Project-004

新冠肺炎呼吸道感染(SARS-CoV-2病毒)已成为一种流行病。它有很高的死亡率 发病率高，特别是在老年患者中。死亡通常是由严重的呼吸道感染引起的 导致急性呼吸窘迫综合征。为了与细胞膜结合，SARS-CoV-2需要S蛋白被切割，或者通过 跨膜丝氨酸蛋白酶，丝氨酸2(TMPRSS2)，或由组织蛋白酶B和L(CatB/L)内体 很复杂。TMPRSS2可以被卡莫司坦甲基化(CM)抑制，但CM是一种刺激物，可能不是理想的 进展性ARDS患者的呼吸道给药。值得注意的是，CatB/L受到内体碱化的抑制 使用氯化铵。作为P01项目HL128192的一部分，我们正在研究呼吸道的有益影响 哮喘和囊性纤维化患者的碱化。我们有一种药物，碱性甘氨酸缓冲液(AGB)，即 在我们的P01项目中正在生产用于吸入。这种药物具有有效的IND，并具有极好的安全性数据。我们 因此测试以确定AGB是否会导致培养的原代人的细胞内碱化 取自P01受试者的呼吸道上皮细胞。的确如此；而且这种药物在体外细胞中耐受性很好 (就像在体内一样)。下一步是确定AGB是否抑制病毒复制(作为一种 在我们的原代人类呼吸道上皮细胞培养中，感染的多样性[MOI]和病毒进入(PCR)的功能。 为此，我们正在与我们的印第安纳大学BSL3病毒学实验室(S·吉尔克和罗宾逊博士)合作，他 预计下周将交付SARS-CoV-2病毒。他们的实验室已经完成了准备工作，特别是在 这个项目的。如果AGB真的抑制了病毒复制，我们会提议与FDA讨论这种可能性 我们可以扩大我们的IND，允许对有呼吸窘迫风险的患者进行试验，这些患者与已知的 CoVID-2呼吸系统疾病。最终，这项试验的结果将被提议包括：死亡率(初级)； 以及ICU住院时间和血氧饱和度指数曲线下面积(次)。在研究中 在这里提出，我们计划实现三个目标。首先，我们将检验SARS-CoV-2 S蛋白的假设 人类原代呼吸道上皮细胞碱化使用银杏叶蛋白可抑制卵裂。第二，我们将测试 AGB暴露可阻止SARS-CoV-2复制和细胞进入原发人类呼吸道的假说 体外培养上皮细胞。第三，我们将进行剂量反应和时间历程实验，以确定是否 使用银杏叶提取物抑制SARS-CoV-2的复制和细胞进入可能是一种现实的治疗方法。

Respiratory infection with COVID-19 (the SARS-CoV-2 virus) has become pandemic. It has significant mortality and high morbidity, particularly among older patients. Death typically results from severe respiratory infection leading to ARDS. To bind to cell membranes, SARS-CoV-2 requires S protein cleavage either by the transmembrane serine protease, serine 2 (TMPRSS2), or by the cathepsin B and L (CatB/L) endosomal complex. TMPRSS2 can be inhibited by camostat methylate (CM), but CM is an irritant and may not be ideal for airway administration in patients with evolving ARDS. Of note, CatB/L is inhibited by endosomal alkalinization using ammonium chloride. As part of P01 project HL128192, we are studying the beneficial effects of airway alkalinization in patients with asthma and cystic fibrosis. We have a drug, alkaline glycine buffer (AGB), that is being produced for inhalation in our P01 project. This drug has an active IND and has excellent safety data. We therefore tested to determine whether AGB would cause intracellular alkalinization in cultured primary human airway epithelial cells obtained from our P01 subjects. It did; and the drug was well-tolerated by the cells in vitro (as it is in vivo). The next step is to determine whether AGB inhibits viral replication (plaque formation as a function of multiplicity of infection [MOI]) and viral entry (PCR) in our primary human airway epithelial cultures. To do this, we are partnering with our Indiana University BSL3 virology lab (Dr.’s Gilk and Robinson) who anticipate delivery of SARS-CoV-2 next week. Their lab has completed preparation, particularly in anticipation of this project. If in fact AGB inhibits viral replication, we would propose to discuss with the FDA the possibility that we could expand our IND, allowing a trial in patients at risk for respiratory distress associated with known COVID-2 respiratory disease. Ultimately, outcomes of this trial would be proposed to include: mortality (primary); as well as ICU length of stay and oxygen saturation index area under the curve (secondary). In the study proposed here, we plan to accomplish three Aims. First we will test the hypothesis that SARS-CoV-2 S-protein cleavage is inhibited by human primary airway epithelial cell alkalinization using AGB. Second, we will test the hypothesis that AGB exposure prevents SARS-CoV-2 replication and cell entry in primary human airway epithelial cells in vitro. Third, we will perform dose-response and time course experiments to determine whether the inhibition of SARS-CoV-2 replication and cell entry using AGB could be a realistic therapy.