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

重组肝精氨酸琥珀酸合酶（rASS）用于治疗脓毒症/终点

• 批准号: 8121317
• 负责人: STANISLAV I SVETLOV
• 金额: $ 29.14万
• 依托单位: BANYAN BIOMARKERS, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8121317
• 关键词: Acute-Phase Proteins; Acute-Phase Reaction; Affinity; Aftercare; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Antioxidants; Apoptosis; Bacillus subtilis; Bacterial Infections; Binding; Biological Assay; Biological Markers; Blood; Blood Circulation; C-reactive protein; CCL2 gene; Cell Culture Techniques; Cell Line; Childhood; Clinical Trials; Development; Diagnostic; Disease; Dose; Drug Formulations; Endotoxemia; Endotoxic Shock; Endotoxins; Ensure; Enzyme-Linked Immunosorbent Assay; Enzymes; Escherichia coli; Failure; Galactosamine; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Growth; Heart; Hepatic; High Pressure Liquid Chromatography; Hour; Human; Human Engineering; Immune response; In Vitro; Inflammation; Injection of therapeutic agent; Injury; Interferon Type II; Interleukin-1; Interleukin-10; Interleukin-6; Kidney; Laboratories; Ligation; Lipopolysaccharides; Liver; Maltose; Mass Spectrum Analysis; Modeling; Monitor; Morbidity - disease rate; Mus; N-terminal; Organ; Organism; Outcome; Outer Mitochondrial Membrane; Pathogenesis; Patients; Phase; Plasma; Preparation; Production; Protein Biosynthesis; Proteins; Protocols documentation; Puncture procedure; RANTES; Recombinants; Reporting; Residual state; Role; Sepsis; Septic Shock; Serum; Staphylococcus aureus; System; TNF gene; Testing; Therapeutic; Time; Treatment Efficacy; Treatment Protocols; Troponin; Validation; argininosuccinate synthase; base; chemokine; combat; cytokine; cytotoxicity; enteropeptidase; hepatotoxin; in vivo; liver-specific protein; macrophage; mortality; mouse model; multiorgan injury; novel; older patient; research study; response; sephadex; septic; urea cycle; vector

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.

描述（由申请方提供）：脓毒性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未得到很详细的了解，而且其治疗，包括抗生素，仍然主要是对症和支持。在循环中响应于内毒素（脂多糖，LPS）释放的内源性蛋白质在很大程度上被认为是损伤的迹象，而很少被认为是生命系统对抗和对抗疾病的尝试。急性时相反应的蛋白质如C-反应蛋白和TNF-α已经常用作全身性炎症和感染性休克的诊断。包括ALT、NGAL和肌钙蛋白在内的其他指标分别指示更特异性的器官损伤，例如肝脏、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们所知。然而，肝损伤的特异性诊断标志物，包括内毒素诱导的肝损伤，仍然难以捉摸。在对包括LPS在内的肝毒素引起的肝损伤进行生物标志物研究的过程中，我们发现尿素循环的酶--氨基琥珀酸合成酶（ASS）是一种高度敏感的标志物，在用LPS/D-半乳糖胺处理后30分钟内大量释放到血液中，在较小程度上，单独用LPS处理后。考虑到LPS和D-gal的组合对肝脏产生显著损伤，ASS（一种外线粒体膜/胞质酶）的释放可以被认为是肝损伤的早期迹象，甚至在血液中ALT或AST增加之前。我们实验室已完成了血浆/血清中高灵敏度诊断ASS SW ELISA的开发和验证。在ASS生物标志物研究期间，我们发现了一个有趣的现象，即重组ASS（rASS）几乎消除由高剂量内毒素诱导的培养物中小鼠巨噬细胞损伤的能力，即使在LPS攻击后1小时加入时，并且减少TNF-α释放（Prima等人，2010年）。另一项研究报告说，ASS实际上是物理结合LPS。在初步实验中，我们能够证明注射rASS显著降低了用高剂量LPS处理的小鼠死亡率（在注射后32小时为50%对100%）。我们假设ASS是内毒素中和系统的天然成分，与其他抗氧化系统协同作用，以保护免受内毒素/脓毒性损伤。该项目的最终目标是开发一种新的，特异性的治疗脓毒症/内毒素血症的能力的基础上，肝琥珀酸合成酶（ASS），以减轻细菌内毒素和异常免疫反应的损伤作用，并减轻人类患者的多器官损伤。在第一阶段，将实现以下具体目标：具体目标1：开发，纯化和表征治疗级，无内毒素的重组人ASS蛋白。(1-4月的项目）。具体目标2。表征rASS制剂的体外抗菌和抗炎活性。(2-12具体目标3：检查rASS在小鼠脓毒症和内毒素休克模型中的治疗功效，并建立可从I期获得的最有效的方案（项目的8-24个月）：通过rASS治疗小鼠内毒素血症/脓毒症的方案。I期里程碑：rASS制剂治疗小鼠模型内毒素血症/脓毒症的验证。治疗级无LPS rASS对于后续的人体临床试验应该是安全的，因为它代表了一种内源性肝脏特异性蛋白。 公共卫生相关性：脓毒性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。我们建议开发一种新的，特异性的治疗内毒素血症/脓毒症的基础上重组肝琥珀酸合成酶（rASS）的能力，以减轻细菌内毒素和异常免疫反应的损伤作用，并减轻多器官损伤。在I期，将生产治疗级、无标签和无内毒素的工程化人重组ASS，并将确定其在小鼠模型中治疗内毒素血症/脓毒症的效率。结果将是用于治疗小鼠模型中内毒素血症/脓毒症的经验证的rASS制剂和方案。