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

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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.