TRIGGERED LOCAL RELEASE OF ACTIVE THROMBOLYTIC AGENTS

由血管血栓形成引起的心血管疾病是导致死亡的主要原因。目前，治疗方法是使用溶栓剂，即纤溶酶原激活剂（PA）溶解血栓。纤溶酶原的活化产生降解纤维蛋白的纤溶酶。然而，纤溶蛋白也会降解凝血因子。因此，溶栓治疗会导致全身产生纤溶酶，有出血的危险。在此之前，我们提出了一种新的、前药物和触发释放的方法，可以在没有出血风险的情况下进行靶向溶栓。该方法由两部分组成：[i]与阴离子肝素连接的纤维蛋白靶向抗体（称为Ab-Hep）；和[ii]阳离子修饰的PA（称为m-PA+）。这两个成分通过静电相互作用连接在一起。由于所用阳离子较小，m-PA+将保持其催化活性。然而，这种活性在与Ab-Hep结合后会被抑制，因为附加的大分子阻断了PA的活性位点。由于鱼精蛋白是一种临床肝素结合解毒剂，它可以安全地用于触发Ab-Hep-m-PA+复合物中m-PA+的释放。因此，该方法将允许注射一种以纤维蛋白为靶点但无活性的PA药物（从而通过中止系统产生纤溶酶来减轻出血风险），随后在纤维蛋白沉积物附近触发活性m-PA+的释放。虽然这是一个全新的项目，数据很少，但之前的申请在提交后不久就得到了NIH的全力支持，这是基于其科学价值和临床意义。然而，由于没有明确说明的原因，NIH将总体拨款期限从4年削减到3年。尽管由于更新时间的缩短，我们的小组取得了显著的进步和突出的生产力。在短短两年的时间里，已发表或提交了20篇手稿和10篇摘要。该项目的体外可行性，特别是在药物前和触发释放特性上，已在血浆中得到证实。在这个新的应用程序中，我们计划以这些有希望的发现为基础，进一步建立这个项目。我们的综合具体目标是：[i]开发对项目至关重要的分析方法；[ii]通过生物或化学方法产生所需的m-PA+；[iii]产生Ab-Hep缀合物；[iv]体外检测Ab-Hep-m-PA+终产物的功能；[v]检测其在大鼠体内的药代动力学特性；并通过兔颈静脉模型和临床模拟犬冠状动脉内血栓形成模型在体内测试其功能。

Cardiovascular diseases which result from thrombosis of blood vessels are a leading cause of deaths. At present, the treatment is dissolution of the thrombus using a thrombolytic agent, namely a plasminogen activator (PA). Activation of plasminogen produces plasmin which degrades fibrin. Plasmin, however, also degrades clotting factors. Thrombolytic therapy which introduces systemic generation of plasmin, therefore, carries the risk of hemorrhage. Previously, we proposed a novel, pro-drug and triggered release approach which could permit targeted thrombolysis without the bleeding risk. The approach consists of two components: [i] a fibrin- targeting antibody linked to an anionic heparin (termed Ab-Hep); and [ii] a cation-modified PA (termed m-PA+). These two components are linked via an electrostatic interaction. Since the used cations are small, m-PA+ would retain its catalytic activity. This activity, however, would be inhibited after binding to Ab-Hep due to blockage of the PA's active site by the appended macromolecules. Since protamine is a clinical heparin- binding antidote, it can be used safely to trigger the release of m-PA+ from the Ab-Hep-m-PA+ complex. Thus, the approach would permit injection of a fibrin-targeting but inactive PA drug (thereby alleviating the bleeding risk by aborting systemic generation of plasmin), and subsequently a triggered release of the active m-PA+ in close proximity of a fibrin deposit. Although it was a brand new project with minimal data, the previous application received full support from NIH shortly after submission based on its scientific merits and clinical significance. For reasons not clearly stated, however, the overall grant period was cut by NIH from 4 to 3 years. Despite being handicapped by a shortened time for renewal, our group has made remarkable progress and outstanding productivity. In a short 2-year period, 20 manuscripts and 10 abstracts have been published or submitted. The in vitro feasibility of the project, particularly on the pro-drug and triggered-release features, have been demonstrated in plasma. In this new application, we plan to build upon those promising findings and further establish the project. Our integrated specific aims are: [i] develop analytical methods essential to the project; [ii] produce the desired m-PA+ by biological or chemical means; [iii] produce the Ab-Hep conjugates; [iv] test the functions of the final Ab-Hep-m-PA+ products in vitro; [v] examine their pharmacokinetic properties in rats; and [vi] test their functions in vivo using a rabbit jugular vein model and a clinically- simulated canine intracoronary thrombosis model.