A Hybrid PARP Inhibitor and Redox Catalyst for Lung Transplantation

描述(申请人提供)：Radikal Treeutics(RTX)正在开发一种新的双功能小分子R-503，旨在预防与原位肺移植相关的肺缺血再灌注损伤(LIRI)。LIRI是由于急性再灌流引起的自由基超氧阴离子合成的改变，随后被转化为强大的毒素，包括过氧亚硝酸盐、过氧化氢和羟基自由基。这些氧化和亚硝化物种诱导DNA单链断裂，激活聚(ADP-核糖)聚合酶(PARP)，这是一种核DNA修复酶，反过来耗尽其底物NAD，导致细胞内能量耗竭、ATP耗尽和组织坏死。PARP激活还可诱导促炎基因的广泛表达，导致血管通透性、肺水肿、中性粒细胞渗透、肺分流和呼吸衰竭。在LIRI损伤的实验模型中，PARP的药理抑制有效地减轻了肺损伤，但其保护程度并不完全，其能否成功临床翻译尚不确定。我们现在建议更彻底地阻断LIRI诱导的组织损伤，利用上游DNA损伤氧化和亚硝化物种的同时和协同去除以及下游PARP的抑制。R-503由两个部分的共价键形成，每个部分都具有组织保护作用：1)PARP抑制物部分，2)作为广谱氧化还原催化剂的富含硫醇的二氢硫辛基(DHL)结构域。DHL部分的加入赋予了R-503独特的性质，使该分子能够关闭PARP依赖和PARP非依赖的氧化还原应激途径。R-503是一种有效的PARP抑制剂(IC50=20 nM)，在体外比单功能PARP抑制剂具有更强的细胞保护作用，在体内具有显著的保护作用，如在小鼠LD100酵母多糖诱导的多器官衰竭和氯气吸入性肺损伤模型中所显示的那样。具体目的：建立双功能PARP抑制剂R-503在实验性大鼠LIRI模型中的优势及体内协同作用。在单侧肺缺血90min和再灌注4h之前，大鼠将接受静脉注射R-503、DHL、单功能PARP抑制剂(INO-1001)或DHL和INO-1001的联合治疗。假动物不会经历缺血，也不会接受药物治疗。我们预计R-503将在以下方面表现出优于INO-1001、DHL及其联合治疗的疗效：1)组织损伤(组织学评分、中性粒细胞渗透水平、脂质过氧化、蛋白亚硝化、血管通透性、PARP激活和细胞凋亡)和动脉供氧；以及2)肺损伤的支气管肺泡灌洗标志物(中性粒细胞渗透水平、肿瘤坏死因子-β、MIP-1β和核核因子-β)。这样的治疗效果有望转化为LIRI的临床终点：1)肺分流减少，2)机械通气持续时间，3)住院时间，4)全因死亡30天和365天。 公共卫生相关性：缺血-再灌注损伤是肺移植后的主要医学并发症，也是导致该人群高死亡率的原因之一。目前还没有批准的预防措施。我们正在开发一种针对这种疾病的基本机制的新药，并将在临床相关的动物模型中测试这种药物。

DESCRIPTION (provided by applicant): Radikal Therapeutics (RTX) is developing a novel bifunctional small molecule, R-503, intended for the prevention of lung ischemia-reperfusion injury (LIRI) associated with orthotopic lung transplantation. LIRI results from an acute reperfusion-induced alteration in the synthesis of the free radical superoxide anion that is subsequently converted into powerful toxins, including peroxynitrite, hydrogen peroxide, and hydroxyl radical. These oxidizing and nitrosating species induce DNA single strand breaks that activate poly(ADP-ribose) polymerase (PARP), a nuclear DNA repair enzyme that in turn depletes its substrate NAD, resulting in an exhaustion of intracellular energetics, ATP depletion, and tissue necrosis. PARP activation also induces a widespread expresion of pro-inflammatory genes that contribute to vascular permeability, lung edema, neutrophil infiltration, pulmonary shunt, and respiratory failure. In experimental models of LIRI injury, pharmacologic inhibition of PARP potently reduces lung injury, but the extent of protection was not complete and its successful clinical translation is uncertain. We now propose a more thorough interruption of LIRI-induced tissue damage, exploiting the simultaneous and synergistic removal of upstream DNA-damaging oxidizing and nitrosating species plus downstream PARP inhibition. R-503 is formed from the covalent linkage of 2 moieties, each with demonstrated tissue protection: 1) a PARP inhibitor moiety, and 2) a thiol-rich dihydrolipoyl ("DHL") domain that acts as a broad-spectrum redox catalyst. The inclusion of the DHL moiety confers unique properties on R-503, allowing the molecule to shut down both PARP-dependent and PARP-independent pathways of redox stress. R-503 is a potent PARP inhibitor (IC50=20 nM), more cytoprotective than a monofunctional PARP inhibitor in vitro, and remarkably protective in vivo, as shown in murine LD100 models of zymosan-induced multiple organ failure and chlorine inhalational lung injury. Specific Aim: Establish the superiority and in vivo synergy of the bifunctional PARP inhibitor R-503 in an experimental rat model of LIRI. Prior to 90 min of unilateral lung ischemia and 4 h of reperfusion, rats will be treated with IV R-503, DHL, a monofunctional PARP inhibitor (INO-1001), or a combination of DHL and INO-1001. A sham animal will not undergo ischemia nor receive drug therapy. We expect that R-503 will exhibit superior efficacy, relative to treatment with INO-1001, DHL, and their combination, with respect to: 1) tissue damage (histology score, levels of neutrophil infiltration, lipid peroxidation, protein nitrosation, vascular permeability, PARP activation, and apoptosis) and arterial oxygenation; and 2) bronchoalveolar lavage markers of pulmonary injury (levels of neutrophil infiltration, concentrations of TNF-¿, MIP-1¿, and nuclear NF-?B). Such treatment effects are expected to translate into clinical endpoints in LIRI of decreased: 1) pulmonary shunt, 2) duration of mechanical ventilation, 3) length of hospitalization, and 4) all-cause 30 and 365 day mortality. PUBLIC HEALTH RELEVANCE: Ischemia-reperfusion injury is a major medical complication following lung transplantation and contributes to the high mortality in this population. At present there are no approved prophylactic measures. We are developing a novel drug that targets the basic mechanisms of this condition and will test this agent in a clinically-relevant animal model.