Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants

CS-8958, a Prodrug of the New Neuraminidase Inhibitor R-125489, Shows Long-Acting Anti-Influenza Virus Activity▿

Two neuraminidase (NA) inhibitors, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu), have been licensed for the treatment of and prophylaxis against influenza. In this paper, the new potent NA inhibitor R-125489 is reported for the first time. R-125489 inhibited the NA activities of various type A and B influenza viruses, including subtypes N1 to N9 and oseltamivir-resistant viruses. The survival effect of R-125489 was shown to be similar to that of zanamivir when administered intranasally in a mouse influenza virus A/Puerto Rico/8/34 infection model. Moreover, we found that the esterified form of R-125489 showed improved efficacy compared to R-125489 and zanamivir, depending on the acyl chain length, and that 3-(O)-octanoyl R-125489 (CS-8958) was the best compound in terms of its life-prolonging effect (P < 0.0001, compared to zanamivir) in the same infection model. A prolonged survival effect was observed after a single administration of CS-8958, even if it was given 7 days before infection. It is suggested that intranasally administered CS-8958 works as a long-acting NA inhibitor and shows in vivo efficacy as a result of a single intranasal administration

Laninamivir octanoate and artificial surfactant combination therapy significantly increases survival of mice infected with lethal influenza H1N1 Virus.

BACKGROUND: Patients with influenza virus infection can develop severe pneumonia and acute respiratory distress syndrome (ARDS) which have a high mortality. Influenza virus infection is treated worldwide mainly by neuraminidase inhibitors (NAIs). However, monotherapy with NAIs is insufficient for severe pneumonia secondary to influenza virus infection. We previously demonstrated that mice infected with a lethal dose of influenza virus develop diffuse alveolar damage (DAD) with alveolar collapse similar to that seen in ARDS in humans. Additionally, pulmonary surfactant proteins were gradually increased in mouse serum, suggesting a decrease in pulmonary surfactant in the lung. Therefore, the present study examined whether combination therapy of NAI with exogenous artificial surfactant affects mortality of influenza virus-infected mice. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c mice were inoculated with several viral doses of influenza A/Puerto Rico/8/34 (PR8) virus (H1N1). The mice were additionally administered exogenous artificial surfactant in the presence or absence of a new NAI, laninamivir octanoate. Mouse survival, body weight and general condition were observed for up to 20 days after inoculation. Viral titer and cytokine/chemokine levels in the lungs, lung weight, pathological analysis, and blood O(2) and CO(2) pressures were evaluated. Infected mice treated with combination therapy of laninamivir octanoate with artificial surfactant showed a significantly higher survival rate compared with those that received laninamivir octanoate monotherapy (p = 0.003). However, virus titer, lung weight and cytokine/chemokine responses were not different between the groups. Histopathological examination, a hydrostatic lung test and blood gas analysis showed positive results in the combination therapy group. CONCLUSIONS/SIGNIFICANCE: Combination therapy of laninamivir octanoate with artificial surfactant reduces lethality in mice infected with influenza virus, and eventually suppresses DAD formation and preserves lung function. This combination could be effective for prevention of severe pneumonia secondary to influenza virus infection in humans, which is not improved by NAI monotherapy

Survival curves and body weights of infected mice with combination therapy or monotherapy.

<p>(A) Survival curves of infected mice with combination therapy of artificial surfactant with laninamivir octanoate are shown. Mice infected with an extremely high dose (3741 MLD₅₀) of PR8 virus were treated with laninamivir octanoate. The mice were additionally administered artificial surfactant (combination therapy, red line) or normal saline solution (monotherapy, blue line) intranasally once daily during 3–14 days postinfection. Significant differences in mouse survival rates between the combination therapy and monotherapy groups were analyzed by the log-rank original method. Experiments were independently repeated three times. Percentage survival in a representative experiment is shown. (B) Mouse body weight of the combination therapy (red line) and monotherapy (blue line) groups is shown. Experiments were independently repeated three times. The percentage of mouse body weight in a representative experiment is shown. Differences in means ± SD are shown.</p

SP-D levels in infected lungs and survival of infected mice treated with artificial surfactant.

<p>(A) SP-D levels in lung homogenates from mice infected with the middle dose (5 MLD₅₀) of PR8 virus were measured by ELISA. Infected mice were sacrificed at the indicated days, and then clarified lung homogenates (<i>n</i> = 5 or 6) were used. Differences in means ± SD and <i>p</i> values are shown. **<i>p</i><0.01, ***<i>p</i><0.001. (B) Survival curves of infected mice administered artificial surfactant are shown. Mice infected with a low dose (2 MLD₅₀) of PR8 virus were additionally administered artificial surfactant (<i>n</i> = 8, red line) or normal saline solution (<i>n</i> = 8, blue line) intranasally once daily during 3–8 days postinfection. Significant differences in the survival rates of groups of mice treated with artificial surfactant or normal saline solution were analyzed by the log-rank original method. Percentage survival is shown.</p

Gross pathology and hydrostatic test of lungs from infected mice with combination therapy or monotherapy.

<p>(A) Gross pathology of mouse lungs with combination therapy is shown. Mice intranasally inoculated with an extremely high dose (3741 MLD₅₀) of PR8 virus were treated with laninamivir octanoate. The mice were additionally administered artificial surfactant (combination therapy, lower panels) or normal saline solution (monotherapy, upper panels) intranasally once daily during 3–14 days postinfection. Five mice in each group were sacrificed at 7 days postinfection, when the mouse survival rate in the monotherapy group was approximately 50%. Arrowheads indicate the area that appeared relatively healthy. (B) Hydrostatic lung test using mouse lungs from the monotherapy (tube number 1–5) and the combination therapy (tube number 6–10) groups is shown. The arrow indicates collapsing lungs (tube numbers 2–5). The arrowhead indicates floating lungs.</p

Histopathology of the lungs of infected mice with combination therapy or monotherapy.

<p>Mice intranasally inoculated with an extremely high dose (3741 MLD₅₀) of PR8 virus were treated with laninamivir octanoate. The mice were additionally administered artificial surfactant (combination therapy, right panels) or normal saline solution (monotherapy, left panels) intranasally once daily during 3–14 days postinfection. Five mice in each group were sacrificed at 7 days postinfection, when mouse survival rate in the normal saline solution group was approximately 50%. Enlarged images are inserted in each panel. (A and B) Hematoxylin and eosin staining, magnification, 40×. (C) The hyaline membrane (arrowheads) was specifically stained pastel purple by Masson's Trichrome (MT) method in alveoli and alveolar ducts throughout the lungs in the control group. Magnification, 200×. (D) Little, if any, hyaline membrane was stained in mouse lungs from the surfactant group. Magnification, 200×. (E) Severe alveolar collapse can be seen. Elastica van Gieson (EVG) staining was used. Magnification, 200×. (F) Mild alveolar collapse can be seen. Magnification, 200×. (G and H) Immunohistochemistry (IHC) using anti-influenza virus polyclonal antibodies is shown. Red indicates influenza virus antigen. Antigen-positive cell debris (arrowheads) is located in the bronchioles. No antigen-positive cells or cell debris were found in pulmonary parenchyma. Magnification, 200×.</p