Intracellular targeting Hsp70 for pulmonary cytoprotection after toxin inhalation

细胞内靶向 Hsp70 用于毒素吸入后的肺细胞保护

基本信息

批准号：
8920321
负责人：
Robert Nishimura
金额：
$ 5万
依托单位：
RUBICON BIOTECHNOLOGY, INC.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-25 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8920321
关键词：
Accidents Address Adverse effects Aerosols Air Alveolar Alveolar Cell Antibodies Binding Biological Assay Breathing Bronchiolo-Alveolar Adenocarcinoma Buffers Cell Death Cell Survival Cells Cerebral Infarction Cessation of life Chemical Industry Chemicals Chimeric Proteins Chromatography Clinical Trials Clinical effectiveness Cytoprotection Cytoprotective Agent DNA Data Analyses Development Dose Drug Formulations Effectiveness Electrophoresis Event Exposure to FDA approved Funding Gases Glutathione Goals Health Heat shock proteins Heat-Shock Proteins 70 Histology Human Hydrogen Peroxide In Situ In Vitro Industrial Accidents Industry Infarction Inhalant dose form Injury Intracellular Transport Lung Mass Spectrum Analysis Maximum Tolerated Dose Measures Mediating Medical Membrane Mission Modeling Molecular Nucleosides Oxidation-Reduction Oxidative Stress Oxygen Pathway interactions Phase III Clinical Trials Phosgene Plants Preparation Prevalence Primates Production Protein Denaturation Pulmonary Edema Radioimmunoconjugate Rattus Reactive Oxygen Species Reagent Research Research Personnel Research Proposals Self Administration Self-Administered Source Specificity Staging Stroke Structure of parenchyma of lung Surface System Testing Therapeutic Time Tissues Toxic effect Toxin Training Treatment Efficacy Weight Weight Gain Work aerosolized base brain tissue cell injury efficacy testing emergency service responder extracellular in vivo innovation liquid formulation novel oxidative damage pre-clinical prevent protein aggregation respiratory small molecule stability testing therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): In 2003, the EPA identified 123 chemical plants in the nation where a terrorist attack or accident could potentially expose more than 1 million people to a cloud of toxic gas. Upon exposure to certain chemicals, a major cause of cell death is from oxidative stress. Current strategies to prevent oxidative stress include the use of small molecule reagents, but they lack specificity and require excessive material to be delivered to the lungs possibly causing dangerous side effects. We developed the Fv fragment of a cell-penetrating antibody, mAb 3E10, as an intracellular transporter to deliver heat-shock protein 70 (Fv-Hsp70) into cells, and we demonstrated cytoprotection against oxidative damage in vitro and in vivo. Our long-term goal is to determine the clinical effectiveness of an aerosol formulation of Fv-Hsp70 that is portable and can be self-administered if exposure to toxic inhalants is suspected. The objective here in the pursuit of this goal is to establish proof-of- principle for effectivenes against phosgene exposure, an unmet medical need given the prevalence of phosgene in the chemical industry. Our central hypothesis is that Fv-Hsp70 will be therapeutically effective in protecting against lung damage as a result of exposure to phosgene gas. The rationale for the proposed research is that the cell penetrating antibody, 3E10, is an intracellular transporter that can deliver Hsp70 directly into cells where it minimizes protein denaturation and aggregation caused by oxidative stress. The antibody binds extracellular DNA and nucleosides, targets that are quite accessible where there are damaged cells, and it penetrates still viable cells through an equilibrative nucleoside salvage pathway. 3E10 is unique in that it penetrates cells without apparent harm and has been administered to humans without evidence of toxicity. Fv-Hsp70 has already been created and shown to be an effective cytoprotectant in vivo, minimizing by 68% of the infarct volume in brain tissue when administered to rats after a stroke. We are requesting funding to achieve the following specific aims: 1) Evaluate the effect of Fv-Hsp70 on survival and barrier integrity in vitro of lung cells exposed to triphosgene (a phosgene simulant) as part of a cell-based potency assay. 2) Establish a product stability profile for the Fv-Hsp70 aerosol and determine the maximum tolerated dose (MTD) in rats. 3) Evaluate the therapeutic efficacy of aerosolized Fv-Hsp70 in vivo with rats exposed to phosgene. A single- dose of Fv-Hsp70 aerosol will be provided either pre-exposure, 30 minutes post-exposure, or 60 minutes post- exposure. The proposed research is significant because it develops a critical therapeutic for an unmet need. The proposed research is innovative because it utilizes a unique antibody-mediated, energy-independent intracellular delivery system for protein therapeutics. Inducing heat shock protein production in vivo can take time, whereas the impact of our product is the rapid delivery of Hsp70 into damaged cells to prevent cell death.

描述（由申请人提供）：2003年，EPA在美国确定了123个化学植物，恐怖袭击或事故可能会使超过100万人暴露于有毒天然气的云中。暴露于某些化学物质后，细胞死亡的主要原因是氧化应激。当前预防氧化应激的策略包括使用小分子试剂，但它们缺乏特异性，需要过多的物质将其输送到肺部可能会引起危险的副作用。我们开发了细胞穿透抗体的FV片段MAB 3E10，作为细胞内转运蛋白，以将热冲蛋白70（FV-HSP70）传递到细胞中，我们证明了在体外和体内抗氧化损伤的细胞保护。我们的长期目标是确定FV-HSP70的气溶胶配方的临床有效性，该公式是便携式的，如果怀疑暴露于有毒吸入剂中，则可以自我管理。追求这一目标的目的是建立针对磷光下暴露的作用的原则证明，鉴于化学工业中富香之元的普遍性，医疗需求未满足。我们的中心假设是，FV-HSP70将在治疗上有效防止因磷酸气体暴露而防止肺损伤。拟议研究的理由是，细胞穿透抗体3E10是一种细胞内转运蛋白，可以将HSP70直接输送到细胞中，在这些细胞中，它可以最大程度地减少氧化应激引起的蛋白质变性和聚集。抗体结合细胞外DNA和核苷，在损坏细胞的情况下易于访问，并且通过平衡的核苷拯救途径穿透了仍然活的细胞。 3E10是独一无二的，因为它可以穿透细胞而没有明显的伤害，并且已经对人类施用而没有毒性证据。 FV-HSP70已经被创建并显示为体内有效的细胞保护剂，当中风后对大鼠施用时，将脑组织中的梗死体积的68％最小化。我们要求资助以实现以下特定目的：1）评估FV-HSP70对暴露于Triphosgene（Phosgene Sibulant）的肺部生存和屏障完整性的影响，作为基于细胞的效力测定的一部分。 2）建立FV-HSP70气溶胶的产品稳定性曲线，并确定大鼠的最大耐受剂量（MTD）。 3）评估体内雾化的FV-HSP70在体内的治疗功效，暴露于Phosgene。暴露前30分钟或暴露后60分钟，将提供一剂FV-HSP70气溶胶。拟议的研究很重要，因为它为未满足的需求开发了关键的治疗方法。拟议的研究具有创新性，因为它利用了独特的抗体介导的蛋白质疗法的抗体介导的，无关的细胞内递送系统。在体内诱导热休克蛋白的产生可能需要时间，而我们产品的影响是将Hsp70迅速递送到受损细胞中以防止细胞死亡。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Development of an acute, short-term exposure model for phosgene.

开发光气急性、短期暴露模型。

DOI：
10.1080/15376516.2019.1636170
发表时间：
2019
期刊：
Toxicology mechanisms and methods
影响因子：
3.2
作者：
Hobson,StephenT;Casillas,RobertP;Richieri,RichardA;Nishimura,RobertN;Weisbart,RichardH;Tuttle,Rick;Reynolds,GlennT;Parseghian,MissagH
通讯作者：
Parseghian,MissagH

Targeted heat shock protein 72 for pulmonary cytoprotection.