The orally available nucleoside analog molnupiravir (MOL) was recently authorized for the treatment of adults with COVID-19 under emergency use. Although MOL demonstrates moderate effectiveness in non-hospitalized patients, it failed to provide therapeutic benefit to those hospitalized with COVID-19. Here, we aim to explain the failure of MOL in hospitalized patients and identify optimal dosing strategies that will improve therapeutic outcomes in an out-patient setting. Human lung A549 cells expressing high levels of ACE2 were infected with USA-WA1/2020 strain of SARS-CoV-2 and exposed to various concentrations of MOL up to 72h post-infection. Cells and viral supernatants were harvested daily for up to 5 days. Antiviral effect was determined by quantifying infectious extracellular and intracellular SARS-CoV-2 burden by plaque assay and cell-to-cell viral spread by flow cytometry. Additionally, UPLC-MS/MS was performed to enumerate intracellular levels of MOL, MOL triphosphate (TP), and cytidine TP. The initiation of therapy heavily influenced MOL activity, as a 48h delay completely abrogated antiviral effect. These adverse effects could be overcome by administering higher exposures of MOL, as concentrations equivalent to 10x EC<sub>95</sub> were able to suppress viral replication even when therapy was delayed by 72 h. The ratio of MOL TP to cytidine TP was stable in SARS-CoV-2 infected cells over time and increased with higher exposures of MOL. These data indicate that MOL therapy fails when initiated later in the infection process due to the increased concentration of intracellular virus in the presence of a stable ratio of MOL TP to cytidine TP. This dilution effect can be overcome by increasing the amount of MOL TP via higher exposures of MOL. These findings explain why MOL therapy failed in hospitalized patients that received treatment &gt;7 days post-symptom onset. MOL should be administered as quickly as possible after infection to yield better therapeutic outcomes.

Optimizing antiviral therapy with nucleoside analogs for the treatment of COVID-19

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ACS Fall 2022

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