Stimulation of the beta3-Adrenoceptor as a novel treatment strategy for anxiety and depressive disorders.

The characterization of the first selective orally active and brain-penetrant beta3-adrenoceptor agonist, SR58611A (amibegron), has opened new possibilities for exploring the involvement of this receptor in stress-related disorders. By using a battery of tests measuring a wide range of anxiety-related behaviors in rodents, including the mouse defense test battery, the elevated plus-maze, social interaction, stress-induced hyperthermia, four-plate, and punished drinking tests, we demonstrated for the first time that the stimulation of the beta3 receptor by SR58611A resulted in robust anxiolytic-like effects, with minimal active doses ranging from 0.3 to 10 mg/kg p.o., depending on the procedure. These effects paralleled those obtained with the prototypical benzodiazepine anxiolytic diazepam or chlordiazepoxide. Moreover, when SR58611A was tested in acute or chronic models of depression in rodents, such as the forced-swimming and the chronic mild stress tests, it produced antidepressant-like effects, which were comparable in terms of the magnitude of the effects to those of the antidepressant fluoxetine or imipramine. Supporting these behavioral data, SR58611A modified spontaneous sleep parameters in a manner comparable to that observed with fluoxetine. Importantly, SR58611A was devoid of side effects related to cognition (as shown in the Morris water maze and object recognition tasks), motor activity (in the rotarod), alcohol interaction, or physical dependence. Antagonism studies using pharmacological tools targeting a variety of neurotransmitters involved in anxiety and depression and the use of mice lacking the beta3 adrenoceptor suggested that these effects of SR58611A are mediated by beta3 adrenoceptors. Taken as a whole, these findings indicate that the pharmacological stimulation of beta3 adrenoceptors may represent an innovative approach for the treatment of anxiety and depressive disorders.

CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus.

In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30-300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction-liquid chromatography-tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K(+), with or without addition of Ca(2+), or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.

Single and multiple doses of rimonabant antagonize acute effects of smoked cannabis in male cannabis users.

RATIONALE: A single 90-mg dose of the cannabinoid CB1 receptor antagonist rimonabant attenuates effects of smoked cannabis in humans. OBJECTIVES: The objective of this study is to evaluate whether repeated daily 40-mg doses of rimonabant can attenuate effects of smoked cannabis to the same extent as a single higher (90 mg) dose. MATERIALS AND METHODS: Forty-two male volunteers received one of three oral drug regimens in a randomized, double blind, parallel group design: (1) 40 mg rimonabant daily for 15 days, (2) placebo for 14 days, then 90 mg rimonabant on day 15, or (3) placebo for 15 days. All participants smoked an active or placebo cannabis cigarette 2 h after medication on days 8 and 15. Subjective effects were measured with visual analog scales and the marijuana-scale of the Addiction Research Center Inventory. RESULTS: Cannabis-induced tachycardia was significantly lower for the 40-mg group on day 8 and for the 40 and 90 mg rimonabant groups on day 15 as compared to placebo. The 40-mg dose significantly decreased peak subjective effects on day 8. Neither the 90-mg nor 40-mg doses significantly decreased peak subjective effects on day 15. Rimonabant treatment did not significantly affect Delta(9)-tetrahydrocannabinnol pharmacokinetics. CONCLUSIONS: Repeated lower daily rimonabant doses (40 mg) attenuated the acute physiological effects of smoked cannabis to a similar degree as a single 90-mg dose; repeated 40-mg doses attenuated subjective effects after 8 but not 15 days.

[New methods in pharmaceutical research: combinatorial chemistry and high throughput screening]. / Les nouvelles méthodes en recherche pharmaceutique: chimie combinatoire et criblage à haut débit.

New lead-identification methodologies such as high-throughput screening and combinatorial chemistry have been integrated into pharmaceutical research over the past 5-10 years. More rational use in the selection of potential preclinical candidates for some difficult targets has increased the chances of success.

[Pharmacology of cannabinoid receptors]. / Pharmacologie des récepteurs cannabinoïdes.

The endocannabinoid (EC) system is a physiological system with an important regulatory role in numerous biological functions, both centrally and peripherally. In certain conditions it can become hyperactive and induce a variety of disorders. The system has two receptor types, designated CB1 and CB2 (present respectively in the CNS and the periphery), as well as endogenous ligands (AEA and 2-AG) and equipment for transporting, synthesizing and degrading them. The discovery of specific CB1 antagonists has opened up interesting new possibilities for the treatment of obesity, diabetes and cardiometabolic risk factors.

The CB1 receptor antagonist rimonabant reverses the diet-induced obesity phenotype through the regulation of lipolysis and energy balance.

We investigated the molecular events involved in the long-lasting reduction of adipose mass by the selective CB1 antagonist, SR141716. Its effects were assessed at the transcriptional level both in white (WAT) and brown (BAT) adipose tissues in a diet-induced obesity model in mice. Our data clearly indicated that SR141716 reversed the phenotype of obese adipocytes at both macroscopic and genomic levels. First, oral treatment with SR141716 at 10 mg/kg/d for 40 days induced a robust reduction of obesity, as shown by the 50% decrease in adipose mass together with a major restoration of white adipocyte morphology similar to lean animals. Second, we found that the major alterations in gene expression levels induced by obesity in WAT and BAT were mostly reversed in SR141716-treated obese mice. Importantly, the transcriptional patterns of treated obese mice were similar to those obtained in the CB1 receptor knockout mice fed a high-fat regimen and which are resistant to obesity, supporting a CB1 receptor-mediated process. Functional analysis of these modulations indicated that the reduction of adipose mass by the molecule resulted from an enhanced lipolysis through the induction of enzymes of the beta-oxidation and TCA cycle, increased energy expenditure, mainly through futile cycling (calcium and substrate), and a tight regulation of glucose homeostasis. These changes accompanied a significant cellular remodeling and contributed to a reduction of the obesity-related inflammatory status. In addition to a transient reduction of food consumption, increases of both fatty acid oxidation and energy expenditure induced by the molecule summate leading to a sustained weight loss. Altogether, these data strongly indicate that the endocannabinoid system has a major role in the regulation of energy metabolism.

Anandamide induced PPARgamma transcriptional activation and 3T3-L1 preadipocyte differentiation.

We investigated the effects of anandamide on peroxisome proliferator-activated receptor gamma (PPARgamma) activity. In two different transactivation systems using either full-length or only the ligand binding domain of PPARgamma, we showed that anandamide, but not palmitoylethanolamide induced transcriptional activation of PPARgamma in a dose dependent manner with an EC50 of 8 microM. In addition, competition binding experiments showed that anandamide but not palmitoylethanolamide binds directly to PPAR-ligand binding domain. We also found that anandamide treatment induced 3T3-L1 fibroblast differentiation into adipocytes. Indeed, anandamide induced triglyceride droplet accumulation and the expression of PPARgamma responsive genes such as CCAAT enhancer binding protein alpha (C-EBPalpha), aP2, PerilipinA and Acrp30. Furthermore, the PPARgamma antagonist (GW9662) inhibited the anandamide-induced 3T3-L1 differentiation confirming that this is a PPARgamma-mediated process. Altogether, these data indicate that anandamide binds PPARgamma and induces cellular PPARgamma signaling.

Targeted inactivation of the neurotensin type 1 receptor reveals its role in body temperature control and feeding behavior but not in analgesia.

Three subtypes of neurotensin receptor have been described, two members of the heptahelical transmembrane domain G protein-coupled receptor superfamily NT-1R and NT-2R, and NT-3R unrelated to this family. We have generated NT-1R deficient (NT-1R(-/-)) mice. NT-1R(-/-) mice were born at the expected Mendelian frequency without obvious abnormalities and they were fertile. The NT-induced analgesia on the writhing induced by phenyl-p-benzoquinone administration remained at wild-type levels in the NT-1R(-/-) mice demonstrating that the NT-1R is not implicated in the analgesic effect of NT in this test. The NT-1R(-/-) mice were hyperthermic; their body temperature was not affected by intracerebroventricular (i.c.v.) administration of NT, contrasting with the hypothermia induced in NT-1R(+/+) mice. NT-1R(-/-) mice showed a small significant increase in body weight compared to the NT-1R(+/+) congeners as early as 10 weeks after birth, correlated with a higher food intake. NT-1R(-/-) mice showed similar spontaneous locomotion to the control littermates, but did not respond to i.c.v. NT-induced hypolocomotion. I.c.v. injection of NT inhibited feeding in fasted wild-type mice, but had no effect on feeding of the NT-1R(-/-) mice. I.c.v. administration of the orexigenic neuropeptide Y (NPY) stimulated feeding to the same extent in both wild-type and NT-1R(-/-) mice. This analysis of NT-1R-deficient mice shows that the NT-1R does not play a role in NT-induced analgesia, but that it is clearly implicated in thermal and feeding regulation, weight control, and NT-induced hypolocomotion.

Modulation of gastric emptying and gastrointestinal transit in rats through intestinal cannabinoid CB(1) receptors.

We studied the delay in gastric emptying and gastrointestinal transit induced by the cannabinoid receptor agonists (+)-WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) and CP 55,940 ((-)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), as prevented by the selective cannabinoid CB(1)-receptor antagonist SR141716 ((N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide)) in rats after systemic or central drug administration. Oral SR141716 showed comparable potency (ID(50) range 1.0-3.9 mg/kg) in antagonizing gastric emptying and gastrointestinal transit delay by (+)-WIN 55,212-2 or CP 55,940. Gastric emptying and gastrointestinal transit delay after intracerebroventricular (i.c.v.) (+)-WIN 55,212-2 was prevented by oral or i.c.v. SR141716, but i.c.v. SR141716 did not significantly reduce the effect of i.p. (+)-WIN 55,212-2. Pertussis toxin prevented the delaying action of i.c.v. (+)-WIN 55,212-2 on both gastric emptying and gastrointestinal transit, but had no effect on (+)-WIN 55,212-2 i.p. These findings are consistent with a primary role of peripheral cannabinoid CB(1) receptor mechanisms in gastrointestinal transit delay by specific agonists.

Research strategies in the pharmaceutical industry to cope with the problems of ageing.

The present research strategies for ageing are focussed around three areas. The first area is the demographic study of the present-day populations and those in the future. The analysis of the evolution of the metropolitan French population from 1950 to 2050 indicates that the proportion of subjects aged greater than 60 years will pass from 16% to 35%. In addition, the level of dependence of these subjects will vary considerably from one to another. The second area is the integration of epidemiological data of the pathologies related to the aged subject. Numerous epidemiological studies of the aged show that the elderly population is in better health, better informed about the diseases that affect them and also financially more secure, which allows them to take better care of themselves. The epidemiological studies of certain dementias show, for example, that if we reduce the delay to when the disease appears by five years, then we reduce the prevalence by a factor of 2. The third area relates to the important new progress in the understanding of the biological mechanisms involved in cellular ageing. The recent progress in the area of cellular biology has started to decode the complex mechanisms underlying cellular ageing. The important understanding of the phenomena of apoptose with the development of relevant biological models of the natural ageing process have anticipated future treatments and, eventually, the prophylactic treatment of specific pathologies of the elderly patient.

[Design and birth of drugs]. / Conceptions et naissances des médicaments.

Today the design of new clinical entities uses highly sophisticated techniques. This design is a step by step procedure: first selection and validation of the therapeutic targets, where the contribution of genomics is very important; then, setting up the different screening tests. This step permits to detect the potentially active molecules, which will be optimised, before being tested in healthy volunteers and then in patients.

Rimonabant reduces obesity-associated hepatic steatosis and features of metabolic syndrome in obese Zucker fa/fa rats.

This study investigated the effects of rimonabant (SR141716), an antagonist of the cannabinoid receptor type 1 (CB1), on obesity-associated hepatic steatosis and related features of metabolic syndrome: inflammation (elevated plasma levels of tumor necrosis factor alpha [TNFalpha]), dyslipidemia, and reduced plasma levels of adiponectin. We report that oral treatment of obese (fa/fa) rats with rimonabant (30 mg/kg) daily for 8 weeks abolished hepatic steatosis. This treatment reduced hepatomegaly, reduced elevation of plasma levels of enzyme markers of hepatic damage (alanine aminotransferase, gamma glutamyltransferase, and alkaline phosphatase) and decreased the high level of local hepatic TNFalpha currently associated with steatohepatitis. In parallel, treatment of obese (fa/fa) rats with rimonabant reduced the high plasma level of the proinflammatory cytokine TNFalpha and increased the reduced plasma level of the anti-inflammatory hormone adiponectin. Finally, rimonabant treatment also improved dyslipidemia by both decreasing plasma levels of triglycerides, free fatty acids, and total cholesterol and increasing the HDLc/LDLc ratio. All the effects of rimonabant found in this study were not or only slightly observed in pair-fed obese animals, highlighting the additional beneficial effects of treatment with rimonabant compared to diet. These results demonstrate that rimonabant plays a hepatoprotective role and suggest that this CB1 receptor antagonist potentially has clinical applications in the treatment of obesity-associated liver diseases and related features of metabolic syndrome.

The cannabinoid CB1 receptor antagonist rimonabant (SR141716) inhibits cell proliferation and increases markers of adipocyte maturation in cultured mouse 3T3 F442A preadipocytes.

Adipocyte cell proliferation is an important process in body fat mass development in obesity. Adiponectin or Acrp30 is an adipocytokine exclusively expressed and secreted by adipose tissue that regulates lipid and glucose metabolism and plays a key role in body weight regulation and homeostasis. Adiponectin mRNA expression in adipose tissue and plasma level of adiponectin are decreased in obesity and type 2 diabetes. In obese rodents, the selective CB(1) receptor antagonist rimonabant reduces food intake and body weight and improves lipid and glucose parameters. We have reported previously that rimonabant stimulated adiponectin mRNA expression in adipose tissue of obese fa/fa rats, by a direct effect on adipocytes. We report here that rimonabant (10-400 nM) inhibits cell proliferation of cultured mouse 3T3 F442A preadipocytes in a concentration-dependent manner. In parallel to this inhibitory effect on preadipocyte cell proliferation, rimonabant (25-100 nM) stimulates mRNA expression and protein levels of two late markers of adipocyte differentiation (adiponectin and glyceraldehyde-3-phosphate dehydrogenase) with a maximal effect at 100 nM, without inducing the accumulation of lipid droplets. Furthermore, treatment of mouse 3T3 F442A preadipocytes with rimonabant (100 nM) inhibits basal and serum-induced p42/44 mitogen-activated protein (MAP) kinase activity. These results suggest that inhibition of MAP kinase activity by rimonabant may be one of mechanisms involved in the inhibition of 3T3 F442A preadipocyte cell proliferation and stimulation of adiponectin and GAPDH expression. The inhibition of preadipocyte cell proliferation and the induction of adipocyte late "maturation" may participate in rimonabant-induced antiobesity effects, particularly the reduction of body fat mass.

The effect of relcovaptan (SR 49059), an orally active vasopressin V1a receptor antagonist, on uterine contractions in preterm labor.

Relcovaptan (SR 49059) is a non-peptide, orally active vasopressin V1a receptor inhibitor. The effect on uterine contractions in 18 women with preterm labor in pregnancy weeks 32-36 was assessed in a double-blind investigation. The inclusion criterion was at least four regular uterine contractions over 30 min as measured by external tocodynamometry. Twelve patients received at random a single oral dose of 400 mg relcovaptan and six received placebo, and contractions were monitored up to 6 h thereafter. Rescue medication (beta-adrenoceptor-stimulating drug) was allowed after 2 h. Before drug administration a mean (+/- SE) of 8.2 +/- 1.4 and 9.7 +/- 1.6 contractions/30 min were recorded in the relcovaptan- and placebo-treated groups, respectively. In the former group, the frequency of uterine contractions started to decrease within the first half hour, and 1.5-2 h after dosing it was steady at 3.2 +/- 0.9 contractions/30 min. Correspondingly, after placebo, 7.8 +/- 2.2 contractions/30 min were recorded, a statistically significant difference (p = 0.017). The activity in the relcovaptan-treated women remained low, whereas in the placebo group inhibited uterine contractions were observed only in women receiving 'rescue' tocolytic treatment. It is concluded that relcovaptan inhibits preterm labor.

Hypersensitization of the Orexin 1 receptor by the CB1 receptor: evidence for cross-talk blocked by the specific CB1 antagonist, SR141716.

In the present study, we observed evidence of cross-talk between the cannabinoid receptor CB1 and the orexin 1 receptor (OX1R) using a heterologous system. When the two receptors are co-expressed, we observed a major CB1-dependent enhancement of the orexin A potency to activate the mitogen-activated protein kinase pathway; dose-responses curves indicated a 100-fold increase in the potency of orexin-mediated mitogen-activated protein kinase activation. This effect required a functional CB1 receptor as evidenced by the blockade of the orexin response by the specific CB1 antagonist, N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716), but also by pertussis toxin, suggesting that this potentiation is Gi-mediated. In contrast to OX1R, the potency of direct activation of CB1 was not affected by co-expression with OX1R. In addition, electron microscopy experiments revealed that CB1 and OX1R are closely apposed at the plasma membrane level; they are close enough to form hetero-oligomers. Altogether, for the first time our data provide evidence that CB1 is able to potentiate an orexigenic receptor. Considering the antiobesity effect of SR141716, these results open new avenues to understand the mechanism by which the molecule may prevent weight gain through functional interaction between CB1 and other receptors involved in the control of appetite.

Permissive role of neurokinin NK(3) receptors in NK(1) receptor-mediated activation of the locus coeruleus revealed by SR 142801.

The present experiments investigated the role of neurokinin-1 (NK(1)) and neurokinin-3 (NK(3)) receptors on the activity of the locus coeruleus (LC)-noradrenergic system by using a dual probe microdialysis technique in anesthetized guinea pigs. The local application in the LC of the selective NK(1) receptor agonists [SAR(9),Met(O(2))(11)]-SP (10 microM) and septide (1 microM) as well as the selective NK(3) receptor agonist senktide (1 microM), enhanced the extracellular norepinephrine (NE) levels in the prefrontal cortex. The enhancing effect of [SAR(9),Met(O(2))(11)]-SP was completely blocked by the peripheral administration of the selective non peptide NK(1) and NK(3) receptor antagonists, GR 205171 (1 mg/kg, i.p.) and SR 142801 (0.1 mg/kg, i.p.), respectively, whereas SR 142806 (0.1 mg/kg, i.p.) the inactive enantiomer of SR 142801 had no effect. Moreover, the [SAR(9),Met(O(2))(11)]-SP-induced increase in LC DOPAC concentrations, is only antagonized by GR 205171. In contrast, only SR 142801 (0.3 mg/kg, i.p.) could block stereoselectively the senktide-evoked increase in NE levels. Both [SAR(9),Met(O(2))(11)]-SP and senktide effects were blocked by local infusion into the LC of SR 142801 (10(-9) M). These results demonstrate that stimulation of NK(1) and NK(3) receptors located in the LC area modulates the activity of the LC-NE system, and that the excitatory effects of NK(1) receptor agonists require NKB/NK(3) receptor activation in the LC.

Expression profile and up-regulation of PRAX-1 mRNA by antidepressant treatment in the rat brain.

A protein associated with the peripheral-type benzodiazepine receptor (PRAX-1) has recently been cloned, but its regional distribution in the central nervous system and its function remain to be clarified. In situ hybridization was carried out to localize PRAX-1 mRNA in the rat brain and revealed a high expression of the transcript in limbic structures such as the CA1 region of the hippocampus, as well as the dentate gyrus, septum, amygdala, and the islands of Calleja. A dense hybridization signal was also observed in the nucleus accumbens, caudate nucleus, olfactory tubercle, pineal gland, and cerebellar cortex. PRAX-1 mRNA expression was largely neuronal; it colocalized with neuron-specific enolase but not glial fibrillary acidic protein. Long-term treatments (21 days) with the neuroleptic haloperidol increased PRAX-1 mRNA expression only in the dentate gyrus, whereas anxiolytic/anticonvulsant diazepam had no effect in any of the hippocampal region studied. Repeated electroconvulsive shock administration significantly enhanced PRAX1 expression in the CA1 subfield and dentate gyrus. Several classes of antidepressant treatment, including serotonin selective reuptake inhibitor (fluoxetine), mixed serotonin- and norepinephrine-uptake inhibitor (imipramine), and monoamine oxidase inhibitors (iproniazid and tranylcypromine), shared this effect. Furthermore, the selective nonpeptide NK2 receptor antagonist (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR48968), which shows an antidepressant profile in animal studies, also enhanced PRAX-1 mRNA expression. These results point to a potential role of PRAX-1 function in the central nervous system and suggest that the up-regulation of PRAX-1 mRNA represents a common action of chronic antidepressant treatment.

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.).

SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride], a new nonpeptide antagonist of the bradykinin B1 receptor: biochemical and pharmacological characterization.

The biochemical and pharmacological properties of a novel non-peptide antagonist of the bradykinin (BK) B(1) receptor, SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride] were evaluated. SSR240612 inhibited the binding of [(3)H]Lys(0)-des-Arg(9)-BK to the B(1) receptor in human fibroblast MRC5 and to recombinant human B(1) receptor expressed in human embryonic kidney cells with inhibition constants (K(i)) of 0.48 and 0.73 nM, respectively. The compound selectivity for B(1) versus B(2) receptors was in the range of 500- to 1000-fold. SSR240612 inhibited Lys(0)-desAr(9)-BK (10 nM)-induced inositol monophosphate formation in human fibroblast MRC5, with an IC(50) of 1.9 nM. It also antagonized des-Arg(9)-BK-induced contractions of isolated rabbit aorta and mesenteric plexus of rat ileum with a pA(2) of 8.9 and 9.4, respectively. Antagonistic properties of SSR240612 were also demonstrated in vivo. SSR240612 inhibited des-Arg(9)-BK-induced paw edema in mice (3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p.). Moreover, SSR240612 reduced capsaicin-induced ear edema in mice (0.3, 3 and 30 mg/kg p.o.) and tissue destruction and neutrophil accumulation in the rat intestine following splanchnic artery occlusion/reperfusion (0.3 mg/kg i.v.). The compound also inhibited thermal hyperalgesia induced by UV irradiation (1 and 3 mg/kg p.o.) and the late phase of nociceptive response to formalin in rats (10 and 30 mg/kg p.o.). Finally, SSR240612 (20 and 30 mg/kg p.o.) prevented neuropathic thermal pain induced by sciatic nerve constriction in the rat. In conclusion, SSR240612 is a new, potent, and orally active specific non-peptide bradykinin B(1) receptor antagonist.

SR147778 [5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide], a new potent and selective antagonist of the CB1 cannabinoid receptor: biochemical and pharmacological characterization.

Based on binding, functional, and pharmacological data, this study introduces SR147778 [5-(4-bromophenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide] as a highly potent, selective, and orally active antagonist for the CB1 receptor. This compound displays nanomolar affinity (Ki = 0.56 and 3.5 nM) for both the rat brain and human CB1 recombinant receptors, respectively. It has low affinity (Ki = 400 nM) for both the rat spleen and human CB2 receptors. Furthermore, it shows no affinity for any of the over 100 targets investigated (IC50 > 1 microM). In vitro, SR147778 antagonizes the inhibitory effects of CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] on both the mouse vas deferens contractions (pA2 value = 8.1) and on forskolin-stimulated adenylyl cyclase activity in the U373 MG cell lines (pA2 value = 8.2) but not in Chinese hamster ovary (CHO) cells permanently expressing the human peripheral cannabinoid receptor (hCB2). SR147778 is able to block the mitogen-activated protein kinase activity induced by CP 55,940 in the CHO cell line expressing human brain cannabinoid receptor (IC50 = 9.6 nM) but was inactive in cells expressing hCB2. After oral administration, SR147778 displaced the ex vivo [3H]-CP 55,940 binding to mouse brain membranes (ED50 = 3.8 mg/kg) with a long duration of action, whereas it did not interact with the CB2 receptor expressed in the mouse spleen. Using different routes of administration, SR147778 (0.3-3 mg/kg) is shown to antagonize pharmacological effects (hypothermia, analgesia, and gastrointestinal transit) induced by R-(+)-(2,3-dihydro-5-methyl-3-[[4-morpholinyl]methyl] pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone in mice. Finally, per se, SR147778 (0.3-10 mg/kg) is able to reduce ethanol or sucrose consumption in mice and rats and food intake in fasted and nondeprived rats.