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.

Efficacy and Safety of Ensitrelvir in Patients With Mild-to-Moderate Coronavirus Disease 2019: The Phase 2b Part of a Randomized, Placebo-Controlled, Phase 2/3 Study.

BACKGROUND: This phase 2b part of a randomized phase 2/3 study assessed the efficacy and safety of ensitrelvir for mild-to-moderate coronavirus disease 2019 (COVID-19) during the Omicron epidemic. METHODS: Patients were randomized (1:1:1) to orally receive ensitrelvir fumaric acid 125 mg (375 mg on day 1) or 250 mg (750 mg on day 1) or placebo once daily for 5 days. The co-primary endpoints were the change from baseline in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) titer on day 4 and time-weighted average change from baseline up to 120 hours in the total score of predefined 12 COVID-19 symptoms. Safety was assessed through adverse events. RESULTS: A total of 341 patients (ensitrelvir 125-mg group: 114; ensitrelvir 250-mg group: 116; and placebo group: 111; male: 53.5-64.9%; mean age: 35.3-37.3 years) were included in the efficacy analyses. The change from baseline in SARS-CoV-2 titer on day 4 was significantly greater with both ensitrelvir doses than with placebo (differences from placebo: -0.41 log10 50% tissue-culture infectious dose/mL; P < .0001 for both). The total score of the 12 COVID-19 symptoms did not show a significant difference between the ensitrelvir groups and placebo group. The time-weighted average change from baseline up to 120 hours was significantly greater with ensitrelvir versus placebo in several subtotal scores, including acute symptoms and respiratory symptoms. Most adverse events were mild in severity. CONCLUSIONS: Ensitrelvir treatment demonstrated a favorable antiviral efficacy and potential clinical benefit with an acceptable safety profile. CLINICAL TRIALS REGISTRATION: Japan Registry of Clinical Trials: jRCT2031210350 (https://jrct.niph.go.jp/en-latest-detail/jRCT2031210350).

A Randomized Phase 2/3 Study of Ensitrelvir, a Novel Oral SARS-CoV-2 3C-Like Protease Inhibitor, in Japanese Patients with Mild-to-Moderate COVID-19 or Asymptomatic SARS-CoV-2 Infection: Results of the Phase 2a Part.

This multicenter, double-blind, phase 2a part of a phase 2/3 study assessed the efficacy and safety of ensitrelvir, a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor, in Japanese patients with mild-to-moderate coronavirus disease 2019 (COVID-19) or asymptomatic SARS-CoV-2 infection. Sixty-nine patients were randomized (1:1:1) to orally receive 5-day ensitrelvir fumaric acid (375 mg on day 1 followed by 125 mg daily, or 750 mg on day 1 followed by 250 mg daily) or placebo and followed up until day 28. The primary outcome was the change from baseline in the SARS-CoV-2 viral titer. A total of 16, 14, and 17 patients in the ensitrelvir 125 mg, ensitrelvir 250 mg, and placebo groups, respectively, were included in the intention-to-treat population (mean age: 38.0 to 40.4 years). On day 4, the change from baseline in SARS-CoV-2 viral titer (log10 50% tissue culture infectious dose/mL) in patients with positive viral titer and viral RNA at baseline was greater with ensitrelvir 125 mg (mean [standard deviation], -2.42 [1.42]; P = 0.0712) and 250 mg (-2.81 [1.21]; P = 0.0083) versus placebo (-1.54 [0.74]); ensitrelvir treatment reduced SARS-CoV-2 RNA by -1.4 to -1.5 log10 copies/mL versus placebo. The viral titer and viral RNA were similar across groups on and after day 6. The median time to infectious viral clearance decreased by approximately 50 h with ensitrelvir treatment. All adverse events were mild to moderate. Ensitrelvir treatment demonstrated rapid SARS-CoV-2 clearance and was well tolerated (Japan Registry of Clinical Trials identifier: jRCT2031210350).

Assessing the fitness of a dual-antiviral drug resistant human influenza virus in the ferret model.

Influenza antivirals are important tools in our fight against annual influenza epidemics and future influenza pandemics. Combinations of antivirals may reduce the likelihood of drug resistance and improve clinical outcomes. Previously, two hospitalised immunocompromised influenza patients, who received a combination of a neuraminidase inhibitor and baloxavir marboxil, shed influenza viruses resistant to both drugs. Here-in, the replicative fitness of one of these A(H1N1)pdm09 virus isolates with dual resistance mutations (NA-H275Y and PA-I38T) was similar to wild type virus (WT) in vitro, but reduced in the upper respiratory tracts of challenged ferrets. The dual-mutant virus transmitted well between ferrets in an airborne transmission model, but was outcompeted by the WT when the two viruses were co-administered. These results indicate the dual-mutant virus had a moderate loss of viral fitness compared to the WT virus, suggesting that while person-to-person transmission of the dual-resistant virus may be possible, widespread community transmission is unlikely.

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model.

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.

Comprehensive assessment of amino acid substitutions in the trimeric RNA polymerase complex of influenza A virus detected in clinical trials of baloxavir marboxil.

BACKGROUND: Baloxavir marboxil (BXM) is an approved drug that selectively targets cap-dependent endonuclease on PA subunit in the RNA polymerase complex of influenza A and B viruses. Amino acid substitutions at position 38 in the PA subunit were identified as a major pathway for reduced susceptibility to baloxavir acid (BXA), the active form of BXM. Additionally, substitutions found at positions E23, A37, and E199 in the PA subunit impact BXA susceptibility by less than 10-fold. METHODS: We comprehensively evaluated the impact of novel amino acid substitutions identified in PA, PB1, and PB2 subunits in BXM clinical trials and influenza sequence databases by means of drug susceptibility and replicative capacity. RESULTS: PA/I38N in A(H1N1)pdm09 and PA/I38R in A(H3N2) were newly identified as treatment-emergent substitutions in the CAPSTONE-2 study. The I38N substitution conferred reduced susceptibility by 24-fold, whereas replicative capacity of the I38N-substituted virus was impaired compared with the wild-type. The I38R-substituted virus was not viable in cell culture. All other mutations assessed in this extensive study did not significantly affect BXA susceptibility (< 2.4-fold change). CONCLUSION: These results provide additional information on the impact of amino acid substitutions in the trimeric viral polymerase complex to BXA susceptibility and will further support influenza surveillance.

Carryover effects of baloxavir acid in human nasopharyngeal/pharyngeal swabs on infectious titer testing of influenza virus.

Baloxavir marboxil (BXM) demonstrated a rapid and profound decline in infectious viral titer 1 day after BXM administration. Rapid reduction in virus titer is a characteristic of BXM. There may be a possibility that drug carryover effects have impacts on the observed antiviral effects due to the poor correlation that was observed between viral titer reduction and alleviation of influenza symptoms. Here, we report possible carryover effects of baloxavir acid (BXA), an active form of BXM, on infectious titer testing. Our findings indicate that there is little impact of BXA carryover on the infectious titer testing.

Baloxavir Marboxil in Japanese Pediatric Patients With Influenza: Safety and Clinical and Virologic Outcomes.

BACKGROUND: We assessed the safety and effectiveness of baloxavir marboxil administration in Japanese children with influenza. METHODS: This open-label study administered 1 weight-adjusted dose of baloxavir to 107 children aged 1-11 years with laboratory-confirmed, febrile influenza virus infection of ≤48 hours duration. RESULTS: Adverse events (AEs) were reported in 34.6% of patients, most commonly vomiting (7.5%); no serious AEs or AEs causing discontinuation occurred. The median time to alleviation of influenza illness was 44.6 hours (95% confidence interval, 38.9-62.5 hours), to resolution of fever was 21.4 hours, and to sustained cessation of infectious viral shedding was 24.0 hours. However, viruses with amino acid substitutions in the viral polymerase acidic protein at position I38 (PA/I38T/M) emerged in 18 of 77 (23.4%) patients. Emergence was associated with longer infectious virus detectability (median time, 180.0 hours) and time to illness alleviation (median, 79.6 vs 42.8 hours in patients without PA/I38T/M-substituted viruses). Among patients with PA/I38T/M-substituted virus emergence, those with baseline hemagglutinin inhibition (HAI) antibody titer <40 experienced delay in time to illness alleviation (median, 85.4 vs 56.0 hours in patients with higher baseline HAI antibody titer). CONCLUSIONS: A single, oral dose of baloxavir marboxil was well tolerated and rapidly reduced viral titers, but the common emergence of PA/I38T/M-substituted viruses warrants consideration of alternative dosing regimens in young children. CLINICAL TRIALS REGISTRATION: Japan Pharmaceutical Information Center Clinical Trials Information (Japic CTI-163417).

Treatment-Emergent Influenza Variant Viruses With Reduced Baloxavir Susceptibility: Impact on Clinical and Virologic Outcomes in Uncomplicated Influenza.

BACKGROUND: Single-dose baloxavir rapidly reduces influenza virus titers and symptoms in patients with uncomplicated influenza, but viruses with reduced in vitro susceptibility due to amino acid substitutions at position 38 of polymerase acidic protein (PA/I38X) sometimes emerge. METHODS: We evaluated the kinetics, risk factors, and effects on clinical and virologic outcomes of emergence of PA/I38X-substituted viruses. RESULTS: Viruses containing PA/I38X substitutions were identified 3-9 days after baloxavir treatment in 9.7% (36/370) of patients, of whom 85.3% had transient virus titer rises. Median time to sustained cessation of infectious virus detection was 192, 48, and 96 hours in the baloxavir recipients with PA/I38X-substituted viruses, without PA/I38X-substituted viruses, and placebo recipients, respectively. The corresponding median times to alleviation of symptoms were 63.1, 51.0, and 80.2 hours, respectively. After day 5, symptom increases occurred in 11.5%, 8.0%, and 13.0%, respectively, and in 8.9% of oseltamivir recipients. Variant virus emergence was associated with lower baseline neutralizing antibody titers. CONCLUSIONS: The emergence of viruses with PA/I38X substitutions following baloxavir treatment was associated with transient rises in infectious virus titers, prolongation of virus detectability, initial delay in symptom alleviation, and uncommonly with symptom rebound. The potential transmissibility of PA/I38X-substituted viruses requires careful study. CLINICAL TRIAL REGISTRATION: NCT02954354.

In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit.

Cap-dependent endonuclease (CEN) resides in the PA subunit of the influenza virus and mediates the critical "cap-snatching" step of viral RNA transcription, which is considered to be a promising anti-influenza target. Here, we describe in vitro characterization of a novel CEN inhibitor, baloxavir acid (BXA), the active form of baloxavir marboxil (BXM). BXA inhibits viral RNA transcription via selective inhibition of CEN activity in enzymatic assays, and inhibits viral replication in infected cells without cytotoxicity in cytopathic effect assays. The antiviral activity of BXA is also confirmed in yield reduction assays with seasonal type A and B viruses, including neuraminidase inhibitor-resistant strains. Furthermore, BXA shows broad potency against various subtypes of influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2). Additionally, serial passages of the viruses in the presence of BXA result in isolation of PA/I38T variants with reduced BXA susceptibility. Phenotypic and genotypic analyses with reverse genetics demonstrate the mechanism of BXA action via CEN inhibition in infected cells. These results reveal the in vitro characteristics of BXA and support clinical use of BXM to treat influenza.

Dietary total antioxidant capacity from different assays in relation to serum C-reactive protein among young Japanese women.

BACKGROUND: The association between dietary total antioxidant capacity (TAC) from different assays and serum C-reactive protein (CRP) has not been assessed in non-Western populations. We examined the association between dietary TAC and serum CRP concentration in young Japanese women using different four TAC assays. METHODS: The subjects were 443 young Japanese women aged 18-22 years. Dietary TAC was assessed with a self-administered diet history questionnaire and the TAC value of each food using the following four assays: ferric reducing ability of plasma (FRAP); oxygen radical absorbance capacity (ORAC); Trolox equivalent antioxidant capacity (TEAC); and total radical-trapping antioxidant parameter (TRAP). Serum CRP concentrations were measured by highly sensitive nephelometry. RESULTS: The major contributor to dietary TAC was green, barley, and oolong tea (FRAP: 53%, ORAC: 45%, TEAC: 36%, and TRAP: 44%). The prevalence of elevated CRP concentrations (≥ 1 mg/L) was 5.6%. TAC from FRAP was inversely associated with serum CRP concentrations (adjusted odds ratio [OR] for elevated CRP concentration in high [compared with low] dietary TAC group: 0.39 [95% confidence interval (CI): 0.16-0.98]; P = 0.04). TAC from ORAC was inversely associated with CRP, although the association was not significant (OR: 0.48 [95% CI: 0.20-1.14]; P = 0.10). TAC from TEAC was inversely associated with CRP (OR: 0.32 [95% CI: 0.12-0.82]; P = 0.02), as was TAC from TRAP (OR: 0.31 [95% CI: 0.12-0.81]; P = 0.02). CONCLUSIONS: Dietary TAC was inversely associated with serum CRP concentration in young Japanese women regardless of assay. Further studies are needed in other populations to confirm these results.

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner.

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.

Establishment of a cynomolgus macaque model of influenza B virus infection.

Pathogenicity of influenza B virus was examined in cynomolgus macaques to establish a macaque model suitable for vaccine and antiviral drug development. We prepared influenza B viruses for inoculation with minimal passages after isolation from patients. Macaques inoculated with influenza B virus showed higher body temperature than that before infection for 6 to 12 days. Virus was detected in nasal, tracheal, and bronchial samples until 6 days after inoculation followed by an increase in neutralizing antibody. High levels of IL-6 and TNF-α in nasal swabs from the infected macaques were correlated with fever. Symptoms and duration of the viral replication would be sufficient to evaluate efficacy of vaccines and antiviral agents. In addition, measurement of immune responses including antibody and cytokine production would provide an immunological rationale in efficacy of vaccines and antiviral agents. The results suggest that cynomolgus macaques are appropriate model animals for research of influenza B virus.

Association between Hardness (Difficulty of Chewing) of the Habitual Diet and Premenstrual Symptoms in Young Japanese Women.

Recent evidence suggests that voluntary rhythmic movements such as chewing may increase blood serotonin and subsequently brain serotonin, which in turn acts to alleviate premenstrual symptoms. In this observational cross-sectional study, we tested the hypothesis that hardness (difficulty of chewing) of the habitual diet (i.e. dietary hardness) is associated with decreased premenstrual symptoms. Subjects were 640 female Japanese dietetic students aged 18-22 years. Dietary hardness was assessed as an estimate of masticatory muscle activity for the habitual diet (i.e. the difficulty of chewing the food). The consumption of a total of 107 foods was estimated by means of a self-administered, comprehensive diet history questionnaire, and masticatory muscle activity during the ingestion of these foods was estimated according to published equations. Menstrual cycle symptoms were assessed using the retrospective version of the Moos Menstrual Distress Questionnaire, from which total score and subscale scores (i.e. pain, concentration, behavioral change, autonomic reactions, water retention, and negative affect) in the premenstrual phase were calculated and expressed as percentages relative to those in the intermenstrual phase. Dietary hardness was not associated with total score in the premenstrual phase (P for trend = 0.48). Further, no association was seen for any subscale score in the premenstrual phase (P for trend = 0.18-0.91). In conclusion, this preliminary study failed to substantiate a hypothesized inverse relationship between hardness of the habitual diet and premenstrual symptoms. Considering the plausibility of the putative mechanism, however, further investigation using more relevant measures of chewing and premenstrual symptoms is warranted.

Total n-3 polyunsaturated fatty acid intake is inversely associated with serum C-reactive protein in young Japanese women.

Little is known about the relation of dietary factors to circulating C-reactive protein (CRP) concentrations in young adults and non-Western populations. We cross-sectionally examined associations between dietary intake and serum CRP concentrations in young Japanese women. The subjects were 443 female Japanese dietetic students aged 18 to 22 years. Dietary intake was assessed with a validated, self-administered, comprehensive, diet history questionnaire. Serum CRP concentrations were measured by highly sensitive nephelometry. The prevalence of elevated CRP (> or = 1 mg/L) was 5.6%. After adjustment for possible confounding factors including body mass index, a significant inverse association was seen between total n-3 polyunsaturated fatty acid intake and elevated CRP. The multivariate adjusted odds ratios of elevated CRP for women with intake below and above the median (1.1% of energy) were 1.00 and 0.33 (95% confidence interval, 0.13-0.82; P = .02), respectively. Intake of eicosapentaenoic acid + docosahexaenoic acid and alpha-linolenic acid was not associated with elevated CRP concentrations (P = .62 and P = .27, respectively). Vitamin C intake was independently inversely associated with elevated CRP, although the association was nonsignificant (P = .10). No clear associations were observed for other dietary factors examined including total fat, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, total dietary fiber, soluble dietary fiber, insoluble dietary fiber, and magnesium; fruits, vegetables, and fish and shellfish; and dietary glycemic load (P = .27 to P = .99). In conclusion, total n-3 polyunsaturated fatty acid intake showed an independent inverse association with elevated serum CRP concentration in a group of young Japanese women.