Prophylactic effect of ensitrelvir in mice infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of coronavirus disease 2019 (COVID-19) and continues to be a major health concern worldwide. Strategies to protect individuals at high risk of COVID-19 are critical but are currently a largely unmet need. We evaluated the oral antiviral drug ensitrelvir, which specifically targets the SARS-CoV-2 3CL protease, for its efficacy as a pre-exposure prophylactic treatment. Aged BALB/c mice were subcutaneously treated with various doses of ensitrelvir 24 h prior to a lethal SARS-CoV-2 challenge infection. Mouse body weight changes, survival rates, and viral titers in the lungs were evaluated, and plasma concentrations of ensitrelvir were determined. A single subcutaneous administration of ensitrelvir at 64 mg/kg or greater 24 h prior to SARS-CoV-2 challenge infection significantly protected aged mice against lethality and inhibited body weight loss. Pharmacokinetic analysis of ensitrelvir in the aged mice suggested that plasma concentrations ≥2.99 µg/mL resulted in a significant prophylactic effect against SARS-CoV-2 infection. In the aged mouse prophylaxis model, SARS-CoV-2 titers were suppressed in the lungs of mice treated with ensitrelvir 24 h prior to challenge infection, suggesting that the prophylactic administration of ensitrelvir exerted its prophylactic effect by suppressing viral proliferation. These findings suggest that ensitrelvir is a candidate drug for pre-exposure prophylactic treatment of individuals at high risk of COVID-19.

Pharmacokinetic and Pharmacodynamic Analysis of the 3CL Protease Inhibitor Ensitrelvir in a SARS-CoV-2 Infection Mouse Model.

The small-molecule antiviral drug ensitrelvir targets the 3C-like protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study evaluated its inhibitory effect on viral replication in a delayed-treatment mouse model and investigated the relationship between pharmacokinetic (PK) parameters and pharmacodynamic (PD) effects. SARS-CoV-2 gamma-strain-infected BALB/c mice were orally treated with various doses of ensitrelvir starting 24 h post-infection. Effectiveness was determined 48 h after first administration based on lung viral titers. Ensitrelvir PK parameters were estimated from previously reported plasma concentration data and PK/PD analyses were performed. Ensitrelvir doses ≥ 16 mg/kg once daily, ≥8 mg/kg twice daily, or ≥8 mg/kg thrice daily for two days significantly reduced lung viral titers compared to that of the vehicle. PK/PD analyses revealed that mean AUC0-48h post-first administration, plasma concentration 48 h post-first administration (C48h), and total time above the target plasma concentration (TimeHigh) were PK parameters predictive of viral titer reduction. In conclusion, ensitrelvir dose-dependently reduced lung SARS-CoV-2 titers in mice, suggesting it inhibited viral replication. PK parameters C48h and TimeHigh were associated with sustained ensitrelvir plasma concentrations and correlated with the reduced viral titers. The findings suggest that maintaining ensitrelvir plasma concentration is effective for exerting antiviral activity against SARS-CoV-2.

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.

Combination treatment with the cap-dependent endonuclease inhibitor baloxavir marboxil and a neuraminidase inhibitor in a mouse model of influenza A virus infection.

OBJECTIVES: Baloxavir marboxil (formerly S-033188) is a first-in-class, orally available, cap-dependent endonuclease inhibitor licensed in Japan and the USA for the treatment of influenza virus infection. We evaluated the efficacy of delayed oral treatment with baloxavir marboxil in combination with a neuraminidase inhibitor in a mouse model of lethal influenza virus infection. METHODS: The inhibitory potency of baloxavir acid (the active form of baloxavir marboxil) in combination with neuraminidase inhibitors was tested in vitro. The therapeutic effects of baloxavir marboxil and oseltamivir phosphate, or combinations thereof, were evaluated in mice lethally infected with influenza virus A/PR/8/34; treatments started 96 h post-infection. RESULTS: Combinations of baloxavir acid and neuraminidase inhibitor exhibited synergistic potency against viral replication by means of inhibition of cytopathic effects in vitro. In mice, baloxavir marboxil monotherapy (15 or 50 mg/kg twice daily) significantly and dose-dependently reduced virus titre 24 h after administration and completely prevented mortality, whereas oseltamivir phosphate treatments were not as effective. In this model, a suboptimal dose of baloxavir marboxil (0.5 mg/kg twice daily) in combination with oseltamivir phosphate provided additional efficacy compared with monotherapy in terms of virus-induced mortality, elevation of cytokine/chemokine levels and pathological changes in the lung. CONCLUSIONS: Baloxavir marboxil monotherapy with 96 h-delayed oral dosing achieved drastic reductions in virus titre, inflammatory response and mortality in a mouse model. Combination treatment with baloxavir acid and oseltamivir acid in vitro and baloxavir marboxil and oseltamivir phosphate in mice produced synergistic responses against influenza virus infections, suggesting that treating humans with the combination may be beneficial.

Evaluation of the repeated dose liver micronucleus assay using young adult rats with cyclophosphamide monohydrate: a report of a collaborative study by CSGMT/JEMS.MMS.

The repeated dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect liver carcinogens, and this assay could be integrated into general toxicological studies. In this study, in order to assess the performance of the assay, cyclophosphamide monohydrate (CP) was tested in a 14-day RDLMN assay. Based on the results of the 4-day repeated dose-finding study, 10 mg/kg/day of CP was selected as the highest dose and the lower doses were set at 5, 2.5, 1.25, and 0.625 mg/kg/day for the 14-day RDLMN assay. On the day after the completion of the dosing period, specimens of hepatocytes and bone marrow cells were prepared and the induction of micronuclei was assessed. No changes were observed in the incidences of micronucleated hepatocytes. Nevertheless, the incidences of micronucleated immature erythrocytes in the bone marrow were increased significantly at CP doses of 1.25 mg/kg/day or more. These findings are consistent with reports that CP induces tumors in various tissues but it does not induce liver tumors.

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

ASP4000, a slow-binding dipeptidyl peptidase 4 inhibitor, has antihyperglycemic activity of long duration in Zucker fatty rats.

ASP4000 ((2S)-1-{[(1R,3S,4S,6R)-6-hydroxy-2-azabicyclo[2.2.1]hept-3-yl]carbonyl}-2-pyrrolidinecarbonitrile hydrochloride) is a novel, potent and selective dipeptidyl peptidase 4 (DPP IV, EC 3.4.14.5) inhibitor (Keiko Tanaka-Amino et al. in Eur J pharmacol 59:444-449, 2008). The aim of the present study was to characterize the kinetic profile of and identify the long duration effect of the antihyperglycemic activity of ASP4000. ASP4000 was found to inhibit human recombinant DPP4 activity with a K(i) of 1.05 nM, a k(on) value of 22.3 x 10(5) M(-1) s(-1), and a k (off) of 2.35 x 10(-3) M(-1) s(-1), with higher affinity than that of vildagliptin. The kinetic studies indicate that both the formation and dissociation of ASP4000/DPP4 complex were faster than those of vildagliptin, and that ASP4000 slow-bindingly inhibits DPP4 with a different mode of inhibition than vildagliptin. In addition, ASP4000 augmented the insulin response and ameliorated the glucose excursion during the oral glucose tolerance test in Zucker fatty rats at 4 h post dosing. ASP4000 is expected to be a promising, long duration DPP4 inhibitor for type 2 diabetes.

ASP4,000, a novel, selective, dipeptidyl peptidase 4 inhibitor with antihyperglycemic activity.

ASP4,000, (2S)-1-{[(1R,3S,4S,6R)-6-hydroxy-2-azabicyclo[2.2.1]hept-3-yl]carbonyl}-2-pyrrolidinecar bonitrile hydrochloride, is a novel dipeptidyl peptidase (DPP) 4 inhibitor. In the present study, we characterized the compound as an oral antidiabetic agent both in vitro and in vivo. ASP4,000 inhibited human recombinant DPP4 with an IC(50) value of 2.25 nM, and the enzyme-kinetic curve indicated that the inhibition type was competitive. In addition, ASP4,000 also potently inhibited DPP4 activity in human, rat, dog, and monkey plasma at concentrations of the order of 10(-9) M, and showed high selectivity against other related enzymes, including DPP8 and DPP9. The antihyperglycemic activity of ASP4,000 in vivo was examined using Zucker fa/fa rats, a type 2 diabetes animal model. A single oral administration of ASP4,000 at doses of 0.03-1 mg/kg suppressed plasma DPP4 activity, and then reduced the glucose level with increasing the active GLP-1 and insulin levels in oral glucose tolerance test. These results indicate that ASP4,000 is a potent, competitive, selective DPP4 inhibitor with antihyperglycemic activity, and could be a promising candidate agent for the treatment of patients with type 2 diabetes.

Anti-inflammatory and potential cancer chemopreventive constituents of the fruits of Morinda citrifolia (Noni).

A new anthraquinone, 1,5,15-tri-O-methylmorindol (1), and two new saccharide fatty acid esters, 2-O-(beta-D-glucopyranosyl)-1-O-hexanoyl-beta-D-gluropyranose (4) and 2-O-(beta-D-glucopyranosyl)-1-O-octanoyl-beta-D-gluropyranose (5), have been isolated from a methanol extract of the fruits of Morinda citrifolia (noni) along with 10 known compounds, namely, two anthraquinones (2, 3), six saccharide fatty acid esters (6-11), an iridoid glycoside (12), and a flavanol glycoside (13). Upon evaluation of six compounds (5-7, 9, 10, and 13) for inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg/ear) in mice, four saccharide fatty acid esters, 5-7 and 9, exhibited potent anti-inflammatory activity, with ID50 values of 0.46-0.79 mg per ear. In addition, when compounds 1-13 were evaluated against the Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA, all of the compounds exhibited moderate inhibitory effects (IC50 values of 386-578 mol ratio/32 pmol TPA).