Triggered Local Release of Active Thrombolytic Agents

描述（由申请人提供）：心血管疾病是由危急位置的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溶栓剂溶解血栓，即纤溶酶原激活剂（PA）。PA剂激活纤溶酶原产生纤溶酶，然后使纤维蛋白降解。然而，纤溶蛋白也会降解其他凝血因子。因此，溶栓治疗会导致全身产生纤溶酶，有出血的风险。在此之前，我们提出了一种新的前药物和触发释放方法（称为Antibody _Targeted_Triggered _electrmodified prodrug . _type_strategy;AT-rEMPTS.），该方法可以实现无出血风险的靶向溶栓。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+）。这两个组件通过静电相互作用自动连接。由于用于PA修饰的阳离子很小，m-PA+将保留其纤溶活性。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的催化位点。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，因此它可以安全有效地用于触发血栓部位Ab-Hep/m-PA+复合物释放m-PA+。因此，该方法将允许给药一种强纤维蛋白靶向但无活性的PA药物，随后在纤维蛋白沉积物附近触发释放活性PA药物。这些特点不仅可以增强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攻击血栓的相关成分而降低出血风险，同时保留其他循环因子。在上一个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，已经发表或提交了30篇同行评审的手稿和17篇摘要。在之前的申请中提出的所有具体目标都已成功实现。最重要的是，在上一个资助期间取得的进展和发现的缺陷导致建立了一个微调的研究计划和战略，包括使用：(i)逆转尝试（称为重新尝试）战略；（ii）计算机模拟技术，以确定t-PA中肽修饰的理想位点；（iii）点突变方法，实现肽与t-PA的位点特异性结合；（iv）现代基于生理的药代动力学（PBPK）方法来优化触发剂的给药时间，以达到有效和安全的溶栓治疗的最大目标。4个广泛整合的特异性目标是：Aim [i]：合成抗纤维蛋白抗体连接的修饰的t-PA复合物（称为Ab/mt-PA），该复合物由聚（Glu）修饰的阴离子t-PA（称为m-PA）和低分子蛋白修饰的阳离子抗体（称为低分子蛋白-Ab）组成；目的[ii]：利用流动型血浆凝块法测定Ab/mt-PA复合物的体外特性；目的[iii]：通过体内靶向药代动力学研究，评估触发剂肝素的最佳给药时间；Aim [iv]：利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reattempts系统的有效性和安全性进行体内评价。

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.