Peroxynitrite Decomposition Catalyst and Nitric Oxide Donor for Endotoxemia

描述（由申请人提供）：自由基一氧化氮（NO）和超氧阴离子的生物合成的改变通常被认为是通过诱导微空洞缺血和直接器官毒性导致脓毒症中广泛的组织损伤的原因。为了纠正这种自由基失衡，Radikal Therapeutics （RTX）正在开发一种一流的小分子药物（R-100），这是一种基于双功能氧化还原的技术，由两个化学部分的共价键形成：1)释放NO的有机硝基血管扩张剂，2)作为活性氧降解三功能催化剂的吡咯烷类氮氧化物。一种超氧化物歧化酶模拟物，一种脱毒过氧化氢的过氧化氢酶模拟物，以及一种过氧亚硝酸盐分解催化剂。在内毒素血症的LD100小鼠模型中，在脂多糖（LPS）攻击后1小时开始用R-100复苏可产生100%的存活率，并伴有对终末器官损伤的几乎完全保护。我们假设R-100优于其两组分功能的总和，并且这两种特性的共价融合成一个单一的分子实体，为脓毒症的治疗创造了强大的商业前景。RTX将通过开展内毒素研究来验证这一假设，以确定小鼠腹腔注射R-100的剂量-反应、时间窗、作用机制和安全性。目的1：将3种剂量水平的R-100在脂多糖（LPS）刺激后1小时与对照进行比较，以确定提供最佳结果的最低剂量（“LDPOO”）。R-100的组织水平和来自心脏、肺、肾和肝脏的代谢物将被测量，以便将血浆和器官药物摄取联系起来。目的2：将R-100的LDPOO剂量与等摩尔剂量的羟甲基proxyl（“HMP”，R-100的氮氧化物成分）、单硝酸异山梨酯（“ISMN”，一种经典的单功能NO供体）以及HMP和ISMN的组合进行比较，以验证双功能化合物（R-100）优于其组分功能的混合物。治疗将在LPS刺激后1小时开始。目标3：我们将通过在LPS刺激后1、2、4和8小时引入R-100来确定治疗时间窗口的持续时间。在给药48小时后检测血清和组织，以确定形态学和生化终点，包括脂质过氧化（f21 -异前列腺素，GSH:GSSG比率），中性粒细胞浸润，3-硝基酪氨酸（3- nt），亚硝酸盐/硝酸盐和聚（adp -核糖）形成，BUN，肌酐，NGAL， AST， ALT，组织学损伤评分，血清TNF-1， HMGB-1, IL-6, TRP14, Trx1, LC8, I:B1, IL-12, MIP-11， CXCL9和CXCL10的浓度。目的4：在4小时的时间内，我们将监测麻醉内毒素中毒小鼠的外周动脉血压和心率，这些小鼠在LPS刺激后1小时接受了载体对照或R-100治疗，以确认治疗方案的血流动力学中性。我们预计R- 100将优于HMP和ISMN，无论是单独使用还是联合使用，并且在LPS攻击后6小时启动时有效，从而验证其作为临床败血症潜在治疗方法的实用性。

DESCRIPTION (provided by applicant): Alterations in the biosynthesis of the free radicals nitric oxide (NO) and superoxide anion are generally accepted to contribute to widespread tissue injury in sepsis via their induction of microvacular ischemia and direct organ toxicity. To correct this free radical imbalance, Radikal Therapeutics (RTX) is developing a first- in-class small molecule drug (R-100), a bifunctional redox-based technology formed from the covalent linkage of 2 chemical moieties: 1) an organic nitrovasodilator that releases NO, and 2) a pyrrolidine nitroxide that acts as a trifunctional catalyst of reactive oxygen species degradation: a superoxide dismutase mimetic, a catalase mimic that detoxifies hydrogen peroxide, and a peroxynitrite decomposition catalyst. In an LD100 murine model of endotoxinemia, resuscitation by R-100 starting 1 h after lipopolysaccharide (LPS) challenge produces 100% survival, accompanied by near complete protection against end-organ injury. We hypothesize that R-100 is superior to the sum of its two component functionalities and that the covalent fusion of these two properties into a single molecular entity creates a strong commercial prospect for therapy of sepsis. RTX will test this hypothesis by carrying out endotoxemic studies to establish the dose-response, time-window, mechanism of action, and safety in mice of intraperitoneal (IP) administered R-100. Aim #1: R-100 at 3 dose levels will be compared to vehicle control 1 h post lipopolysaccharide (LPS) challenge, in order to establish the lowest dose providing optimal outcome ("LDPOO"). Tissue levels of R-100 and metabolites from heart, lung, kidney, and liver will be measured, in order to relate plasma and organ drug uptake. Aim #2: the LDPOO dose of R-100 will be compared to equimolar doses of hydroxymethylproxyl ("HMP", the nitroxide component of R-100), isososorbide mononitrate ("ISMN", a classic monofunctional NO donor), and the combination of HMP and ISMN, in order to verify that a bifunctional compound (R-100) is superior to a mixture of its component functionalities. Treatment will be initiated 1 h after LPS challenge. Aim #3: We will determine the duration of the therapeutic time window by introducing R-100 1, 2, 4, and 8 h after LPS challenge. Serum and tissue will be examined 48 h post LPS dosing in each of the above Aims for determination of morphologic and biochemical endpoints, including lipid peroxidation (F21-isoprostane, GSH:GSSG ratio), neutrophil infiltration, 3- nitrotyrosine (3-NT), nitrite/nitrate, and poly(ADP-ribose) formation, BUN, creatinine, NGAL, AST, ALT, histology injury score, and serum concentrations of TNF-1, HMGB-1, IL-6, TRP14, Trx1, LC8, I:B1, IL-12, MIP-11, CXCL9, and CXCL10. Aim #4: Over a 4 h period, we will monitor peripheral arterial blood pressure and heart rate in anesthetized endotoxinemic mice treated with vehicle control or R-100 administered 1 h after LPS challenge, in order to confirm the hemodynamic neutrality of the treatment regimen. We expect that R- 100 will be superior to HMP and ISMN, alone and in combination, and will be effective when initiated 6 h after LPS challenge, thereby verifying its utility as a potential therapeutic for clinical sepsis. PUBLIC HEALTH RELEVANCE: Septic shock resulting from acute bowel perforation and infection is a major cause of mortality. At present, there is no approved therapy for this condition and prognosis is uniformly poor. We are developing a novel drug that targets the basic mechanisms of septic shock, and has proven effective in experimental models of septic inflammation. We will now test this agent in a series of investigations in order to determine the optimal dose, to confirm the mechanism of action, and to establish the window of opportunity after disease onset within which therapy may be initiated.