Department of Pharmacodynamics, Medical University of Bialystok, Bialystok, Poland (B.K., J.M., J.S., A.J., D.P., A.M.); Faculty of Chemistry, Jagiellonian University, Krakow, Poland (K.K., M.N., K.S.); and Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji, Hyogo, Japan (S.-I.Y.)
Department of Pharmacodynamics, Medical University of Bialystok, Bialystok, Poland (B.K., J.M., J.S., A.J., D.P., A.M.); Faculty of Chemistry, Jagiellonian University, Krakow, Poland (K.K., M.N., K.S.); and Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, Himeji, Hyogo, Japan (S.-I.Y.).
J Pharmacol Exp Ther. 2020 Apr;373(1):51-61. doi: 10.1124/jpet.119.262931. Epub 2020 Jan 14.
Bleeding resulting from the application of low-molecular-weight heparins (LMWHs) may be treated with protamine sulfate, but this treatment lacks efficiency; its action against antifactor Xa activity is limited to ∼60%. Moreover, protamine sulfate can cause life-threatening hypersensitivity reactions. We developed diblock heparin-binding copolymer (HBC), which can neutralize the anticoagulant activity of parenteral anticoagulants. In the present study, we explored the safety profile of HBC and its potential to reverse enoxaparin, nadroparin, dalteparin, and tinzaparin in human plasma and at in vivo conditions. HBC-LMWH complexes were characterized using zeta potential, isothermal titration calorimetry, and dynamic light scattering. The rat cardiomyocytes and human endothelial cells were used for the assessment of in vitro toxicity. Male Wistar rats were observed for up to 4 days after HBC administration for clinical evaluation, gross necropsy, and biochemistry and histopathological analysis. Rats were treated with LMWHs alone or followed by short-time intravenous infusion of HBC, and bleeding time and antifactor Xa activity were measured. HBC completely reversed antifactor Xa activity prolonged in vitro by all LMWHs with an optimal weight ratio of 2.5:1. The complexes of HBC-LMWHs were below 5 µm. We observed no effects on the viability of cardiovascular cells treated with HBC at concentrations up to 0.05 mg/ml. Single doses up to 20 mg/kg of HBC were well tolerated by rats. HBC completely reversed the effects of LMWHs on bleeding time and antifactor Xa activity in vivo after 20 minutes and retained ∼80% and ∼60% of reversal activity after 1 and 2 hours, respectively. Well-documented efficacy and safety of HBC both in vitro and in vivo make this polymer a promising candidate for LMWHs reversal. SIGNIFICANCE STATEMENT: Over the last decade, there has been significant progress in developing antidotes for the reversal of anticoagulants. Until now, there has been no effective and safe treatment for patients with severe bleeding under low-molecular-weight heparin therapy. Based on our in vitro and in vivo studies, heparin-binding copolymer seems to be a promising candidate for neutralizing all clinically relevant low-molecular-weight heparins.
低分子肝素（LMWH）应用引起的出血可用硫酸鱼精蛋白治疗，但该治疗方法效率低下；其对抗因子 Xa 活性的作用仅限于约 60%。此外，硫酸鱼精蛋白可引起危及生命的过敏反应。我们开发了二嵌段肝素结合嵌段共聚物（HBC），可中和肠外抗凝剂的抗凝活性。在本研究中，我们探索了 HBC 的安全性概况及其逆转依诺肝素、那屈肝素、达肝素和亭扎肝素在人血浆中和体内条件下的潜在作用。使用zeta 电位、等温滴定量热法和动态光散射法对 HBC-LMWH 复合物进行了表征。使用大鼠心肌细胞和人内皮细胞评估体外毒性。雄性 Wistar 大鼠在给予 HBC 后观察 4 天，进行临床评估、大体解剖和生物化学及组织病理学分析。大鼠单独用 LMWH 治疗或在 LMWH 后短时间静脉内输注 HBC，测量出血时间和抗因子 Xa 活性。HBC 以 2.5:1 的最佳重量比完全逆转了所有 LMWH 体外延长的抗因子 Xa 活性。HBC-LMWH 复合物的直径小于 5 µm。在高达 0.05 mg/ml 的浓度下，用 HBC 处理的心血管细胞的活力没有受到影响。高达 20 mg/kg 的 HBC 单次剂量对大鼠的耐受性良好。HBC 在 20 分钟后完全逆转了 LMWH 对出血时间和抗因子 Xa 活性的体内作用，在 1 小时和 2 小时后分别保留了约 80%和 60%的逆转活性。HBC 在体外和体内的良好疗效和安全性使该聚合物成为 LMWH 逆转的有前途的候选药物。
在过去的十年中，在开发抗凝剂逆转的解毒剂方面取得了重大进展。到目前为止，对于接受低分子肝素治疗的严重出血患者，还没有有效的治疗方法。基于我们的体外和体内研究，肝素结合嵌段共聚物似乎是一种有前途的候选药物，可以中和所有临床相关的低分子肝素。
