TRIGGERED LOCAL RELEASE OF ACTIVE THROMBOLYTIC AGENTS

描述：(改编自研究人员摘要)心血管疾病 仍然是工业化国家的主要死亡原因。这个 这些疾病的根本原因是危重部位血栓形成。 血管。一种治疗方法是溶解血栓。 使用溶栓剂，即纤溶酶原激活剂。激活 纤溶酶原导致纤溶酶的形成，从而降解纤维蛋白。 不幸的是，纤溶酶是一种非特异性的蛋白水解酶，也能降解其他 循环凝血因子。因此，溶栓疗法，它引入了 全身产生过量的纤溶酶，有出血的风险。这个 因此，对高度改进的溶栓剂的追求仍然存在。这个 直接引用《科学》杂志上一篇广为人知的评论文章中的“理想”代理人 (243：51,1989)，应该是：“有能力选择性地攻击部件 同时避免循环中的凝血蛋白和血小板“。 我们提出了一种新的方法，可以满足这些标准。这个 该方法包括一个由两个组分组成的大型蛋白质复合体：A 与肝素偶联的纤维蛋白靶向抗体和纤溶酶原 活化剂(PA)用季铵盐衍生化(QAS)。两个人 各组分通过阴离子之间的静电相互作用连接 肝素和阳离子QAS种类。由于QA是小离子，所以 QAS修饰的PA将完全保留其溶栓活性。这项活动， 然而，在与抗体成分结合后将被抑制， 主要是由于酶的催化部位被附加的 肝素大分子。由于鱼精蛋白是一种临床肝素拮抗剂 由于对肝素有更强的亲和力，可以安全地使用它来触发 QAS改性的PA从大的复合体中释放出来。因此， 方法将允许给药强烈的纤维蛋白靶向，但 非活性溶栓复合体，随后触发释放 活性PA药物在纤维蛋白沉淀物附近。这些功能 不仅会增强溶栓剂的效力和特异性， 而且还可以通过允许药剂选择性地 攻击血栓的成分，同时保留循环中的蛋白质。 在本方案中，我们计划使用链激酶和t-PA作为两种模型 以检验该方法的可行性。我们的具体目标 是：(1)制备抗体-PA复合体；(2)检测其靶向性 和体外纤溶功能；(3)检测其 在大鼠体内的药代动力学/生物分布特性；(4)测试其 体内纤溶功能，使用兔颈静脉模型；以及(5) 测试它们在再通闭塞冠状动脉方面的有效性，使用 临床刺激犬冠状动脉内血栓形成模型。

DESCRIPTION: (Adapted from investigator's abstract) Cardiovascular diseases remain the leading cause of deaths in industrialized countries. The underlying cause in these diseases is thrombosis of critically situated blood vessels. One approach to the treatment is dissolution of the thrombus with thrombolytic agents, namely plasminogen activators. Activation of plasminogen results in the formation of plasmin which degrades fibrin. Unfortunately, plasmin is a non-specific protease that also degrades other circulating clotting factors. Thus thrombolytic therapy, which introduces systemic generation of excess plasmin, carries the risk of hemorrhage. The quest for highly improved thrombolytic agents therefore remains. The "ideal" agent, quoted directly from a highly publicized review in Science (243:51,1989), should: "be capable of selectively attacking the components while sparing the circulating clotting proteins and platelet". We propose a novel approach which could fulfill these criteria. The approach comprises a large protein complex of two components: a fibrin-targeting antibody conjugated with heparin, and a plasminogen activator (PA) derivatized with quaternary ammonium species (QAS). The two components are linked via an electrostatic interaction between the anionic heparin and the cationic QAS species. Since QAS are small ions, the QAS-modified PA will fully retain its thrombolytic activity. This activity, however, will be inhibited after binding with the antibody component, primarily due to the blockage of the enzyme's catalytic site by the appended heparin macromolecule. Since protamine is a clinical heparin antagonist with a much stronger affinity to heparin, it can be used safely to trigger the release of the QAS-modified-PA from the large complex. Thus, the approach would permit the administration of a strong fibrin-targeting but inactive thrombolytic complex, and subsequently a triggered release of the active PA drug in close proximity of the fibrin deposit. These features would not only enhance the potency and specificity of a thrombolytic agent, but also alleviate the bleeding risk by allowing the agent to selectively attack the components of a thrombus while sparing the circulating proteins. In this proposal, we plan to use streptokinase and t-PA as two model compounds to examine the feasibility of the approach. Our specific aims are: (1) to prepare the antibody-PA complexes; (2) to test their targeting and fibrinolytic functions in vitro; (3) to examine their pharmacokinetic/biodistribution properties in rats; (4) to test their fibrinolytic function in vivo, using a rabbit jugular vein model; and (5) to test their efficacy in recanalizing occluded coronary arteries, using a clinically-stimulated canine intracoronary thrombosis model.