Peptide inhibitors of oxidative heme toxicity in acute hemolysis

急性溶血中氧化血红素毒性的肽抑制剂

• 批准号: 9751956
• 负责人: NEEL KUMAR KRISHNA
• 金额: $ 18.75万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751956
• 关键词: Acute; Acute Renal Failure with Renal Papillary Necrosis; Animal Model; Antioxidants; Autoimmune hemolytic anemia; Bilirubin; Binding; Biological Assay; Blood Chemical Analysis; Cells; Cessation of life; Classical Complement Pathway; Complement; Complement 1q; Complement Activation; Complement Inactivators; Creatinine; Cysteine; Data; Deposition; Development; Disease; Dose; Drug Metabolic Detoxication; Erythrocyte Transfusion; Erythrocytes; Free Radical Formation; Functional disorder; Goals; Heme; Hemoglobin; Hemoglobin concentration result; Hemolysis; Hemolytic Anemia; Histopathology; Human; Immunohistochemistry; In Vitro; Inflammation; Injury; Kidney; Kidney Failure; Laboratories; Lead; Mediating; Medical; Methemoglobin; Modeling; Molecular; Organ; Outcome; Oxidation-Reduction; Oxidative Stress; Oxides; Pathologic; Peptides; Peroxidases; Pharmaceutical Preparations; Phenotype; Play; Process; Property; Publishing; Rattus; Reaction; Reactive Oxygen Species; Renal tubule structure; Research; Research Project Grants; Role; Sickle Cell Anemia; Sulfhydryl Compounds; System; Therapeutic; Tissues; Toxic effect; Transfusion; Weight; Wistar Rats; base; clinically significant; in vitro Assay; in vitro activity; in vivo; inflammatory milieu; inhibiting antibody; inhibitor/antagonist; nephrotoxicity; novel; novel therapeutics; oxidative damage; prevent; prophylactic; renal damage; research clinical testing; transfusion medicine

Acute or severe hemolysis result from the release of significant amounts of toxic free heme leading to organ, tissue and cellular damage manifested in a variety of pathological conditions such as acute hemolytic transfusion reactions (AHTR). In the field of transfusion medicine, AHTR, while rare, is a clinically significant concern. AHTR-mediated RBC hemolysis releases hemoglobin (Hb) into the vasculature which promotes a pro-oxidative and pro-inflammatory environment. Additionally, free heme is exquisitely nephrotoxic leading to renal failure through a number of mechanisms including oxidative damage and heme deposition in the renal tubules. Given the highly destructive properties of cell free heme, development of an inhibitor to detoxify its oxidative properties remains a major unmet medical need. Our laboratory has developed peptide constructs known as Peptide Inhibitors of Complement C1 (PIC1) that inhibit antibody-initiated, classical complement pathway mediated destruction of human RBC in vitro as well as in a rat model of AHTR. Recently, we have discovered that PIC1 can inhibit the peroxidase activity of RBC lysates and purified human Hb in in vitro assays via an antioxidant mechanism mediated by two cysteine residues contained within the PIC1 sequence. Given its dual ability to inhibit complement-mediated RBC destruction and neutralize Hb peroxidase activity, PIC1 provides an exceptional opportunity to mitigate the AHTR disease process. The experimental focus of this project is to assess inhibition of heme peroxidase toxicity of PIC1 in our well-defined rat model as well as define the mechanism by which PIC1 exerts its inhibitory effect on Hb with the long range goal of producing a therapeutic molecule for clinical testing in humans. Our specific hypothesis is that PIC1 will inhibit Hb peroxidase activity in vivo via its reactive cysteine residues, thus limiting toxic free radical formation and inflammation minimizing downstream sequelae, including kidney failure and death. Specific Aim 1 will evaluate the efficacy of PIC1 in preventing heme-mediated tissue and organ injury created by infusing RBC lysates in the AHTR rat model. Following optimization of the amount of RBC lysates required to induce a reliable phenotype, prophylactic and rescue administration of PIC1 in the animal model will be assessed. Readouts will consist of various blood chemistry markers as well as kidney damage evaluated by histopathology. Specific Aim 2 will evaluate the mechanism of PIC1 peroxidase inhibitory activity. PIC1 acts as an antioxidant to inhibit peroxidase activity of Hb via a redox mechanism involving the two cysteine residues. We will evaluate the role each of these cysteine residues play in this activity in vitro and in vivo. First we will utilize derivatives of PIC1 in which one or both cysteine residues are substituted to assess Hb peroxidase inhibitory/antioxidant activity in vitro. Secondly, we will determine the efficacy of selected PIC1 derivatives on inhibition of heme toxicity in the animal model. Successful accomplishment of these aims will establish PIC1 as a novel therapeutic to detoxify free heme in the setting of acute hemolysis such as AHTR, hemolytic anemia and sickle cell disease.

急性或严重溶血是由于释放大量有毒游离血红素导致器官， 组织和细胞损伤表现在各种病理状况中，例如急性溶血性 输血反应（AHTR）。在输血医学领域，AHTR虽然罕见，但却是临床上重要的 关心AHTR介导的RBC溶血将血红蛋白（Hb）释放到血管系统中， 促氧化和促炎症环境。此外，游离血红素具有很强的肾毒性， 肾功能衰竭通过多种机制，包括氧化损伤和血红素沉积在肾脏 小管鉴于无细胞血红素的高度破坏性，开发一种抑制剂来对其解毒， 氧化性能仍然是一个主要的未满足的医疗需求。我们的实验室已经开发了肽结构 称为补体C1肽抑制剂（PIC 1），其抑制抗体起始的经典补体 在体外以及在AHTR的大鼠模型中的人RBC的通路介导的破坏。最近我们 发现PIC 1在体外可抑制红细胞裂解物和纯化的人血红蛋白的过氧化物酶活性 通过由PIC 1序列中包含的两个半胱氨酸残基介导的抗氧化机制进行测定。 鉴于其抑制补体介导的RBC破坏和中和Hb过氧化物酶活性的双重能力， PIC 1为缓解AHTR疾病过程提供了一个特殊的机会。实验重点 本项目是评估PIC 1在我们定义明确的大鼠模型中对血红素过氧化物酶毒性的抑制作用，以及 定义PIC 1对Hb发挥抑制作用的机制，其长期目标是产生一种抑制作用， 用于人类临床试验的治疗分子。我们的具体假设是，PIC 1将抑制Hb 通过其反应性半胱氨酸残基在体内具有过氧化物酶活性，从而限制有毒自由基的形成， 炎症最小化下游后遗症，包括肾衰竭和死亡。具体目标1将评估 PIC 1在预防血红素介导的组织和器官损伤中的功效，所述组织和器官损伤是通过将RBC裂解物输注到 AHTR大鼠模型。在优化诱导可靠的免疫应答所需的RBC裂解物的量之后， 将评估动物模型中PIC 1的表型、预防性和补救性给药。读数将 包括各种血液化学标志物以及通过组织病理学评估的肾损伤。具体 目的2探讨PIC 1抑制过氧化物酶活性的机制。PIC 1作为一种抗氧化剂， 通过涉及两个半胱氨酸残基的氧化还原机制，血红蛋白的过氧化物酶活性。我们将评估 这些半胱氨酸残基中的每一个都在体外和体内发挥这种活性。首先，我们将利用PIC 1的衍生物， 其中一个或两个半胱氨酸残基被取代以评估Hb过氧化物酶抑制/抗氧化活性， 体外其次，我们将确定所选择的PIC 1衍生物对血红素毒性抑制的功效， 动物模型这些目标的成功实现将使PIC 1成为一种新型的解毒治疗药物。 在急性溶血如AHTR、溶血性贫血和镰状细胞病的情况下的游离血红素。