This project is aimed at developing novel synthetic compounds capable of stabilizing platelet membranes. One of the major thrusts is directed at aiding individuals especially predisposed to thromboembolic complicating resulting from their blood's exposure to biomaterials like implanted prosthetic devices and extracorporeal equipment. In this proposal, novel molecular entities will be obtained using a three-pronged approach: (i) because of the importance of enantioselectivity in drug action, each of the 3 most active compounds from their previous investigation will be resolved into its 3 stereoisomers. The individual enantiomers and diastereomers will be tested for their inhibitory effects on human blood platelet aggregation in vitro, induced by human alpha-thrombin, collagen, and epinephrine. (ii) Racemic analogues of the 3 compounds will be synthesized in order to extend their duration of action, and (iii) hydrophbiaity and electron density will be optimized in structural components not previously examined. Compounds showing reasonable potency in preliminary screening will be evaluated for their effects in vitro, (a) on cytosolic ionized calcium ([Ca. 2+]) concentrations, (b) on levels of platelet factor 4 (PF-4), platelet factor 3 (PF-3) and serotonin, and (c) for acute toxicity in vivo, in mice. In cooperation with Dr. Larry V. McIntire, at Rice University, the impact of selected compounds will be studied on the kinetics of human blood platelet adhesion and thrombus growth effected by collagen and biomaterials in his parallel plate flow chamber system. One or two highly active and least toxic compounds will be tested for platelet aggregation inhibitory activity, ex vivo, in dogs. The pharmacokinetics of the same compounds will be evaluated in mice and dogs, and bioavailibility in dogs. Structure of metabolites found in urine will be established using HPLC-MS. Due to the chiral environment of many biological systems, delineation of the enantio-selectivity of these synthetic compounds is expected to uncover increased antithrombotic potency which, at the same time, could register lesser toxicity. Identification of the structural features associated with metabolic inactivation would provide leads to the eventual design of highly active molecules with optimal duration of action. Relating structural features of the synthetic compounds to their influence on thrombocyte response should permit interpretation of human blood platelet response patterns in terms of chemical parameters, and to 'fine- tune' molecular segments toward optimal activity.
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