Kallikrein Inhibitors as Antiinflammatory Therapeutics.

Abstract 3179

Poster Board III-118

Plasma kallikrein is a novel target for antiinflammatory inhibitor design since it plays a role in the initiation of the intrinsic clotting, fibrinolysis, bradykinin-forming and neutrophil activation. The goal of this study was to synthesize a potent and selective inhibitor of kallikrein for the prevention and/or treatment of hyperfibrinolytic states, systemic inflammation, sepsis as well as cardiopulmonary bypass. Using a focus library of compounds, UK-097820, CP-013118 and PF-04886847 were found not to be cytotoxic and had the ability to abolish the hydrolysis of S2302 by kallikrein both in fluid phase and on the surface-mediated activation. In a competition study, PF-04886847 (3 mM) reduced the formation of a complex between C1-inhibitor and kallikrein by 40% following the activation of prekallikrein (kallikrein zymogen) on bound high molecular weight kininogen (HK) to human pulmonary artery endothelial cell (HPAEC). It was also mechanistically important to distinguish between direct kallikrein inhibition caused by the binding of kallikrein inhibitor to kallikrein versus the inhibitor's indirect effect on HK, prekallikrein or perturbation of the membrane function. The binding of PK to HK or HK to HPAEC surface was unaffected by the kallikrein inhibitors. To determine the selectivities of the kallikrein inhibitors, the effects of UK-097820, CP-013118 and PF-04886847-00 on factor XI and factor XII were examined. PF-04886847 was found to be a potent and selective inhibitor of kallikrein at a concentration ranging from 1 to 3 mM. BK stimulates cultured endothelial cells to release various mediators. The effects of the kallikrein inhibitors on the generation of bradykinin (BK), nitric oxide (NO) and prostacyclin and intracellular calcium signaling were determined. The generation of BK, NO formation and 6-keto-PGF_1a release were blocked by PF-04886847, indicating that PF-04886847 might have potential applications for controlling unwanted inflammation provoked by BK-induced endothelium activation. In situ study, PF-04886847 blocked PK activation on intact aorta precontracted by phenylephrine. Taken together, we propose that PF-04886847 treatment might be beneficial in tissue injury and have potential clinical application.