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

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.

S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and ameliorates COVID-19 severity in hamsters.

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity, but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (Mpro, also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to sub-micromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern (VOCs), including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), possesses remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

Ensitrelvir is effective against SARS-CoV-2 3CL protease mutants circulating globally.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health concern worldwide. Ensitrelvir (S-217622) has been evaluated as an antiviral treatment for COVID-19, targeting SARS-CoV-2 3C-like protease (3CLpro). Ensitrelvir has been reported to have comparable antiviral activity against some of the SARS-CoV-2 variants: alpha, beta, gamma, delta, and omicron (BA.1.18). In this paper, we describe that ensitrelvir is effective against newly emerging SARS-CoV-2 variants and globally prevalent 3CLpro mutations. Ensitrelvir exhibited comparable antiviral activity against SARS-CoV-2 variants, including recently emerging ones: omicron (BA1.1, BA.2, BA.2.75, BA.4, BA.5, BQ.1.1, XBB.1, and XE), mu, lambda, and theta. Genetic surveillance of SARS-CoV-2 3CLpro, the target of ensitrelvir, was conducted using a public database and identified 11 major 3CLpro mutations circulating globally (G15S, T21I, T24I, K88R, L89F, K90R, P108S, P132H, A193V, H246Y, and A255V). The 3CLpro mutation from proline to histidine at amino acid position 132 was especially identified in the omicron variant, with prevalence of 99.69%. Enzyme kinetic assay revealed that these 3CLpro mutants have enzymatic activity comparable to that of the wild type (WT). Next, we assessed the inhibitory effect of ensitrelvir against mutated 3CLpro, with it showing inhibitory effects similar to that against the WT. These in vitro data suggest that ensitrelvir will be effective against currently circulating SARS-CoV-2 variants, including omicron variants and those carrying 3CLpro mutations, which emerging novel SARS-CoV-2 variants could carry.

Efficacy of ensitrelvir against SARS-CoV-2 in a delayed-treatment mouse model.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19) and a devastating worldwide health concern. Development of safe and effective treatments is not only important for interventions during the current pandemic, but also for providing general treatment options moving forward. We have developed ensitrelvir, an antiviral compound that targets the 3C-like protease of SARS-CoV-2. In this study, a delayed-treatment mouse model was used to clarify the potential in vivo efficacy of ensitrelvir. METHODS: Female BALB/cAJcl mice of different ages were infected with the SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021) or mouse-adapted SARS-CoV-2 MA-P10 and then 24 h post-infection orally administered various doses of ensitrelvir or vehicle. Viral titres and RNA levels in the lungs were quantified using VeroE6/TMPRSS2 cells and RT-qPCR, respectively. Body weight loss, survival, lung weight, cytokine/chemokine production, nucleocapsid protein expression and lung pathology were evaluated to investigate the in vivo efficacy of ensitrelvir. RESULTS: Based on infectious viral titres and viral RNA levels in the lungs of infected mice, ensitrelvir reduced viral loads in a dose-dependent manner. The antiviral efficacy correlated with increased survival, reduced body weight loss, reduced pulmonary lesions and suppression of inflammatory cytokine/chemokine levels. CONCLUSIONS: This was the first evaluation of the in vivo anti-SARS-CoV-2 efficacy of ensitrelvir in a delayed-treatment mouse model. In this model, ensitrelvir demonstrated high antiviral potential and suppressed lung inflammation and lethality caused by SARS-CoV-2 infection. The findings support the continued clinical development of ensitrelvir as an antiviral agent to treat patients with COVID-19.