Alpha B crystallin as a novel therapeutic for SIRS and Sepsis

描述(申请人提供)：Alpha B晶体蛋白(CryAB)是小热休克蛋白家族的成员，最近被证明是一种广泛的抗炎剂，在多发性硬化症、类风湿性关节炎、缺血-再灌注损伤和中风的动物模型中有效。这一建议的基础是确定CryAB在内毒素中毒和细菌性败血症模型中是否有效。这项资助的目的首先是证明CryAB，而不是在残基120处有点突变的CryAB，可以(I)以剂量依赖的方式降低注射内毒素和(Ii)小鼠血清IL-6、血清淀粉样蛋白A和C反应蛋白的水平。与未经治疗的动物相比，注射致死剂量的内毒素可延长动物的寿命。第二个目的是通过比较炎症细胞因子的调节与小鼠激活蛋白C治疗观察到的结果来确定CryAB的效力之间的相关性。激活蛋白C是唯一被批准用于治疗脓毒症的药物，任何预期的治疗方法都必须同等或更有效。此外，我们建议确定CryAB和激活的蛋白C是否可以通过这两种蛋白的共同作用而发挥协同作用。最后一个目的是检测注射脂多糖后不同时间小鼠血浆中CryAB的水平，以确定该蛋白是否是疾病严重程度的标志，并支持CryAB在调节炎症方面具有天然作用的假设。此外，还将监测创伤患者血浆中的CryAB水平，以确定该蛋白是否可用作SIRS进展为感染性休克的标志。 CryAB的作用模式似乎与该蛋白作为伴侣的角色有关。小分子热休克蛋白结合部分未折叠蛋白并降低其在炎症部位的浓度，从而限制聚集和炎症刺激的能力。使用质谱仪，我们已经证明了CryAB以一种温度依赖的方式与50到75种不同的血浆蛋白质结合，其中绝大多数是急性期蛋白质或补体或凝血级联的成员。对这些下拉实验的上清液进行分析表明，许多血浆蛋白的浓度明显降低。这一数据为CryAB与大量尚未达到临床里程碑的潜在疗法在这种复杂疾病中区分开来提供了科学基础。我们认为，αB晶体蛋白的广泛特异性可以使该蛋白成为治疗脓毒症和全身炎症反应综合征的有效药物。 与公共卫生相关：败血症是一种复杂的炎症调节失调，由宿主对细菌感染或严重创伤的反应引起，占重症监护病房死亡人数的一半以上，每年超过20万人死亡。在过去的二十年里，超过30种不同的抗炎药临床试验都失败了。这项建议旨在提供临床前支持，即一种新的生物治疗药物，αB晶体蛋白，可以成为治疗脓毒症的有效药物。这种乐观情绪的智力框架是，与先前临床试验中使用的候选药物相比，蛋白质调节血浆中广泛的炎症介质的能力导致这种复杂疾病的血清标志物显着减少。

DESCRIPTION (provided by applicant): Alpha B crystallin (Cryab), a member of the small heat shock protein family, recently has been shown to be a broad based anti-inflammatory agent effective in animal models of multiple sclerosis, rheumatoid arthritis, ischemia-reperfusion injury, and stroke. The basis of this proposal is to establish whether Cryab is effective in models of LPS toxemia and bacterial sepsis. The aims of the grant are first to demonstrate that Cryab, and not Cryab with a point mutation at residue 120, can (i.) reduce in a dose dependent manner serological levels of IL-6, serum amyloid A, and C reactive protein in mice injected with LPS and (ii.) extend the lifetime of animals administered lethal doses of LPS compared with untreated animals. The second aim is to establish the relevance of the potency of Cryab by comparing the modulation of inflammatory cytokines with that observed for treatment with murine activated protein C. Activated protein C is the only approved drug for sepsis and any prospective therapeutic must be equally or more potent. In addition, we propose to determine whether Cryab and activated protein C could act synergistically by the co-administrating of the two proteins. The last aim is to assay the levels of Cryab in murine plasma at various times after injection with LPS to determine whether the protein is a marker of the severity of the disease and also support the hypothesis that Cryab has a natural role in modulating inflammation. In addition, Cryab levels in plasma from human trauma patients will be monitored to determine whether this protein can be used as a marker for progression of SIRS into septic shock. The mode of action of Cryab appears to be linked with the protein's role as a chaperone. The ability of the small heat shock proteins to bind partially unfolded proteins and lower their concentration at sites of inflammation and consequently limit aggregation and the inflammatory stimulus. Using mass spectrometry we have demonstrated that Cryab binds between 50 and 75 different plasma proteins in a temperature dependent fashion, with the vast majority being acute phase proteins or members of the complement or coagulation cascades. Analyzing the supernatants of these pull down experiments demonstrate that the concentration of many of the plasma proteins was reduced significantly. This data provides scientific foundation that Cryab can be distinguished from the large number of potential therapeutics that have not achieved their clinical milestones in this complex disease. We believe that the broad specificity of alpha B crystallin could enable the protein to be an effective therapeutic for sepsis and SIRS. PUBLIC HEALTH RELEVANCE: Sepsis, a complex dysregulation of inflammation resulting from the host response to a bacterial infection or severe trauma, is responsible for over half of the deaths in intensive care units accounting for more than 200,000 deaths per year. Over 30 different clinical trials of anti-inflammatory agents have failed during the past twenty years. This proposal seeks to provide preclinical support that a novel biotherapeutic, alpha B crystallin, can be an effective drug for sepsis. The intellectual framework for this optimism is the protein's capacity to modulate a far wider range of inflammatory mediators in plasma than the drug candidates used in previous clinical trials resulting in significant reductions of the serological markers of this complex disease.