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

严重COVID-19患者的临床结果仍然很差，因为缺乏高效的 对这些人的治疗。为他们找到更好的补救办法是一个重要的利基市场，也是一个亟待解决的问题。 需要的该临床前项目的目的是证实联合α-1- 抗胰蛋白酶（AAT）+依诺肝素（一种低分子肝素）抗SARS-CoV-2感染及其 后果确定AAT +依诺肝素联合治疗的疗效将提供必要的基础 用于未来的临床试验，目标是为严重的COVID-19患者提供有效的治疗。 我们有新的生物学证据支持基于人工智能的分子建模 依诺肝素与AAT协同抑制TMPRSS 2（一种细胞表面蛋白酶， SARS-CoV-2蛋白），并降低原代人气道上皮细胞（hAEc）中的SARS-CoV-2负荷 和单核细胞衍生的巨噬细胞（MDM）。因为AAT和依诺肝素都包含一整套 拮抗严重COVID-19的其他致病机制的活性-包括抗炎， 抗血栓形成，促自噬（已知杀死MERS-CoV）和内皮细胞保护-我们假设 AAT +依诺肝素联合用药（与单独用药相比）在缓解SARS方面最有效- CoV-2感染及其后果 我们将使用三种互补的模型来阐明AAT、依诺肝素和联合用药的疗效。 均针对SARS-CoV-2感染：（i）原发性hAEc，因为它们表达高水平的ACE 2，暴发性气道炎 疾病发生，纤毛hAEc的感染和其防御的破坏启动了进入细胞的门户， 下呼吸道/肺泡导致COVID-19肺炎;（ii）来自AAT缺陷型 个体在接受常规静脉内AAT之前和之后立即接受，因为巨噬细胞 是COVID-19中观察到的过度炎症反应的关键协调;以及（iii）两种鼠模型， 一个具有野生型AAT，另一个具有敲除的AAT。 目的1：在原发性hAEc中确定AAT、依诺肝素以及两者降低SARS的机制。 CoV-2感染及其后果方法：hAEc将感染SARS-CoV-2，然后不感染 治疗或用AAT、依诺肝素或两者的组合治疗，并测定病毒载量、自噬 流量、促炎细胞因子和hAEc活力和屏障完整性。 目的2：在巨噬细胞中确定AAT（体内给药）±依诺肝素减轻 SARS-CoV-2感染。方法：感染在自体血浆中培养的人MDM-由AAT制备- 在AAT输注±离体依诺肝素之前和之后缺乏个体-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）产生。 目的3：确定AAT、依诺肝素和AAT与依诺肝素的联合是否减轻了小鼠的SARS-CoV-2感染 两者方法：培育转基因（Tg）小鼠以表达具有或不具有AAT缺失的人ACE 2（以 分别代表AAT充满和AAT缺乏的人）将不治疗或治疗 与AAT、依诺肝素或其组合沿着SARS-CoV-2感染。从肺和脾，我们 将量化病毒载量和巨噬细胞，树突状细胞，CD 4 + T细胞和CD 8 + T细胞的表型， 两个器官，以及分析肺上皮和内皮损伤，SARS-CoV-2的共定位 与气道和肺泡上皮细胞，和中性粒细胞胞外陷阱（NET）的形成（MET和 涉及严重COVID-19免疫血栓形成的NET）。 确定AAT +依诺肝素组合的疗效将为未来的临床试验提供基础 目标是为患有严重COVID-19的退伍军人和非退伍军人提供更有效的治疗。

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.