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NEUROPHARMACOLOGY

Radioligand Binding Properties and Pharmacological Characterization of 6-Amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198), a High-Affinity, Selective, and Noncompetitive Antagonist of Metabotropic Glutamate Receptor Type 1

Neuroscience Research, Pharmacology Laboratories (A.K., T.T., S.T., T.W., Y.N., Y.S., M.O.), Molecular Medicine Laboratory, Molecular Laboratories (S.K.), and Chemistry Laboratory I, Chemistry Laboratories (S.H.), Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Japan

Received March 31, 2005; accepted June 14, 2005.

	Abstract

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Metabotropic glutamate receptor type 1 (mGluR1) is thought to play important roles in the neurotransmission and pathogenesis of several neurological disorders. Here, we describe the radioligand binding properties and pharmacological effects of a newly synthesized, high-affinity, selective, and noncompetitive mGluR1 antagonist, 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198). YM-298198 inhibited glutamate-induced inositol phosphate production in mGluR1-NIH3T3 cells with an IC₅₀ of 16 ± 5.8 nM in a noncompetitive manner. Its radiolabeled form, [³H]YM-298198, bound to mGluR1-NIH3T3 cell membranes with a K_D of 32 ± 8.5 nM and a B_max of 2297 ± 291 fmol/mg protein. In ligand displacement experiments using rat cerebellum membrane, an existing noncompetitive mGluR1 antagonist 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) competitively displaced [³H]YM-298198 binding, although glutamate and other mGluR1 ligands acting on a glutamate site failed to inhibit [³H]YM-298198 binding, suggesting that YM-298198 binds to CPCCOEt (allosteric) binding sites but not to glutamate (agonist) binding sites. Specificity was demonstrated for mGluR1 over mGluR subtypes 2 to 7, ionotropic glutamate receptors, and other receptor, transporter, and ion channel targets. In in vivo experiments, orally administered YM-298198 showed a significant analgesic effect in streptozotocin-induced hyperalgesic mice at doses (30 mg/kg) that did not cause Rotarod performance impairment, indicating that it is also useful even for in vivo experiments. In conclusion, YM-298198 is a newly synthesized, high-affinity, selective, and noncompetitive antagonist of mGluR1 that will be a useful pharmacological tool due to its highly active properties in vitro and in vivo. Its radiolabeled form [³H]YM-298198 will also be a valuable tool for future investigation of the mGluR1.

Recently, several mGluR1-specific antagonists have been discovered. 7-(Hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) is an antagonist that is structurally different from phenylglycines or glutamate (Litschig et al., 1999). It inhibits receptor activity without affecting the binding affinity of glutamate, i.e., in a noncompetitive manner. BAY36-7620 and R214127 are more potent noncompetitive mGluR1 antagonists with inverse agonist activity (Carroll et al., 2001; Lavreysen et al., 2003). Although intravenous administration of BAY36-7620 has been reported to protect neurons from degeneration in the acute subdural hematoma model, as well as to attenuate pentylenetetrazole-induced convulsions, the compound is not highly centrally active (De Vry et al., 2001; Lavreysen et al., 2004). Thus, there remains a need for novel mGluR1 antagonists with higher affinity and better bioavailability for the future investigation of mGluR1s. In this context, YM-298198 was designed and synthesized as a novel mGluR1 antagonist. In the present study, radioligand binding properties and pharmacological profiles of YM-298198 were investigated through in vitro and in vivo experiments.

	Materials and Methods

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Materials. YM-298198 and YM-193167 (Fig. 1) were synthesized at Yamanouchi Pharmaceutical Co., Ltd. (Ibaraki, Japan). A hydrochloride salt of YM-298198 is used as YM-298198. [³H]YM-298198 (82 Ci/mmol) was prepared by GE Healthcare (Piscataway, NJ; for a detailed description of its synthesis, see [³H]YM-298198 synthesis below). [³H]Myo-inositol and [³H]quisqualate were obtained from GE Healthcare. (R,S)-a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [(RS)-AMPA], L-2-amino-4-phosphonobutyrate (L-AP4), (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid [(1S,3R)-ACPD], (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG], (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), CPCCOEt, (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG], and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide were obtained from Tocris Cookson Inc. (Anawa Trading SA, Zurich, Switzerland). Glutamate, 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-[5H-2,3]-benzodiazepine (GYKI 52466), kainate, dizocilpine [(+)-MK-801], and N-methyl-D-aspartic acid (NMDA) were obtained from Sigma-Aldrich (St. Louis, MO). Quisqualate was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Glycine was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Tissue culture reagents were obtained from Invitrogen (Carlsbad, CA), Nissui (Tokyo, Japan), or Sigma-Aldrich. Streptozotocin was purchased from Wako Pure Chemicals (Osaka, Japan). All other reagents were obtained from standard suppliers.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Chemical structures of [3H]YM-298198 (T, tritium), YM-193167, and CPCCOEt.

[³H]YM-298198 Synthesis. 6-Amino-N-cyclohexyl-3-methylthiazolo[3,2-a]benzimidazole-2-carboxamide dihydrochloride 1.7 hydrate, a desmethyl derivative of YM-298198 (17.5 mg), was dissolved in 1 ml of N,N-dimethylformamide, and 7.5 mg of sodium hydride was added with stirring for 30 min at 0°C. The flask was then frozen in liquid nitrogen and evacuated to hard vacuum, and [³H]methyl iodide (5 Ci at 85 Ci/mmol) was distilled to it. The solution was allowed to thaw to room temperature and was stirred for 30 min. The reaction mixture was then partitioned between ethyl acetate and water, and the organic layer was collected. The aqueous layer was extracted with further portions of ethyl acetate, and the organic fractions were combined. A portion of the crude product, 250 mCi, was purified by high-performance liquid chromatography on an Ultrasphere ODS column eluting with a water/methanol/hydrochloric acid gradient system. The required product was collected, evaporated to dryness, and, after 2 x 2 ml of ethanol was evaporated off to remove traces of hydrochloric acid, redissolved in ethanol. The high-performance liquid chromatography purification was repeated twice to obtain the required purity. The resulting [³H]YM-298198 had a radiochemical purity of 98.5% and a specific activity of 82 Ci/mmol.

Animals. Male Wistar rats (Japan SLC, Hamamatsu, Japan) were used for preparation of cerebellum membrane fraction. Male ICR mice were purchased from Japan SLC for the nociceptive test and Rotarod performance assessment. They were given free access to solid food and water in an animal room maintained at 23 ± 2C° and 55 ± 10% humidity with a 13:11-h day/night cycle. All experiments were performed according to the regulations of the Animal Ethical Committee of Yamanouchi Pharmaceutical Co., Ltd.

Cell Culture and Membrane Preparation. NIH3T3 cells stably expressing rat mGlu1R (mGluR1-NIH) and HEK293 cells stably expressing rat mGluR5a (mGluR5-HEK) were obtained as described previously (Kawabata et al., 1996) and cultured with Dulbecco's modified Eagle's medium with 10% dialyzed fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin. Chinese hamster ovary (CHO)-dhfr^– cells stably expressing rat mGluR2, 3, 4a, 6, and 7b, received as gifts from S. Nakanishi (Kyoto University, Kyoto, Japan), were cultured with Dulbecco's modified Eagle's medium or -minimum essential medium with 10% dialyzed fatal bovine serum, 1% L-proline, 100 units/ml penicillin G, and 100 µg/ml streptomycin.

The cells cultured in 15-cm culture dishes were rinsed with Ca²⁺- and Mg²⁺-free Dulbecco's phosphate-buffered saline and removed from the dishes by scraping. The removed cells were homogenized with 2 mM HEPES, 10 mM EDTA, pH 7.4 (approximately 6 ml per dish) using a Polytron PTA 10-TS homogenizer (level 5, for 15 s, three times). The homogenate was centrifuged at 40,000g for 30 min at 4°C. The resulting pellet was resuspended with 2 mM HEPES, 10 mM EDTA, pH 7.4, and centrifuged at 40,000g for 30 min at 4°C. The final pellet was homogenized in 20 mM HEPES, 10 mM EDTA, pH 7.4, using a glass-Teflon homogenizer and stored in aliquots at –80°C until use. For the rat cerebellum membrane preparation, 11- to 12-week-old male Wistar rats were decapitated, and the cerebella were immediately dissected. The cerebella were homogenized with a Teflon-glass potter homogenizer in 10 volumes of 0.32 M sucrose and centrifuged at 900g for 15 min. After centrifugation, the resulting supernatant was further centrifuged at 15,000g for 20 min. The resulting pellet was washed twice by centrifugation at 15,000g for 20 min in 5 mM Tris-HCl, pH 7.4. The final pellet was homogenized in 50 mM Tris-HCl, pH 7.4, using a glass-Teflon homogenizer and stored in aliquots at –80°C until use.

IPs Accumulation Assay. Inositol phosphates (IPs) formation was measured according to the procedures described by Aramori and Nakanishi (1992). Briefly, mGluR1-NIH cells were seeded on 24-well plates at a density of 1 x 10⁵ cells/well and cultured overnight in a CO₂ incubator (37°C, 5% CO₂). The wells were labeled with [³H]myoinositol (3 µCi/ml) and cultured overnight. The labeled cells were washed with PBS (137 mM NaCl, 2.68 mM KCl, 1.05 mM MgCl₂, 0.9 mM CaCl₂, 8.1 mM Na₂HPO₄, and 1.47 mM KH₂PO₄) and incubated for 20 min at 37°C. The cells were further incubated with antagonists in PBS, including 10 mM LiCl (PBS-Li) for 20 min at 37°C. A reaction was started by exchanging the mixture with 10 mM LiCl in PBS including agonists/antagonists. After incubation (20 min at 37°C), the reaction was terminated by exchanging the reaction mixture with 1 ml of ice-cold 0.2 M perchloric acid. After standing (1–3 h at 4°C), 100 µl of 2 N KOH and 50 µl of 100 mM EDTA disodium salt were added into each well and mixed. One milliliter of reaction extract was added to AG1 x 8 columns (Bio-Rad, Hercules, CA) and washed with 7 ml of wash solution (5 mM sodium tetraborate and 60 mM sodium formate). IPs were extracted by adding 4 ml of elution solution (0.1 M formate and 1 M ammonium formate), and the whole elutes were collected into liquid scintillation vials. The radioactivity of the elutes was counted using a liquid scintillation counter. EC₅₀ and IC₅₀ values were calculated from the nonlinear regression analysis using SAS version 8.2.

cAMP Accumulation Assay. Cyclic adenosine monophosphate (cAMP) accumulation was measured according to the procedures described by Tanabe et al. (1992). Briefly, Chinese hamster ovary cells expressing rat mGluR2, 3, 4a, 6, and 7b were seeded at a density of 5 x 10⁴ cells/well on 24-well plates and cultured overnight in a CO₂ incubator (37°C, 5% CO₂). The wells were washed twice with PBS-3-isobutyl-1-methylxanthine (137 mM NaCl, 2.68 mM KCl, 1 mM MgCl₂, 0.85 mM CaCl₂, 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, and 1 mM isobutylmethylxantine) and incubated for 20 min at 37°C. After incubation, a reaction was started by adding a reaction mixture (PBS-3-isobutyl-1-methylxanthine including agonists/antagonists) and incubated for 10 min at 37°C. The reaction was terminated by exchanging the reaction mixture with 900 µl of ice-cold 0.2 M percholic acid. After the mixture had stood for 1 to 3 h at 4°C, 100 µl of 1.8 M KOH was added into the wells and mixed. The cyclic AMP level was determined using Biotrak cAMP EIA System (GE Healthcare).

Intracellular Ca²⁺ Mobilization. Intracellular Ca²⁺ mobilization was measured according to the procedures described by Kawabata et al. (1996).

[³H]YM-298198 Binding Assay. The binding assay was performed by the method of Thomsen et al. (1993) with minor modifications. Briefly, cell membranes or rat cerebellum membranes (20–50 µg of protein), [³H]YM-298198, and competitors were incubated at 25°C for 30 min in 0.1 ml of 50 mM Tris-HCl, pH 7.4, containing 2.5 mM CaCl₂. For the determination of nonspecific binding, 10 µM YM-193167 was used. The incubation was terminated by rapid filtration through Whatman GF/B filters. Protein concentration was determined using Bio-Rad DC protein assay (Bio-Rad) in which bovine serum albumin was used as a standard. The specific binding was defined as a portion of the total binding, which was replaced by 10 µM YM-193167. The dissociation constant (K_D) and binding site density (B_max) were calculated using Scatchard analysis using SAS version 8.2. The IC₅₀ values were calculated from the nonlinear regression analysis using SAS version 8.2. The values of the apparent equilibrium dissociation constant of inhibitors (K_i) were calculated by the method of Cheng and Prusoff (1973).

[³H]Quisqualate Binding Assay. The binding assay was performed by the method of Mutel et al. (2000) with minor modifications. Briefly, cell membranes (30–50 µg of protein), [³H]quisqualate, and competitors were incubated at 25°C for 60 min in 0.1 ml of 50 mM Tris-HCl, pH 7.4, containing 2.5 mM CaCl₂. For the determination of nonspecific binding, 1 mM glutamate was used. The incubation was terminated by rapid filtration through Whatman GF/B filters. The protein concentration was determined using BioRad DC protein assay (Bio-Rad) in which bovine serum albumin was used as a standard. The specific binding was defined as a portion of the total binding, which was replaced by 1 mM glutamate.

Specificity against Other Sites. The studies for the investigation of the affinity to ionotropic glutamate receptors, other known neurotransmitter receptors, ion channels, transporters, and enzymes were performed at NovaScreen (Hanover, MD), using published protocols.

Nociceptive Test. The tail-pinch test was conducted with a modification of the previously reported method (Takagi et al., 1966). Briefly, the mice were tested by pinching their tail base with forceps (500g pressure), and nociceptive response latency (seconds) was measured with a cut-off time of 10 s to prevent tissue damage. The nociceptive response was indicated by biting of the forceps. An increase of the mechanical nociception was then induced by a modification of the previously reported method (Kamei et al., 1992). Briefly, the mice were deprived of food but provided with water for about 18 h. They were then treated with single intraperitoneal administration of streptozotocin at 200 mg/kg body weight (approximately 20g at the streptozotocin administration). Streptozotocin was dissolved in citrate buffer solution adjusted to pH 4.5 and administered immediately. A control group of age-matched mice received administration of the vehicle. Two weeks after the streptozotocin or vehicle treatment, the mice treated with streptozotocin showed significantly shorter nociceptive response latency (median latency 1.85 s, Q1 1.15 and Q3 5.09 s, n = 52) than mice treated with the vehicle (median latency 2.97 s, Q1 2.2 and Q3 7.88 s, n = 42, p < 0.01 with Wilcoxon rank sum test), indicating that the mice treated with streptozotocin also showed increased mechanical nociception under our experimental conditions. Predrug response latency was measured on the 14th day after streptozotocin treatment, and the mice that showed latency shorter than 3 s were provided for the drug evaluation. The mice were allocated to three groups (14 mice per group) such that the differences in the averages of predrug nociceptive response latency among the groups were small. On the 15th day after streptozotocin treatment, postdrug response latency was measured 45 min after oral administration of YM-298198 at an administration volume of 10 ml/kg. The analgesic efficacy of the drugs was represented as prolongation of the nociceptive response latency, measured up to 10 s, obtained from individual scores and calculated according to the following formula: postdrug response latency – predrug response latency.

Rotarod Performance Assessment. Rotarod performance was assessed to evaluate the drug effect on motor coordination. The Rotarod apparatus (KN-75; Natsume Seisakusho, Tokyo, Japan) comprised a drum that rotated at a speed of 5 revolutions per min. Mice were placed individually on the rotating drum. The time in seconds at which each animal fell from the drum was recorded for up to 120 s using a stopwatch and indicated as performance time. Mice that show a 120-s performance time were selected for drug evaluation (n = 8/group) by a predrug test session. In the drug evaluation session, Rotarod performance time was measured three times, up to 120 s, 45 min after oral administration of the test compound, and the mean was adopted as the performance time for each animal.

Statistics. The competition study of CPCCOEt for [³H]YM-298198 binding was statistically analyzed using a one-way analysis of variance followed by Dunnett's test. The results for analgesic effect in the nociceptive test and performance time in the Rotarod test were statistically analyzed using a nonparametric Kruskal-Wallis H test followed by Steel's test. Probability values less than 0.05 were considered significant.

	Results

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Antagonistic Activity at mGluR1-NIH Cells. YM-298198 inhibited glutamate-induced IP production with an IC₅₀ value of 16 ± 5.8 nM (n = 3; Fig. 2A), whereas CPCCOEt inhibited it with an IC₅₀ value of 6.3 ± 0.48 µM. YM-298198 did not affect the basal IP production up to 10 µM (data not shown). YM-298198 concentration dependently reduced the maximal IP production response to glutamate (Fig. 2, B and C). YM-298198 also concentration dependently increased the EC₅₀ value of glutamate, whereas it did not affect the Hill coefficient of the glutamate concentration-response curves (Fig. 2, D and E).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Antagonist profiles of YM-298198. A, inhibition curve of glutamate (100 µM)-induced IP formation in mGluR-NIH cells. Data are presented as the mean ± S.E.M. of triplicates within one experiment. B, concentration-response curve of glutamate, alone or together with 10, 30, or 100 nM YM-298198. Data are the mean ± S.E.M. of triplicates within one experiment. C to E, maximal IP production, log EC50 values, and Hill coefficient of indicated concentration-response curves, respectively. Data are the mean ± S.E.M. of three independent experiments.

Radioligand Binding Study Using [³H]YM-298198. Specific binding of 10 nM [³H]YM-298198 was observed for rat mGluR1-NIH membranes, although no specific [³H]YM-298198 (33 nM) binding was observed for native NIH3T3 cell membranes (data not shown). The nonspecific binding was 48% of the total binding under the conditions of a concentration of 10 nM [³H]YM-298198 in 30 µg of protein per assay (data not shown). Specific [³H]YM-298198 binding was only faint for mGluR5-HEK cell membranes, despite the assurance of the same [³H]quisqualate binding level as seen for mGluR1-NIH cell membranes (Fig. 3). Saturation binding with [³H]YM-298198 to mGluR1-NIH membranes showed that YM-298198 binds in a saturable manner to a single affinity state of this receptor with a K_D value of 32 ± 8.5 nM and a B_max value of 2297 ± 291 fmol/mg protein as determined by Scatchard analysis (n = 3; Fig. 4). Competition experiments were performed to evaluate the pharmacology of the site labeled by [³H]YM-298198 on mGluR1-NIH membranes. YM-298198 and YM-193167 displaced [³H]YM-298198 binding with K_i values of 19 ± 2.2 and 6.6 ± 0.27 nM, respectively (Table 3). CPCCOEt also displaced [³H]YM-298198 binding with the K_i value of 33 ± 8.3 µM. mGluR1 ligands that bind glutamate binding site [glutamate, quisqualate, (1S,3R)-ACPD, (S)-3,5-DHPG, and (S)-4C3HPG], on the other hand, did not displace [³H]YM-298198 binding up to 1 mM.

View larger version (14K): [in this window] [in a new window]   Fig. 3. [3H]YM-298198 binding to mGluR1-NIH and mGluR5-HEK membranes. The insert figure shows [3H]quisqualate binding to mGluR1-NIH and mGluR5-HEK membranes. For each experiment, data points were determined in triplicate.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Representative saturation binding plot and Scatchard plot of [3H]YM-298198 binding to mGluR1-NIH membranes. Data points were determined in duplicate.

Binding Study Using Rat Cerebellum Membranes. To analyze the binding of YM-29818 to native mGluR1, we performed a [³H]YM-298198 binding assay on rat cerebellum membranes. When 45 µg of protein per assay was used, the nonspecific binding was 30% of the total binding at a concentration of 10 nM [³H]YM-298198 (data not shown). [³H]YM-298198 showed affinity to rat cerebellum membranes with a K_D value of 22 ± 1.8 nM and a B_max value of 4552 ± 80 fmol/mg protein (Fig. 5; Table 4). For the investigation of the binding site of [³H]YM-298198 on rat cerebellum membranes, we performed saturation experiments on the [³H]YM-298198 binding in the absence and presence of a reference compound CPCCOEt. The addition of CPCCOEt caused an increase in the K_D value in a concentration-dependent manner (Fig. 5; Table 4). CPCCOEt, on the other hand, did not affect the B_max value. Competition experiments were performed to evaluate the pharmacology of the site labeled by [³H]YM-298198 on rat cerebellum membranes (Table 5). YM-298198 and YM-193167 displaced [³H]YM-298198 bindings with K_i values of 20 ± 1.6 and 4.4 ± 0.70 nM, respectively. CPCCOEt also displaced [³H]YM-298198 binding with a K_i value of 13 ± 0.78 µM. Other ionotropic/metabotropic glutamate receptor ligands tested, including mGluR1 ligands that bind glutamate binding sites [glutamate, quisqualate, (1S,3R)-ACPD, (S)-3,5-DHPG, and (S)-4C3HPG], did not displace [³H]YM-298198 binding up to 1 mM.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Representative Scatchard plot and saturation binding plot of [3H]YM-298198 binding to rat cerebellum membranes in the absence and presence of CPCCOEt (10 and 30 µM). Data points were determined in triplicate.

Analgesia and Rotarod Performance Assessment. Oral administration of YM-298198 prolonged nociceptive response latency in streptozotocin-induced hyperalgesic mice. The effect at a dose of 30 mg/kg p.o. was statistically significant (Fig. 6). YM-298198 did not affect motor coordination assessed with Rotarod performance time in mice up to 30 mg/kg p.o., on the other hand, whereas haloperidol significantly reduced the Rotarod performance time at 3 mg/kg p.o. (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Analgesic effects of YM-298198 on streptozotocin-induced hyperalgesia. YM-298198 was orally administrated 45 min before the test. Prolonged latency was calculated by (postdrug response latency) – (predrug response latency). Each point represents the prolonged latency for individual animal. Horizontal bar represents median latency. **, p < 0.01 significantly different from the control (Kruskal-Wallis H test followed by Steel's test).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Lack of motor coordination impairment by oral administration of YM-298198. Haloperidol or YM-298198 was orally administrated at the indicated dose (milligrams per kilogram) 45 min before the test. Each point represents the time on rod for individual animal. Horizontal bar represents median latency. **, p < 0.01 significantly different from the control (Kruskal-Wallis H test followed by Steel's test).

	Discussion

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The tritium-labeled form of YM-298198, [³H]YM-298198, showed saturable binding with high affinity (K_D = 32 ± 8.5 nM), which was almost the same as the IC₅₀ values for glutamate-induced IP production inhibition, suggesting that this labeled form is a useful radioligand in the mGluR1 investigation. The ligands that act on glutamate binding sites of mGluR1, including endogenous agonist glutamate, failed to inhibit [³H]YM-298198 binding to mGluR1-NIH membranes, although CPCCOEt successfully inhibited binding. Because it has been reported that CPCCOEt does not affect [³H]glutamate binding to rat mGluR1 membranes (Litschig et al., 1999) and that it acts on Thr815 and Ala818 in transmembrane domain VII of mGluR1, rather than the glutamate binding domain, it is suggested that the YM-298198 binding site is close to the CPCCOEt (allosteric) site rather than the glutamate (agonist) site. A saturation experiment with [³H]YM-298198 using rat cerebellum membrane suggests that [³H]YM-298198 specifically labels native mGluR1 on native brain cell membrane, because the K_D value for [³H]YM-298198 at the cerebellum membrane is almost the same as that at mGluR1-NIH cells. CPCCOEt fully and competitively inhibited [³H]YM-298198 binding to cerebellar membranes with a K_i value of 13 ± 0.78 µM, which was similar to its K_i at mGluR1-NIH membranes, although agonist site ligands including glutamate failed to inhibit it. The results for the cerebellum membrane also support the conclusion that YM-298198 binds to the CPCCOEt (allosteric) site rather than the glutamate (agonist) site.

YM-291898 showed an obvious antinociceptive effect in streptozotocin-induced hyperalgesic mice, indicating that YM-298198 is also highly active in in vivo models even with oral treatment. It has been reported that mutant mice lacking mGluR1 show an increase in the nociceptive threshold (Corsi et al., 1996) and that intrathecal administration of mGluR1 antisense oligonucleotides shows an analgesic effect in various animal pain models (Fundytus et al., 2001, 2002; Noda et al., 2003). The analgesic activity of YM-298198 in the present study accords well with these previous findings. YM-298198 did not affect motor coordination assessed by the Rotarod test, although mGluR1 knockout mice reportedly showed ataxic behavior (Aiba et al., 1994). It is possible that mGluRs are involved mainly in the development but not the maintenance of neuronal conditions related to motor coordination. In vivo studies with YM-298198 suggest that specific mGluR1 antagonists would be useful in the treatment of pain conditions.

In conclusion, YM-298198 is a newly synthesized, high-affinity, selective, and noncompetitive antagonist of mGluR1, and it will be a useful pharmacological tool due to its highly active properties in vitro and in vivo. Its radiolabeled form [³H]YM-298198 will also be a valuable tool for future investigation of mGluR1.

	Footnotes

 
doi:10.1124/jpet.105.087171.

ABBREVIATIONS: mGluR, metabotropic glutamate receptor; CPCCOEt, 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester; BAY 36-7620, (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on; R214127, 1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone; YM-298198, 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide; YM-193167, 6-{[(2-methoxyethyl)amino]methyl}-N-methyl-N-neopentylthiazolo[3,2-a]benzoimidazole-2-carboxamide dihydrochloride; (RS)-AMPA, (R,S)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; L-AP4, L-(+)-2-amino-4-phosphonobutylic acid; (1S,3R)-ACPD, (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; (S)-4C3HPG, (S)-4-carboxy-3-hydroxyphenylglycine; L-CCG-I, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine; (S)-3,5-DHPG, (S)-3,5-dihydroxyphenylglycine; GYKI 52466, 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-[5H-2,3]-benzodiazepine; (+)-MK-801, dizocilpine; NMDA, N-methyl-D-aspartic acid; HEK, human embryonic kidney; IP, inositol phosphate; PBS, phosphate-buffered saline.

Address correspondence to: Dr. Masamichi Okada, Neuroscience Research, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, 305-8585, Japan. E-mail: masamichi.okada{at}jp.astellas.com

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