Vol. 298, Issue 1, 376-385, July 2001

BMS-229724 Is a Tight-Binding Inhibitor of Cytosolic Phospholipase A₂ That Acts at the Lipid/Water Interface and Possesses Anti-Inflammatory Activity in Skin Inflammation Models

James R. Burke, Lynda B. Davern, Paul L. Stanley, Kurt R. Gregor, Jacques Banville, Roger Remillard, John W. Russell, Patrick J. Brassil, Mark R. Witmer, Graham Johnson, Jeffrey A. Tredup and Kenneth M. Tramposch

Dermatology Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Buffalo, New York (L.B.D., P.L.S., K.M.T.); and Candiac, Quebec, Canada (J.B., R.R.), Wallingford, Connecticut (P.J.B., G.J., J.W.R.), Princeton, New Jersey (J.R.B., K.R.G., M.R.W., J.A.T.)

Cytosolic phospholipase A₂ (cPLA₂) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA₂ (IC₅₀ = 2.8 µM) in that it did not inhibit secreted phospholipase A₂ in vitro, nor phospholipase C and phospholipase D in cells. The compound was active in inhibiting arachidonate and eicosanoid production in U937 cells, neutrophils, platelets, monocytes, and mast cells. With a synthetic covesicle substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (K_I*^app) was determined to be 1 · 10⁵ mol% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.35 mol%. The unit of concentration in the interface is mole fraction (or mol%), which is related to the surface concentration of substrate, rather than bulk concentration that has units of molarity. Thus, BMS-229724 represents a novel inhibitor of cPLA₂, which partitions into the phospholipid bilayer and competes with phospholipid substrate for the active site. This potent inhibition of the enzyme translated into anti-inflammatory activity when applied topically (5%, w/v) to a phorbol ester-induced chronic inflammation model in mouse ears, inhibiting edema and neutrophil infiltration, as well as prostaglandin and leukotriene levels in the skin. In hairless guinea pigs, BMS-229724 was active orally (10 mg/kg) in a UVB-induced skin erythema model in hairless guinea pigs.