Optimal preclinical models for human dose projection of SARS-CoV-2 small molecule direct-acting antivirals

The coronavirus disease 2019 (COVID-19) pandemic, driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the urgent need for effective therapeutics, particularly as limitations of existing antiviral treatments persist. Establishing the proper strategy to set target concentrations for human dose projection is essential for any novel drug development project. In this study, we investigated the translatability of various preclinical models, specifically in vitro A549-hACE2 cells and air-liquid interface human nasal epithelial cultures, and in vivo Syrian golden hamsters and K18-hACE2 transgenic mice, using 3-chymotrypsin-like protease (3CL^pro) inhibitors as model direct-acting antivirals. We assessed the minimal efficacious concentrations in these models of both ensitrelvir (ETV) and nirmatrelvir (NTV) co-dosed with ritonavir (Paxlovid) and compared this to clinical human data. Notably, in vitro models and the K18-hACE2 mice proved to be the most predictive for human efficacious exposures. Interestingly, lower efficacious exposures were needed in the more severe K18-hACE2 mouse model than in the Syrian golden hamster model for mild infection. Overall, we recommend applying a factor of 4-fold to the in vitro 90% effective concentration (EC₉₀) for a conservative estimate of human efficacious exposure and recommend validation using K18-hACE2 mice. These insights are critical for guiding the development of effective SARS-CoV-2 therapeutics and optimizing clinical trial design.