Why Molnupiravir Fails in Hospitalized Patients

Figures

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  FIG 1. Evaluating the influence of therapy initiation on EIDD-1931 antiviral activity. A549 cells that stably expressed ACE2 were mixed with SARS-CoV-2 (MOI = 10 to 5) and inoculated into the hollow fiber infection model (HFIM). EIDD-1931 therapy (tx) was administered into hollow fiber (HF) cartridges at concentrations equivalent to 0.146 μg/mL (A), 0.324 μg/mL (B), and 3.5 μg/mL (C) beginning at 0 h (tx at 0 h), 24 h (tx at 24 h), 48 h (tx at 48 h), or 72 h (tx at 72 h) postinfection. HF cartridges were sampled daily, and the infectious viral burden was quantified by plaque assay on Vero E6 cells. Data points are reported in log10 PFU per mL and represent the mean of two biological replicates ± one standard deviation. The dashed line corresponds to the plaque assay limit of detection (2 log10 PFU/mL). Two independent studies were conducted.

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  FIG 2. Cytotoxicity of EIDD-1931 on A549 cells that stably express ACE2. ACE2-expressing A549 cells were seeded into 96-well plates at a concentration of 5,000 cells per well. EIDD-1931 was added to cells 24 h after plating and incubated for an additional 72 h. Cell health and metabolism were measured using a WST-1 assay. A 1 mg/mL concentration of novobiocin was used as a positive control for cytotoxicity and cells that did not receive EIDD-1931 served as a negative control. Bars correspond to the mean ± one standard deviation of six biological replicates. Three independent assays were conducted.

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  FIG 3. Influence of therapy delay when human pharmacokinetic profiles were simulated for EIDD-1931. (A) A549 cells that stably expressed ACE2 were mixed with SARS-CoV-2 (MOI = 10 to 5) and inoculated into the hollow fiber infection model (HFIM). EIDD-1931 was administered into hollow fiber (HF) cartridges to mimic human pharmacokinetic profiles associated with 800 mg once daily (Q24h) or 400 mg twice daily (Q12h). Therapy was initiated 24 h (tx at 24 h) or 48 h (tx at 48 h) after infection for both dosage regimens. One HF cartridge did not receive the drug, serving as a no-treatment control, and one cartridge received a high dose (3.5 μg/mL) of EIDD-1931 as a continuous infusion served as a positive control. Cartridges were sampled daily, and the infectious viral burden was measured by plaque assay on Vero E6 cells. Data points correspond to the mean ± one standard deviation of two biological replicates and the dashed line signifies the plaque assay limit of detection. Two independent studies were conducted. (B) Medium samples from the central reservoir of each HFIM system were taken multiple times throughout the first 48 h to ensure the desired PK profiles were achieved. Concentrations…

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  FIG 4. The effect of therapy initiation delays on intracellular viral production and spread as well as EIDD-1931 metabolism. Tissue culture flasks seeded with ACE2 expressing A549 cells were infected with SARS-CoV-2 at an MOI = 0.03. EIDD-1931 was added to cell monolayers at concentrations equivalent to 0.648 μg/mL and 3.24 μg/mL at 0 h, 24 h, 48 h, or 72 h postinfection. Control flasks that did not receive drugs served as a negative control. Viral supernatants and cells were harvested daily beginning 24 h posttherapy initiation for up to 120 h postinfection. (A) The concentration of extracellular infectious viral burden was determined by performing plaque assays on viral supernatant samples. (B) Flow cytometry was performed on a portion of harvested cells to determine the amount of cell-to-cell viral spread over time. Cells were fixed and stained with an antibody specific to the SARS-CoV-2 nucleocapsid (NP) protein. (C) The intracellular infectious viral burden was determined by performing a plaque assay on a portion of the harvested cells. The intracellular viral burden is reported as log10 PFU per antigen (NP) positive cells. (D) The ratio of EIDD-1931 triphosphate (TP) to CTP…

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  FIG 5. Summary of key experimental findings. This illustration summarizes the key experiments and the corresponding findings conducted in this study. The outline of our overall mechanism explaining the loss of antiviral activity with the delay of therapy initiation is shown in the bottom right panel.