Barbieri Christopher M, Wang Xinkang, Wu Weizhen, Zhou Xueping, Ogawa Aimie M, O'Neill Kim, Chu Donald, Castriota Gino, Seiffert Dietmar A, Gutstein David E, Chen Zhu
In 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.
In 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
J Pharmacol Exp Ther. 2017 Mar;360(3):466-475. doi: 10.1124/jpet.116.238493. Epub 2016 Dec 29.
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 ED 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 ED 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.
凝血因子 XII（FXII）在血栓形成中起关键作用。目前尚不清楚实现疗效所需的 FXIIa 酶占有率水平以及 FXIIa 抑制对脑栓塞的影响。为了解决这些问题，我们制备了一种选择性活化 FXII（FXIIa）抑制剂，即重组人白蛋白标记的突变型 Infestin-4（rHA-Mut-inf）。rHA-Mut-inf 的效力与原始野生型 HA-Infestin-4 相当（人 FXIIa 抑制常数分别为 0.07 和 0.12 nM），对因子 Xa（FXa）和纤溶酶的选择性显著提高。rHA-Mut-inf 结合 FXIIa，但不结合 FXII 酶原，并竞争性抑制 FXIIa 蛋白酶活性。因此，其作用模式类似于典型的小分子抑制剂。使用 rHA-Mut-inf 进行的血浆转移和活化部分凝血活酶时间（aPTT）研究表明，在人、非人灵长类动物和兔中，为实现假定的 aPTT 加倍目标，计算得出的 FXIIa 酶占有率超过 99.0%。在兔中同时评估了 rHA-Mut-inf 对颈动脉血栓形成和大脑中动脉微栓塞信号（MES）的影响。观察到对动脉血栓形成和 MES 均有剂量依赖性抑制作用。血栓形成的半数有效剂量（ED），对于综合血流，静脉注射 rHA-Mut-inf 为 0.17 mg/kg，对于血栓重量为 ０.16 mg/kg；MES 的 ED 为 0.06 mg/kg。体外 aPTT 与疗效相关。总之，我们的研究结果表明，FXIIa 活性位点抑制剂需要非常高的酶占有率，这突出了在人体中实现疗效所需的高效力和精确选择性。我们的 MES 研究表明，靶向 FXIIa 可能为与血栓栓塞事件相关的中风预防提供一种有前景的策略。
