BT200,一种聚乙二醇化形式的适体BT100,抑制血管性血友病因子(VWF)与血小板糖蛋白GPIb的结合,预防食蟹猴动脉血栓形成。正在开发用于动脉血栓形成的二级预防,例如中风或心肌梗塞。预期BT200对血栓形成的抑制提供治疗益处。然而,可能有意外出血(例如,附带创伤),其中需要逆转剂。为了满足这一需求,BT101,一种互补适体,已被开发为特异性抑制BT100和BT200功能。
为了表征BT101在体外和体内的作用。
使用聚丙烯酰胺凝胶电泳评估BT101和核心适体BT100之间的直接相互作用。使用酶联免疫吸附测定进一步表征BT200与纯化的人VWF的结合和VWF活性的抑制。通过血小板功能分析仪和全血中的聚集度测定法测量VWF依赖性血小板功能。此外,BT101的体内药代动力学特征及其逆转BT200活性的能力,在食蟹猴中进行了评估。
BT101以1:1的比例与核心适体BT100结合,抑制BT200与纯化的人VWF的结合,并在体外逆转BT200诱导的VWF活性和VWF依赖性血小板功能的抑制。给猴子静脉注射后,BT101在几分钟内逆转BT200诱导的VWF活性和血小板功能的影响,不会造成任何不良影响。
本研究的结果表明BT101是BT200的有效逆转剂。
BT200, a pegylated form of the aptamer BT100, inhibits binding of von Willebrand factor (VWF) to platelet glycoprotein GPIb, preventing arterial thrombosis in cynomolgus monkeys. It is being developed for secondary prevention of arterial thrombosis such as stroke or myocardial infarction. Inhibition of thrombogenesis by BT200 is expected to provide a therapeutic benefit. However, there may be unexpected bleeding (eg, incidental trauma) in which a reversal agent is required. To address this need, BT101, a complementary aptamer, has been developed to specifically inhibit BT100 and BT200 function.
To characterize the effects of BT101 both in vitro and in vivo.
The direct interaction between BT101 and the core aptamer BT100 was evaluated using polyacrylamide gel electrophoresis. The binding of BT200 to purified human VWF and inhibition of VWF activity was further characterized using enzyme-linked immunosorbent assay. VWF-dependent platelet function was measured by the platelet function analyzer and aggregometry in whole blood. In addition, both the in vivo pharmacokinetic profile of BT101 as well as its ability to reverse BT200 activity, were evaluated in cynomolgus monkeys.
BT101 bound to the core aptamer BT100 at a 1:1 ratio, inhibited BT200 binding to purified human VWF, and reversed BT200-induced inhibition of both VWF activity and VWF-dependent platelet function in vitro. After intravenous injection to monkeys, BT101 reversed BT200-induced effects on VWF activity and platelet function within minutes, without causing any adverse effects.
The results of this study demonstrate that BT101 is an effective reversal agent for BT200.