Project-004

COVID-19（SARS-CoV-2病毒）的呼吸道感染已成为大流行。它的死亡率很高 高发病率，尤其是老年患者。严重的呼吸道感染通常会导致死亡 导致ARDS。为了与细胞膜结合，SARS-CoV-2需要S蛋白裂解， 跨膜丝氨酸蛋白酶，丝氨酸2（TMPRSS 2），或由组织蛋白酶B和L（CatB/L）内体 复杂. TMPRSS2可以被卡莫司他甲基化物（CM）抑制，但是CM是刺激物并且可能不是理想的。 呼吸道给药治疗进展中的ARDS患者。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HL 128192的一部分，我们正在研究气道的有益作用， 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）， 在我们的P01项目中被生产用于吸入。该药物具有活性IND，并具有良好的安全性数据。我们 因此，测试以确定AGB是否会引起培养的原代人细胞内碱化 从我们的P01受试者获得的气道上皮细胞。确实如此，而且这种药物在体外细胞中耐受性良好 (as它在体内）。下一步是确定AGB是否抑制病毒复制（空斑形成作为一种免疫抑制剂）。 感染复数的功能[MOI]）和病毒进入（PCR）。 为了做到这一点，我们正在与我们的印第安纳州大学BSL 3病毒学实验室（博士' s Gilk和罗宾逊）谁 预计SARS-CoV-2将于下周交付。他们的实验室已经完成了准备工作，特别是在预期 of this project项目.如果事实上AGB抑制病毒复制，我们将建议与FDA讨论 我们可以扩大我们的IND，允许在与已知疾病相关的呼吸窘迫风险患者中进行试验 COVID-2呼吸道疾病。最终，本试验的结局将包括：死亡率（主要）; 以及ICU住院时间和血氧饱和度指数曲线下面积（次要）。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 切割被使用AGB的人原代气道上皮细胞碱化所抑制。第二，我们将测试 AGB暴露阻止SARS-CoV-2复制和细胞进入人原代气道假设 体外培养上皮细胞。第三，我们将进行剂量反应和时间过程实验，以确定是否 使用AGB抑制SARS-CoV-2复制和细胞进入可能是一种现实的疗法。

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.