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: 8249357
• 负责人: STANISLAV I SVETLOV
• 金额: $ 28.96万
• 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 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8249357
• 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; public health relevance; 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（由申请人提供）：败血症并发症引起的多器官损伤和失败导致明显的发病率和死亡率，特别是在患有严重细菌感染的儿科和老年患者中。内毒性休克/败血症的发病机制尚未详细了解，此外，其疗法（包括抗生素）在很大程度上仍然是症状和支持。响应内源毒素（脂多糖，LPS）释放的内源性蛋白在很大程度上被认为是损害的迹象，很少是生活系统来抵消和应对疾病的尝试。急性期反应的蛋白质（例如C反应蛋白和TNF-A）经常被用作全身炎症和败血性休克的诊断。其他，包括ALT，NGAL和肌钙蛋白在内，分别表明了更具体的器官损害，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已长期闻名。然而，包括内毒素诱导在内的肝损伤的特定诊断标记仍然难以捉摸。在针对包括LPS在内的肝毒素的生物标志物研究过程中，我们发现尿素周期的酶Argininoscication合成酶（ASS）是一种高度敏感的标记物，在使用LPS/D-甲乳糖苷和单独的LPS治疗后30分钟内，在血液中大量释放出大量的血液释放。鉴于LPS和D-GAL的组合会对肝脏产生重大损害，因此屁股的释放是一种外部线粒体膜/胞质酶，可以被认为是肝损伤的早期迹象，甚至是血液中ALT或AST的增加。在我们的实验室中，已经完成了血浆/血清中高度敏感的诊断屁股SW ELISA的开发和验证。在ASS生物标志物研究中，我们发现了一种有趣的现象，即重组屁股（RASS）几乎消除了由高剂量的内毒素引起的培养中小鼠巨噬细胞损害的能力，即使在LPS挑战后增加了1小时，并减少TNF-A释放（Prima等，2010，2010）。另一项研究报告说，屁股实际上是在物理上结合了LP。在试点实验中，我们能够证明注射RASS可以显着降低用高剂量LPS​​治疗的小鼠死亡率（注射后32小时，在50％vs.100％）。我们假设ASS是内和毒素中和系统的自然组成部分，该系统与其他抗氧化剂系统一起起作用，以防止内毒素/化粪池损伤。该项目的最终目标是基于肝非毒素习惯蛋白合成酶（ASS）改善细菌内毒素和异常免疫反应的造成伤害作用的能力，并减轻人类患者的多器官损伤，从而开发出一种新型的败血症/内毒素治疗的新型疗法。在第一阶段，将实现特定目标：具体目标1：开发，纯化和表征治疗级，无内毒素的重组人屁股蛋白。 （该项目的1-4个月）。特定的目标2。在体外表征RASS制剂的抗细菌和抗炎活性。 （该项目的2-12个月）具体目标3：检查小鼠败血症和内毒性休克模型中的RASS治疗功效，并建立可从I阶段的最有效方案（项目的8-24个月）从RASS中的I阶段：内毒素/败血症的治疗方案传递。第一阶段的里程碑：在小鼠模型中治疗内毒素血症/败血症的RASS公式的验证。治疗等级，无LPS的RAS应该是随后的人类临床试验安全的，因为它代表了内源性肝特异性蛋白。 公共卫生相关性：化粪池并发症引起的多器官损伤和失败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偶联合酶（RASS）来改善细菌内毒素和异常免疫反应的造成伤害作用的能力，为内毒素/败血症开发一种新型的，特异性的治疗方法。在第一阶段，将产生一种治疗等级，无标记和无内毒素的人重组屁股，并将在小鼠模型中建立其治疗内毒素/败血症的效率。结果将是一种经过验证的RASS公式和方案，用于治疗小鼠模型中内毒素/败血症。