PARP Inhibitor and Redox Catalyst for Ventilatory Trauma

描述(申请人提供)：大容量机械通气(MV)通过产生有毒的自由基和氧化剂，破坏肺泡毛细血管膜，触发促炎基因表达，导致肺动脉高压和呼吸衰竭，从而导致致命的肺部并发症，称为呼吸机诱导的肺损伤(VILI)。为了满足这一未得到满足的医疗需求，我们正在开发一种新型的双功能小分子R-503，它：1)通过广谱氧化还原催化防止过氧亚硝酸盐的产生，2)竞争性抑制核DNA修复酶多聚(ADP-核糖)聚合酶1(“PARP-1”)。在酵母多糖诱导的小鼠多器官衰竭和Cl2吸入性肺损伤模型中，在酵母多糖滴注或Cl2吸入后开始复苏，可使中性粒细胞的渗入减少65%，肺组织学损伤减少80%(p&lt；0.001)。基于炎症和氧化还原应激在VILI病理生理学中的中心地位，我们假设PARP-1在肺组织中高度激活，R-503干预后损伤将减轻。具体目的#1：建立大鼠高潮气量(TV)MV呼气末正压(PEEP)为3cmH2O的模型，建立R-503的药效学模型。TV最初设置为30毫升/公斤，然后降低到最大吸气压(PIP)为30厘米水。R-503(0、10、30和80 mg/kg)将在大容量机械通气开始后1小时开始治疗。阴性对照组采用低通气量(TV=6mL/kg，PEEP=3cmH2O)。阳性对照组给予大容量机械通气加甲基强的松龙(30 mg/kg)静脉滴注。对照组给予等质量剂量的N-乙酰半胱氨酸(NAC)静脉注射(30 mg/kg)。肺力学将每小时进行一次评估，以确定：1)动态呼吸系统顺应性，2)动态压力容量环得出的阻力和滞后，以及3)静态顺应性，包括根据静态充气和放气气道压力曲线的形状得出的下拐点和上拐点。我们将通过跟踪心率、平均动脉压和动脉压变化来监测血流动力学状态。在MV开始后4h，即安乐死前，测定动脉血气，然后行左肺支气管肺泡灌洗(BALF)，以确定气体交换和二氧化碳分压/分钟容量比。将分析BALF的细胞密度、LDH释放、促炎蛋白表达和蛋白质含量。将分析右肺的组织学损伤、湿/干比、氧化还原应激生化标志物(F2β-异前列腺素，GSH/GSSG比值)、中性粒细胞浸润(髓过氧化物酶；“MPO”)、促炎蛋白表达，以及过氧亚硝酸盐诱导的3-硝基酪氨酸(3-NT)和多聚(ADP-核糖)形成的免疫组织化学证据。肺中R-503浓度将与预后指标相关联，从而构建帕金森病模型。

DESCRIPTION (provided by applicant): High volume mechanical ventilation (MV) induces a lethal pulmonary complication, termed "ventilator induced lung injury" (VILI), via the generation of toxic free radical and oxidant species that damage the alveolocapillary membrane, trigger pro-inflammatory gene expression, and induce pulmonary arterial hypertension and respiratory failure. To address this unmet medical need, we are developing a novel bifunctional small molecule, R-503, that: 1) prevents the generation of peroxynitrite via broad-spectrum redox catalysis, and 2) competitively inhibits the nuclear DNA repair enzyme poly(ADP-ribose) polymerase 1 ("PARP- 1"). In murine LD100 models of zymosan-induced multiple organ failure (MOF) and Cl2 inhalational lung injury, resuscitation with R-503 begun after the onset of zymosan instillation or Cl2 inhalation reduced neutrophil (PMN) infiltration by 65% and histologic lung injury by 80% (p<0.001). Based upon the centrality of inflammation and redox stress to the pathophysiology of VILI, we hypothesize that PARP-1 is highly activated in the lung and that injury will be attenuated after R-503 intervention. Specific Aim #1: Establish the pharmacodynamic (PD) profile of R-503 in a rat model of VILI Sprague-Dawley rats will be subjected to high tidal volume (TV) MV with positive end expiratory pressure (PEEP) of 3 cm H2O. TV will be initially set at 30 mL/kg and then reduced to limit peak inspiratory pressure (PIP) to 30 cm H2O. Treatment with R-503 (0, 10, 30, and 80 mg/kg) will be initiated 1 h after the onset of high volume MV. A negative control group will undergo low-volume ventilation (TV=6 mL/kg, PEEP=3 cm H2O). A positive control group will undergo high-volume MV plus therapy with IV methylprednisolone (30 mg/kg). A comparator group will be administered IV N-acetylcysteine (NAC) at an equivalent mass dose (30 mg/kg). Lung mechanics will be evaluated hourly, allowing for determination of: 1) dynamic respiratory system compliance, 2) resistance and hysteresivity derived from dynamic pressure volume loops, and 3) static compliance, including lower and upper inflection points derived from the shape of the static inflation and deflation airway pressure curves. We will monitor hemodynamic status by tracking heart rate, mean arterial pressure, and arterial pulse pressure variation. Arterial blood gas determination followed by bronchoalveolar lavage (BALF) of the left lung will be undertaken 4 h after the onset of MV, directly before euthanasia, allowing for determination of gas exchange and the PCO2/Minute Volume Ratio. The BALF will be analyzed for cellularity, LDH release, pro-inflammatory protein expression, and protein content. The right lung will be analyzed for histologic injury, wet/dry ratio, biochemical markers of redox stress (F2¿-isoprostane, GSH/GSSG ratio), PMN infiltration (myeloperoxidase; "MPO"), pro-inflammatory protein expression, and immunohistochemical evidence of peroxynitrite-induced 3- nitrotyrosine (3-NT) and poly(ADP-ribose) formation. Lung concentrations of R-503 will be correlated with outcome measures, so as to construct a PD profile.