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）：化脓性并发症导致的多器官损伤和衰竭会导致显着的发病率和死亡率，特别是对于患有严重细菌感染的儿童和老年患者。内毒素休克/败血症的发病机制尚未得到详细了解，而且其治疗（包括抗生素）仍然主要是对症和支持性的。内毒素（脂多糖，LPS）在循环中释放的内源性蛋白质在很大程度上被认为是损伤的标志，很少被视为生命系统抵抗和对抗疾病的尝试。急性期反应蛋白，如 C 反应蛋白和 TNF-a，经常被用作全身炎症和感染性休克的诊断。其他指标，包括 ALT、NGAL 和肌钙蛋白，分别指示更具体的器官损伤，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所知。然而，肝损伤（包括内毒素引起的肝损伤）的具体诊断标志物仍然难以捉摸。在针对包括LPS在内的肝毒素引起的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基琥珀酸合酶（ASS）是一种高度敏感的标志物，并在治疗后30分钟内大量释放到血液中。 LPS/D-半乳糖胺，以及在较小程度上单独的 LPS。鉴于 LPS 和 D-gal 的组合会对肝脏产生显着损害，ASS（一种线粒体外膜/胞质酶）的释放可以被认为是肝损伤的早期迹象，甚至早于血液中 ALT 或 AST 的增加。我们的实验室已完成血浆/血清中高灵敏度诊断 ASS SW ELISA 的开发和验证。在 ASS 生物标志物研究过程中，我们发现了一个有趣的现象，即重组 ASS (rASS) 几乎可以消除高剂量内毒素对培养物中小鼠巨噬细胞造成的损伤（即使是在 LPS 攻击后 1 小时添加），并减少 TNF-a发布（Prima 等人，2010）。另一项研究报告称 ASS 实际上与 LPS 发生物理结合。在初步实验中，我们能够证明，注射 rASS 显着降低了用高剂量 LPS 治疗的小鼠死亡率（注射后 32 小时为 50% vs. 100%）。我们假设 ASS 是内毒素中和系统的天然组成部分，它与其他抗氧化系统协同作用，以防止内毒素/脓毒症损伤。该项目的最终目标是基于肝精氨酸琥珀酸合酶（ASS）的能力，开发一种针对败血症/内毒素血症的新型特异性疗法，以改善细菌内毒素和异常免疫反应的有害影响，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、无内毒素重组人 ASS 蛋白。 （项目的 1-4 个月）。具体目标 2. 表征 rASS 制剂的体外抗菌和抗炎活性。 （项目 2-12 个月）具体目标 3：检查小鼠脓毒症和内毒素休克模型中 rASS 的治疗效果，并建立最有效的方案（项目 8-24 个月）第一阶段可交付成果：内毒素血症的治疗方案/rASS 小鼠败血症。第一阶段里程碑：验证 rASS 制剂用于治疗小鼠模型中的内毒素血症/败血症。治疗级、不含 LPS 的 rASS 对于后续的人体临床试验来说应该是安全的，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：脓毒症并发症引起的多器官损伤和衰竭仍然是发病和死亡的重要原因。我们建议基于重组肝精氨酸琥珀酸合酶（rASS）的能力，开发一种针对内毒素血症/败血症的新型特异性疗法，以改善细菌内毒素和异常免疫反应的有害影响，并减轻多器官损伤。在第一阶段，将生产治疗级、无标签和内毒素的工程化人类重组 ASS，并将确定其在小鼠模型中治疗内毒素血症/败血症的效率。结果将是经过验证的 rASS 制剂和方案，用于治疗小鼠模型中的内毒素血症/败血症。