SB-203347, AN INHIBITOR OF 14-KDA PHOSPHOLIPASE A(2), ALTERS HUMAN NEUTROPHIL ARACHIDONIC-ACID RELEASE AND METABOLISM AND PROLONGS SURVIVALIN MURINE ENDOTOXIN-SHOCK

Phospholipase A(2) (PLA(2)) catalyzes the hydrolysis of the sn-2 fatty acyl group [predominately arachidonic acid (AA)] of membrane phosphol ipids, the products of which are further metabolized, forming a variet y of eicosanoids and/or platelet-activating factor. PLA(2) activity is significantly enhanced during inflammation and therefore offers an in triguing target in designing anti-inflammatory drugs. SB 203347 (2-[2- [3,5-bis (trifluoromethyl) sulfonamido]-4-trifluoromethylphenoxy] benz oic acid) potently inhibits rh type II 14 kDa PLA(2) (IC50 = 0.5 mu M) but exhibits a 40-fold weaker inhibition of 85 kDa PLA(2) (IC50 = 20 mu M) using [H-3]-AA E. coli as substrate. A specific interaction with rh type II 14 kDa PLA(2) was confirmed both by observing the pH depen dence of its IC50 and by demonstrating linear inhibition in a ''scooti ng'' kinetic model using radiolabeled phospholipid reporter substrate in a 1,2-dimyristoyl phosphatidylmethanol vesicle. Before evaluating t he effect of SB 203347 on AA metabolism in intact human neutrophil, we showed that it fully inhibits PLA(2) activity in acid extracted intac t human neutrophil homogenate (IC50 = 4.7 mu M). SB 203347 inhibited A 23187-induced intact human neutrophil AA mass release in a concentrati on-dependent manner (IC50 = 1 mu M), which coincided with reductions i n the biosynthesis of platelet-activating factor (IC50 = 1.5 mu M) and leukotriene B-4 (IC50 = 2.3 mu M). Finally, SB 203347 prolonged survi val in a mouse model of endotoxin shock delivered i.p. Taken together, the data support a role of cellular 14 kDa PLA, in the formation of A A-derived proinflammatory lipid mediator. Further, SB 203347 proved ef ficacious in prolonging the survival of mice injected with endotoxin, which indicates the participation of 14 kDa PLA(2) in an in vivo model where lipid mediators have been implicated.