In vitro pharmacological profile of a novel structural class of oxytocin antagonists

A number of structurally novel cyclic hexapeptides have been characterized as potent and selective oxytocin (OT) antagonists in vitro. As a representative of this class of compounds, L-366,948 [[cyclo(L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl- L-pipecolyl-D- histidyl)]] exhibited a high binding affinity (Ki, low nanomolar) for OT receptors in rat (uterus and mammary) and primate (pregnant rhesus and human myometrium) tissue with a several hundred-fold binding selectivity vs. rat arginine vasopressin (AVP)-V1 (liver) and AVP-V2 (kidney medulla) receptors. In functional assays, L-366,948 was a pure OT antagonist, blocking both OT-stimulated contraction of the isolated rat uterus (pA2, 8.5) and phosphatidylinositol turnover in uterine slices (IC50, 40 vs. 3 nM OT), with no evidence of partial agonist activity. L-366,948 was comparatively weak as an antagonist of AVP-induced contraction of the isolated rat tail artery (AVP-V1 receptor) and AVP-stimulated adenylate cyclase (AVP-V2 receptor) activity in rat kidney medulla and did not influence prostaglandin F2 alpha- or bradykinin-induced contractions of the isolated rat uterus. L-366,948 and related compounds described in this report represent new experimental tools for the study of the pharmacology and physiology of OT.     

A novel selective allosteric modulator potentiates the activity of native metabotropic glutamate receptor subtype 5 in rat forebrain

We found that N-[4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl]-2-hydroxybenzamide (CPPHA), is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). CPPHA alone had no agonist activity and acted as a selective positive allosteric modulator of human and rat mGluR5. CPPHA potentiated threshold responses to glutamate in fluorometric Ca(2+) assays 7- to 8-fold with EC(50) values in the 400 to 800 nM range, and at 10 microM shifted mGluR5 agonist concentration-response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine (DHPG) 4- to 7-fold to the left. The only effect of CPPHA on other mGluRs was weak inhibition of mGluR4 and 8. Neither CPPHA nor the previously described 3,3'-difluorobenzaldazine (DFB) affected [(3)H]quisqualate binding to mGluR5, but although DFB partially competed for [(3)H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding, CPPHA had no effect on the binding of this 2-methyl-6-(phenylethynyl)-pyridine analog to mGluR5. Although the binding sites for the two classes of allosteric modulators seem to be different, these different allosteric sites can modulate functionally and mechanistically similar allosteric effects. In electrophysiological studies of brain slice preparations, it had been previously shown that activation of mGluR5 receptors by agonists increased N-methyl-D-aspartate (NMDA) receptor currents in the CA1 region of hippocampal slices. We found that CPPHA (10 microM) potentiated NMDA receptor currents in hippocampal slices induced by threshold levels of DHPG, whereas having no effect on these currents by itself. Similarly, 10 microM CPPHA also potentiated mGluR5-mediated DHPG-induced depolarization of rat subthalamic nucleus neurons. These results demonstrate that allosteric potentiation of mGluR5 increases the effect of threshold agonist concentrations in native systems.     

A novel family of potent negative allosteric modulators of group II metabotropic glutamate receptors

Group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, play a number of important roles in mammalian brain and represent exciting new targets for certain central nervous system disorders. We now report synthesis and characterization of a novel family of derivatives of dihydrobenzo[1,4]diazepin-2-one that are selective negative allosteric modulators for group II mGluRs. These compounds inhibit both mGluR2 and mGluR3 but have no activity at group I and III mGluRs. The novel mGluR2/3 antagonists also potently block mGluR2/3-mediated inhibition of the field excitatory postsynaptic potentials at the perforant path synapse in hippocampal slices. These compounds induce a rightward shift and decrease the maximal response in the glutamate concentration-response relationship, consistent with a noncompetitive antagonist mechanism of action. Furthermore, radioligand binding studies revealed no effect on binding of the orthosteric antagonist [(3)H]LY341495 [2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid]. Site-directed mutagenesis revealed that a single point mutation in transmembrane V (N735D), previously shown to be an important residue for potentiation activity of the mGluR2 allosteric potentiator LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine], is not critical for the inhibitory activity of negative allosteric modulators of group II mGluRs. However, this single mutation in human GluR2 almost completely blocked the enhancing activity of biphenyl-indanone A, a novel allosteric potentiator of mGluR2. Our data suggest that these two positive allosteric modulators of mGluR2 may share a common binding site and that this site may be distinct from the binding site for the new negative allosteric modulators of group II mGluRs.     

In vitro pharmacological characterization of UP 269-6, a novel nonpeptide angiotensin II receptor antagonist

Summary— The in vitro pharmacology of UP 269-6, a novel nonpeptide angiotensin II antagonist, was examined in radioligand binding and functional isolated tissue assays. UP 269-6 bound selectively to AT₁ receptors as evidenced by the inhibition of specific [¹²⁵I] Sar¹, Ile⁸-AII binding in rat adrenal membranes (IC₅₀ = 35.8 nM) and in cultured vascular smooth muscle cells (IC₅₀ = 23.8 nM). UP 269-6 displayed a very high selectivity for the AT₁ compared to the AT₂ receptor subtype (IC₅₀ > 10,000 nM). UP 269-6 inhibited the AII-induced contraction of isolated rabbit aortic strips. The pattern of AII antagonism suggested competitive antagonism at low concentrations (10⁻¹⁰, 3 × 10⁻¹⁰, 10⁻⁹ M) of UP 269-6 and insurmountable antagonism at higher concentrations (3 × 10⁻⁹, 10⁻⁸, 3 × 10⁻⁸ M). Based on the calculated p A₂ values, UP 269-6 (9.86 ± 0.25) was an angiotensin II receptor antagonist as potent as L-158,809 (9.82 ± 0.37) and much more potent than losartan (7.96 ± 0.38). UP 269-6 was devoid of affinity (IC₅₀ > 10,000 nM) for many other receptors, ion channels and uptake sites, demonstrating its high specificity for AII receptors. Furthermore, this compound did not affect the contractile response to KCl or phenylephrine in rabbit aorta and exhibited no effect on angiotensin converting enzyme activity. These data demonstrate that UP 269-6 is a highly potent, selective and specific AT₁ receptor antagonist.     

In vitro characterization of benzodiazepine receptor agonists, antagonists, inverse agonists and agonist/antagonists

Using an extensively washed membrane preparation and standardized incubation conditions, the actions of benzodiazepine (BZ) receptor ligands were evaluated on [3H]flunitrazepam [+/- 10 microM gamma-aminobutyric acid (GABA)], [3H]muscimol (+/- 2.5 microM etazolate) and [35S]butyl bicyclophosphorothionate (TBPS) binding. Classical BZ receptor agonists stimulated [35S]TBPS binding and [3H]muscimol binding in the presence of etazolate. These agents also possessed ratios for [3H]flunitrazepam binding in the absence and presence of GABA (GABA ratio) of 2 to 5. BZ antagonists and inverse agonists had GABA ratios less than 1 and did not alter, or reduced, both [35S]TBPS and [3H]muscimol (+etazolate) binding. The nonsedating BZ agonist/antagonist agents CGS 9896, CL 218872, PK 8165 and PK 9084 all possessed GABA ratios between 1.1 and 1.4 and only stimulated [35S]TBPS and [3H]muscimol (+etazolate) binding to approximately 50% of the level of classical BZ agonists. The BZ partial agonists CGS 9895 and RU 39419 both were unique in that they possessed GABA ratios of 1 or less, stimulated [35S]TBPS binding and had no effect on [3H]muscimol binding (+etazolate). Therefore, by monitoring the major components of the BZ receptor complex (BZ receptor, GABA receptor and chloride channel), we were able to distinguish between different BZ drugs and to support suggestions that these drugs act via unique BZ receptor populations which possess differential couplings to the GABA receptor and chloride channel.     

Morphine in combination with metabotropic glutamate receptor antagonists on schedule-controlled responding and thermal nociception

The present study examined the interactive effects of morphine in combination with metabotropic glutamate (mGlu) receptor antagonists on schedule-controlled responding and thermal nociception. Drug interaction data were examined with isobolographic and dose-addition analysis. Morphine, the mGlu1 receptor antagonist JNJ16259685 [(3,4-dihydro-2H-pyrano-[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone], the mGlu5 receptor antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride], and the mGlu2/3 receptor antagonist LY341495 [(2S)-2-amino-2-[(1S,2S-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] all decreased rates of schedule-controlled responding. JNJ16259685/morphine, MPEP/morphine, and LY341495/morphine mixtures produced additive effects on this endpoint. Morphine also produced dose-dependent antinociception in the assay of thermal nociception, whereas JNJ16259685, MPEP, and LY341495 failed to produce an effect. In this assay, JNJ16259685 and LY341495 potentiated the antinociceptive effects of morphine, whereas MPEP/morphine mixtures produced additive effects. These results suggest that an mGlu1 and an mGlu2/3 receptor antagonist, but not an mGlu5 receptor antagonist, selectively enhance the antinociceptive effects of morphine. In addition, these data confirm that the behavioral effects of drug mixtures depend on the endpoint under study.     

A novel selective positive allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects in rat behavioral models

We found that 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). In Chinese hamster ovary cells expressing human mGluR5, CDPPB potentiated threshold responses to glutamate in fluorometric Ca2+ assays more than 7-fold with an EC50 value of approximately 27 nM. At 1 microM, CDPPB shifted mGluR5 agonist concentration response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine 3- to 9-fold to the left. At higher concentrations, CDPPB exhibited agonist-like activity on cells expressing mGluR5. No other activity was observed on any other mGluR or cell type at concentrations up to 10 microM. CDPPB had no effect on [3H]quisqualate binding to mGluR5 but did compete for binding of [3H]methoxyPEPy, an analog of the selective mGluR5 negative allosteric modulator MPEP. CDPPB was found to be brain penetrant and reversed amphetamine-induced locomotor activity and amphetamine-induced deficits in prepulse inhibition in rats, two models sensitive to antipsychotic drug treatment. These results demonstrate that positive allosteric modulation of mGluR5 produces behavioral effects, suggesting that such modulation serves as a viable approach to increasing mGluR5 activity in vivo. These effects are consistent with the hypothesis that allosteric potentiation of mGluR5 may provide a novel approach for development of antipsychotic agents.     

Two novel and selective nonimidazole histamine H3 receptor antagonists A-304121 and A-317920: I. In vitro pharmacological effects

Histamine H3 receptor (H3R) antagonists enhance neurotransmitter release and are being developed for the treatment of a variety of neurological and cognitive disorders. Many potent histamine H3R antagonists contain an imidazole moiety that limits receptor selectivity and the tolerability of this class of compounds. Here we present the in vitro pharmacological data for two novel piperazine amide ligands, A-304121 [4-(3-((2R)-2-aminopropanoyl-1-piperazinyl)propoxy)phenyl)cyclopropylmethanone] and A-317920 [N-((1R)-2-(4-(3-(4-(cyclopropylcarbonyl)phenoxy)propyl)-1-piperazinyl)-1-methyl-2-oxo-ethyl-)-2-furamide], and compare them with the imidazole H3R antagonists ciproxifan, clobenpropit, and thioperamide. Both A-304121 and A-317920 bind potently to recombinant full-length rat H3R(pKi values = 8.6 and 9.2, respectively) but have lower potencies for binding the full-length human H3R (pKi values = 6.1 and 7.0, respectively). A-304121 and A-317920 are potent antagonists at rat H3R in reversing R-alpha-methylhistamine [(R)-alpha-MeHA] inhibition of forskolin-stimulated cAMP formation (pKb values = 8.0 and 9.1) but weak antagonists at human H3Rs in cyclase (pKb values = 6.0 and 6.3) and calcium mobilization (pKb values = 6.0 and 7.3) assays in cells co-expressing Galphaqi5-protein. Both compounds potently antagonize native H3Rs by blocking histamine inhibition of potassium-evoked [3H]histamine release from rat brain cortical synaptosomes (pKb values = 8.6 and 9.3) and (R)-alpha-MeHA reversal of electric field-stimulated guinea pig ileum contractions (pA2 values = 7.1 and 8.3). A-304121 and A-317920 are also more efficacious inverse agonists in reversing basal guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding at the human H3R (pEC50 values = 5.7 and 7.0) than are the imidazole antagonists. These novel and selective piperazine amides represent useful leads for the development of H3R antagonist therapeutic agents.     

In vitro and in vivo pharmacological characterization of JTE-907, a novel selective ligand for cannabinoid CB2 receptor

JTE-907 [N-(benzo[1,3]dioxol-5-ylmethyl)-7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxamide] was evaluated in vitro and in vivo as a novel selective ligand for cannabinoid receptor of peripheral type (CB2). The compound binds with high affinity to human CB2 or mouse CB2 expressed on CHO cell membrane and to rat CB2 on splenocytes. The K(i) affinities for human, mouse, and rat CB2 were 35.9, 1.55, and 0.38 nM, respectively. The selectivity ratio for the CB2 receptors compared with central nervous type receptors (CB1) of human (expressed on CHO cells), and mouse and rat CB1 on cerebellum were 66, 684, and 2760, respectively. JTE-907 showed concentration-dependent increase of forskolin-stimulated cAMP production in CHO cells expressing human and mouse CB2 in vitro, i.e., JTE-907 behaved as an inverse agonist, which is in contrast to Win55212-2 that reduces cAMP as an agonist. JTE-907 dosed orally inhibited carrageenin-induced mouse paw edema dose dependently. The same in vivo effect was observed with other cannabinoid receptor ligands such as SR144528, Delta(9)-tetrahydrocannabinol (THC), and Win55212-2. This is the first report that a CB2-selective inverse agonist, JTE-907, has an anti-inflammatory effect in vivo, and how the inverse agonist showed the same effect as Win55212-2 and Delta(9)-THC is discussed.     

A potent and selective metabotropic glutamate receptor 4 positive allosteric modulator improves movement in rodent models of Parkinson's disease

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGluR4) have been proposed as a novel therapeutic approach for the treatment of Parkinson's disease. However, evaluation of this proposal has been limited by the availability of appropriate pharmacological tools to interrogate the target. In this study, we describe the properties of a novel mGluR4 PAM. 5-Methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine (ADX88178) enhances glutamate-mediated activation of human and rat mGluR4 with EC(50) values of 4 and 9 nM, respectively. The compound is highly selective for mGluR4 with minimal activities at other mGluRs. Oral administration of ADX88178 in rats is associated with high bioavailability and results in cerebrospinal fluid exposure of >50-fold the in vitro EC(50) value. ADX88178 reverses haloperidol-induced catalepsy in rats at 3 and 10 mg/kg. It is noteworthy that this compound alone has no impact on forelimb akinesia resulting from a bilateral 6-hydroxydopamine lesion in rats. However, coadministration of a low dose of l-DOPA (6 mg/kg) enabled a robust, dose-dependent reversal of the forelimb akinesia deficit. ADX88178 also increased the effects of quinpirole in lesioned rats and enhanced the effects of l-DOPA in MitoPark mice. It is noteworthy that the enhancement of the actions of l-DOPA was not associated with an exacerbation of l-DOPA-induced dyskinesias in rats. ADX88178 is a novel, potent, and selective mGluR4 PAM that is a valuable tool for exploring the therapeutic potential of mGluR4 modulation. The use of this novel tool molecule supports the proposal that activation of mGluR4 may be therapeutically useful in Parkinson's disease.     

Positive and negative allosteric modulation of metabotropic glutamate receptors: emerging therapeutic potential

Metabotropic glutamate receptors (mGluRs) modulate neuronal activity in the central and peripheral nervous systems, and since their discovery have attracted considerable attention as putative therapeutic targets for a range of neurological and psychiatric disorders. A number of competitive agonists and antagonists acting at the N-terminal glutamate binding site have been identified, the majority of which are conformationally constrained or substituted amino acid analogues. These ligands have greatly facilitated investigation of the physiological and pathological roles of the receptor family. However, their utility and therapeutic potential has been restricted by relatively poor bioavailability and central nervous system (CNS) penetration, as well as limited chemical tractability and, generally, a lack of selectivity for individual mGluRs. Recently, a number of non-competitive mGluR ligands have been identified which bind within the receptor transmembrane heptahelical domain. These include both positive and negative allosteric modulators. Positive allosteric modulators do not exhibit intrinsic agonism but facilitate agonist-mediated receptor activity. Negative allosteric modulators include both non-competitive antagonists and inverse agonists. Allosteric modulation offers the potential for improved selectivity, particularly for individual receptors within the mGluR family, and enhanced chemical tractability relative to competitive agonists/antagonists. In addition, positive allosteric modulation provides a distinct, and perhaps superior, profile to receptor agonism, offering the potential for facilitation of physiologically appropriate receptor activation with reduced liability for receptor desensitisation and/or tolerance. Thus, the emerging field of positive and negative allosteric modulation of the mGluR family offers considerable promise for the development of novel therapeutics.     

Group I metabotropic glutamate receptor antagonists block secondary thermal hyperalgesia in rats with knee joint inflammation.

Activation of ionotropic glutamate receptors has been shown previously to be essential for the development of secondary thermal hyperalgesia. The present study assessed involvement of group I metabotropic glutamate receptors (mGlu) in both the induction and maintenance phases of secondary thermal hyperalgesia initiated by knee joint inflammation in rats. The dose dependence of each drug in antagonism of thermal hypersensitivity was demonstrated in pre- and post-treatment paradigms. Knee joint inflammation was induced by injection of kaolin and carrageenan. Four hours later the paw withdrawal latencies were significantly shorter than baseline values. Rats were pretreated by spinal microdialysis infusion of group I mGlu receptor antagonists, LY393053 [(+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid], LY367385 [(S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid], or AIDA [(R,S)-1-aminoindan-1,5-dicarboxylic acid/UPF 523] before knee joint injection.The paw withdrawal latencies measured 4 h after the injection were significantly longer in the presence of group I mGlu receptor antagonists than those of the artificial cerebrospinal fluid-treated arthritic control group. Post-treatment with the group I mGlu receptor antagonists LY367385 and AIDA allowed significant recovery of the paw withdrawal latencies after the onset of the knee joint inflammation. The knee joint inflammation itself was not affected by either treatment. The results of the present study indicate that secondary thermal hyperalgesia can be effectively attenuated during both the development and maintenance phases of acute knee joint inflammation by spinal application of specific group I mGlu receptor antagonists.     

In vitro and in vivo pharmacological characterization of BIIL 284, a novel and potent leukotriene B(4) receptor antagonist

BIIL 284 is a new LTB(4) receptor antagonist. It is a prodrug and has negligible binding to the LTB(4) receptor. However, ubiquitous esterases metabolize BIIL 284 to the active metabolites BIIL 260 and BIIL 315, the glucuronidated form of BIIL 260. Both metabolites have high affinity to the LTB(4) receptor on isolated human neutrophil cell membranes with K(i) values of 1.7 and 1.9 nM, respectively. On vital human neutrophilic granulocytes K(i) was around 1 nM. BIIL 260 and BIIL 315 interact with the LTB(4) receptor in a saturable, reversible, and competitive manner. BIIL 260 and its glucuronide BIIL 315 also potently inhibited LTB(4)-induced intracellular Ca(2+) release in human neutrophils (IC(50) values of 0.82 and 0.75 nM, respectively) as measured with Fura-2. High efficacy of BIIL 284 has been demonstrated in various in vivo models. BIIL 284 inhibited LTB(4)-induced mouse ear inflammation with ED(50) = 0.008 mg/kg p.o., LTB(4)-induced transdermal chemotaxis in guinea pigs with ED(50) = 0.03 mg/kg p.o., LTB(4)-induced neutropenia in various species (monkey: ED(50) = 0.004 mg/kg p.o.), and LTB(4)-induced Mac1-expression in monkeys (ED(50) = 0.05 mg/kg p.o. in Tylose). Full blockade of LTB(4) receptors over 24 h was achieved by 0.3 mg/kg BIIL 284 after single oral dose as measured by LTB(4)-induced neutropenia or Mac1-expression in the monkey model. BIIL 284 is an unusually potent and long-acting orally active LTB(4) antagonist, and is therefore under clinical development as a novel anti-inflammatory principle.     

Therapeutic effects of metabotropic glutamate receptor 5 positive allosteric modulator CDPPB on phencyclidine‐induced cognitive deficits in mice

This study was undertaken to examine the effects of CDPPB (3‐cyano‐N‐(1,3‐diphenyl‐1H‐pyrazol‐5‐yl)benzamide), a positive allosteric modulator (PAM) of metabotropic glutamate receptor 5 (mGlu₅), on cognitive deficits in mice after repeated administration of the N‐methyl‐D‐aspartate (NMDA) receptor antagonist phencyclidine (PCP). In the novel object recognition test, PCP (10 mg/kg/day for 10 days)‐induced cognitive deficits in mice were not improved by a single administration of CDPPB (10 mg/kg/day). However, PCP (10 mg/kg/day for 10 days)‐induced cognitive deficits in mice were significantly improved by subsequent subchronic (14 days) administration of CDPPB (10 mg/kg/day), but not of CDPPB (1.0 mg/kg/day). This study suggests that PCP‐induced cognitive deficits in mice are improved by subsequent subchronic administration of CDPPB. Therefore, mGlu₅ PAMs would be potential therapeutic drugs for cognitive deficits in schizophrenia.     

Involvement of metabotropic glutamate 5 receptor in visceral pain

Metabotropic glutamate 5 receptor (mGluR5) antagonists are effective in animal models of inflammatory and neuropathic pain. The involvement of mGluR5 in visceral pain pathways from the gastrointestinal tract is as yet unknown. We evaluated effects of mGluR5 antagonists on the colorectal distension (CRD)-evoked visceromotor (VMR) and cardiovascular responses in conscious rats, and on mechanosensory responses of mouse colorectal afferents in vitro. Sprague-Dawley rats were subjected to repeated, isobaric CRD (12 x 80 mmHg, for 30s with 5 min intervals). The VMR and cardiovascular responses to CRD were monitored. The mGluR5 antagonists MPEP (1-10 micromol/kg, i.v.) and MTEP (1-3 micromol/kg, i.v.) reduced the VMR to CRD dose-dependently with maximal inhibition of 52+/-8% (p<0.01) and 25+/-11% (p<0.05), respectively, without affecting colonic compliance. MPEP (10 micromol/kg, i.v.) reduced CRD-evoked increases in blood pressure and heart rate by 33+/-9% (p<0.01) and 35+/-8% (p<0.05), respectively. Single afferent recordings were made from mouse pelvic and splanchnic nerves of colorectal mechanoreceptors. Circumferential stretch (0-5 g force) elicited slowly-adapting excitation of action potentials in pelvic distension-sensitive afferents. This response was reduced 55-78% by 10 microM MTEP (p<0.05). Colonic probing (2g von Frey hair) activated serosal splanchnic afferents; their responses were reduced 50% by 10 microM MTEP (p<0.01). We conclude that mGluR5 antagonists inhibit CRD-evoked VMR and cardiovascular changes in conscious rats, through an effect, at least in part, at peripheral afferent endings. Thus, mGluR5 participates in mediating mechanically evoked visceral nociception in the gastrointestinal tract.     

L-homocysteine sulfinic acid and other acidic homocysteine derivatives are potent and selective metabotropic glutamate receptor agonists

Moderate hyperhomocysteinemia is associated with several diseases, including coronary artery disease, stroke, Alzheimer's disease, schizophrenia, and spina bifida. However, the mechanisms for their pathogenesis are unknown but could involve the interaction of homocysteine or its metabolites with molecular targets such as neurotransmitter receptors, channels, or transporters. We discovered that L-homocysteine sulfinic acid (L-HCSA), L-homocysteic acid, L-cysteine sulfinic acid, and L-cysteic acid are potent and effective agonists at several rat metabotropic glutamate receptors (mGluRs). These acidic homocysteine derivatives 1) stimulated phosphoinositide hydrolysis in the cells stably expressing the mGluR1, mGluR5, or mGluR8 (plus Galpha(qi9)) and 2) inhibited the forskolin-induced cAMP accumulation in the cells stably expressing mGluR2, mGluR4, or mGluR6, with different potencies and efficacies depending on receptor subtypes. Of the four compounds, L-HCSA is the most potent agonist at mGluR1, mGluR2, mGluR4, mGluR5, mGluR6, and mGluR8. The effects of the four agonists were selective for mGluRs because activity was not discovered when L-HCSA and several other homocysteine derivatives were screened against a large panel of cloned neurotransmitter receptors, channels, and transporters. These findings imply that mGluRs are candidate G-protein-coupled receptors for mediating the intracellular signaling events induced by acidic homocysteine derivatives. The relevance of these findings for the role of mGluRs in the pathogenesis of homocysteine-mediated phenomena is discussed.     

Activation of metabotropic glutamate receptor 1 dimers requires glutamate binding in both subunits

Group I metabotropic glutamate receptors (mGluRs) form stable, disulfide-linked homodimers. Lack of a verifiably monomeric mGluR1 mutant has led to difficulty in assessing the role of dimerization in the molecular mechanism of mGluR1 activation. The related GABA(B) receptor exhibits striking intradimer cross talk (ligand binding at one subunit effectively produces G protein activation at the other), but it is unclear whether group I mGluRs exhibit analogous cross talk. Signaling of heterologously expressed mGluR1 was examined in isolated rat sympathetic neurons by measuring glutamate-mediated inhibition of native calcium currents. To examine mGluR1 activity when only one dimer subunit has access to glutamate ligand, wildtype mGluR1 was coexpressed with mGluR1 Y74A, a mutant with impaired glutamate binding, and the activity of the heterodimer (mutant/wild type) was examined. The mGluR1 Y74A mutant alone had a dose-response curve that was shifted by about 2 orders of magnitude. The half-maximal dose of glutamate shifted from 1.3 (wild-type mGluR1) to about 450 (mGluR1 Y74A) microM. However, the maximal effect was similar. Wild-type mGluR1 was expressed with excess Y74A mGluR1 to generate a receptor population consisting largely of mutant homodimers and mutant/wild-type heterodimers but without detectable wild-type homodimers. Under these conditions, no glutamate-mediated calcium current inhibition was observed below approximately 300 microM glutamate, although wild-type mGluR1 protein was detectable with immunofluorescence. These data suggest that mutant/wild-type heterodimeric receptors are inactive at ligand concentrations favoring glutamate association with receptor dimers at only one subunit.     

In vivo pharmacological characterization of indiplon, a novel pyrazolopyrimidine sedative-hypnotic

Indiplon (NBI 34060; N-methyl-N-[3-[3-(2-thienylcarbonyl)-pyrazolo[1,5-alpha]pyrimidin-7-yl]phenyl]acetamide), a novel pyrazolopyrimidine and high-affinity allosteric potentiator of GABA(A) receptor function, was profiled for its effects in rodents after oral administration. In mice, indiplon inhibited locomotor activity (ED(50) = 2.7 mg/kg p.o.) at doses lower than the nonbenzodiazepine hypnotics zolpidem (ED(50) = 6.1 mg/kg p.o.) and zaleplon (ED(50) = 24.6 mg/kg p.o.), a sedative effect that was reversed by the benzodiazepine site antagonist flumazenil. Indiplon inhibited retention in the mouse passive avoidance paradigm over a dose range and with a temporal profile that coincided with its sedative activity. Indiplon, zolpidem, and zaleplon were equally effective in inhibiting locomotor activity in the rat and produced dose-related deficits on the rotarod. In a rat vigilance paradigm, indiplon, zolpidem, and zaleplon produced performance deficits over a dose range consistent with their sedative effects, although indiplon alone showed no significant increase in response latency. Indiplon produced a small deficit in the delayed nonmatch to sample paradigm at a dose where sedative effects became apparent. Indiplon was active in the rat Vogel test of anxiety, but it showed only a sedative profile in the mouse open field test. The pharmacokinetic profile of indiplon in both rat and mouse was consistent with its pharmacodynamic properties and indicated a rapid T(max), short t(1/2), and excellent blood-brain barrier penetration. Therefore, indiplon has the in vivo profile of an efficacious sedative-hypnotic, in agreement with its in vitro receptor pharmacology as a high-affinity allosteric potentiator of GABA(A) receptor function, with selectivity for alpha1 subunit-containing GABA(A) receptors.