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. 2012;7(8):e42419.
doi: 10.1371/journal.pone.0042419. Epub 2012 Aug 1.

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

Masaya Fukushi  1 , Makoto Yamashita, Tohru Miyoshi-Akiyama, Shuku Kubo, Kenji Yamamoto, Koichiro Kudo
Affiliations

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

Masaya Fukushi et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors declare that Makoto Yamashita and Shuku Kubo are employed by Biological Research Laboratories, Daiichi Sankyo Co., Ltd. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. SP-D levels in infected lungs and survival of infected mice treated with artificial surfactant.
(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 (n = 5 or 6) were used. Differences in means ± SD and p values are shown. **p<0.01, ***p<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 (n = 8, red line) or normal saline solution (n = 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.
Figure 2
Figure 2. Survival curves and body weights of infected mice with combination therapy or monotherapy.
(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.
Figure 3
Figure 3. Virus titers in the lungs and lung weights of infected mice with combination therapy or monotherapy.
(A) Virus titers in the lung homogenates of infected mice 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 bars) or normal saline solution (monotherapy, blue bars) intranasally once daily during 3–14 days postinfection. Four mice in each group were sacrificed at the indicated days postinfection and their lung homogenates were used for a plaque-forming assay. Mean ± SD viral titers are shown. The # symbol indicates under the detection limit. (B) Lung weights of four mice in the combination therapy (red bars) or monotherapy (blue bars) groups are shown. Mice were sacrificed at the indicated days postinfection and their lung weights were measured. Differences in means ± SD are shown.
Figure 4
Figure 4. Cytokine and chemokine expression in lungs from infected mice with combination therapy or monotherapy.
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, red bars) or normal saline solution (monotherapy, blue bars) intranasally once daily during 3–14 days postinfection. Three mice from each group were sacrificed at the indicated days postinfection and their lung homogenates were used to analyze the expression of 22 cytokines and chemokines using the MILLIPLEX MAP Panel. Only 12 cytokines and chemokines are shown; the remainder was under the limit of detection for the assay. The concentration in each panel is pg/ml. Differences in means ± SD are shown.
Figure 5
Figure 5. Gross pathology and hydrostatic test of lungs from infected mice with combination therapy or monotherapy.
(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.
Figure 6
Figure 6. Histopathology of the lungs of infected mice with combination therapy or monotherapy.
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×.
Figure 7
Figure 7. Blood O2 and CO2 pressures of infected mice with combination therapy or monotherapy.
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, red lines) or normal saline solution (monotherapy, blue lines) intranasally once daily during 3–14 days postinfection. Mice (n = 3–6) in each group were sacrificed at the indicated days postinfection. Mice in each group were sacrificed at 7 days postinfection, when the mouse survival rate in the monotherapy group was approximately 50%. Blood was collected and immediately measured for O2 (left panel) and CO2 (right panel) pressures using the i-STAT portable blood gas analyzer. Differences in means ± SD and p values are shown. *p<0.05.
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The Japan Initiative for Global Research Network on Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.