Viral titers in the lungs of infected mice treated with zanamivir or CS-8958 7 days before influenza virus infection. Mice were intranasally administered zanamivir at 4.8 μmol/kg (closed squares), 14 μmol/kg (closed circles), or 43 μmol/kg (closed triangles) (a); CS-8958 at 0.18 μmol/kg (closed squares), 0.53 μmol/kg (closed circles), or 1.6 μmol/kg (closed triangles) (b); or saline (open circles; a and b) 7 days before infection with influenza A virus (A/PR/8/34, 500 PFU). The viral titers (log₁₀ PFU/lungs) in the mouse lungs on days 1, 2, 3, and 4 are shown. Zanamivir doses of 4.8, 14, and 43 μmol/kg correspond to 1.6, 4.7, and 14 mg/kg, respectively; and CS-8958 doses of 0.18, 0.53, and 1.6 μmol/kg correspond to 0.085, 0.25, and 0.76 mg/kg, respectively. Each plot represents the mean ± standard deviation (n = 3). (Inset) AUCs (in percent) for zanamivir (closed circles) and CS-8958 (open circles) are plotted against the dose. The quantitative differences between the compounds were estimated to be 32.8 (P = 0.0028) by the parallel-line assay (ANCOVA).

In vivo efficacy of prophylactic oral administration of oseltamivir phosphate and intranasal administration of CS-8958 in a mouse/influenza A virus infection model. Mice were infected with influenza A virus (A/PR/8/34, 100 PFU/mouse) on day 0 (n = 8). Oseltamivir phosphate at 110 mg/kg (a) and CS-8958 at 0.17 mg/kg (0.37 μmol/kg) (b) were administered at 12 h (open circles), 1 day (open squares), 4 days (open triangles), or 7 days (multiplication signs) before infection. Saline (closed circles) was administered intranasally at 7 days before infection. The number of surviving mice was monitored until 20 dpi. The prolonged survival effects for all groups administered CS-8958 were significant (P < 0.0001 for administration 12 h, 1 day, and 4 days before infection and P = 0.0106 for administration 7 days before infection). The survival effects for groups that received oseltamivir phosphate at 12 h and 1 day before infection were significant (P < 0.0001 and P = 0.0304, respectively).

In vivo efficacy of repeated administration of oseltamivir phosphate and a single administration of CS-8958 in a mouse influenza A virus infection model. Mice were infected with influenza A virus (A/PR/8/34, 100 PFU) on day 0 (0 h) (n = 10). For the group treated with oseltamivir phosphate, the oral administrations started at 11 hpi and were then continued twice daily until 120 hpi (twice daily for 5 days), and saline was administered intranasally at 11 hpi (a). For the group treated with CS-8958, the intranasal administration of CS-8958 was performed at 11 hpi, followed by the oral administration of saline twice daily for 5 days on the same schedule as the oseltamivir phosphate-treated group (b). For the control group, the intranasal administration of saline was performed at 11 hpi, followed by the oral administration of saline twice daily for 5 days on the same schedule as the oseltamivir phosphate-treated group. The doses of oseltamivir phosphate were 0.12 mg/kg (multiplication signs), 0.37 mg/kg (plus signs), 1.1 mg/kg (open triangles), 3.3 mg/kg (open diamonds), and 10 mg/kg (open squares); and the doses of CS-8958 were 0.012 μmol/kg (multiplication signs), 0.037 μmol/kg (closed circles), 0.11 μmol/kg (closed triangles), 0.33 μmol/kg (closed diamonds), and 1.0 μmol/kg (closed squares). Open circles (a and b), controls. CS-8958 doses of 0.012, 0.037, 0.11, 0.33, and 1.0 μmol/kg correspond to 0.0057, 0.017, 0.052, 0.16, and 0.47 mg/kg, respectively. The number of surviving mice was monitored until 20 dpi.

Virus titers in the nasal washes of infected ferrets treated by the repeated administration of oseltamivir phosphate and a single intranasal administration of zanamivir or CS-8958. The virus titers (log₁₀ PFU/ml) in the nasal washes of ferrets treated by the twice-daily oral administration from 4 hpi of 25 mg/kg of oseltamivir phosphate (dotted lines, squares) or by the single intranasal administration at 4 hpi of 0.5 μmol/kg (0.17 mg/kg) of zanamivir (dotted lines, triangles), 0.05 μmol/kg (0.024 mg/kg; solid lines, triangles) or 0.5 μmol/kg (0.24 mg/kg; solid lines, squares) of CS-8958, or saline (solid lines, open circles) are shown. The virus titers (log₁₀ PFU/ml of nasal wash) were measured at 1, 2, and 3 dpi. Each plot represents the mean ± standard deviation (n = 5). No viruses were detected in the nasal washes of one ferret in the saline-treated group, three of the ferrets in the oseltamivir-treated group, and four of ferrets in the zanamivir-treated group and both of the CS-8958 groups at 1 dpi and in the nasal washes of one of the ferrets in the high-dose CS-8958 group at 2 dpi. In those cases, the mean viral titers were calculated by using their lower limits of detection, and the standard deviation values were not calculated. By using the mean viral titers, oseltamivir phosphate, zanamivir, a low dose of CS-8958, and a high dose of CS-8958 showed statistically significant reductions in viral titers from 1 to 2 dpi (P = 0.0013, <0.0001, 0.0081, and < 0.0001, respectively.)

In vivo efficacy of prophylactic intranasal administration of zanamivir or CS-8958 in mouse/influenza A virus infection model. Mice were infected with influenza A virus (A/PR/8/34, 500 PFU/mouse) on day 0 (n = 12). Zanamivir was intranasally administered at 0 μmol/kg (open circles; saline), 5.2 μmol/kg (multiplication signs), 10 μmol/kg (open triangles), 21 μmol/kg (open diamonds), or 42 μmol/kg (open squares) (a) and CS-8958 was intranasally administered at 0 μmol/kg (open circles; saline), 0.20 μmol/kg (multiplication signs), 0.39 μmol/kg (closed triangles), 0.78 μmol/kg (closed diamonds), or 1.5 μmol/kg (closed squares) (b) 7 days before infection. Zanamivir doses of 5.2, 10, 21, and 42 μmol/kg correspond to 1.7, 3.3, 7.0, and 14 mg/kg, respectively; and CS-8958 doses of 0.20, 0.39, 0.78, and 1.5 μmol/kg correspond to 0.095, 0.18, 0.37, and 0.71 mg/kg, respectively. The number of surviving mice was monitored until 20 dpi. The differences were not significant for the group treated with zanamivir at 10 μmol/kg and were significant for the groups treated with zanamivir at 5.2, 21, and 42 μmol/kg (P = 0.0102, P = 0.0029, and P < 0.0001, respectively) compared with the results for the saline group. The differences in the results were not significant for the groups treated with CS-8958 at 0.20 and 0.39 μmol/kg and were significant for the groups treated with CS-8958 at 0.78 and 1.5 μmol/kg (P < 0.0001 for both) compared with the results for the saline-treated group.

In vivo efficacy of prophylactic intranasal administration of zanamivir or CS-8958 in a mouse/influenza B virus infection model. Mice were infected with influenza B virus (B/Hong Kong/5/72, 1,500 PFU/mouse) on day 0 (n = 11). Zanamivir was intranasally administered at 0 μmol/kg (open circles; saline), 3.3 μmol/kg (open triangles), 9.8 μmol/kg (closed squares), or 29 μmol/kg (closed circles) (a) and CS-8958 was intranasally administered at 0 μmol/kg (open circles; saline), 0.16 μmol/kg (open triangles), 0.49 μmol/kg (closed squares), or 1.5 μmol/kg (closed circles) (b) 7 days before infection. Zanamivir doses of 3.3. 9.8, and 29 μmol/kg correspond to 1.1, 3.3, and 9.6 mg/kg, respectively; and CS-8958 doses of 0.16, 0.49, and 1.5 μmol/kg of correspond to 0.076, 0.23, and 0.71 mg/kg, respectively. The number of surviving mice was monitored until 20 dpi. Differences were significant for the groups treated with zanamivir at 3.3, 9.8, and 29 μmol/kg (P = 0.0223, P = 0.0046, and P < 0.0001, respectively) compared with the results for the saline-treated group. The differences were not significant for the group treated with CS-8958 at 0.16 μmol/kg and were significant for the groups treated with CS-8958 at 0.49 and 1.5 μmol/kg (P < 0.0001 for both) compared with the results for the saline-treated group.

Virus titers in the lungs of infected mice treated with a single administration or repeated intranasal administrations of zanamivir or CS-8958. The virus titers (log₁₀ PFU/lungs) in the mouse lungs after saline administration (closed circles), a single administration (open triangles) or repeated administrations (closed triangles) of zanamivir, and a single administration (open squares) or repeated administrations (closed squares) of CS-8958 are shown. The dose of both compounds used was 0.5 μmol/kg (0.17 mg/kg and 0.24 mg/kg for zanamivir and CS-8958, respectively). The virus titers were measured at 35, 59, and 83 h after infection with A/PR/8/34 (30 PFU) at 0 h. The repeated administrations started at 11 hpi and were then given twice daily. For the single administration, the compound was dosed at 11 hpi, and saline was then administered twice daily. Each plot represents the mean ± standard deviation (n = 3). No statistically significant differences were observed between a single administration of CS-8958 and repeated administrations of CS-8958 or between a single administration of CS-8958 and repeated administrations of zanamivir.

Virus titers in the lungs of the infected mice treated with the repeated oral administration of oseltamivir phosphate or a single intranasal administration of CS-8958. Virus titers (log₁₀ PFU/lungs) in the mouse lungs after saline administration (closed circles), the repeated administration of oseltamivir phosphate at 1 mg/kg (open triangles) or 10 mg/kg (closed triangles), and a single administration of CS-8958 at 0.057 μmol/kg (open squares) or 0.17 μmol/kg (closed squares) are shown. The virus titers were measured at 35, 59, and 83 h after infection with A/PR/8/34 (30 PFU) at 0 h. For the repeated administrations, oral administration of oseltamivir phosphate and then the intranasal administration of saline were performed at 11 hpi, followed by the oral administration of saline twice daily. For the single administration, CS-8958 was intranasally administered at 11 hpi and saline was orally administered on the same schedule indicated above. For the controls, saline was administered by both routes at 11 hpi and was orally administered at the rest of the time points. The CS-8958 dose of 0.17 μmol/kg corresponds to 0.080 mg/kg. Each plot represents the mean ± standard deviation (n = 3).

Virus titers in the lungs of oseltamivir-resistant or -sensitive virus-infected mice treated by the repeated oral administration of oseltamivir or a single intranasal administration of CS-8958. Mice infected with oseltamivir-sensitive A/Yokohama/67/2006 clone 1 (a) or H274Y oseltamivir-resistant A/Yokohama/67/2006 clone 11 (b) were orally administered oseltamivir phosphate twice daily from 13 hpi with 1.0 mg/kg (dotted lines, triangles) or 10 mg/kg (dotted lines, squares), were intranasally administered CS-8958 once at 13 hpi at 0.057 μmol/kg (0.027 mg/kg; solid lines, triangles) or 0.17 μmol/kg (0.080 mg/kg; solid lines, squares), or were administered saline (solid lines, open circles). The viral titers (log₁₀ PFU/lungs) in the lungs were measured at 37, 61, and 85 hpi, and the lower limit of detection was 2.4 (log₁₀ PFU/lungs). Each plot represents the mean ± standard deviation (n = 3). For the plot indicated by “#,” the viral titer was calculated to be 2.4 (log₁₀ PFU/lungs) for 1 out of 3 mice because no viruses were recovered from the mouse lungs.