 7, A and B). In a time-course analysis, TASP0433864 (10−30 mg/kg) showed significant dose-time interactions (F16,160 = 4.61, P < 0.001) on the ketamine-induced hyperactivity of locomotion (Fig. 7A). TASP0433864 dose-dependently inhibited the ketamine-induced hyperlocomotion (F2,20 = 5.12, P < 0.05), showing a significant inhibition at 30 mg/kg compared with the vehicle (P < 0.05) (Fig. 7B).
In addition, the administration of methamphetamine at a dose of 1 mg/kg significantly increased locomotor and rearing activities in rats (all P < 0.001) (Fig. 8). In a time-course analysis, TASP0433864 (10−100 mg/kg) showed significant dose-time interactions (F33,308 = 8.89, P < 0.001; F33,308 = 8.16, P < 0.001) on the methamphetamine-induced hyperactivities of locomotion and rearing, respectively (Fig. 8, A and C). TASP0433864 dose-dependently inhibited the methamphetamine-induced hyperlocomotion (F3,28 = 20.36, P < 0.001), showing a significant inhibition at 10 mg/kg or more compared with the vehicle (all P < 0.001) (Fig. 8B). Simultaneously, TASP0433864 also inhibited the methamphetamine-increased rearing in a dose-dependent manner (F3,28 = 18.91, P < 0.001), showing a significant inhibition at 10 mg/kg or more compared with the vehicle (all P < 0.001) (Fig. 8D).
As shown in Table 3, TASP0433864 exposure levels in the plasma, brain, and CSF dose-dependently increased at 1 hour after the administration of TASP0433864 in the pharmacokinetic follow-up study.
The pathophysiology of schizophrenia is known to involve not only subcortical excessive dopaminergic transmission, but also cortical hyperglutamatergic transmission resulting from a reduction in GABAergic interneuron activity through NMDA receptor hypofunction (Krystal et al., 2003; Laruelle et al., 2003). In the present study, we demonstrated that TASP0433864, a newly synthesized selective mGlu2 receptor PAM, normalized brain metabolic hyperactivity and γ EEG hyperactivity in the cortex induced by the blockade of NMDA receptor (a situation that may mimic the pathophysiologic conditions of schizophrenia) and improved the locomotor hyperactivities in both glutamatergic and dopaminergic models of schizophrenia.
When examined using in vitro studies, TASP0433864 exhibited a potentiation effect on glutamate-increased [35S]GTPγS binding to both rat and human recombinant mGlu2 receptors. The PAM activity of TASP0433864 at mGlu2 receptor was also confirmed using a cell-based assay, where TASP0433864 enhanced the inhibitory effect of glutamate on forskolin-stimulated cAMP formation. In contrast, TASP0433864 did not affect some of the other mGlu receptor subtypes, including mGlu3 receptor, both in the presence and absence of glutamate, and did not exhibit an apparent agonist activity on mGlu2 receptor. These results indicate that TASP0433864 selectively enhances the stimulating effect of endogenous glutamate on mGlu2 receptor activity without affecting mGlu2 receptor activity on its own, unlike orthosteric mGlu2/3 receptor agonists, although we have not fully addressed the effects of TASP0433864 on mGlu1, -4, and -7. Of note, we confirmed that TASP0433864 did not show activity at mGlu1 receptor up to 10 μM in a preliminary study (Ca2+ mobilization assay).
We next addressed whether TASP0433864 exerts PAM activity on native mGlu2 receptors using an electrophysiologic study in rat hippocampal MPP-DG synapses, where mGlu2 receptor is abundantly expressed at presynaptic terminals (Shigemoto et al., 1997; Wright et al., 2013). In the present study, the mGlu2/3 receptor agonist DCG-IV reduced the fEPSP amplitude in a concentration-dependent manner, with an EC50 value of 175 nM, which was consistent with the findings of previous reports (Kilbride et al., 1998; Kew et al., 2001). Moreover, DCG-IV increased the PPR in a concentration-dependent manner, meaning that DCG-IV decreased glutamate release from the presynaptic terminals (Kilbride et al., 1998). The effects of DCG-IV on both the fEPSP amplitude and the PPR were antagonized by LY341495 in a concentration-dependent manner, demonstrating that the activation of mGlu2/3 receptors inhibits excitatory synaptic transmission through the inhibition of glutamate release from the presynaptic terminals. Under this condition, TASP0433864 potentiated the effects of DCG-IV on both the fEPSP amplitude and the PPR in a concentration-dependent manner. Given that the inhibition of MPP-DG synaptic transmission by DCG-IV has been reported to be largely due to the activation of mGlu2 receptor (Kew et al., 2001), these results suggest that TASP0433864 exerts PAM activity on native mGlu2 receptor, leading to the suppression of glutamate release and excitatory neurotransmission. Interestingly, TASP0433864 by itself altered neither the fEPSP amplitude nor the PPR in this study. This result may be explained by the fact that glutamate released from the presynaptic terminals in response to an electrical stimulus is insufficient to activate mGlu2 autoreceptor, at least in our experimental protocol.
We then examined whether TASP0433864, through its mGlu2 receptor PAM activity, was capable of reversing the aberrant neurotransmission resulting from cortical disinhibition using two neurophysiologic methods in vivo. In the present study, the 2-DG uptake and the γ band oscillation power were used as indexes of brain metabolic activity and local neuronal network activity, respectively. Subanesthetic doses of NMDA receptor antagonists increased the indexes of both cortical activity as evident by the increased 2-DG uptake and γ band oscillation power, which was consistent with the findings of previous reports (Pinault, 2008; Dedeurwaerdere et al., 2011; Hiyoshi et al., 2014). These alterations of cortical activity induced by NMDA receptor blockade are likely to reflect the hyperexcitability of pyramidal neurons through GABAergic disinhibition (Krystal et al., 2003; Gunduz-Bruce, 2009), which is associated with an increase in extracellular glutamate in the cortex (Moghaddam et al., 1997; Moghaddam and Adams, 1998). TASP0433864 showed a wide range of inhibitory effects on the memantine-elicited 2-DG uptake and the MK-801– and ketamine-increased γ oscillation power, suggesting that TASP0433864 inhibits the excessive activation of cortical pyramidal neurons. In consideration of the effects of TASP0433864 on hippocampal fEPSP, a reduction in glutamate release from presynaptic terminals likely contributes to the normalization of cortical hyperglutamatergic state. In a preliminary study, TASP0433864 slightly but dose-dependently reduced both the baseline of 2-DG uptake and γ oscillation power with the same effective doses (data not shown). Therefore, we cannot fully exclude the possibility that TASP0433864 also affects the normal neurotransmission mediated by glutamate. It should be noted that the measurements of 2-DG uptake or γ band EEG power observed in the present study can be translated into a positron emission tomography with 2-[fluorine-18]-fluoro-2-deoxy-d-glucose or a quantitative EEG in clinical study, respectively (Breier et al., 1997; Sanacora et al., 2014). Hence, the alternation of cortical activity may be a translatable biomarker for mGlu2 receptor PAMs.
Finally, we confirmed the antipsychotic activity of TASP0433864 using both NMDA receptor antagonist and dopaminergic psychostimulant models. In mice, TASP0433864 significantly inhibited ketamine-induced hyperlocomotion at a dose of 30 mg/kg, which was consistent with an effective dose for the cortical hyperactivity (the memantine-increased 2-DG uptake) in mice. Given that subanesthetic doses of ketamine reportedly increase glutamate release in the PFC (Moghaddam et al., 1997), the counteraction of TASP0433864 on locomotor hyperactivity in the ketamine model may be attributable to the inhibition of excessive glutamate release in the cortex induced by NMDA receptor blockade. In rats, TASP0433864 at a dose of 10 mg/kg also inhibited both horizontal and vertical locomotor activities increased by methamphetamine. Of note, the exposure level of TASP0433864 in CSF after the administration of TASP0433864 at 10 mg/kg was 498 nM, which was well over the EC50 values for rat mGlu2 receptor PAM activity in vitro, indicating that the antipsychotic effect of TASP0433864 is mediated through mGlu2 receptor PAM activity. Moreover, because the effective dose of TASP0433864 in the dopaminergic psychosis model was practically in accordance with that in the γ EEG hyperactivity model in rats, the mechanism for the antipsychotic effect of TASP0433864 in this model may also be attributed to the suppression of cortical hyperactivity, which is also supported by a previous report that methamphetamine increased the glutamate level in the PFC, but not the nucleus accumbens, in a microdialysis study (Shoblock et al., 2003). In addition, the inhibitory effect of TASP0433864 was more sensitive with regard to the methamphetamine-induced vertical (rearing) and horizontal hyperactivities compared with the ketamine-induced hyperlocomotion. Given that the methamphetamine-induced hyperlocomotion and the increase in rearing activity are assumed to be associated with an increase in the dopamine level in the nucleus accumbens and striatum (Camp et al., 1994; Swanson et al., 1997; Thiel et al., 1999), in addition to the suppression of cortical hyperactivity, a dopaminergic modulation by TASP0433864 in the subcortical region might also contribute to the inhibition of locomotor hyperactivity, although we cannot rule out a difference in the exposure level of TASP0433864 between mice and rats. It might also be noted that TASP0433864 displays, in addition to the mGlu2 receptor PAM activity, a moderate enzyme inhibitory activity against MAO-B, with an IC50 value of 0.59 μM. Consequently, the involvement of MAO-B in the action of TASP0433864 cannot simply be ignored. However, the inhibition of MAO-B is unlikely to be involved in the actions of TASP0433864 because MAO-B inhibition has been reported to be ineffective in an animal model for schizophrenia (Tatsuta et al., 2005). Moreover, TASP0433864 demonstrated agonist activity for mGlu5 receptor at a high concentration (10 μM). However, mGlu5 receptor agonist activity may not contribute to neurophysiologic and antipsychotic effects of TASP0433864, because it was absent up to 1 μM.
In conclusion, we used TASP0433864, a newly synthesized selective mGlu2 receptor PAM, to demonstrate that mGlu2 receptor potentiation ameliorates excessive cortical hyperactivity presumably through the suppression of glutamate overflows, which mimic the pathophysiologic states observed in schizophrenia, and that this mechanism may be responsible for the antipsychotic activity in animal models of schizophrenia. Our results raise the possibility that selective mGlu2 receptor PAMs might provide a novel approach to the treatment of disorders with an underlying pathophysiology of hyperglutamatergic neurotransmission, including schizophrenia, and that TASP0433864 might be a useful pharmacologic tool for examining the further potential of mGlu2 receptor PAM for the treatment of disorders.
The authors thank Dr. Takeshi Aoki for obtaining complementary data for in vitro studies, and Katsuya Iwata for measurements of TASP0433864 in samples. The authors also thank Dr. Shigetada Nakanishi of Osaka Bioscience Institute for providing the CHO cell lines expressing rat mGlu2 receptor.
Participated in research design: Hiyoshi, Marumo, Hikichi, Tomishima.
Conducted experiments: Hiyoshi, Marumo, Hikichi, Tomishima, Iida.
Contributed new reagents or analytic tools: Tamita, Urabe, Yasuhara.
Performed data analysis: Hiyoshi, Marumo, Hikichi, Tomishima.
Wrote or contributed to the writing of the manuscript: Hiyoshi, Karasawa, Chaki.
- Received July 24, 2014.
- Accepted October 1, 2014.
- artificial cerebrospinal fluid
- analysis of variance
- Chinese hamster ovary
- cerebrospinal fluid
- dentate gyrus
- 2-deoxyglucose 6-phosphate
- field excitatory postsynaptic potential
- γ band oscillation
- guanosine 5′-O-(3-[35S]thio)triphosphate
- (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid
- monoamine oxidase B
- metabotropic glutamate
- (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate
- medial perforant path
- N-methyl-d-aspartic acid
- positive allosteric modulator
- prefrontal cortex
- paired-pulse ratio
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics