Proinflammatory role of vasopressin through V1b receptors in hapten-induced experimental colitis in rodents: implication in IBD.

Vasopressin and its receptors modulate several gut functions, but their role in intestinal inflammation is unknown. Our aims were to determine 1) the localization of V1b receptors in human and rodent colon, 2) the role of vasopressin and V1b receptors in experimental colitis using two approaches: V1b⁻(/)⁻ mice and a selective V1b receptor antagonist, SSR149415, and 3) the mechanisms involved. V1b receptors were localized in normal and inflamed colon from humans and rats. Experimental colitis was induced in rats and mice and some groups were treated before or after colitis induction with oral SSR149415 (3-30 mg/kg). Other groups of mice were submitted to dehydration to increase vasopressin plasma levels, prior to colitis induction. Body weight, damage scores, MPO, and TNF-α tissue levels were determined. Finally, colonic segments of wild-type (WT) and V1b⁻(/)⁻ mice were mounted in Ussing chambers and paracellular permeability in response to vasopressin was studied. V1b receptors were expressed in enterocytes and ganglia cells of the enteric nervous system of human and rat intestine. Expression levels were independent from inflammatory status. Colitis was less severe in rodents treated by either preventive or curative SSR149415 and in V1b⁻(/)⁻ mice. 2,4,6-Trinitrobenzene sulfonic acid induced a strong mortality in dehydrated animals that was reversed by preventive SSR149415 or mast cell stabilizer. Vasopressin significantly increased paracellular permeability in WT, but not in V1b⁻(/)⁻ mice. Preincubation of colon tissues with SSR149415 abolished the vasopressin effect. Similarly, vasopressin had no effect in colonic preparations from WT mice pretreated with mast cell stabilizers. Vasopressin, through V1b receptor interaction, has proinflammatory properties linked to mast cell activation and downstream alterations of the colonic epithelial barrier. These findings underline the potential interest of V1b receptor blockers in gut inflammatory diseases.

Differential Virtual Screening (DVS) with Active and Inactive Molecular Models for Finding and Profiling GPCR Modulators: Case of the CCK1 Receptor.

We discovered a constitutively activating mutation (CAM) V308E for the neurotensin NT1 receptor. Molecular dynamics (MD) performed for the CAM NT1-V308E exhibiting a high spontaneous activity, and for the wild-type NT1 without basal activity, show dramatic conformational changes for the CAM. To test if the two MD models could be valuable active and inactive templates for building molecular models for other class-A GPCR, supposed active and inactive models were built by homology for the cholecystokinin CCK1 receptor. Virtual screening of a corporate library with 250 000 compounds was performed with the two CCK1 models, and a differential virtual screening analysis (DVS), led us to isolate 250 predicted agonists and 250 predicted antagonists. The two sets were merged and the compounds were tested in CCK1 agonist and antagonist cellular assays. An excellent correlation was obtained between predictions and biological results. The effective profiling provided by DVS with active and inactive molecular models, opens new perspectives for finding agonists and antagonists for other class-A GPCR, notably for orphan GPCRs for which no ligands are known.

Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand.

[(3)H]SSR-149415 is the first tritiated nonpeptide vasopressin V(1b) receptor (V(1b)R) antagonist ligand. It was used for studying rodent (mouse, rat, hamster) and human V(1b)R from native or recombinant origin. Moreover, a close comparison between the human and the mouse V(1b)R was performed using SSR-149415/[(3)H]SSR-149415 in binding and functional studies in vitro. [(3)H]SSR-149415 binding was time-dependent, reversible, and saturable. Scatchard plot analysis gave a single class of high-affinity binding sites with apparent equilibrium dissociation constant (K(d)) approximately 1 nM and maximum binding density (B(max)) values from 7,000 to 300,000 sites/cell according to the cell line. In competition experiments, [(3)H]SSR-149415 binding was stereospecific and dose-dependently displaced by reference peptide and nonpeptide arginine vasopressin (AVP)/OT ligands following a V(1b) rank order of affinity: SSR-149415 = AVP > dCha > dPen > dPal > dDavp > SSR-126768A > SR-49059 > SSR-149424 > OT > SR-121463B. Species differences between human, rat, mouse, and hamster V(1b)R were observed. Autoradiography studies with [(3)H]SSR-149415 on rat and human pituitary showed intense specific labeling confined to corticotroph cells and absence of labeling in the other tissues examined. SSR-149415 potently and stereospecifically antagonized the AVP-induced inositol phosphate production and intracellular Ca(2+) increase (EC(50) from 1.83 to 3.05 nM) in recombinant cell lines expressing either the mouse or the human V(1b)R. AVP (10(-7) M) exposure of AtT20 cells expressing mouse or human EGFP-tagged V(1b)R induced their rapid internalization. Preincubation with 10(-6) M SSR-149415 counteracted the internalization process. Moreover, recycling of internalized receptors was observed upon 10(-6) M SSR-149415 treatment. Thus SSR-149415/[(3)H]SSR-149415 are unique tools for studying animal and human V(1b)R.

In vitro and in vivo pharmacological characterization of ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino]-phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640), a new potent and selective human beta3-adrenoceptor agonist for the treatment of preterm labor.

Ethyl-4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl)amino] phenoxy]propyl) amino]cyclohexyl]benzoate hydrochloride (SAR150640) was characterized as a new potent and selective beta(3)-adrenoceptor agonist for the treatment of preterm labor. SAR150640 and its major metabolite, the corresponding acid 4-[trans-4-[((2S)-2-hydroxy-3-[4-hydroxy-3[(methylsulfonyl) amino] phenoxy]propyl)amino]cyclohexyl]benzoic acid (SSR500400), showed high affinity for beta(3)-adrenoceptors (K(i) = 73 and 358 nM) and greater potency than (-)-isoproterenol in increasing cAMP production in membrane preparations from human neuroblastoma cells (SKNMC), which express native beta(3)-adrenoceptors (pEC(50) = 6.5, 6.2, and 5.1, respectively). SAR150640 and SSR500400 also increased cAMP production in membrane preparations from human uterine smooth muscle cells (UtSMC), which also express native beta(3)-adrenoceptors (pEC(50) = 7.7 and 7.7, respectively). In these cells, SAR150640 dose-dependently inhibited oxytocin-induced intracellular Ca(2+) mobilization and extracellular signal-regulated kinase 1/2 phosphorylation. SAR150640 and SSR500400 had no beta(1)- or beta(2)-agonist or antagonist activity in guinea pig atrium and trachea, or in human isolated atrium and bronchus preparations. Both compounds concentration-dependently inhibited spontaneous contractions in human near-term myometrial strips, with greater potency than salbutamol and 4-[3-[(1,1-dimethylethyl)-amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP12177) (pIC(50) = 6.4, 6.8, 5.9, and 5.8, respectively), but with similar potency to (-)-isoproterenol and atosiban (oxytocin/vasopressin V(1)a receptor antagonist). SAR150640 also inhibited the contractions induced by oxytocin and prostaglandin F(2alpha). In vivo, after intravenous administration, SAR150640 (1 and 6 mg/kg), but not atosiban (6 mg/kg), dose-dependently inhibited myometrial contractions in conscious unrestrained female cynomolgus monkeys, with no significant effects on heart rate or blood pressure. In contrast, salbutamol (50 and 250 microg/kg) had no inhibitory effect on uterine contractions, but it dose-dependently increased heart rate. These findings indicate a potential for the therapeutic use of SAR150640 in mammals during preterm labor.

Pancreatic vasopressin V1b receptors: characterization in In-R1-G9 cells and localization in human pancreas.

Vasopressin (AVP) receptors present in In-R1-G9 cells, a hamster glucagon-secreting alpha-pancreatic cell line, were characterized using SSR-149415, a selective nonpeptide V1b receptor antagonist, and reference AVP compounds. Binding experiments, using [3H]AVP as a ligand, identified a single population of high-affinity binding sites. SSR-149415 competitively inhibited this binding and exhibited nanomolar and stereospecific affinity for these sites. The affinity of various AVP/oxytocin ligands confirmed a V1b binding profile. In functional studies, AVP was a potent stimulant in inducing intracellular Ca2+ increase, glucagon secretion, and cell proliferation. These effects were fully antagonized by SSR-149415 with a nanomolar potency, whereas its diasteroisomer as well as two selective V1a and V2 receptor antagonists were much less potent. Additionally, the order of potency of AVP agonists and antagonists was in agreement with V1b-mediated effects. By RT-PCR, we confirmed the presence of V1b receptor mRNA in both In-R1-G9 cells and in human pancreas. The distribution pattern of V1b receptors investigated in human pancreas by immunohistochemistry showed strong labeling in islets of Langerhans, and colocalization studies indicated that this receptor was expressed in alpha-glucagon, beta-insulin, and somatostatin pancreatic cells. Thus, in In-R1-G9 cells, AVP mediates intracellular Ca2+ increase, glucagon secretion, and cell proliferation by activating V1b receptors, and these effects are potently antagonized by SSR-149415. Moreover, the presence of V1b receptors also found in human Langerhans islets could suggest hormonal control of AVP in human pancreas.

Anxiolytic- and antidepressant-like effects of the non-peptide vasopressin V1b receptor antagonist, SSR149415, suggest an innovative approach for the treatment of stress-related disorders.

The limbic localization of the arginine vasopressin V(1b) receptor has prompted speculation as to a potential role of this receptor in the control of emotional processes. To investigate this possibility, we have studied the behavioral effects of SSR149415, the first selective and orally active non-peptide antagonist of vasopressin V(1b) receptors, in a variety of classical (punished drinking, elevated plus-maze, and light/dark tests) and atypical (fear/anxiety defense test battery and social defeat-induced anxiety) rodent models of anxiety, and in two models of depression [forced swimming and chronic mild stress (CMS)]. When tested in classical tests of anxiety, SSR149415 produced anxiolytic-like activity at doses that ranged from 1 to 30 mg/kg (i.p. or p.o.), but the magnitude of these effects was overall less than that of the benzodiazepine anxiolytic diazepam, which was used as a positive control. In contrast, SSR149415 produced clear-cut anxiolytic-like activity in models involving traumatic stress exposure, such as the social defeat paradigm and the defense test battery (1-30 mg/kg, p.o.). In the forced swimming test, SSR149415 (10-30 mg/kg, p.o.) produced antidepressant-like effects in both normal and hypophysectomized rats. Moreover, in the CMS model in mice, repeated administration of SSR149415 (10 and 30 mg/kg, i.p.) for 39 days improved the degradation of the physical state, anxiety, despair, and the loss of coping behavior produced by stress. These findings point to a role for vasopressin in the modulation of emotional processes via the V(1b) receptor, and suggest that its blockade may represent a novel avenue for the treatment of affective disorders.

Structure and stereochemistry of the active metabolite of clopidogrel.

Clopidogrel (SR25990C, PLAVIX) is a potent antiplatelet drug, which has been recently launched and is indicated for the prevention of vascular thrombotic events in patients at risk. Clopidogrel is inactive in vitro, and a hepatic biotransformation is necessary to express the full antiaggregating activity of the drug. Moreover, 2-oxo-clopidogrel has been previously suggested to be the essential key intermediate metabolite from which the active metabolite is formed. In the present paper, we give the evidence of the occurrence of an in vitro active metabolite after incubation of 2-oxo-clopidogrel with human liver microsomes. This metabolite was purified by liquid chromatography, and its structure was studied by a combination of mass spectometry (MS) and NMR experiments. MS results suggested that the active metabolite belongs to a family of eight stereoisomers with the following primary chemical structure: 2-[1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene]acetic acid. Chiral supercritical fluid chromatography resolved these isomers. However, only one of the eight metabolites retained the biological activity, thus underlining the critical importance of associated absolute configuration. Because of its highly labile character, probably due to a very reactive thiol function, structural elucidation of the active metabolite was performed on the stabilized acrylonitrile derivative. Conjunction of all our results suggested that the active metabolite is of S configuration at C 7 and Z configuration at C 3-C 16 double bound.

SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]- 4-piperidinyl)-2-methylpropanamide], a centrally active nonpeptide antagonist of the tachykinin neurokinin-1 receptor: I. biochemical and pharmacological characterization.

The biochemical and pharmacological properties of a novel antagonist of the tachykinin neurokinin 1 (NK1) receptor, SSR240600 [(R)-2-(1-[2-[4-[2-[3,5-bis(trifluoromethyl)phenyl]acetyl]-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl]-4-piperidinyl)-2-methylpropanamide], were evaluated. SSR240600 inhibited the binding of radioactive substance P to tachykinin NK1 receptors in human lymphoblastic IM9 cells (K(i) = 0.0061 nM), human astrocytoma U373MG cells (K(i) = 0.10 nM), and human brain cortex (IC50 = 0.017 nM). It also showed subnanomolar affinity for guinea pig NK1 receptors but was less potent on rat and gerbil NK1 receptors. SSR240600 inhibited [Sar(9),Met(O2)(11)]substance P-induced inositol monophosphate formation in human astrocytoma U373MG cells with an IC50 value of 0.66 nM (agonist concentration of 100 nM). It also antagonized substance P-induced contractions of isolated human small bronchi with a pIC50 value of 8.6 (agonist concentration of 100 nM). The compound was >100- to 1000-fold more selective for tachykinin NK1 receptors versus tachykinin NK2 or NK3 receptors as evaluated in binding and in vitro functional assays. In vivo antagonistic activity of SSR240600 was demonstrated on tachykinin NK1 receptor-mediated hypotension in dogs (3 and 10 microg/kg i.v.), microvascular leakage (1 and 3 mg/kg i.p.), and bronchoconstriction (50 and 100 microg/kg i.v.) in guinea pigs. It also prevented citric acid-induced cough in guinea pigs (1-10 mg/kg i.p.), an animal model in which central endogenous tachykinins are suspected to play a major role. In conclusion, SSR240600 is a new, potent, and centrally active antagonist of the tachykinin NK1 receptor, able to antagonize various NK1 receptor-mediated pharmacological effects in the periphery and in the central nervous system.

Biochemical and pharmacological activities of SSR 146977, a new potent nonpeptide tachykinin NK3 receptor antagonist.

SSR 146977 is a potent and selective antagonist of the tachykinin NK3 receptor. In Chinese hamster ovary cells expressing the human tachykinin NK3 receptor, SSR 146977 inhibited the binding of radioactive neurokinin B to NK3 receptors (Ki = 0.26 nM), senktide (10 nM) induced inositol monophosphate formation (IC50 = 7.8-13 nM), and intracellular calcium mobilization (IC50 = 10 nM). It antagonized [MePhe7]neurokinin B induced contractions of guinea pig ileum (pA2 = 9.07). Senktide (30 nM) induced firing rate increase of noradrenergic neurons in the guinea pig locus coeruleus and dopaminergic neurons in the guinea pig substantia nigra was also blocked by SSR 146977 (50 and 100 nM, respectively). In vivo, in the respiratory system, SSR 146977 inhibited bronchial hyperresponsiveness to acetylcholine, bronchial microvascular permeability hypersensitivity to histamine (doses of 0.1-1 mg/kg i.p.), and cough (doses of 0.03-1 mg/kg i.p.) provoked by citric acid in guinea pigs. In the central nervous system, SSR 146977 inhibited turning behaviour (ID50 = 0.2 mg/kg i.p. and 0.4 mg/kg p.o.) and prevented the decrease of locomotor activity (10 and 30 mg/kg i.p) mediated by the stimulation of NK3 receptors in gerbils. In guinea pigs, SSR 146977 antagonized senktide-induced acetylcholine release in the hippocampus (0.3 and 1 mg/kg i.p) and norepinephrine release in the prefrontal cortex (0.3 mg/kg i.p.). It also prevented haloperidol-induced increase of the number of spontaneously active dopamine A10 neurons (1 and 3 mg/kg i.p.).

Characterization of (2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415), a selective and orally active vasopressin V1b receptor antagonist.

(2S,4R)-1-[5-Chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide (SSR149415), the first selective, nonpeptide vasopressin V1b receptor antagonist yet described, has been characterized in vitro and in vivo. SSR149415 showed competitive nanomolar affinity for animal and human V1b receptors and exhibited much lower affinity for rat and human V1a, V2, and oxytocin receptors. Moreover, this compound did not interact with a large number of other receptors, enzymes, or ion channels. In vitro, SSR149415 behaved as a full antagonist and potently inhibited arginine vasopressin (AVP)-induced Ca2+ increase in Chinese hamster ovary cells expressing rat or human V1b receptors. The in vivo activity of SSR149415 has been studied in several models of elevated corticotropin secretion in conscious rats. SSR149415 inhibited exogenous AVP-induced increase in plasma corticotropin, from 3 mg/kg i.p. and 10 mg/kg p.o. upwards. Similarly, this compound antagonized AVP-potentiated corticotropin release provoked by exogenous corticoliberin at 3 mg/kg p.o. The effect lasted for more than 4 h at 10 mg/kg p.o. showing a long-lasting oral effect. SSR149415 (10 mg/kg p.o.) also blocked corticotropin secretion induced by endogenous AVP increase subsequent to body water loss. Moreover, 10 mg/kg i.p SSR149415 inhibited plasma corticotropin elevation after restraint-stress in rats by 50%. In the four-plate test, a mouse model of anxiety, SSR149415 (3 mg/kg p.o. upwards) displayed anxiolytic-like activity after acute and 7-day repeated administrations. Thus, SSR149415 is a potent, selective, and orally active V1b receptor antagonist. It represents a unique tool for exploring the functional role of V1b receptors and deserves to be clinically investigated in the field of stress and anxiety.

SSR126768A (4-chloro-3-[(3R)-(+)-5-chloro-1-(2,4-dimethoxybenzyl)-3-methyl-2-oxo-2,3-dihydro-1H-indol-3-yl]-N-ethyl-N-(3-pyridylmethyl)-benzamide, hydrochloride): a new selective and orally active oxytocin receptor antagonist for the prevention of preterm labor.

4-chloro-3-[(3R)-(+)-5-chloro-1-(2,4-dimethoxybenzyl)-3-methyl-2-oxo-2,3-dihydro-1H-indol-3-yl]-N-ethyl-N-(3-pyridylmethyl)benzamide, hydrochloride (SSR126768A), a new potent and selective, orally active oxytocin (OT) receptor antagonist was characterized in several biochemical and pharmacological models. In binding studies, SSR126768A showed nanomolar affinity for rat and human recombinant and native OT receptors (K(i) = 0.44 nM) and exhibited much lower affinity for V(1a), V(1b), and V(2) receptors. In addition, it did not interact with a large number of other receptors, enzymes, and ion channels (1 microM). In autoradiographic experiments performed on at-term human pregnant uterus sections, SSR126768A dose dependently displaced [I(125)]d(CH(2))(5)[Tyr(Me)(2), Thr(4), Orn(8) (125)I-Tyr-NH(2)(9)]VT in situ labeling to OT receptors highly expressed in these tissues. In functional studies, SSR126768A behaved as a full antagonist and potently antagonized OT-induced intracellular Ca(2+) increase (K(i) = 0.50 nM) and prostaglandin release (K(i) = 0.45 nM) in human uterine smooth muscle cells. In rat isolated myometrium, OT-induced uterine contractions were competitively antagonized by SSR126768A (pA(2) = 8.47). Similarly, in human pregnant myometrial strips, SSR126768A inhibited the contractile uterine response to OT. In conscious telemetrated rats, oral administration of SSR126768A (1-10 mg/kg) produced a competitive inhibition of the dose response to OT on uterine contractions up to 24 h at 3 mg/kg p.o.; no tachyphylaxis was observed after 4-day repeated treatment. Finally, SSR126768A (30 mg/kg p.o.) significantly delayed parturition in pregnant rats in labor similar to ritodrine (10 mg/kg p.o.). Thus, SSR126768A is a potent, highly selective, orally active OT receptor antagonist with a long duration of action. This molecule could find therapeutic application as a tocolytic agent for acute and chronic oral management of preterm labor.

4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-thiazol-2-amine hydrochloride (SSR125543A): a potent and selective corticotrophin-releasing factor(1) receptor antagonist. I. Biochemical and pharmacological characterization.

4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1- (3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-thiazol-2-amine hydrochloride (SSR125543A), a new 2-aminothiazole derivative, shows nanomolar affinity for human cloned or native corticotrophin-releasing factor (CRF)(1) receptors (pK(i) values of 8.73 and 9.08, respectively), and a 1000-fold selectivity for CRF(1) versus CRF(2 alpha) receptor and CRF binding protein. SSR125543A antagonizes CRF-induced stimulation of cAMP synthesis in human retinoblastoma Y 79 cells (IC(50) = 3.0 +/- 0.4 nM) and adrenocorticotropin hormone (ACTH) secretion in mouse pituitary tumor AtT-20 cells. SSR125543A is devoid of agonist activity in these models. Its brain penetration was demonstrated in rats by using an ex vivo [(125)I-Tyr(0)] ovine CRF binding assay. SSR125543A displaced radioligand binding to the CRF(1) receptor in the brain with an ID(50) of 6.5 mg/kg p.o. (duration of action >24 h). SSR125543A also inhibited the increase in plasma ACTH levels elicited in rats by i.v. CRF (4 microg/kg) injection (ID(50) = 1, 5, or 5 mg/kg i.v., i.p., and p.o., respectively); this effect lasted for more than 6 h when the drug was given orally at a dose of 30 mg/kg. SSR125543A (10 mg/kg p.o.) reduced by 73% the increase in plasma ACTH levels elicited by a 15-min restraint stress in rats. Moreover, SSR125543A (20 mg/kg i.p.) also antagonized the increase of hippocampal acetylcholine release induced by i.c.v. injection of 1 microg of CRF in rats. Finally, SSR125543A reduced forepaw treading induced by i.c.v. injection of 1 microg of CRF in gerbils (ID(50) = approximately 10 mg/kg p.o.). Altogether, these data indicate that SSR125543A is a potent, selective, and orally active CRF(1) receptor antagonist.