SummaryLike ticlopidine, the ADP receptor antagonist clopidogrel is inactive in vitro and must be administered i.v. or orally to exhibit antiaggregatory and antithrombotic activities. We have previously shown that hepatic metabolism is necessary for activity. This study demonstrates that an active metabolite can be generated from human liver microsomes incubated with clopidogrel. Using several analytical methodologies (LC/MS, NMR, chiral supercritical fluid chromatography), we have identified its structure. In vitro, this highly unstable compound, different from that formed from ticlopidine, exhibited all the biological activities of clopidogrel observed ex vivo: Irreversible inhibition of the binding of 33P-2MeS-ADP to washed human platelets (IC50 = 0.53 µM), selective inhibition of ADP-induced platelet aggregation (IC50 = 1.8 µM) and ADP-induced adenylyl cyclase down-regulation. The irreversible modification of the ADP-receptor site which is responsible for the biological activity could be explained by the formation of a disulfide bridge between the reactive thiol group of the active metabolite and a cysteine residue of the platelet ADP receptor. Abbreviations: ADP: adenosine 5’diphosphate; 2-MeS-ADP: 2-methylthioadenosine-5’-diphosphate; Bmax: maximum binding capacity; IC50: concentration which inhibits 50% of the activity; Kd: dissociation constant; LC/MS: Liquid chromatography coupled to mass spectrometry; NMR: Nuclear magnetic resonance

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxinsensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the G i protein. Furthermore, SR 141716A inhibited guanosine 5-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the G i protein and consequently abrogates most of the G i -mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the G i protein. This supports the notion that the G i protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.

ABSTRACT: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-oxoclopidogrel 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.

The limbic localization of the arginine vasopressin V1b 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 light͞dark tests) and atypical (fear͞anxiety 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 mg͞kg (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 anxiolyticlike activity in models involving traumatic stress exposure, such as the social defeat paradigm and the defense test battery (1-30 mg͞kg, p.o.). In the forced swimming test, SSR149415 (10 -30 mg͞kg, 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 mg͞kg, 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.A rginine vasopressin (AVP) is a cyclic nonapeptide that is synthesized centrally in the hypothalamus. Although it participates in the hypothalamic-pituitary-adrenal axis, regulating pituitary ACTH (corticotropin) secretion by potentiating the stimulatory effects of corticotropin releasing factor (CRF), extrahypothalamic AVP-containing neurons have been characterized in the rat, notably in the medial amygdala, that innervate limbic structures such as the lateral septum and the ventral hippocampus (1). In these latter structures, AVP was suggested to act as a neurotransmitter, exerting its action by binding to specific G protein-coupled receptors, i.e., V 1a and V 1b (2-4), which are widely distributed in the central nervous system, including the septum, cortex, and hippocampus (2, 5).Because the pioneering studies of David De Wied and colleagues (6, 7), it has been widely accepted that AVP is involved in various types of behavioral processes (8). Early work paid attention to the possible role of this peptide in learning and memory, in particular with regard to avoidance behavior (for review, see ref. 9), but also in antypiresis, scent marking, and social communication (for reviews, see refs. 10 and 11). For instance, several studies showed that centrally administered AVP in rats reverses drug-induced memory loss and affects long-term memory processes, improvi...

In this work, the 100-kDa neurotensin (NT) receptor previously purified from human brain by affinity chromatography (Zsü rger, N., Mazella, J., and Vincent, J. P. (1994) Brain Res. 639, 245-252) was cloned from a human brain cDNA library. This cDNA encodes a 833-amino acid protein 100% identical to the recently cloned gp95/ sortilin and was then designated NT3 receptor-gp95/sortilin. The N terminus of the purified protein is identical to the sequence of the purified gp95/sortilin located immediately after the furin cleavage site. The binding of iodinated NT to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized extracts of COS-7 cells transfected with the cloned cDNA was saturable and reversible with an affinity of 10 -15 nM. The localization of the NT3 receptor-gp95/sortilin into intracellular vesicles was in agreement with previous results obtained with the purified receptor and with gp95/sortilin. Affinity labeling and binding experiments showed that the 110-kDa NT3 receptor can be partly transformed into a higher affinity (K d ‫؍‬ 0.3 nM) 100-kDa protein receptor by cotransfection with furin. This 100-kDa NT receptor corresponded to the mature form of the receptor. The NT3/gp95/sortilin protein is the first transmembrane neuropeptide receptor that does not belong to the superfamily of G-protein-coupled receptors. The neuropeptide neurotensin (NT)1 exerts distinct central and peripheral effects in mammals (see Ref. 1 for review). Central administration of NT modulates dopaminergic transmission and triggers hypothermic and naloxone-insensitive analgesic responses, whereas in the periphery, NT induces hypotension, decreases gastric acid secretion, and activates lipid digestion (1). Both central and peripheral actions of NT are initiated by association of the peptide to specific receptors located on the plasma membrane of target cells. Two different NT receptors have been cloned in the last years and shown to belong to the family of G-protein-coupled receptors (2-5). The use of the recently developed nonpeptide NT antagonist SR48692 (6) allowed us to demonstrate that the NT-induced modulation of midbrain dopaminergic pathways could be attributed to the high affinity NT receptor (NTR1) that was cloned first (2). However, SR48692 was unable to block a series of NT effects including central analgesia and hypothermia (7), suggesting that these effects could be mediated either by the lately cloned low affinity NT2 receptor (4, 5) or by another form of NT receptor not yet cloned.We previously described the solubilization and purification of a 100-kDa NT receptor from mouse and human brain (8 -10). This 100-kDa receptor protein has been observed in primary cultures of neurons from embryonic mice together with the NT1 receptor and shown to be implicated in the internalization mechanism of NT (11). The 100-kDa protein is initially localized in an intracellular vesicular compartment and appears to the plasma membrane only after the NT-induced sequestration of the NT1 receptor, which is initially pres...