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

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...

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