In vitro and in vivo evaluation of the main protease inhibitor FB2001 against SARS-CoV-2

FB2001 is a drug candidate that targets the main protease of SARS-CoV-2 via covalently binding to cysteine 145. In this study, we evaluated the inhibitory activities of FB2001 against several SARS-CoV-2 variants in vitro and in vivo (in mice), and we also evaluated the histopathological analysis and immunostaining of FB2001 on lung and brain which have been rarely reported. The results showed that FB2001 exhibited potent antiviral efficacy against several current SARS-CoV-2 variants in Vero E6 cells, namely, B.1.1.7 (Alpha): EC(50) = 0.39 ± 0.01 μM, EC(90) = 0.75 ± 0.01 μM; B.1.351 (Beta): EC(50) = 0.28 ± 0.11 μM, EC(90) = 0.57 ± 0.21 μM; B.1.617.2 (Delta): EC(50) = 0.27 ± 0.05 μM, EC(90) = 0.81 ± 0.20 μM; B.1.1.529 (Omicron): EC(50) = 0.26 ± 0.06 μM and EC(50) = 0.042 ± 0.007 μM (in the presence of a P-glycoprotein inhibitor). FB2001 remained potent against SARS-CoV-2 replication in the presence of high concentrations of human serum, which indicating that human serum had no significant effect on the in vitro inhibitory activity. Additionally, this inhibitor exhibited an additive effect against SARS-CoV-2 when combined with Remdesivir. Furthermore, FB2001 significantly reduced the SARS-CoV-2 copy numbers and titers in the lungs and brains in vivo, and alleviated the pathological symptoms. In addition, FB2001 could alleviated local bleeding, erythrocyte overflow, edema, and inflammatory cell infiltration in brain tissue, and inhibitors reducing viral titers and improving inflammation in the brain have been rarely reported. A physiologically based pharmacokinetic model was established and verified to predict the FB2001 concentration in human lungs. When FB2001 was administered at 200 mg twice a day for 5 days, the observed C(trough ss) in plasma and predicted C(trough ss) of lung total concentration were 0.163 and 2.5 μg/mL, which were approximately 9 and 132-fold higher than the EC(50) of 0.019 μg/mL (0.042 μM) against Omicron variant. Taken together, our study suggests that FB2001 is a promising therapeutic agent in COVID-19 treatment and can be combined with remdesivir to achieve improved clinical outcomes. Owing to its good safety and tolerability in healthy human (NCT05197179 and NCT04766931), FB2001 has been approved for Phase II/III clinical trial (NCT05445934).