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Published on: June 29, 2015
Factor XIIa as a Novel Target for Thrombosis: Target Engagement Requirement and Efficacy in a Rabbit Model of
Christopher M Barbieri1, Xinkang Wang1, Weizhen Wu1
1In Vitro Pharmacology (C.M.B., A.M.O., K.O., D.C.) and Cardiometabolic Diseases (X.W., W.W., X.Z., G.C., D.A.S., D.E.G., Z.C.), Merck & Co., Inc., Kenilworth, New Jersey.
Abstract:
Coagulation Factor XII (FXII) plays a critical role in thrombosis. What is unclear is the level of enzyme occupancy of FXIIa that is needed for efficacy and the impact of FXIIa inhibition on cerebral embolism. A selective activated FXII (FXIIa) inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf), was generated to address these questions. rHA-Mut-inf displayed potency comparable to the original wild-type HA-Infestin-4 (human FXIIa inhibition constant = 0.07 and 0.12 nM, respectively), with markedly improved selectivity against Factor Xa (FXa) and plasmin. rHA-Mut-inf binds FXIIa, but not FXII zymogen, and competitively inhibits FXIIa protease activity. Its mode of action is hence akin to typical small-molecule inhibitors. Plasma shift and aPTT studies with rHA-Mut-inf demonstrated that calculated enzyme occupancy for FXIIa in achieving a putative aPTT doubling target in human, nonhuman primate, and rabbit is more than 99.0%. The effects of rHA-Mut-inf in carotid arterial thrombosis and microembolic signal (MES) in middle cerebral artery were assessed simultaneously in rabbits. Dose-dependent inhibition was observed for both arterial thrombosis and MES. The ED50 of thrombus formation was 0.17 mg/kg i.v. rHA-Mut-inf for the integrated blood flow and 0.16 mg/kg for thrombus weight; the ED50 for MES was 0.06 mg/kg. Ex vivo aPTT tracked with efficacy. In summary, our findings demonstrated that very high enzyme occupancy will be required for FXIIa active site inhibitors, highlighting the high potency and exquisite selectivity necessary for achieving efficacy in humans. Our MES studies suggest that targeting FXIIa may offer a promising strategy for stroke prevention associated with thromboembolic events.
Insights
Achieving high enzyme occupancy (>99%) of activated Factor XII (FXIIa) is crucial for effective inhibition. Targeting FXIIa shows promise for preventing stroke and thromboembolic events.
Area of Science:
- Biochemistry
- Pharmacology
- Thrombosis Research
Background:
- Coagulation Factor XII (FXII) is implicated in thrombosis.
- The required enzyme occupancy of FXIIa for efficacy and its effect on cerebral embolism remain unclear.
Purpose of the Study:
- To investigate the enzyme occupancy needed for FXIIa inhibition efficacy.
- To assess the impact of FXIIa inhibition on cerebral embolism.
Main Methods:
- Developed a selective FXIIa inhibitor, recombinant human albumin-tagged mutant Infestin-4 (rHA-Mut-inf).
- Assessed inhibitor potency, selectivity, binding kinetics, and mechanism of action.
- Evaluated enzyme occupancy in plasma and aPTT studies across species.
- Investigated effects on carotid arterial thrombosis and microembolic signals (MES) in rabbits.
Main Results:
- rHA-Mut-inf demonstrated high potency and selectivity for FXIIa.
- Over 99.0% FXIIa enzyme occupancy was required to double aPTT in humans, nonhuman primates, and rabbits.
- rHA-Mut-inf dose-dependently inhibited arterial thrombosis and MES in rabbits.
- Ex vivo aPTT correlated with observed efficacy.
Conclusions:
- Very high enzyme occupancy is necessary for FXIIa active site inhibitors.
- Potent and selective FXIIa inhibitors are required for human efficacy.
- Targeting FXIIa is a potential strategy for stroke prevention in thromboembolic events.
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