Baloxavir marboxil, a novel cap-dependent endonuclease inhibitor potently suppresses influenza virus replication and represents therapeutic effects in both immunocompetent and immunocompromised mouse models

Abstract

Baloxavir marboxil (BXM) is an orally available small molecule inhibitor of cap-dependent endonuclease (CEN), an essential enzyme in the initiation of mRNA synthesis of influenza viruses. In the present study, we evaluated the efficacy of BXM against influenza virus infection in mouse models. Single-day oral administration of BXM completely prevented mortality due to infection with influenza A and B virus in mice. Moreover, 5-day repeated administration of BXM was more effective for reducing mortality and body weight loss in mice infected with influenza A virus than oseltamivir phosphate (OSP), even when the treatment was delayed up to 96 hours post infection (p.i.). Notably, administration of BXM, starting at 72 hours p.i. led to significant decrease in virus titers of >2-log10 reduction compared to the vehicle control within 24 hours after administration. Virus reduction in the lung was significantly greater than that observed with OSP. In addition, profound and sustained reduction of virus titer was observed in the immunocompromised mouse model without emergence of variants possessing treatment-emergent amino acid substitutions in the target protein. In our immunocompetent and immunocompromised mouse models, delayed treatment with BXM resulted in rapid and potent reduction in infectious virus titer and prevention of signs of influenza infection, suggesting that BXM could extend the therapeutic window for patients with influenza virus infection regardless of the host immune status.

Conflict of interest statement

All authors are employees of Shionogi and Co., Ltd or Shionogi TechnoAdvance Research, Co., Ltd, an affiliation of Shionogi. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Therapeutic efficacy of single-day oral administration of BXM in a lethal infection mouse model.

Nine to ten mice per group were intranasally infected with (A) 1.38 × 103 or (B) 4.42 × 104 TCID50 virus suspension of A/PR/8/34, or (C) 3.30 × 105 or (D) 1.98 × 106 TCID50 virus suspension of B/HK/5/72 and then orally administered BXM (0.05, 0.5, 5, or 50 mg/kg) bid for 1 day, OSP (5 or 50 mg/kg) bid for 5 days, or vehicle bid for 1 day. Mice were examined daily for survival through 14 or 21 days p.i.. The survival time in groups treated with BXM for 1 day was significantly prolonged compared to those in the vehicle-treated group (*, P < 0.05; **, P < 0.001; ***, P < 0.001) or OSP (5 mg/kg bid)-treated group (†, P < 0.0005).

Fig 2. Inhibitory effects of single-day oral administration of BXM on virus titers in lungs in a lethal infection mouse model.

Five mice per group were intranasally infected with (A) 1.38 × 103 or (B) 4.42 × 104 TCID50 virus suspension of A/PR/8/34, or (C) 3.30 × 105 or (D) 1.98 × 106 TCID50 virus suspension of B/HK/5/72 and then orally administered BXM (0.05, 0.5, 5, or 50 mg/kg), OSP (5 or 50 mg/kg), or vehicle bid for 1 day. The virus titers in lungs on day 1 p.i. were measured by TCID50 method. Each bar represents the mean ± SD of 5 mice. The limit of quantification (1.50 log10 TCID50/mL) is indicated by a dotted line. In the A/PR/8/34-infected mouse model, significant differences in virus titers were observed in BXM (except for 0.05 mg/kg bid at the infectious dose of 1.38 × 103 TCID50)- and OSP-treated groups in comparison with the vehicle-treated group (*, P < 0.05; **, P < 0.001; ***, P < 0.0001). Significant differences in virus titers were also observed between BXM- and OSP-treated groups (†, P < 0.05; ††, P < 0.001; †††, P < 0.0001 vs OSP 5 mg/kg bid; §§§, P < 0.0001 vs OSP 50 mg/kg bid). In the B/HK/5/72-infected mouse model, significant differences in virus titers were observed in BXM (except for 0.5 mg/kg bid at the infectious dose of 1.98 × 106 TCID50)- and OSP (at the infectious dose of 3.30 × 105 TCID50)-treated groups in comparison with the vehicle-treated group (*, P < 0.05; **, P < 0.001; ***, P < 0.0001). Significant differences in virus titers were also observed between BXM- and OSP-treated groups (†††, P < 0.0001 vs OSP 5 mg/kg bid; §§§, P < 0.0001 vs OSP 50 mg/kg bid).

Fig 3. Therapeutic efficacy of delayed administration of BXM against lethal influenza A virus infection in mice.

Ten mice per group infected with A/PR/8/34 (1.38 × 103 TCID50) were treated orally with BXM (1.5 or 15 mg/kg), OSP (5 mg/kg), or vehicle bid for 5 days from (A) 24, (B) 48, (C)72, or (D) 96 hours p.i.. Mice were monitored daily for survival and body weight through 28 days p.i.. Treatment periods are indicated by the gray zones. Significant differences in survival time were observed in groups treated with BXM from 24, 48, 72, and 96 hours p.i. in comparison with the vehicle-treated group (**, P < 0.01; ***, P < 0.001). The survival time in groups treated with OSP from 24 and 48 hours p.i. was significantly prolonged compared to that in the vehicle-treated group (***, P < 0.0001). The survival time of the group that received BXM starting at 72 and 96 hours p.i. was significantly prolonged compared to that of the groups treated with OSP at a dose of 5 mg/kg bid (†, P < 0.05; ††, P < 0.01; †††, P < 0.0001).

Fig 4. Effects of delayed administration of BXM on body weight change following influenza virus infection in mice.

Mice infected with A/PR/8/34 (1.38 × 103 TCID50) were orally treated with BXM (1.5 or 15 mg/kg), OSP (5 mg/kg), or vehicle bid for 5 days from (A, E) 24, (B, F) 48, (C, G) 72, or (D, H) 96 hours p.i. and monitored daily for body weight up to 28 days p.i.. Uninfected mice (n = 5) were also monitored daily for body weight as a control. Significant differences in body weight were observed in groups treated with BXM and OSP in comparison with vehicle-treated group on the indicated days (*, P < 0.05; **, P < 0.01; ***, P < 0.0001). The groups treated with BXM from 24, 48, 72 or 96 hours p.i. showed significantly less body weight loss than the OSP-treated group on the indicated days (†, P<0.05; ††, P < 0.01, †††, P < 0.0001).

Fig 5. Inhibitory effects of delayed administration of BXM on virus replication in mice.

Mice infected with A/PR/8/34 (1.38 × 103 TCID50) were orally treated with BXM (1.5 or 15 mg/kg), OSP (5 mg/kg), or vehicle bid daily up to 5 days from 72 hours p.i. and were euthanized on the indicated days. Treatment period is indicated by the gray zone. Virus titers in the lungs were measured by the TCID50 method. The limit of quantification (1.50 log10 TCID50/mL) is indicated by a dotted line. Each point represents the mean ± SD of 6 to 8 mice except points that indicated virus titer on days 8 and 10 in mice treated with OSP (N = 1), in which only one mouse survived. Virus titers in mice treated with BXM and OSP on days 8 and 10 were at or lower than the quantification limit. Significant differences in virus titers were observed in BXM- and OSP-treated groups in comparison with the vehicle-treated group on the days 4 and 6, and day 4 p.i., respectively (**, P < 0.01; ***, P < 0.0001). Significant differences in virus titers were also observed between BXM- and OSP-treated groups on days 4 and 6 p.i. (†††, P < 0.0001).

Fig 6. Effects of BXM on the virus titers and body weight changes in immunocompromised mice infected with influenza A virus.

Five mice per group infected with A/PR/8/34 (100 TCID50) were treated orally with BXM (1.5, 15 or 50 mg/kg), OSP (5 or 50 mg/kg), or vehicle bid for 5 days from 120 hours p.i.. The mice were monitored daily for body weight up to day 10 p.i. and their lungs were harvested on days 5, 6, 7, 8, 9 and 10 p.i. to determine virus titers. Treatment periods are indicated by the gray zones. (A) A significant reduction in virus titer from days 5 to 10 p.i. was observed in all groups administered BXM in comparison with the group administered the vehicle or OSP at doses of 5 and 50 mg/kg bid (*, P < 0.0001 vs vehicle; †, P < 0.0001 vs OSP 5 mg/kg bid; ‡, P < 0.0001 vs OSP 50 mg/kg bid). (B) All groups treated with BXM showed significantly less reduction of body weight compared with the vehicle-treated group or OSP-treated groups (*, P < 0.0001 vs vehicle; †, P < 0.0005; ††, P < 0.0001 vs OSP 5 mg/kg bid; ‡, P < 0.005, ‡‡, P < 0.0001 vs OSP 50 mg/kg bid).