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

摘要新冠肺炎(2019年冠状病毒病)继续成为全球性的健康危机。所做的努力 为新冠肺炎开发疗法是全球性的，也是史无前例的。莫鲁匹拉韦、尼马瑞韦和 Redesivir就是这种努力的成功例子。从机制上讲，尼马瑞韦抑制肿瘤的复制。 SARS-CoV-2靶向病毒主要蛋白酶(MPRO)，而莫诺普拉韦和雷米索韦靶向RNA-De-R。 悬垂型RNA聚合酶(RdRp)。Molnupiravir导致RdRp引入广泛的病毒错误 基因组，导致致命的突变。相反，redesivir导致停顿和诱导链终止。 尼马瑞韦是细胞色素P450 3A4(CYP3A4)的强健底物，其氧化作用代表 失活。相比之下，Molnupiravir和redesivir是前药，需要对其进行初始水解 抗病毒活性。我们已经证明，雷米昔韦是由羧酸酯酶-1(CES1)水解的，而 Molnupiravir由CES2提供。我们还证明了瑞德韦是CES2的不可逆转的抑制剂。此外, 我们的初步研究表明，Molnupiravir下调了CYP3A4的表达。新冠肺炎 症状与多个器官有关，但在严重程度上主要与肺系统有关。 该项目的中心假设是，Molnupiravir、nirmatrelvir和redesivir相互作用地影响他们的 疗效取决于组合和交付策略。具体目标是：(1)确定 Molnupiravir、Nirmatrelvir和redesivir之间基于代谢的相互作用，以及(2)发育器官-- 通过纳米制剂进行靶向传递。将对大量人体样本进行新陈代谢测试，以 确定个体的可变性。人的原代肺和肝细胞将用CES或CYP3A4处理 诱导剂或抑制剂，并测试这些新冠肺炎药物的新陈代谢变化。这些药物将会是 在细胞中一起孵育，他们的代谢相互作用将被评估。为了开发纳米配方， 将合成脂质包裹的磷酸钙(LCP)纳米颗粒，并测试其与 以及细胞对母体新冠肺炎药物及其代谢物的摄取/保留。纳米配方 将通过气管内测试制剂的药代动力学和器官靶向性优势。 行政管理。为了将感染潜力与治疗潜力联系起来，单细胞RNA测序 将进行(ScRNAseq)以确定表达感染受体的细胞是否 为这些新冠肺炎药物提供适当的代谢酶。该项目的科学前提是强有力的 而且很有创意。其独创性源于莫诺普拉韦之间的药理协同作用的新颖性， Nirmatrelvir和redesivir取决于联合和给药策略。该项目还将建立一个 针对病毒感染细胞的药物选择(例如，病毒复制)的框架应基于 关于受感染的细胞是否表达药物代谢酶来确保所选药物的疗效。 此外，待研究的药物具有广泛的抗病毒活性。因此，这个项目将 不仅对新冠肺炎，而且对未来的大流行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.