Involvement of subtype 1 metabotropic glutamate receptors in apoptosis and caspase-7 over-expression in spinal cord of neuropathic rats.

The effect of the non-selective, 1-aminoindan-1,5-dicarboxylic acid (AIDA), and selective (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl) methanone (JNJ16259685), metabotropic glutamate subtype 1 (mGlu1) receptor antagonists, on rat sciatic nerve chronic constrictive injury (CCI)-induced hyperalgesia, allodynia, spinal dorsal horn apoptosis, and gliosis was examined at 3 and 7 days post-injury. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), nestin, GFAP, and caspase-7 mRNA in the dorsal horn spinal cord by 3 days post-CCI. At 7 days post-CCI, only over-expression of bcl-2, nestin and GFAP mRNA was observed. Administration of AIDA reduced thermal hyperalgesia and mechanical allodynia at 3 and 7 days post-CCI; administration of JNJ16259685 reduced thermal hyperalgesia at 3 and 7 days post-CCI, but not mechanical allodynia. AIDA decreased the mRNA levels of bax, apaf-1, GFAP and caspase-7 genes. JNJ16259685 increased the mRNA levels of bcl-2 and GFAP gene, and decreased APAF-1 and caspases-7 genes. Inhibiting mGlu1 receptors also reduced TUNEL-positive profiles and immunohistochemical reactivity for caspase-7. We report here that despite inhibiting CCI-induced over-expression of pro-apoptotic genes in the spinal cord dorsal horn, the selective mGlu1 receptor antagonist JNJ16259685 exerted only a slight and transient allodynic effect. Moreover, JNJ16259685, but not the non-selective AIDA, increased astrogliosis which may account for its decreased analgesic efficacy. This study provides evidence that the contemporary and partial blockade of group I and likely ionotropic glutamate receptors may be a more suitable therapy than selective blockade of mGlu1 subtype receptors condition to decrease neuropathic pain symptoms.     

Estrogen receptors and type 1 metabotropic glutamate receptors are interdependent in protecting cortical neurons against β-amyloid toxicity

We examined the interaction between estrogen receptors (ERs) and type 1 metabotropic glutamate receptors (mGlu1 receptors) in mechanisms of neurodegeneration/neuroprotection using mixed cultures of cortical cells challenged with β-amyloid peptide. Both receptors were present in neurons, whereas only ERα but not mGlu1 receptors were found in astrocytes. Addition of 17β-estradiol (17βE2) protected cultured neurons against amyloid toxicity, and its action was mimicked by the selective ERα agonist, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) as well as by a cell-impermeable bovine serum albumin conjugate of 17βE2. The selective ERβ agonist, diarylpropionitrile (DPN), was only slightly neuroprotective. The mGlu1/5 receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), was also neuroprotective against amyloid toxicity, and its action was abolished by the mGlu1 receptor antagonist, (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ 16259685). Neuroprotection by 17βΕ2 or PPT (but not DPN) and DHPG was less than additive, suggesting that ERα and mGlu1 receptors activate the same pathway of cell survival. More important, neuroprotection by 17βΕ2 was abolished not only by the ER antagonist fulvestrant (ICI 182,780) but also by JNJ 16259685, and neuroprotection by DHPG was abolished by ICI 182,780. ERα and mGlu1 receptors were also interdependent in activating the phosphatidylinositol-3-kinase pathway, and pharmacological blockade of this pathway abolished neuroprotection by 17βE2, DHPG, or their combination. These data provide the first evidence that ERα and mGlu1 receptors critically interact in promoting neuroprotection, information that should be taken into account when the impact of estrogen on neurodegeneration associated with central nervous system disorders is examined.     

Effects of mGlu1 receptor blockade on anxiety-related behaviour in the rat lick suppression test

Rationale Group I metabotropic glutamate receptor antagonists, which block both the mGlu1 and mGlu5 receptors, have been shown to have anxiolytic effects in the lick suppression test in rats.Objective The anxiolytic potential of the selective mGlu1 antagonist 3,4-dihydro-2H-pyrano[2,3]-quinolin-7-yl)(cis-4-methoxycyclohexyl)methanone (JNJ16259685) was investigated and compared with the mGlu5 antagonist MPEP.Methods Anxiety-related behaviour was assessed in lick suppression and in the elevated zero maze in rats. Non-specific effects on pain threshold, water intake and locomotor activity were also measured.Results Acute administration of JNJ16259685 or MPEP increased the number of licks (lowest active dose 2.5 mg/kg IP for each compound). JNJ16259685 did not increase water intake or reduce acute pain threshold, suggesting that the anxiolytic-like properties are specific. However, acute administration decreased locomotor activity. The effects of chronic administration of JNJ16259685 over 14 days (5 mg/kg bid) on lick suppression were comparable to those seen after acute administration, arguing against development of behavioural tolerance or sensitisation. Yet, there was a tendency for an increase in locomotor activity after cessation of chronic treatment. Acute co-administration of both JNJ16259685 and MPEP had additive effects on the number of licks. No anxiolytic-like properties of JNJ16259685 were observed in the elevated zero maze.Conclusion Our data suggest that the anxiolytic-like effects induced by group I metabotropic glutamate receptor antagonists are mediated through both mGlu1 and mGlu5 receptors. Rather than producing a general anxiolytic-like effect, the effects seen following mGlu1 antagonism seem task-dependent, as prominent effects were seen in a conflict procedure, but not in a task based on spontaneous exploration.     

JNJ16259685, a selective mGlu1 antagonist, suppresses isolation-induced aggression in male mice

mGlu1 receptors are present in brain regions involved in aggression modulation. This study examines the effects of 3-4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl-(cis-4-methoxycyclohexyl)-methanone (JNJ16259685; 0.125, 0.25, 0.5, 1, 2, 4 and 8 mg/kg, i.p), a selective antagonist of the mGlu1 receptors, on agonistic interactions between male mice. Individually housed mice were exposed to anosmic "standard opponents" 30 min after drug administration. Ten minutes of diadic interactions was staged between a singly housed and an anosmic mouse in a neutral area. The encounters were videotaped and the accumulated time allocated by subjects to ten broad behavioural categories was estimated using an ethologically based analysis. JNJ16259685 (all doses) produced a significant reduction of offensive behaviours (threat and attack), without affecting immobility. These findings suggest for the first time a role for mGlu1 receptors in aggression regulation.     

Metabotropic glutamate 1 receptor distribution and occupancy in the rat brain: a quantitative autoradiographic study using [3H]R214127

We used the selective metabotropic glutamate (mGlu) 1 receptor antagonist [3H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone ([3H]R214127) to investigate the distribution of mGlu1 receptor binding sites in rat brain. We found high mGlu1 receptor binding in the cerebellum, thalamus, dentate gyrus and medial central gray, moderate binding within the CA3 of the hippocampus and hypothalamus, and low mGlu1 receptor binding in the basal ganglia and cortex. The mGlu1 receptor is also present in variable degree in the dorsal lateral septal nucleus, amygdala, interpeduncular nucleus and median raphe nucleus. Additionally, we employed [3H]R214127 autoradiography as a means of investigating the occupancy of central mGlu1 receptors following in vivo administration of mGlu1 receptor antagonists that prevent binding of this radioligand. We found that the mGlu1 receptor antagonist (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), administered subcutaneously (s.c.) at 10 mg/kg, only occupied about 30% of cerebellar and thalamic mGlu1 receptors. The mGlu1/5 receptor antagonist 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390) exhibited a relatively high potency in occupying mGlu1 receptors in rat cerebellum (ED50 = 0.75 mg/kg, s.c.) and thalamus (ED50 = 0.63 mg/kg, s.c). In the future, this method can be employed to gain more insight into the in vivo profile and central activity of potential therapeutic agents that act upon the mGlu1 receptor.     

3H]R214127: a novel high-affinity radioligand for the mGlu1 receptor reveals a common binding site shared by multiple allosteric antagonists

R214127 was shown to be a potent and noncompetitive metabotropic glutamate 1 (mGlu1) receptor-selective antagonist. The kinetics and pharmacology of [(3)H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127) binding to rat mGlu1a receptor Chinese hamster ovary (CHO)-dhfr(-) membranes was investigated, as well as the distribution of [(3)H]R214127 binding in rat brain tissue and sections. Specific binding to rat mGlu1a receptor CHO-dhfr(-) membranes was approximately 92% of total and was optimal at 4 degrees C. Full association was reached within 5 min, and [(3)H]R214127 bound to a single binding site with an apparent K(D) of 0.90 +/- 0.14 nM and a B(max) of 6512 +/- 1501 fmol/mg of protein. Inhibition experiments showed that [(3)H]R214127 binding was completely blocked by 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390), (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), and 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), but was not displaced by competitive mGlu1 receptor ligands such as glutamate and quisqualate, suggesting that R214127, NPS 2390, BAY 36-7620, and CPCCOEt bind to the same site or mutually exclusive sites. Experiments using rat cortex, striatum, hippocampus and cerebellum revealed that [(3)H]R214127 labeled a single high-affinity binding site (K(D) approximately 1 nM). B(max) values were highest in the cerebellum (4302 +/- 2042 fmol/mg of protein) and were 741 +/- 48, 688 +/- 125, and 471 +/- 68 fmol/mg of protein in the striatum, hippocampus, and cortex, respectively. The distribution of [(3)H]R214127 binding in rat brain was investigated in more detail by radioligand autoradiography. A high density of binding sites was detected in the molecular layer of the cerebellum. Moderate labeling was seen in the CA3 and dentate gyrus of the hippocampus, thalamus, olfactory tubercle, amygdala, and substantia nigra reticulata. The cerebral cortex, caudate putamen, ventral pallidum, and nucleus accumbens showed lower labeling. The high affinity and selectivity of [(3)H]R214127 for mGlu1 receptors renders this compound the ligand of choice to study the native mGlu1 receptor in brain.     

Potent and specific action of the mGlu1 antagonists YM-298198 and JNJ16259685 on synaptic transmission in rat cerebellar slices

BACKGROUND AND PURPOSE: Specific and selective inhibitors for mGlu1 receptors are presently inadequate. A new generation of non-competitive mGlu1 antagonists with low nanomolar potencies is emerging. We evaluated two new compounds, YM-298198 and JNJ16259685, for effectiveness, potency and specificity for the first time in a brain slice preparation. EXPERIMENTAL APPROACH: Patch-clamp recording of Purkinje neurones in cerebellar slices were obtained. The slow mGlu1-mediated EPSP was used to establish a concentration-response curve. Fast excitatory synaptic inputs were tested for non-specific effects. KEY RESULTS: YM-298198 and JNJ16259685 inhibited the synaptic activation of mGlu1 in a concentration-dependent manner (IC(50) values of 24 nM and 19 nM, respectively). The antagonists were slow to inhibit and to reverse on washout, probably due to their lipophilic nature. There were no non-specific effects on fast AMPA receptor-mediated synaptic transmission in the cerebellum. CONCLUSIONS AND IMPLICATIONS: These compounds are more than a thousand-fold more potent than previously available compounds. Their selectivity and specificity will be very useful for studying the role of mGlu1 receptors both in vitro and in vivo.     

Effects of RPR 118723, a novel antagonist at the glycine site of the NMDA receptor, in vitro

RPR 118723 ((8-chloro-5-methyl-2,3-dioxo-1,4-dihydro-5H-indeno[1, 2-b]pyrazin-5-yl) acetic acid) was previously reported to exhibit potent affinity for the glycine site of the N-methyl-D-aspartate (NMDA) receptor-channel complex in the nanomolar range (K(i)=3.1+/-0. 8 nM). We now report on the effects of RPR 118723 in two functional tests reflecting the interaction between the glycine site and the NMDA receptor. First, RPR 118723 potently inhibited [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) binding in the presence of NMDA (IC(50)=3.5+/-0.4 nM). Second, RPR 118723 antagonized the NMDA-induced increase in [3H]dopamine release in mouse striatal slices (IC(50)=8.0+/-1.1 nM). In both experimental models, an excess of glycine reversed the effect of RPR 118723. These results show that RPR 118723 interferes functionally in the nanomolar range with the glycine site coupled to the NMDA receptor in vitro. The blockade of the glycine site with RPR 118723 may be useful for the therapy of the disorders linked to excessive NMDA stimulation.     

Effects of T-82, a new quinoline derivative, on cholinesterase activity and extracellular acetylcholine concentration in rat brain

The effects of T-82 (2-[2-(1-benzylpiperidin-4-yl)ethyl]-2,3-dihydro-9-methoxy-1H-pyrrolo [3,4-b]quinolin-1-one hemifumarate), a new quinoline derivative, on acetylcholinesterase (AChE) activity and acetylcholine (ACh) release were compared with those of the well-known cholinesterase inhibitors tacrine and E2020. T-82, tacrine and E2020 all concentration-dependently inhibited AChE in rat brain homogenate (IC50 = 109.4, 84.2 and 11.8 nM, respectively). In addition, although tacrine strongly inhibited butyrylcholinesterase (BuChE), T-82 and E2020 showed only weak activity on BuChE in human plasma. In ex vivo experiments, intraperitoneal administration of T-82 at a dose of 30 mg/kg inhibited AChE activity in the hippocampus, frontal cortex and parietal cortex of rats. The effect of T-82 on the extracellular ACh concentration in rat brain was measured using in vivo microdialysis. T-82 at doses of 10 and 30 mg/kg, i.p. increased the extracellular ACh concentration in the hippocampus and striatum in a dose-dependent manner. These findings suggest that T-82 activates the central cholinergic system by selectively inhibiting AChE activity, while weakly affecting peripheral BuChE activity, and that T-82 increases the extracellular ACh concentration in the brain, which is followed by inhibited AChE activity.     

(S)-3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates metabotropic glutamate receptors on primary afferent terminals in the neonatal rat spinal cord

(S)-3,4-Dicarboxyphenylglycine (DCPG) has been tested on cloned human mGlu1-8 receptors individually expressed in AV12-664 cells co-expressing a rat glutamate/aspartate transporter and shown to be a potent and selective mGlu8a receptor agonist (EC(50) value 31+/-2 nM, n=3) with weaker effects on the other cloned mGlu receptors (EC(50) or IC(50) values >3.5 microM on mGlu1-7).Electrophysiological characterisation on the neonatal rat spinal cord preparation revealed that (S)-3,4-DCPG depressed the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) giving a biphasic concentration-response curve showing EC(50) values of 1.3+/-0.2 microM (n=17) and 391+/-81 microM (n=17) for the higher and lower affinity components, respectively. The receptor mediating the high-affinity component was antagonised by 200 microM (S)-alpha-methyl-2-amino-4-phosphonobutyrate (MAP4, K(D) value 5.4+/-1.5 microM (n=3)), a group III metabotropic glutamate (mGlu) receptor antagonist. The alpha-methyl substituted analogue of (S)-3,4-DCPG, (RS)-3,4-MDCPG (100 microM), antagonised the effects of (S)-3,4-DCPG (K(D) value 5.0+/-0.4 microM, n=3) in a similar manner to MAP4. (S)-3,4-DCPG-induced depressions of the fDR-VRP in the low-affinity range of the concentration-response curve were potentiated by 200 microM (S)-alpha-ethylglutamate (EGLU), a group II mGlu receptor antagonist, and were relatively unaffected by MAP4 (200 microM). However, depressions of the fDR-VRP mediated by the AMPA selective antagonist (R)-3,4-DCPG were not potentiated by EGLU, suggesting that the low-affinity component of the concentration-response curve for (S)-3,4-DCPG is not due to antagonism of postsynaptic AMPA receptors. It is suggested that the receptor responsible for mediating the high-affinity component is mGlu8. The receptor responsible for mediating the low-affinity effect of (S)-3,4-DCPG has yet to be identified but it is unlikely to be one of the known mGlu receptors present on primary afferent terminals or an ionotropic glutamate receptor of the AMPA or NMDA subtype.     

Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.     

Synthesis, pharmacological characterization, and molecular modeling of heterobicyclic amino acids related to (+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (LY354740): identification of two new potent, selective, and systemically active agonists for group II metabotropic glutamate receptors

As part of our ongoing research program aimed at the identification of highly potent, selective, and systemically active agonists for group II metabotropic glutamate (mGlu) receptors, we have prepared novel heterobicyclic amino acids (-)-2-oxa-4-aminobicyclo[3.1. 0]hexane-4,6-dicarboxylate (LY379268, (-)-9) and (-)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795, (-)-10). Compounds (-)-9 and (-)-10 are structurally related to our previously described nanomolar potency group II mGlu receptor agonist, (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740 monohydrate, 5), with the C4-methylene unit of 5 being replaced with either an oxygen atom (as in (-)-9) or a sulfur atom (as in (-)-10). Compounds (-)-9 and (-)-10 potently and stereospecifically displaced specific binding of the mGlu2/3 receptor antagonist ([3H]LY341495) in rat cerebral cortical homogenates, displaying IC50 values of 15 +/- 4 and 8.4 +/- 0.8 nM, respectively, while having no effect up to 100 000 nM on radioligand binding to the glutamate recognition site on NMDA, AMPA, or kainate receptors. Compounds (-)-9 and (-)-10 also potently displaced [3H]LY341495 binding from membranes expressing recombinant human group II mGlu receptor subtypes: (-)-9, Ki = 14.1 +/- 1.4 nM at mGlu2 and 5.8 +/- 0.64 nM at mGlu3; (-)-10, Ki = 40.6 +/- 3.7 nM at mGlu2 and 4.7 +/- 1.2 nM at mGlu3. Evaluation of the functional effects of (-)-9 and (-)-10 on second-messenger responses in nonneuronal cells expressing human mGlu receptor subtypes demonstrated each to be a highly potent agonist for group II mGlu receptors: (-)-9, EC50 = 2.69 +/- 0.26 nM at mGlu2 and 4.58 +/- 0.04 nM at mGlu3; (-)-10, EC50 = 3.91 +/- 0.81 nM at mGlu2 and 7.63 +/- 2. 08 nM at mGlu3. In contrast, neither compound (up to 10 000 nM) displayed either agonist or antagonist activity in cells expressing recombinant human mGlu1a, mGlu5a, mGlu4a, or mGlu7a receptors. The agonist effects of (-)-9 and (-)-10 at group II mGlu receptors were not totally specific, however, as mGlu6 agonist activity was observed at high nanomolar concentrations for (-)-9 (EC50 = 401 +/- 46 nM) and at micromolar concentrations (EC50 = 2 430 +/- 600 nM) for (-)-10; furthermore, each activated mGlu8 receptors at micromolar concentrations (EC50 = 1 690 +/- 130 and 7 340 +/- 2 720 nM, respectively). Intraperitoneal administration of either (-)-9 or (-)-10 in the mouse resulted in a dose-related blockade of limbic seizure activity produced by the nonselective group I/group II mGluR agonist (1S,3R)-ACPD ((-)-9 ED50 = 19 mg/kg, (-)-10 ED50 = 14 mg/kg), indicating that these molecules effectively cross the blood-brain barrier following systemic administration and suppress group I mGluR-mediated limbic excitation. Thus, heterobicyclic amino acids (-)-9 and (-)-10 are novel pharmacological tools useful for exploring the functions of mGlu receptors in vitro and in vivo.     

Face-washing behavior induced by the group I metabotropic glutamate receptor agonist (S)-3,5-DHPG in mice is mediated by mGlu1 receptor

It is known for the non-selective group I metabotropic glutamate (mGlu) receptors agonist (S)-3,5-dihydroxyphenylglycine (S-3,5-DHPG) to cause convulsions, which are mediated by mGlu1 receptor. However, the behavioral changes other than convulsions caused by (S)-3,5-DHPG have not been well studied. The purpose of the present study was to explore the behavioral changes elicited by activation of group I mGlu receptors with (S)-3,5-DHPG and to clarify which, mGlu1 receptor or mGlu5 receptor, is responsible for such behavior. (S)-3,5-DHPG at doses of 3-30 nmol caused characteristic face-washing behavior. This behavioral change was inhibited by both the competitive mGlu1 receptor antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-4-carboxyphenylglycine (S-4CPG) and the non-competitive mGlu1 receptor antagonist, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide (FTIDC), but not by the mGlu5 receptor antagonist 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), the mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), the mGlu2/3 receptor antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), the N-methyl-d-asparate (NMDA) receptor antagonist 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), or the competitive non-NMDA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX). These findings indicate that face-washing behavior is due to selective activation of mGlu1 receptor by (S)-3,5-DHPG, and that the face-washing behavior induced by (S)-3,5-DHPG in mice can be used for in vivo testing of the antagonistic potency of both competitive and non-competitive mGlu1 receptor antagonists.     

Synthesis, in vitro pharmacology, structure-activity relationships, and pharmacokinetics of 3-alkoxy-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent and selective group II metabotropic glutamate receptor antagonists

Novel group II metabotropic glutamate receptor (mGluR) antagonists, 3-alkoxy-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives 11 and 12, were discovered by the incorporation of a hydroxy or alkoxyl group onto the C-3 portion of selective and potent group II mGluR agonist 5, (1R,2S,5R,6R)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid. Among these compounds, (1R,2R,3R,5R,6R)-2-amino-3-(3,4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (-)-11be (MGS0039) was a highly selective and potent group II mGluR antagonist with the best pharmacokinetic profile. Compound (-)-11be exhibited high affinities for mGlu 2 (Ki = 2.38 +/- 0.40 nM) and mGlu 3 (4.46 +/- 0.31 nM) but low affinity for mGluR 7 (Ki = 664 +/- 106 nM), and potent antagonist activities for mGlu 2 (IC50 = 20.0 +/- 3.67 nM) and mGluR 3 (IC50 = 24.0 +/- 3.54 nM) but much less potent antagonist activities for mGlu 4 (IC50 = 1740 +/- 1080 nM), mGlu 6 (IC50 = 2060 +/- 1270 nM), mGlu 1 (IC50 = 93300 +/- 14600 nM), and mGluR 5 (IC(50) = 117000 +/- 38600 nM). No significant agonist activities of (-)-11be were found for mGluRs 2, 3, 4, 6, 1, and 5 (EC50 > 100,000 nM). Furthermore, (-)-11be exhibited dose-dependent oral absorption (plasma C(max): 214 +/- 56.7, 932 +/- 235, and 2960 +/- 1150 ng/mL for 3 mg/kg, 10 mg/kg, and 30 mg/kg, po, respectively) and acceptable blood-brain barrier penetration (brain C(max): 13.2 ng/mL for 10 mg/kg, p.o. 6 h). In this paper, we report the synthesis, in vitro pharmacological profile, and structure-activity relationships (SARs) of 3-alkoxy-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives 11 and 12, and pharmacokinetic profiles of several typical compounds.     

Characterization of neuronal cholecystokinin receptor by L-364,718 in Auerbach's plexus

The release of [3H]acetylcholine [( 3H]ACh) from Auerbach's plexus and the contraction of longitudinal muscle strips in response to the administration of cholecystokinin (CCK) were measured and recorded simultaneously. The peripheral CCK receptor antagonist, 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-1H- indole-2-carboxamide (L-364,718), antagonized the ACh releasing effect of CCK in a dose-dependent manner. The IC50 value and the dissociation constant (KD) were 41.0 +/- 2.0 pM and 0.06 +/- 0.01 nM, respectively. These results suggest that L-364,718 is a very potent antagonist of the neuronal CCK receptors.     

The role of the 5-HT1D receptor as a presynaptic autoreceptor in the guinea pig

The present study investigated the role of the 5-hydroxytryptamine (5-HT, serotonin)1D receptor as a presynaptic autoreceptor in the guinea pig. In keeping with the literature, the 5-HT1B selective antagonist, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro [furo[2,3-f]indole-3,4'-piperidine]oxalate (SB224289) potentiated [3H]5-HT outflow from pre-labelled slices of guinea pig cerebral cortex confirming its role as a presynaptic autoreceptor in this species. In addition, the 5-HT1D receptor-preferring antagonists, 1-[2-[4-(6-fluoro-1H-indol-3-yl)-3,6-dihydro-2H-pyridin-1-yl]-ethyl]-3-pyridin-4-yl-methyl-tetrahydro-pyrimidin-2-one (LY367642), (R)-1-[2-(4-(6-fluoro-1H-indol-3-yl-)-3,6-dihydro-1(2H)-pyridinyl)ethyl]-3,4-dihydro-1H-2-benzopyran-6-carboxamide (LY456219), (S)-1-[2-(4-(6-fluoro-1H-indol-3-yl-)-3,6-dihydro-1(2H)-pyridinyl)ethyl]-3,4-dihydro-1H-2-benzopyran-6-carboxamide (LY456220) and 1-[2-[4-(4-fluoro-benzoyl)-piperidin-1-yl]-ethyl]-3,3-dimethyl-1,2-dihydro-indol-2-one (LY310762), potentiated [3H]5-HT outflow from this preparation with potencies (EC50 values=31-140 nM) in the same range as their affinities for the guinea pig 5-HT1D receptor (Ki values=100-333 nM). The selective 5-HT1D receptor agonist, R-2-(4-fluoro-phenyl)-2-[1-[3-(5-[1,2,4]triazol-4-yl-1H-indol-3-yl)-propyl]-piperidin-4-ylamino]-ethanol dioxylate (L-772,405), inhibited [3H]5-HT outflow. In microdialysis studies, administration of either SB224289 or LY310762 at 10 mg/kg by the intraperitoneal (i.p.) route, potentiated the increase in extracellular 5-HT concentration produced by a maximally effective dose of the selective serotonin re-uptake inhibitor, fluoxetine (at 20 mg/kg i.p.). In addition, the 5-HT1D receptor-preferring antagonist and 5-HT transporter inhibitor, LY367642 (at 10 mg/kg i.p.), elevated extracellular 5-HT concentrations to a greater extent than a maximally effective dose of fluoxetine. It is concluded that the 5-HT1D receptor, like the 5-HT1B receptor, may be a presynaptic autoreceptor in the guinea pig.     

Cloning and expression of canine glucagon receptor and its use to evaluate glucagon receptor antagonists in vitro and in vivo

Metabotropic glutamate receptor 1 (mGlu(1) receptor) has been suggested to play an important role in pain transmission. In this study, the effects of a newly-synthesized mGlu(1) receptor antagonist, (R)-N-cycloheptyl-6-({[(tetrahydro-2-furyl)methyl]amino}methyl)thieno[2,3-d]pyrimidin-4-ylamine (YM-230888), were examined in a variety of rodent chronic pain models in order to characterize the potential analgesic profile of mGlu(1) receptor blockade. YM-230888 bound an allosteric site of mGlu(1) receptor with a K(i) value of 13+/-2.5 nM and inhibited mGlu(1)-mediated inositol phosphate production in rat cerebellar granule cells with an IC(50) value of 13+/-2.4 nM. It showed selectivity for mGlu(1) versus mGlu(2)-mGlu(7) subtypes and ionotropic glutamate receptors. YM-230888 recovered mechanical allodynia with an ED(50) value of 8.4 mg/kg p.o. in L5/L6 spinal nerve ligation models. It also showed antinociceptive response at doses of 10 and 30 mg/kg p.o. in streptozotocin-induced hyperalgesia models. In addition, it significantly reduced pain parameters at a dose of 30 mg/kg p.o. in complete Freund's adjuvant-induced arthritic pain models. Although YM-230888 showed no significant effect on rotarod performance time at doses of 10 or 30 mg/kg p.o., it significantly decreased it at a dose of 100 mg/kg p.o. On the other hand, YM-230888 showed no significant sedative effect in locomotor activity measurement up to 100 mg/kg p.o. These results suggest that the blockade of mGlu(1) receptors is an attractive target for analgesics. YM-230888 has potential as a new analgesic agent for the treatment of various chronic pain conditions. In addition, YM-230888 may be a useful tool for the investigation of mGlu(1) receptors.     

Dual inhibitors of thymidylate synthase and dihydrofolate reductase as antitumor agents: design, synthesis, and biological evaluation of classical and nonclassical pyrrolo[2,3-d]pyrimidine antifolates(1)

We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine, 22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction using iodine. For the classical analogue 4, the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC(50) = 90 nM) and human DHFR (IC(50) = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogues were only inhibitors of human TS with IC(50) values of 0.23-26 microM.     

Characterization of angiotensin II antagonism displayed by Ib, a novel nonpeptide angiotensin AT(1) receptor antagonist

The pharmacologic profile of Ib, 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a novel nonpeptide angiotensin AT(1) receptor antagonist, was investigated by receptor-binding studies, functional in vitro assays with rabbit and rat aorta, and in vivo experiments in rats. Ib inhibited [(125)I] angiotensin II binding to AT(1) receptors in rat liver membranes (K(i)=2.5+/-0.5 nM) and did not interact with AT(2) receptors in bovine cerebellar membranes. In functional studies with rat and rabbit aorta, Ib inhibited the contractile response to angiotensin II (pD(2)' value: 7.43 and 7.29, respectively) with a significant reduction in the maximum. In pithed rats, Ib inhibited the angiotensin II induced pressor response in a dose-related manner. After intravenous administration, Ib produced a dose-dependent antihypertensive effects in spontaneously hypertensive rats and renal hypertensive rats. These results suggest that Ib is a potent angiotensin AT(1) selective receptor antagonist with a mode of insurmountable antagonism.