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

由于缺乏高效的治疗手段，重症新冠肺炎患者的临床结果仍然很差。 对这类人的治疗。为他们找到更好的治疗方法是一个重要的利基市场，也是一个迫在眉睫的问题 需要。这一临床前项目的目的是证实联合应用α-1-β的治疗效果。 抗胰蛋白酶+低分子肝素依诺肝素抗SARS-CoV-2感染及其临床意义 后果。确定AAT+依诺肝素的联合疗效将提供必要的基础 用于未来的临床试验，目标是对患有严重新冠肺炎的人采用有效的治疗方法。 我们有基于人工智能的分子建模支持的新生物证据 依诺肝素与AAT协同抑制TMPRSS2(一种激活尖峰的细胞表面蛋白酶 SARS-CoV-2蛋白)和降低原代人呼吸道上皮细胞的SARS-CoV-2载量 和单核细胞来源的巨噬细胞(MDM)。因为AAT和依诺肝素都包含了 拮抗严重新冠肺炎其他发病机制的活性-包括抗炎、 抗血栓、促自噬(已知可杀死MERS冠状病毒)和内皮细胞保护--我们假设 AAT+依诺肝素联合用药在缓解SARS方面将是最有效的(与单独用药相比)。 冠状病毒2型感染及其后果。 我们将使用三个互补的模型来阐明AAT、依诺肝素和联合应用 抗SARS-CoV-2感染：(I)原发hAEc，因为它们表达高水平的ACE2，暴发性呼吸道 疾病发生时，纤毛虫hAEc的感染和对其防御的破坏启动了进入 下呼吸道/肺泡导致新冠肺炎肺炎；(Ii)来自AAT缺陷的MDM+血浆 在接受常规静脉注射AAT之前和之后立即接受AAT的个体，因为巨噬细胞 是新冠肺炎所见的高炎症反应的关键协调者；以及(Iii)两个小鼠模型， 一个是野生型AAT，另一个是AAT基因敲除。 目的1：初步确定AAT、依诺肝素及两者降低SARS的作用机制。 冠状病毒2型感染及其后果。方法：hAEc将感染SARS-CoV-2，然后是NO 用AAT、依诺肝素或两者的组合进行治疗或治疗，并检测病毒载量，自噬 通量、促炎细胞因子、hAEc活性和屏障完整性。 目的2：确定AAT(体内给药)±依诺肝素在巨噬细胞中减轻作用的机制 SARS-CoV-2感染。方法：感染培养在AAT制备的自体血浆中的人MDM- AAT输注±体外依诺肝素感染SARS-CoV-2前后缺陷者的检测 病毒载量、自噬通量和促炎细胞因子/巨噬细胞外陷阱(METS)的产生。 目的3：确定AAT、依诺肝素及其联合应用是否能减轻小鼠SARS-CoV-2感染 两者都有。方法：培育表达人ACE2的转基因(TG)小鼠，可缺失或不缺失AAT(TO) 分别代表AAT完全和AAT缺陷的人类)要么不治疗，要么治疗 AAT、依诺肝素或联合应用合并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、CD4+T细胞和CD8+T细胞的表型 两个器官，以及分析肺上皮和内皮损伤，共定位的SARS-CoV-2 有呼吸道和肺泡上皮细胞，并形成中性粒细胞细胞外陷阱(NETS)(Met和 与严重新冠肺炎的免疫血栓形成有关的NETS)。 确定AAT+依诺肝素的联合疗效将为未来的临床试验提供基础 目的是为患有严重新冠肺炎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.