Establishing the Therapeutic Efficacy of Alpha-1-Antitrypsin and Enoxaparin Against COVID-19

确定 Alpha-1-抗胰蛋白酶和依诺肝素针对 COVID-19 的治疗效果

• 批准号: 10588400
• 负责人: EDWARD D CHAN
• 金额: --
• 依托单位: VA EASTERN COLORADO HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10588400
• 关键词: 2019-nCoV; ACE2; Airway Disease; Alveolus; Anti-Inflammatory Agents; Artificial Intelligence; Autologous; Autophagocytosis; Biochemical; Biological; Biological Assay; Blood Vessels; Breeding; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; COVID-19; COVID-19 impact; COVID-19 pneumonia; Cell Survival; Cell surface; Cells; Clinical; Clinical Trials; Cytoprotection; Data; Dendritic Cells; Endothelial Cells; Endothelium; Enoxaparin; Enzymes; Epithelial Cells; Epithelium; Foundations; Future; Goals; Human; Individual; Infection; Inflammatory; Inflammatory Response; Inflammatory Response Pathway; Infusion procedures; Injury; Intravenous; Knock-out; Left; Low-Molecular-Weight Heparin; Lung; Macrophage; Middle East Respiratory Syndrome Coronavirus; Modeling; Mus; Organ; Pathogenicity; Patient-Focused Outcomes; Peptide Hydrolases; Phenotype; Plasma; Pre-Clinical Model; Production; Property; Proteins; SARS-CoV-2 infection; SARS-CoV-2 inhibitor; SARS-CoV-2 spike protein; Safety; Spleen; TMPRSS2 gene; Testing; Therapeutic Effect; Thrombosis; Transgenic Mice; Treatment Efficacy; Veterans; Viral Load result; Virus; airway epithelium; alpha 1-Antitrypsin; alpha 1-Antitrypsin Deficiency; alveolar epithelium; antagonist; cell injury; cell type; cytokine; cytotoxicity; effective therapy; efficacious treatment; extracellular; immunothrombosis; in vivo; molecular modeling; monocyte; mouse model; neutrophil; novel; pre-clinical; prevent; severe COVID-19; synergism; therapeutic evaluation; thrombotic

The clinical outcome for patients with severe COVID-19 remains poor due to the lack of highly efficacious treatment for such individuals. Finding a better remedy for them is an important niche and immediate unmet need. The aim of this pre-clinical project is to substantiate the therapeutic effect of combined alpha-1- antitrypsin (AAT) + enoxaparin (a low molecular weight heparin) against SARS-CoV-2 infection and its consequences. Establishing the efficacy of AAT + enoxaparin combination will provide a necessary foundation for future clinical trials with the goal of employing effective therapy for those with severe COVID-19. We have novel biological evidence supported by Artificial Intelligence-based molecular modeling that enoxaparin synergizes with AAT to inhibit TMPRSS2 (a cell surface protease that activates the spike protein of SARS-CoV-2) and to reduce SARS-CoV-2 burden in primary human airway epithelial cells (hAEc) and monocyte-derived macrophages (MDM). Because both AAT and enoxaparin embrace a panoply of activities that antagonize other pathogenic mechanisms of severe COVID-19 – including anti-inflammatory, anti-thrombotic, pro-autophagy (known to kill MERS-CoV), and endothelial cell protection – we hypothesize that the AAT + enoxaparin combination will be most effective (compared to each alone) in mitigating SARS- CoV-2 infection and its consequences. We will use three complementary models to elucidate the efficacy of AAT, enoxaparin, and combination of both against SARS-CoV-2 infection: (i) primary hAEc since they express high ACE2 levels, fulminant airway disease occurs, and infection of ciliated hAEc and breach of their defense initiates a portal of entry into the lower airways / alveoli to cause COVID-19 pneumonia; (ii) MDM + plasma derived from AAT-deficient individuals immediately before and immediately after receiving routine intravenous AAT since macrophages are key orchestrators of the hyper-inflammatory response seen with COVID-19; and (iii) two murine models, one with wildtype AAT and another with AAT knocked out. Aim 1: Determine in primary hAEc the mechanisms by which AAT, enoxaparin, and both reduce SARS- CoV-2 infection and its consequences. Approach: hAEc will be infected with SARS-CoV-2 followed by no treatment or treatment with AAT, enoxaparin, or combination of both and assayed for viral load, autophagic flux, pro-inflammatory cytokines, and hAEc viability and barrier integrity. Aim 2: Determine in macrophages the mechanisms by which AAT (given in vivo) ± enoxaparin mitigate SARS-CoV-2 infection. Approach: infect human MDM cultured in autologous plasma – prepared from AAT- deficient individuals before and after AAT infusions ± ex vivo enoxaparin – with SARS-CoV-2 and determine viral load, autophagic flux, and pro-inflammatory cytokine / macrophage extracellular trap (METs) production. Aim 3: Determine if SARS-CoV-2 infection of mice is mitigated by AAT, enoxaparin, and combination of both. Approach: transgenic (Tg) mice bred to express human ACE2 with or without deletion of AAT (to represent humans who are AAT-replete and AAT-deficient, respectively) will either be left untreated or treated with AAT, enoxaparin, or the combination along with SARS-CoV-2 infection. From the lungs and spleens, we will quantify viral load and the phenotypes of macrophages, dendritic cells, CD4+ T cells, and CD8+ T cells in both organs, as well as analyze the lungs for epithelial and endothelial injury, co-localization of SARS-CoV-2 with airway and alveolar epithelial cells, and neutrophil extracelluar trap (NETs) formation (both METs and NETs implicated in immunothrombosis of severe COVID-19). Establishing the efficacy of AAT + enoxaparin combination will provide a foundation for future clinical trials with the goal of employing more effective therapy for veterans and non-veterans with severe COVID-19.

由于缺乏高效的治疗方法，重症 COVID-19 患者的临床结果仍然很差 为这些人寻找更好的治疗方法是一个重要的利基市场，但目前尚未得到满足。 该临床前项目的目的是证实联合α-1-的治疗效果。 抗胰蛋白酶（AAT）+依诺肝素（一种低分子量肝素）对抗 SARS-CoV-2 感染及其 确定 AAT + 依诺肝素组合的疗效将为建立必要的基础。 进行未来的临床试验，目标是对重症 COVID-19 患者采用有效的治疗方法。 我们拥有基于人工智能的分子模型支持的新颖的生物学证据 依诺肝素与 AAT 协同抑制 TMPRSS2（一种细胞表面蛋白酶，可激活刺突蛋白） SARS-CoV-2 蛋白）并减少原代人气道上皮细胞 (hAEc) 中的 SARS-CoV-2 负担 因为 AAT 和依诺肝素都含有多种成分。 对抗严重 COVID-19 的其他致病机制的活性 - 包括抗炎、 抗血栓、促自噬（已知可杀死中东呼吸综合征冠状病毒）和内皮细胞保护——我们勇敢 AAT + 依诺肝素组合在缓解 SARS 方面最为有效（与单独使用相比） CoV-2 感染及其后果。 我们将使用三个互补模型来阐明 AAT、依诺肝素及其组合的功效 两者均能对抗 SARS-CoV-2 感染：(i) 原发性 hAEc，因为它们表达高 ACE2 水平、暴发性气道 疾病发生，纤毛 hAEc 的感染和其防御的破坏启动了进入 (ii) MDM + 源自 AAT 缺陷的血浆 接受常规静脉注射 AAT 之前和之后立即进行巨噬细胞治疗的个体 是 COVID-19 过度炎症反应的关键协调者；以及 (iii) 两种小鼠模型， 一个带有野生型 AAT，另一个带有 AAT 敲除。 目标 1：确定原发性 hAEc 中 AAT、依诺肝素以及两者减少 SARS-的机制 CoV-2 感染及其后果：hAEc 会感染 SARS-CoV-2，然后不会感染。 治疗或用 AAT、依诺肝素或两者组合治疗，并检测病毒载量、自噬 通量、促炎细胞因子、hAEc 活力和屏障完整性。 目标 2：确定巨噬细胞中 AAT（体内给予）±依诺肝素缓解的机制 SARS-CoV-2 感染方法：感染在自体血浆中培养的人类 MDM（由 AAT- 制备）。 AAT 输注前后有缺陷的个体±离体依诺肝素 – 与 SARS-CoV-2 并确定 病毒载量、自噬通量和促炎细胞因子/巨噬细胞胞外陷阱 (MET) 的产生。 目标 3：确定 AAT、依诺肝素及其组合是否可以减轻小鼠的 SARS-CoV-2 感染 方法：培育表达人类 ACE2 的转基因 (Tg) 小鼠，并删除或不删除 AAT。 分别代表 AAT 充足和 AAT 缺乏的人类）将不予治疗或接受治疗 与 AAT、依诺肝素或联合用药与 SARS-CoV-2 感染一起，我们从肺部和脾脏中检测到。 将量化病毒载量以及巨噬细胞、树突状细胞、CD4+ T 细胞和 CD8+ T 细胞的表型 两个器官，并分析肺部的上皮和内皮损伤、SARS-CoV-2 的共定位 与气道和肺泡上皮细胞以及中性粒细胞胞外陷阱 (NET) 形成（MET 和 NETs 与严重 COVID-19 的免疫血栓形成有关）。 确定 AAT + 依诺肝素组合的疗效将为未来的临床试验奠定基础 目标是为患有严重 COVID-19 的退伍军人和非退伍军人采用更有效的治疗。