Efficacy comparison of 3CL protease inhibitors ensitrelvir and nirmatrelvir against SARS-CoV-2 in vitro and in vivo

Abstract

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become established in the human population, making the need to develop safe and effective treatments critical. We have developed the small-molecule antiviral ensitrelvir, which targets the 3C-like (3CL) protease of SARS-CoV-2. This study evaluated the in vitro and in vivo efficacy of ensitrelvir compared with that of another SARS-CoV-2 3CL PI, nirmatrelvir.

Methods: Cultured cells, BALB/cAJcl mice and Syrian hamsters were infected with various SARS-CoV-2 strains, including the ancestral strain WK-521, mouse-adapted SARS-CoV-2 (MA-P10) strain, Delta strain and Omicron strain. Ensitrelvir efficacy was compared with that of nirmatrelvir. Effective concentrations were determined in vitro based on virus-induced cytopathic effects, viral titres and RNA levels. Lung viral titres, nasal turbinate titres, body-weight changes, and animal survival were also monitored.

Results: Ensitrelvir and nirmatrelvir showed comparable antiviral activity in multiple cell lines. Both ensitrelvir and nirmatrelvir reduced virus levels in the lungs of mice and the nasal turbinates and lungs of hamsters. However, ensitrelvir demonstrated comparable or better in vivo efficacy than that of nirmatrelvir when present at similar or slightly lower unbound-drug plasma concentrations.

Conclusions: Direct in vitro and in vivo efficacy comparisons of 3CL PIs revealed that ensitrelvir demonstrated comparable in vitro efficacy to that of nirmatrelvir in cell culture and exhibited equal to or greater in vivo efficacy in terms of unbound-drug plasma concentration in both animal models evaluated. The results suggest that ensitrelvir may become an important resource for treating individuals infected with SARS-CoV-2.

Figure 1.

In vivo efficacy of ensitrelvir compared with that of nirmatrelvir in a mouse lethality model. Female BALB/cAJcl mice were nasally infected with the mouse-adapted SARS-CoV-2 (MA-P10) strain and then orally administered various doses of ensitrelvir (ETV), nirmatrelvir (NTV) or vehicle (0.5% MC solution) every 12 h (twice daily) for 5 days. The first administration was performed 1 day p.i. (a) Mouse survival was monitored daily. n = 5 mice/group. P values were calculated using log-rank test versus vehicle. *P < 0.05 and **P < 0.001. (b) Body-weight values are presented as a percentage of initial body weight through to Day 14 p.i. The graph bars represent the mean ± SD of 5 mice/group. (c) Lung viral titres were determined using TCID₅₀ assays on the days p.i. indicated. Each point represents the mean ± SD of n = 4–5 mice. The dashed line represents the lower limit of quantification (LLOQ) of 1.80 log₁₀ TCID₅₀/mL. P values were calculated using Tukey’s test. *P < 0.05 versus vehicle; **P < 0.01 versus vehicle; ***P < 0.001 versus vehicle; †P < 0.05 versus 1000 mg/kg NTV; ††P < 0.01 versus 1000 mg/kg NTV; †††P < 0.001 versus 1000 mg/kg NTV. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

In vivo efficacy of ensitrelvir compared with that of nirmatrelvir against Omicron BA.2 in a hamster infection model. Syrian hamsters were intranasally infected with 1.0 × 10⁴ TCID₅₀ of SARS-CoV-2 Omicron BA.2. The hamsters were treated twice a day at an 8 h/16 h interval for 5 days starting 1 day p.i. with various doses of oral ensitrelvir (ETV), subcutaneous nirmatrelvir (NTV) or vehicle (0.5% MC solution). Hamsters were monitored daily. (a) Body-weight values are presented as a percentage of initial body weight through to Day 14 p.i. The graph bars represent the mean ± SD. n = 4/infected group; n = 3/non-infected group. (b) Hamster lungs (lower airway) and nasal turbinates (upper airway) were harvested and prepared 2 and 4 days p.i. Viral titres were determined using TCID₅₀ assays. Each point represents the mean ± SD of n = 6–8/group. The dashed line represents the LLOQ of 1.80 log₁₀ TCID₅₀/mL. P values were calculated using Tukey’s test. **P < 0.01 versus vehicle; ***P < 0.001 versus vehicle; ^###P < 0.01 versus 12.5 mg/kg ETV; ^§P < 0.05 versus 250 mg/kg NTV; †P < 0.05 versus 750/250 mg/kg NTV; ††P<0.01 versus 750/250 mg/kg NTV. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.