Triggered Local Release of Active Thrombolytic Agents

描述(由申请人提供)：心血管疾病是由危急血管血栓形成引起的，是导致死亡的主要原因。目前，标准的治疗方法是使用一种溶栓剂，即纤溶酶原激活剂(PA)溶解血栓。纤溶酶原被PA激活剂激活后产生纤溶酶，然后纤溶酶降解纤维蛋白。然而，纤溶酶也会降解其他凝血因子。溶栓治疗会导致全身产生纤溶酶，因此存在出血的风险。在此之前，我们提出了一种新的、亲药物和触发释放的方法(称为抗体靶向触发电修饰前药。类型策略；AT-rEMPTS)。这可以在没有出血风险的情况下进行有针对性的血栓溶解。该方法由两部分组成：[i]一种与阴离子肝素相连的纤维蛋白靶向抗体(称为Ab-Hep)；[ii]一种阳离子修饰的PA(称为m-PA+)。这两个组件通过静电相互作用自动链接。由于用于PA改性的阳离子很小，m-PA+将保持其纤溶活性。然而，这种活性在与抗体-Hep结合后会受到抑制，这是因为附加的大分子堵塞了PA的催化部位。由于鱼精蛋白是一种临床肝素拮抗剂，具有无与伦比的肝素结合亲和力，因此可以安全有效地触发血栓部位的抗体-Hep/m-PA+复合体释放m-PA+。因此，该方法将允许给药一种强烈的纤维蛋白靶向但无活性的PA药物，随后在纤维蛋白沉积附近触发活性PA药物的释放。这些特性不仅可以增强PA药物的效力和纤维蛋白选择性，还可以通过允许药物特异性地攻击血栓的相关成分而避免其他循环因素，从而降低出血风险。在前一批赠款期间，取得了显著的进展和突出的生产力。因此，在不到4年的时间里，出版或提交了30份同行评议的手稿和17份摘要。前一次申请中提出的所有具体目标都已成功实现。最重要的是，在前一个赠款期间取得的进展和发现的缺陷导致制定了一项微调的研究计划和战略，包括使用：(I)反向尝试(称为重试)策略；(Ii)计算机模拟技术来确定t-PA中进行肽修饰的理想位置；(Iii)点突变方法，以实现将肽与t-PA结合的部位特异性；以及(Iv)现代生理学药代动力学(PBPK)方法，以优化触发剂的剂量时间，以实现有效和安全的溶栓治疗的最大目标。4个广泛集成的具体目标是：目的[I]：合成由聚(Glu)修饰的阴离子t-PA(称为m-PA)和LMWP修饰的阳离子抗体(称为LMWP-Ab)组成的抗纤维蛋白抗体连接的、修饰的t-PA复合体(称为Ab/mt-PA)；目的[II]：使用流动血浆凝块分析法体外表征抗体/mt-PA复合体；目的[iii]：体内靶向药代动力学研究，以评估肝素的最佳给药时间；目的[IV]：使用大鼠下腔静脉血栓形成模型和犬冠状动脉内血栓形成模型对重试系统的有效性和安全性进行体内评估。

DESCRIPTION (provided by applicant): Cardiovascular diseases, which result from thrombosis of critically situated blood vessels, are a leading cause of deaths. At present, the standard treatment is by dissolution of the thrombus using a thrombolytic agent, namely a plasminogen activator (PA). Activation of plasminogen by the PA agent produces plasmin, which then degrades fibrin. Plasmin, however, also degrades other clotting factors. Thrombolytic therapy, which introduces systemic generation of plasmin, therefore carries the risk of bleeding. Previously, we proposed a novel, pro-drug and triggered-release approach (termed Antibody _Targeted_Triggered _Electrically Modified _Prodrug._Type_Strategy;AT-rEMPTS.) which could permit targeted thrombolysis without the risk of bleeding. The approach consists of 2 components: [i] a fibrin-targeting antibody linked to an anionic heparin (termed Ab-Hep); and [ii] a cation-modified PA (termed m-PA+). These 2 components are linked automatically via an electrostatic interaction. Since the cations used for PA modification are small, m-PA+ would retain its fibrinolytic activity. This activity, however, would be inhibited after binding to Ab-Hep, due to blockage of the PA's catalytic site by the appended macromolecules. Since protamine is a clinical heparin antagonist with an unmatched heparin-binding affinity, it can be used safely and effectively to trigger the release of m-PA+ from the Ab-Hep/m-PA+ complex at site of the thrombus. Therefore, the approach would permit administration of a strong fibrin-targeting but inactive PA drug, and subsequently a triggered release of the active PA drug in close proximity of the fibrin deposit. Such features would not only enhance the potency and fibrin-selectivity of the PA drug, but also attenuate the bleeding risk by permitting the drug to specifically attack the concerned components of a thrombus while sparing other circulating factors. Remarkable progress and outstanding productivity have been achieved during the previous grant period. As a consequence, 30 peer-reviewed manuscripts and 17 abstracts have been published or submitted within a period of less than 4 years. All the specific aims proposed in the previous application have been successfully accomplished. Most importantly, progress made and pitfalls identified during the previous grant period led to the establishment of a fine-tuned research plan and strategy, including the use of: (i) reversed ATTEMPTS (termed re-ATTEMPTS) strategy;(ii) computer simulation technology to identify the ideal site in t-PA for peptide modification;(iii) point-mutation methodology to achieve site-specific incorporation of peptide to t-PA; and (iv) modern physiologically-based pharmacokinetics(PBPK) approach to optimize the dosing time of the triggering agent to achieve the utmost goal of effective and safe thrombolytic therapy. The 4 broadly integrated Specific Aims are: Aim [i]: syntheses of an anti-fibrin antibody-linked, modified-t-PA complex (termed Ab/mt-PA)consisting of a poly(Glu)-modified anionic t-PA (termed m-PA) and a LMWP-modified cationic antibody (termed LMWP-Ab); Aim [ii]: in vitro characterization of the Ab/mt-PA complex using a flow-type plasma clot assay; Aim [iii]: in vivo investigation of the targeting pharmacokinetics for assessing the optimal dosing time for the triggering agent heparin; and Aim [iv]: in vivo evaluation of the efficacy and safety of the re-ATTEMPTS system using both the rat IVC thrombosis model and the canine intra-cornonary thrombosis model.