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

Abstract

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.

© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Figures

Figure 1.

Inhibitory effect of baloxavir acid (BXA) in combination with NAIs on CPE in cultured cells infected with influenza A virus. MDCK cells were infected with A/PR/8/34 (H1N1) in the presence of various concentrations of the indicated compounds and cell viability was assessed at 2 days post-infection. The combination effects are shown by isobologram plot (a) and MacSynergy analysis (b). For the isobologram plot, the EC50 of each substance alone and at a fixed concentration of the other were determined. (DA/A + B)/DA and (DB/A + B)/DB were plotted on the x- and y-axes, respectively; DA is the EC50 of substance A alone, DB is the EC50 of substance B alone, DA/A + B is the concentration of substance A giving 50% inhibition in combination with substance B, and DB/A + B is the concentration of substance B giving 50% inhibition in combination with substance A. LAN, laninamivir; OSA, oseltamivir acid; PER, peramivir trihydrate; ZAN, zanamivir hydrate.

Figure 2.

Virus titre (a) and lung weight (b) in mice infected with influenza virus and treated with antiviral. Data are presented as mean ± SD. The dotted line represents the lower limit of virus detection (a) or mean lung weight in uninfected, inoculated, control mice at day 7 (lower line) or normal uninoculated mice (upper line) (b). There were n = 5 mice per group. Differences between groups were analysed using Dunnett’s test for baloxavir marboxil (BXM) comparisons or the t-test for oseltamivir phosphate (OSP) and combination comparisons: aP < 0.05 versus vehicle, bP < 0.01 versus vehicle, cP < 0.0001 versus vehicle, dP < 0.05 versus OSP 10, eP < 0.01 versus OSP 10, fP < 0.0001 versus OSP 10, gP < 0.05 versus BXM 0.5 and hP < 0.0001 versus BXM 0.5. BXM, baloxavir marboxil; OSP, oseltamivir phosphate. Dosages shown for antiviral compounds are in units of mg/kg.

Figure 3.

Survival rate (a) and change in body weight (b) in mice infected with influenza virus and treated with antiviral. Mice with a −30% change in body weight were euthanized according to humane endpoints and considered dead. For body weight, data are presented as mean ± SD. If a mouse died, the current body weight as a proportion of initial body weight at all timepoints after death was regarded as 70% (−30%). There were n = 10 mice per group. Differences between groups were analysed by log-rank test for survival and one-way ANOVA for body weight: aP < 0.05 versus vehicle, bP < 0.01 versus vehicle, cP < 0.0001 versus vehicle, dP < 0.05 versus OSP 10/50 (for body weight, day 5 and day 7 versus OSP 10), eP < 0.01 versus OSP 10/50 (for survival, BXM 1.5 versus OSP 10; for body weight, day 7 versus OSP 10), fP < 0.0001 versus OSP 10/50 (for body weight, day 5 versus OSP 10), gP < 0.05 versus BXM 0.5 and hP < 0.01 versus BXM 0.5. BXM, baloxavir marboxil; OSP, oseltamivir phosphate. Dosages shown for antiviral compounds are in units of mg/kg.

Figure 4.

Cytokine and chemokine levels in the lungs of mice infected with influenza virus and treated with antiviral. Data are presented as mean ± SD. The lower dotted line represents the mean level in uninfected control mice, and the upper dotted line represents the mean level in infected mice at day 4 (immediately before starting treatment). There were n = 5 mice per group. Differences between groups were analysed using Dunnett’s test for baloxavir marboxil (BXM) comparisons or the t-test for oseltamivir phosphate (OSP) and combination comparisons: aP < 0.05 versus vehicle, bP < 0.01 versus vehicle, cP < 0.0001 versus vehicle, dP < 0.05 versus OSP 10, eP < 0.01 versus OSP 10, fP < 0.0001 versus OSP 10, gP < 0.05 versus BXM 0.5, hP < 0.01 versus BXM 0.5 and iP < 0.0001 versus BXM 0.5. Dosages shown for antiviral compounds are in units of mg/kg.

Figure 5.

Lung pathology at day 7 in mice infected with influenza virus on day 0 and treated with antiviral on days 4–8. Sections of lung were stained with haematoxylin and eosin and observed with light microscopy (a) and scanning electron microscopy (b). Representative sections are presented. BXM, baloxavir marboxil; OSP, oseltamivir phosphate.