Recombinant hepatic argininosuccinate synthase (rASS) for treatment of sepsis/end

描述(由申请人提供)：败血症并发症导致的多器官损伤和衰竭导致显著的发病率和死亡率，尤其是在患有严重细菌感染的儿科和老年患者中。内毒素休克/脓毒症的发病机制尚不十分清楚，其治疗方法，包括抗生素，仍在很大程度上是对症和支持的。循环中释放的内源性蛋白对内毒素(内毒素，LPS)的反应在很大程度上被认为是损伤的迹象，很少被认为是生命系统对抗和抗击疾病的尝试。急性期反应蛋白如C-反应蛋白、肿瘤坏死因子-α等常被用于全身炎症和感染性休克的诊断。其他的，包括ALT、NGAL和肌钙蛋白，分别表明更具体的器官损伤，如肝脏、肾脏和心脏。肝脏在急性期蛋白质合成中的特殊作用早已为人所知。然而，肝损伤的特定诊断标志，包括内毒素诱导，仍然难以捉摸。在对包括内毒素在内的肝毒素所致肝损伤的生物标志物研究过程中，我们发现尿素循环酶精氨酸琥珀酸合成酶(ASS)是一种高度敏感的标志物，经内毒素/D-氨基半乳糖处理后30分钟内在血液中大量释放，仅在30分钟内释放。鉴于内毒素和D-半乳糖会对肝脏产生显著的损害，ASS的释放，一种外部线粒体膜/胞浆酶，可以被认为是肝脏损伤的早期迹象，甚至在血液中ALT或AST升高之前。本实验室已建立并验证了一种高灵敏度的血浆/血清ASS软件酶联免疫吸附试验。在ASS生物标志物研究中，我们发现了一个有趣的现象，重组ASS(RASS)能够几乎消除高剂量内毒素对培养的小鼠巨噬细胞造成的损害，即使在脂多糖攻击后1小时添加，并减少肿瘤坏死因子-α的释放(Prima等人，2010年)。另一项研究报告说，ASS实际上与内毒素有物理上的结合。在初步实验中，我们能够证明注射RASS显著降低了高剂量内毒素治疗的小鼠死亡率(注射后32h为50%比100%)。我们假设，ASS是内毒素中和系统的天然组成部分，与其他抗氧化剂系统协同作用，以保护免受内毒素/败血症的侮辱。该项目的最终目标是开发一种基于肝脏精氨酸琥珀酸合成酶(ASS)能力的新型、特异的脓毒症/内毒素血症治疗方法，以减轻细菌内毒素的损伤效应和异常的免疫反应，并减轻人类患者的多器官损伤。在第一阶段，将实现以下特定目标：特定目标1：开发、纯化和鉴定一种治疗级、无内毒素的重组人驴蛋白。(项目的1-4个月)。具体目的2.确定RASS制剂的体外抗菌和抗炎活性。(项目2-12个月)具体目标3：检验RASS对小鼠败血症和内毒素休克模型的治疗效果，并建立第一阶段可交付的最有效方案(项目8-24个月)：RASS对小鼠内毒素血症/败血症的治疗方案。第一阶段里程碑：RASS配方在小鼠模型中用于治疗内毒素血症/败血症的有效性。治疗级，无内毒素的RAS对于后续的人类临床试验应该是安全的，因为它代表一种内源性肝脏特异性蛋白。 公共卫生相关性：败血症并发症引起的多器官损伤和衰竭仍然是发病率和死亡率的重要原因。我们建议基于重组肝精氨酸琥珀酸合成酶(RASS)减轻细菌内毒素的损伤效应和异常的免疫反应，并减轻多器官损伤的能力，开发一种新的、特异的内毒素血症/脓毒症治疗方法。在第一阶段，将生产一种治疗级、无标签和无内毒素的重组人ASS，并建立其治疗小鼠内毒素血症/败血症模型的有效性。结果将是一种有效的RAS配方和方案，用于治疗小鼠模型的内毒素血症/败血症。

DESCRIPTION (provided by applicant): Multiorgan injury and failure due to septic complications result in significant morbidity and mortality, especially in pediatric and elderly patients with severe bacterial infections. Pathogenesis of endotoxic shock/sepsis has not been understood in great detail, moreover its therapy, including antibiotics, remains largely symptomatic and supportive. The endogenous proteins released in circulation in response to endotoxin (lipopolysaccharide, LPS) have been largely considered as a sign of damage, and rarely as an attempt of the living system to counteract and combat the disease. The proteins of acute phase response such as C-reactive protein and TNF-a, have been frequently used as diagnostics of systemic inflammation and septic shock. Others, including ALT, NGAL, and troponin are indicative of more specific organ damage such as liver, kidney, and heart, respectively. The particular role for liver in acute phase protein synthesis has been known for long time. Yet, specific diagnostic markers of liver injury, including induced by endotoxin, remains elusive. In the course of biomarker study for liver injury in response to hepatotoxins including LPS, we found that an enzyme of urea cycle, argininosuccinate synthase (ASS), is a highly sensitive marker and is released in large amounts in blood within 30 minutes after treatment with LPS/D-galactosamine and, to a lesser extent, LPS alone. Given that combination of LPS and D-gal produces significant damage to the liver, the release of ASS, an outer mitochondrial membrane/cytosolic enzyme, can be considered an early sign of hepatic injury, preceding even the increase of ALT or AST in blood. The development and validation of a highly sensitive diagnostic ASS SW ELISA in plasma/serum has been completed in our laboratory. During ASS biomarker studies, we discovered an intriguing phenomenon, the ability of recombinant ASS (rASS) to nearly abolish the damage to mouse macrophages in culture induced by high doses of endotoxin, even when added 1 hour after LPS challenge, and reduce TNF-a release (Prima et al., 2010). Another study reported that ASS actually physically binds LPS. In pilot experiments, we were able to demonstrate that injection of rASS significantly decreased mice mortality treated with high doses of LPS (50 % vs. 100 % at 32 h post-injection). We hypothesize that ASS is a natural component of the endotoxin neutralization system that acts in concert with other antioxidant systems to protect from endotoxin/septic insults. Ultimate Goal of the project is to develop a novel, specific therapy for sepsis/endotoxemia based on the ability of hepatic argininosuccinate synthase (ASS) to ameliorate injurious effects of bacterial endotoxins and aberrant immune response, and mitigate multiple organ injury in human patients. In phase I, following specific aims will be accomplished: Specific Aim 1: Develop, purify and characterize a therapeutic-grade, endotoxin-free recombinant human ASS protein. (1-4 months of the project). Specific Aim 2. Characterize anti-bacterial and anti-inflammatory activity of rASS formulations in vitro. (2-12 month of the project) Specific Aim 3: Examine rASS treatment efficacy in models of sepsis and endotoxic shock in mice and establish the most efficient protocol (8-24 months of the project) Deliverable from phase I: Treatment protocol for endotoxemia/sepsis in mice by rASS. Phase I Milestone: Validation of rASS formulation for treatment of endotoxemia/sepsis in mouse models. Therapeutic grade, LPS-free rASS should be safe for subsequent human clinical trials since it represents an endogenous liver-specific protein. PUBLIC HEALTH RELEVANCE: Multiorgan injury and failure due to septic complications remains a significant cause of morbidity and mortality. We propose to develop a novel, specific therapy for endotoxemia/sepsis based on the ability of recombinant hepatic argininosuccinate synthase (rASS) to ameliorate injurious effects of bacterial endotoxins and aberrant immune response, and mitigate multiple organ injury. In phase I, a therapeutic grade, tag-and endotoxin-free engineered human recombinant ASS will be produced and its efficiency to treat endotoxemia/sepsis in mouse models will be established. Outcome will be a validated rASS formulation and protocol for treatment of endotoxemia/sepsis in mouse models.