Metabolism-based interactions and organ-targeted delivery of molnupiravir, nirmatrelvir and remdesivir

摘要COVID-19（2019冠状病毒病）仍然是一场全球健康危机。的努力 开发COVID-19的治疗方法是世界范围内前所未有的。莫努匹拉韦、Nirmatrelvir和 remdesivir是这种努力的成功例子。从机制上讲，Nirmatrelvir抑制了 SARS-CoV-2通过靶向病毒主要蛋白酶（Mpro），而莫努匹韦和Remdesivir靶向RNA-de- 悬垂RNA聚合酶（RdRp）。Molnupiravir导致RdRp引入病毒的广泛错误 基因组，导致致命的诱变。相比之下，Remdesivir引起暂停并诱导链终止。 Nirmatrelvir是细胞色素P450 3A 4（CYP 3A 4）的稳定底物，CYP 3A 4的氧化作用代表 失活相比之下，莫努匹拉韦和瑞德西韦是前药，并且需要初始水解以用于它们的合成。 抗病毒活性。我们已经表明，Remdesivir被羧酸酯酶-1（CES 1）水解，而 莫努匹拉韦（CES 2）。我们还表明，Remdesivir是CES 2的不可逆抑制剂。此外，本发明还提供了一种方法， 我们的初步研究已经证明，莫努匹韦下调CYP 3A 4的表达。COVID-19 症状与多个器官有关，但就严重程度而言，主要与肺系统有关。 该项目的中心假设是，莫努匹拉韦、Nirmatrelvir和Remdesivir相互影响其 有效性取决于组合和递送策略。具体目的是：（1）确定 莫努匹拉韦、Nirmatrelvir和Remdesivir之间基于代谢的相互作用，以及（2）开发器官- 通过纳米制剂的靶向递送。将对大量人体样本进行代谢测试， 确定个体差异。人原代肺和肝细胞将用CES或CYP 3A 4处理 诱导剂或抑制剂，并检测这些COVID-19药物的代谢改变。这些药物将 在细胞中一起孵育并评估它们的代谢相互作用。为了开发纳米制剂， 将合成脂质包被的磷酸钙（LCP）纳米颗粒，并测试其与 以及母体COVID-19药物及其代谢产物的细胞摄取/保留。纳米配方 制剂将通过肠内给药检测药代动力学和器官靶向优势 局为了将感染潜力与治疗效力联系起来，单细胞RNA测序 将进行scRNAseq检测，以确定表达感染受体的细胞是否配备有 这些新型冠状病毒药物的代谢酶。该项目的科学前提很强 和原创性。其独创性源于莫努匹拉韦之间药理学协同作用的新奇， Nirmatrelvir和Remdesivir取决于组合和递送策略。该项目还将建立一个 框架是选择靶向病毒感染细胞的药物（例如，病毒复制）应基于 受感染的细胞是否表达药物代谢酶，以确保所选药物的疗效。 此外，待研究的药剂具有广谱抗病毒活性。因此，该项目将 不仅对COVID-19，而且对未来的大流行病具有广泛的临床意义。

Abstract COVID-19 (coronavirus disease of 2019) continues to be a global health crisis. The efforts of developing therapeutics for COVID-19 are worldwide and unprecedented. Molnupiravir, nirmatrelvir and remdesivir are successful examples of such efforts. Mechanistically, nirmatrelvir inhibits the replication of SARS-CoV-2 by targeting viral main protease (Mpro), whereas molnupiravir and remdesivir target RNA-de- pendent RNA-polymerase (RdRp). Molnupiravir causes RdRp to introduce widespread errors of the viral genome, leading to lethal mutagenesis. In contrast, remdesivir causes to pause and induce chain termination. Nirmatrelvir is a robust substrate of cytochrome P450 3A4 (CYP3A4), and oxidation by CYP3A4 represents inactivation. In contrast, molnupiravir and remdesivir are prodrugs and require initial hydrolysis for their antiviral activities. We have shown that remdesivir is hydrolyzed by carboxylesterase-1 (CES1), whereas molnupiravir by CES2. We have also shown that remdesivir is an irreversible inhibitor of CES2. In addition, our Preliminary Study has demonstrated that molnupiravir downregulates CYP3A4 expression. COVID-19 symptoms are related to multiple organs, but largely associated with the pulmonary system in terms of severity. The central hypothesis of this project is that molnupiravir, nirmatrelvir and remdesivir interactively impact their efficacy depending on a combination and delivery strategy. The Specific Aims are: (1) to ascertain metabolism-based interactions among molnupiravir, nirmatrelvir and remdesivir, and (2) to develop organ- targeting delivery via nanoformulation. A large number of human samples will be tested for their metabolism to ascertain individual variability. Human primary lung and liver cells will be treated with a CES or CYP3A4 inducer or suppressor and tested for altered metabolism of these COVID-19 drugs. These drugs will be incubated together in cells and their metabolism interactions will be assessed. To develop nanoformulation, lipid-coated calcium phosphate (LCP) nanoparticles will be synthesized and tested for the incorporation with and cellular uptake/retention of the parent COVID-19 drugs and their metabolites. Nanoformulated preparations will be tested for pharmacokinetic and organ-targeted superiority through intratracheal administration. To connect infectious potential with therapeutic potency, single cell RNA sequencing (scRNAseq) will be performed to determine whether cells expressing the receptor for infection are equipped with proper metabolizing enzymes for these COVID-19 drugs. The scientific premise of the project is strong and original. The originality stems from the novelty of pharmacological synergy among molnupiravir, nirmatrelvir and remdesivir depending on a combination and delivery strategy. The project will also establish a framework that selection of a drug targeting virally infected cells (e.g., viral replication) should be made based on whether the infected cells express drug-metabolizing enzymes that ensure the efficacy of the selected drug. In addition, the agents to be studied have a broad spectrum of antiviral activities. Therefore, this project will have a broad clinical significance not only for COVID-19 but also for future pandemics.