Triggered Local Release of Active Thrombolytic Agents

描述（由申请人提供）：心血管疾病是由关键血管血栓形成引起的，是导致死亡的主要原因。目前，标准治疗是通过使用血栓溶解剂，即纤溶酶原激活剂（PA）溶解血栓。纤溶酶原被PA试剂激活产生纤溶酶，纤溶酶然后降解纤维蛋白。然而，纤溶酶也降解其他凝血因子。因此，溶栓治疗会引起纤溶酶的全身性产生，从而带来出血的风险。以前，我们提出了一种新的前药和触发释放方法（称为抗体_靶向_触发_电修饰_前药。Type_Strategy;AT-rEMPTS.）这可以允许靶向溶栓而没有出血的风险。该方法由2个组分组成：[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，由于附加的大分子阻断了PA的催化位点，这种活性在与Ab-Hep结合后将被抑制。由于鱼精蛋白是一种具有无与伦比的肝素结合亲和力的临床肝素拮抗剂，因此可以安全有效地用于触发血栓部位的Ab-Hep/m-PA+复合物释放m-PA+。因此，该方法将允许施用强纤维蛋白靶向但无活性的PA药物，并且随后在纤维蛋白存款附近触发活性PA药物的释放。这些特征不仅可以增强PA药物的效力和纤维蛋白选择性，而且还可以通过允许药物特异性攻击血栓的相关成分而减少出血风险，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出色的生产力。因此，在不到4年的时间内，出版或提交了30份同行评审的手稿和17份摘要。先前申请中提出的所有具体目标都已成功实现。最重要的是，在上一个资助期内取得的进展和发现的缺陷导致制定了一个微调的研究计划和战略，包括使用：（称为re-ATTEMPTS）策略;（ii）计算机模拟技术以鉴定用于肽修饰的t-PA中的理想位点;（iii）点突变方法以实现肽与t-PA的位点特异性掺入;以及（iv）现代生理学基础的药代动力学（PBPK）方法，以优化触发剂的给药时间，从而实现有效和安全的血栓溶解治疗的最大目标。4个广泛整合的特定目的是：目的[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），由聚（Glu）修饰的阴离子t-PA组成，（称为m-PA）和LMWP修饰的阳离子抗体（称为LMWP-Ab）;目的[ii]：使用流式血浆凝块试验对Ab/mt-PA复合物进行体外表征;目的[iii]：体内靶向药代动力学研究，用于评估触发剂肝素的最佳给药时间;以及目的[iv]：使用大鼠IVC血栓形成模型和犬冠状动脉内血栓形成模型体内评价re-ATTEMPTS系统的有效性和安全性。

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.