RECOMBINANT HUMAN SECRETORY PHOSPHOLIPASE A(2) RELEASED THROMBOXANE FROM GUINEA-PIG BRONCHOALVEOLAR LAVAGE CELLS - IN-VITRO AND EX-VIVO EVALUATION OF A NOVEL SECRETORY PHOSPHOLIPASE A(2) INHIBITOR

The primary objective of this study was to develop a functional assay that could provide rapid and reliable information on some pharmacologi c characteristics of a novel inhibitor of human secretory phospholipas e A(2) (sPLA(2)). Guinea pig bronchoalveolar ravage (BAL) fluid, conta ining predominantly macrophages, eosinophils and epithelial cells, rel eased thromboxane A(2), as measured by thromboxane B-2, in a concentra tion-dependent manner on exposure to recombinant human sPLA(2) (rh-sPL A(2)). Similarly, n-formyl-L-methionyl-L-leucyl-L-phenylalanine (n-F-M et-Leu-Phe) or arachidonic acid also released this lipid mediator. Ind omethacin, a cyclooxygenase inhibitor, blocked synthesis of thromboxan e in response to these agents. p-Bromophenacylbromide-inactivated rh-s PLA(2) was substantially less effective than the untreated enzyme in c ausing release of thromboxane. LY311727 is a potent indole-derived inh ibitor of the isolated enzyme (IC50 = 23 nM). Incubation of this agent with BAL cells, just before addition of rh-sPLA(2), reduced release o f thromboxane with an IC50 = 1.8 x 10(-6) M. Specificity for sPLA(2) w as demonstrated in that LY311727, unlike indomethacin, did not reduce synthesis and subsequent release of thromboxane A(2) in response to ar achidonic acid. Using this technique as a basis, we determined whether LY311727 could sufficiently accumulate in lung after i.v. administrat ion to inhibit rh-sPLA(2)-induced thromboxane A(2) release from BAL ce lls. The compound, given i.v. to guinea pigs 5 min before collecting B AL fluid, produced a dose-dependent inhibition of rh-sPLA(2) with an E D(50) = 50 mg/kg. Thus, new in vitro and ex vivo assays were developed that permit functional evaluation of novel sPLA(2) inhibitors. These techniques should serve as secondary assays for evaluation of human sP LA(2) inhibitory activity from a chemical series and in addition provi de initial data related to metabolic stability and distribution to the lung.