Augmentation of Antioxidant Defenses by Immunotargeting

描述(申请人提供)：血管氧化应激是许多肺部疾病的关键组成部分，但其治疗不充分，部分原因是抗氧化剂未达到向内皮细胞(EC)的最佳输送。以前的研究表明：1)抗血小板内皮细胞黏附分子(PECAM)的抗体可以通过一种新的内吞途径将药物输送到EC，不同于笼蛋白和小窝介导的内吞作用；2)抗氧化酶(AOE)过氧化氢酶(AOE)与抗PECAM抗体在静脉注射后聚集在肺内，在某些动物模型中保护肺免受氧化性肺损伤。这项翻译赠款的目的是将这一有希望的策略的保护效果的持续时间和有效性提高到临床显著水平。我们假设：i)通过优化其设计和控制细胞内转运和溶酶体降解，可以延长结合物的活性；ii)靶向向EC输送额外的AOES将允许增强对氧化应激的保护；以及iii)优化的AOE结合物的肺靶向将对高氧具有保护作用。我们将从以下几个方面对此进行测试：1)明确抗PECAM结合物在EC中的代谢机制。我们将检验内吞和运输涉及PECAM、Na-H交换器(NHE)和细胞骨架重排的假设，这些重排可以被辅助药物影响以延长偶联持续时间；2)设计额外的AOE的靶向。为了加强保护，将产生串联的超氧化物歧化酶/过氧化氢酶(用于解毒02-和H202)和1-CysPrx过氧化还蛋白(用于解毒H202和脂质过氧化)。它们的组成、活性、EC的摄取和保护将在细胞培养中进行研究，而它们的药代动力学和肺靶向性将在幼稚动物和氧化性肺损伤动物中进行测定；3)在动物模型中评价这些结合物的保护作用。在H202诱导的小鼠肺血管内皮细胞急性损伤模型中，将比较新的结合物的效果和持续时间，并研究AOE的靶向方案和保护机制。最后，将测试最佳结合物对高氧性肺损伤的保护作用。这项建议的总体目标是优化血管AOE靶向的策略，最终启动该策略向临床领域的转换。

DESCRIPTION (provided by applicant): Vascular oxidative stress is a key component of many lung diseases, but its treatment is inadequate, in part due to sub-optimal delivery of antioxidants to endothelial cells (EC). Previous studies have shown that: 1) antibodies to Platelet Endothelial Adhesion Molecule (PECAM) permit intracellular delivery of drugs to EC via a novel endocytotic pathway distinct from clathrin- and caveoli-mediated endocytosis; and 2) the antioxidant enzyme (AOE) catalase conjugated with anti-PECAM accumulates in the lungs after IV injection and protects against oxidative lung injury in some animal models. The aim of this translational grant is to enhance the duration and effectiveness of protective effects of this promising strategy to clinically significant levels. We hypothesize that: i) the activity of the conjugates can be prolonged by optimization of their design and by manipulating intracellular trafficking and lysosomal degradation; ii) targeted delivery of additional AOEs to EC will permit enhanced protection from oxidative stress; and iii) pulmonary targeting of optimized AOE conjugates will be protective against hyperoxia. We will test this in the following Specific Aims: 1) define the mechanisms of metabolism of anti-PECAM conjugates in EC. We will test the hypothesis that endocytosis and trafficking involve the cytosolic domain of PECAM, Na+-H+ exchangers (NHE) and rearrangements of cytoskeleton, which can be affected by auxiliary agents to prolong duration of conjugates; 2) design targeting of additional AOE. To enhance protection, tandem SOD/catalase (to detoxify 02- and H202) and 1-CysPrx peroxiredoxin (to detoxify H202 and lipid peroxides) conjugates will be produced. Their composition, activities, EC uptake and protection will be studied in cell culture, while their pharmacokinetics and pulmonary targeting will be determined in naive animals and animals with oxidative lung injury; and 3) evaluate protective effects of the conjugates in animal models. The effectiveness and duration of the effects of new conjugates will be compared and regimens of AOE targeting and mechanisms of protection will be studied in a model of acute EC injury induced by H202 in the pulmonary vasculature in mice. Finally, protection against hyperoxic lung injury by best conjugates will be tested. The overall goal of this proposal is to optimize strategies for vascular AOE targeting, to ultimately initiate translation of this strategy into the clinical domain.