Combinations of L-N^G-monomethyl-arginine and oseltamivir against pandemic influenza A virus infections in mice

Abstract

L-N^G-monomethyl-arginine (L-NMMA) is an experimental compound that suppresses nitric oxide production in animals. The compound was combined with oseltamivir to treat lethal influenza A/California/04/2009 (H1N1) pandemic virus infections in mice. Treatments were given twice a day for five days starting 4 h (oseltamivir, by oral gavage) or three days (L-NMMA, by intraperitoneal route; corresponding to the time previously reported for nitric oxide induction in the animals) after infection. Low doses of oseltamivir were used in order to demonstrate synergy or antagonism. Oseltamivir monotherapy protected 70% of mice from death at 1 mg/kg/day. L-NMMA (40 and 80 mg/kg/day) was ineffective alone in preventing mortality. Compared to oseltamivir treatment alone, L-NMMA combined with oseltamivir was synergistically effective (as evaluated by three-dimensional MacSynergy analysis), resulting in survival increases from 20 to 70% when 40 or 80 mg/kg/day of L-NMMA was combined with 0.3 mg/kg/day of oseltamivir, and from 70 to 100% survival increases when these doses were combined with 1 mg/kg/day of oseltamivir. These data demonstrate that a nitric oxide inhibitor such as L-NMMA has the potential to be beneficial when combined with oseltamivir in treating influenza virus infections.

Influenza viruses in nature are in a state of constant change in their genetic makeup, resulting in viruses with modified proteins that help them to evade neutralization by antibodies or antiviral drugs in infected hosts. Thus, they represent a constant threat to humans. For example, in 2013, a new influenza A H7N9 virus emerged in China that caused lethal infections in humans.¹ The human population has no natural or acquired immunity to H7N9 viruses. And as is increasingly common among influenza A viruses, the novel H7N9 virus is resistant to the drugs amantadine and rimantadine, but sensitive to neuraminidase inhibitors and favipiravir.² In the last decade, influenza viruses resistant to the neuraminidase inhibitor oseltamivir have been more commonly isolated.³ Influenza viruses whose origins are from unique viral recombinations are generally the ones responsible for pandemic outbreaks. As such, a new emerging virus could be adamantane-resistant or -sensitive, neuraminidase inhibitor-resistant or -sensitive, or any combination of resistance or sensitivity to these two classes of commonly used inhibitors. Prior to 2009, the H1N1 virus population was increasingly oseltamivir resistant but the pandemic virus that emerged was oseltamivir sensitive (but adamantane resistant).³

Because of the emergence of viruses that are resistant to clinically approved drugs, other classes of influenza virus inhibitors are being investigated. For example, favipiravir represents an inhibitor of influenza viral RNA polymerase⁴ that is progressing through clinical trials. Antiviral compounds have also been tested experimentally in combination against experimental infections in mice^5–8 and in humans.^9,10 Another area of pursuit is targeting the host, which often involves the immune system, rather than the virus as a means of improving the outcome of influenza virus infections. Severe influenza infections are characterized by a dramatic increase in the inflammatory response, including a hypercytokinemia, often termed the “cytokine storm.” The hyperinflammatory response is due to a robust immune response generated against the virus.¹¹ Compounds that may ameliorate this response but still allow immunological control of the virus infection are desirable.

From studies we completed in the past^12,13 and from examples in the scientific literature,^11,14–18 it appears that certain immunomodulatory compounds provide a protective benefit by monotherapy to treat influenza virus infections in mice. However, complete protection from mortality may not be attainable, as evidenced by examples from a recent review where partial protection was afforded by treatment.¹⁹ One experimental immunomodulatory compound that has been the subject of our recent research is L-N^G-monomethyl-arginine (L-NMMA).

L-NMMA is an inhibitor of nitric oxide production.²⁰ Influenza H2N2 virus-infected mice treated intraperitoneally with the compound (at 80 mg/kg/day for five days starting three days after virus challenge) exhibited a 50% survival rate compared to no survival of untreated mice.²¹ The treatments were reported to reduce detectable nitric oxide in lungs and to reduce lung histopathology in infected animals. A recent report indicated that mice infected with the reconstructed 1918 influenza A H1N1 virus and treated with L-NMMA lived longer than untreated animals.²² Thus, protection from influenza virus infection in mice by L-NMMA has been documented.

Because immunomodulatory agents generally are not sufficiently effective alone to treat influenza virus infections, they may be more effective if used in combination with an approved antiviral drug, such as oseltamivir, which is the current mainstay of anti-influenza treatment for humans. The immunomodulatory compound should reduce the immunopathogenesis, while the antiviral drug reduces the virus burden. This was demonstrated with the combination of compound AAL-R and oseltamivir.¹⁴ In order to determine whether combination drug treatment is beneficial in mice, it is necessary to use sub-optimal doses of oseltamivir. Otherwise, an overwhelmingly potent effect of oseltamivir alone will mask any added benefit of the second compound. This is the approach used herein. However, in the clinic, the goal is to provide the maximum benefit in order to rapidly ameliorate symptoms and restore wellbeing. In this report, we present results of the evaluation of L-NMMA, used alone and in combination with oseltamivir, to treat oseltamivir-sensitive pandemic influenza A H1N1 2009 virus infections in mice.

The influenza A/California/04/2009 (H1N1pdm) strain used in the present studies was originally obtained from Elena Govorkova (St. Jude Children’s Research Hospital, Memphis, TN) and adapted to mice by a published procedure.²³ The virus was amplified in MDCK cells (purchased from the American Type Culture Collection, Manassas, VA, USA), then titrated in BALB/c mice to determine an appropriate lethal challenge dose.

L-NMMA (in its acetate form) was purchased from Cayman Chemical (Ann Arbor, MI) and was dissolved in sterile saline for parenteral administration. Oseltamivir phosphate (referred to as oseltamivir) was purchased as 75 mg Tamiflu® capsules from a local pharmacy. Since the capsules contain excipients, entire capsules (minus the shell) were used in preparation of the drug for animal treatments.

Female, 18–20 g BALB/c, mice were obtained from Charles River Laboratories (Wilmington, MA) for this study. The animals were maintained on standard rodent chow and tap water ad libitum. Antiviral studies were initiated within 72 h after receipt of the mice. The mice were anesthetized by intraperitoneal (i.p.) injection of ketamine/xylazine (50/5 mg/kg) followed by infection intranasally with a 90 -µl suspension of influenza virus. Mice were infected with approximately 10^4.3 50% cell culture infectious doses (CCID₅₀)/mouse, which equated to three 50% mouse lethal challenge doses (3 mL D₅₀). Treatment regimens varied depending upon the compound that was administered. Treatments with oseltamivir (0.3 and 1 mg/kg/day) were given orally (p.o., by gavage) twice a day (at 12 h intervals) for five days starting 4 h after virus exposure. This regimen has been used successfully to treat influenza virus infections in mice in the past.^8,24 L-NMMA (40 and 80 mg/kg/day) was administered i.p. once a day for five days starting three days after infection, reported to correspond to the time of nitric oxide induction in lungs of infected mice, using doses and treatment routes that have been successful in treating influenza virus infections in the past.^21,22 In order to make all treatments equal in terms of inducing stress, mice (including placebos) received both p.o. and i.p. treatments of either test compounds: water (p.o.) or saline (i.p.). The mice were weighed prior to treatment (day 0), followed by weighing every other day thereafter. The animals were weighed in groups rather than individually. Ten mice per drug-treated group and 20 placebos were followed for survival for 21 days.

In order to assess toxicity, groups of five uninfected mice were treated with L-NMMA at 80 mg/kg/day (i.p.) combined with oseltamivir at 1 mg/kg/day (p.o.) by the above treatment regimen to determine tolerability to the combination therapy described above. Studies reported in the literature have already demonstrated that the singly administered compounds are not toxic at the levels administered in this study.^21,22,24,25 Toxicity was evaluated by visual observation (ruffling of fur, lethargy, paralysis, incontinence, repetitive circular motion, and aggression) and body weight loss over 21 days.

On day 6 of the infection, five mice per group were sacrificed to determine lung virus titers. This entailed removal of lungs from sacrificed animals, homogenizing each in cell culture medium (MEM), and freezing the tissue homogenates at −80°C. Later, titrations were conducted by endpoint dilution method²⁶ in 96-well microplates of MDCK cells. Virus-containing homogenates were serially diluted from 10⁻¹ to 10⁻⁸ in 10-fold increments. The presence of virus was determined in wells after six days.

Statistical analyses were performed for the data from survival curves and body weights during the infection. Survival curves were plotted by the Kaplan-Meier method and analyzed by the Mantel-Cox log-rank test. Pairwise comparisons of survivor curves (placebo vs. treatment) were subsequently analyzed by the Gehan-Breslow-Wilcoxon test, with the relative significance adjusted to a Bonferroni-corrected significance threshold for the number of treatment comparisons done. Body weight curves were analyzed by one-way analysis of variance (ANOVA), with Tukey’s multiple comparisons test. Analyses were made using Prism® 6.0 software (GraphPad Inc., San Diego, CA).

Drug–drug interactions were analyzed by the three-dimensional model of Prichard and Shipman,²⁷ using the MacSynergy II software program (kindly provided by Mark Prichard, University of Alabama at Birmingham, AL) at 95% confidence limits. Descriptions of antagonistic, additive, or synergistic interactions using this computer model have been described for data, and represented as percentages.²⁸ Briefly, <25, 25–50, 50–100, and >100 µm² unit % calculated values in either a positive or negative direction using the software are defined as insignificant synergy or antagonism (indifference), minor synergy or antagonism, moderate synergy or antagonism, or strong synergy or antagonism, respectively. The reported values represent the net volume of synergy (total volume of synergy minus total volume of antagonism) for each set of data. The volume of synergy or antagonism is essentially a compilation of the percentages above or below the baseline. The expected value for each combination, assuming no interaction, is zero (i.e. no change either above or below expected values (baseline).

The compounds were evaluated in combination for possible toxic interactions in uninfected animals. L-NMMA (80 mg/kg/day) was used in combination with oseltamivir (1 mg/kg/day), which caused no overt toxicity as evidenced by no significant decreases in body weight or abnormal appearance of the mice relative to placebo-treated animals (Figure S1). In other studies we have reported, oseltamivir was safely administered to mice up to 300 mg/kg/day.²⁹ In addition, L-NMMA has been safely administered to mice up to 80 mg/kg/day.^21,30,31 Thus, the 80-mg/kg/day dose of L-NMMA was chosen as the highest dose for the present antiviral investigations.

The treatment of influenza A (H1N1pdm) virus-infected mice was performed using L-NMMA and oseltamivir. L-NMMA alone at 80 mg/kg/day did not protect mice from influenza-induced death. Treatment with oseltamivir alone at 1 mg/kg/day provided 70% protection from mortality, (Figures 1(a) and 2(a)). Combining 80 mg/kg/day of L-NMMA with oseltamivir (1 mg/kg/day) increased protection from 70% to 100% (Figure 1(a)), which was not statistically significant (P > 0.05)). A 20% survival rate was evident in the group treated with 0.3 mg/kg/day of oseltamivir compared to 15% survival in the placebo group (i.e. essentially no protection compared to placebo). L-NMMA treatment of 80 mg/kg/day increased the efficacy of the 0.3-mg/kg/day dose of oseltamivir to 70% protection (P < 0.05, compared to oseltamivir alone at 0.3 mg/kg/day). Body weight comparisons for the experiment are shown in Figure 1(b). Treatment with 80 mg/kg/day of L-NMMA combined with oseltamivir at 1 mg/kg/day gave the best protection from infection-induced weight loss. Analysis of body weights on days 7–21 (during recovery from the infection) demonstrated that the combination treatment containing 80 mg/kg/day of L-NMMA and 1 mg/kg/day of oseltamivir was significantly higher (P < 0.05) compared to oseltamivir alone at 1 mg/kg/day. Body weights for the oseltamivir (1 mg/kg/day) group were less than that of the lower (0.3 mg/kg/day) dosage group. These data suggest that many of the survivors in the 1-mg/kg/day group (seven animals) were quite ill at the peak of infection and recovered slowly, whereas all but 2 (i.e. the healthiest) of the mice treated with the 0.3 mg/kg/day dose died. No significant body weight differences were observed in mice treated with L-NMMA and oseltamivir (0.3 mg/kg/day) compared to oseltamivir (0.3 mg/kg/day) alone.

Figure 1.

Effects of L-NMMA (80 mg/kg/day), alone or combined with oseltamivir (Osl) on survival (A) and body weights (B) of mice infected with influenza A/California/04/09 (H1N1pdm) virus. Treatments with L-NMMA (i.p.) were given once a day for 5 d starting 72 h after virus exposure. Oseltamivir (p.o.) was administered twice a day (at 12 h intervals) for 5 d starting 4 h after virus challenge. There were 10 animals per drug treated group and 20 placebos. Values in parentheses are mg/kg/day doses. **P < 0.01, ***P < 0.001, compared to placebo for survival. ^↔P < 0.05, compared to oseltamivir (0.3 mg/kg/day) alone for survival. ^↕P < 0.05, compared to oseltamivir (1 mg/kg/day) alone for body weight.

Figure 2.

Effects of L-NMMA (40 mg/kg/day), alone or combined with oseltamivir (Osl) on survival (A) and body weights (B) of mice infected with influenza A/California/04/09 (H1N1pdm) virus. Treatments with L-NMMA (i.p.) were given once a day for 5 d starting 72 h after virus exposure. Oseltamivir (p.o.) was administered twice a day (at 12 h intervals) for 5 d starting 4 h after virus challenge. There were 10 animals per drug treated group and 20 placebos. Values in parentheses are mg/kg/day doses. **P < 0.01, ***P < 0.001, compared to placebo. ^↕P < 0.05, L-NMMA plus 1 mg/kg/day oseltamivir compared to 1 mg/kg/day oseltamivir alone for body weight.

L-NMMA alone at 40 mg/kg/day provided no protection from death relative to placebo-treated mice. Combining 40 mg/kg/day of L-NMMA with 1 mg/kg/day of oseltamivir increased survival from 70% to 100% (Figure 2(a)), which was not statistically significant (P > 0.05)). The 40-mg/kg/day dose of L-NMMA combined with 0.3 mg/kg/day of oseltamivir provided no benefit relative to oseltamivir alone (which was an ineffective dose). Body weight comparisons indicated that treatments with L-NMMA plus 1 mg/kg/day of oseltamivir were significantly higher (P < 0.05) compared to oseltamivir alone at the 1-mg/kg/day dose (Figure 2(b)), suggesting a reduction in morbidity. Using 0.3 mg/kg/day of oseltamivir where very few mice survived, body weights were similar between groups treated with or without L-NMMA.

Three-dimensional analysis of the survival data showed a volume of synergy of 110, indicative of strong synergy for drug combination treatments compared to oseltamivir monotherapy (Figure 3). The only drug combination treatment regimen that did not result in improvement in survival was 40 mg/kg/day of L-NMMA combined with 0.3 mg/kg/day of oseltamivir. Perhaps, a rendering of strong synergy overstates the data. The volume of synergy is calculated over the entire surface, although, as was demonstrated, the only combination that was significantly different from monotherapy was 80 mg/kg/day of L-NMMA plus 0.3 mg/kg/day of oseltamivir. Two other combinations resulted in 100% survival compared to 70% survival with oseltamivir alone, which were not significant. However, 100% puts a cap on how high of an effect that can be achieved. The only way to achieve significance in this case is to use larger group sizes, which was not done in these studies.

Figure 3.

Three-dimensional plot of the interaction of L-NMMA and oseltamivir used alone and in combination to treat an influenza A/California/04/09 (H1N1pdm) virus infection in mice. “Percent from expected” values above and below zero are suggestive of synergy and antagonism, respectively. The volume of synergy for this figure is 110, indicative of strong synergy (per interpretation by Iluyshina et al., 2008). The data for this figure were derived from Figures 1(a) and 2(a), except for oseltamivir used at 0.1 mg/kg/day which results are not presented in depth due to lack of effect. The evaluation was made by MacSynergy.²⁷

The amount of virus detected in lungs of mice at six days after infection in treated animals is shown in Figure S2. There were no significant differences among treatment groups for the single time point that was analyzed. We also preformed cytokine/chemokine analysis on the same lung homogenate samples using a multiplex array (Quansys Biosciences, Logan, UT), but did not detect differences between the drug combination treatments compared to oseltamivir alone for 16 cytokines/chemokines that were assayed (data not shown). For these studies, we also considered determining nitric oxide levels in infected lungs with and without L-NMMA treatment. Akaike et al.²¹ demonstrated there were differences between treated and untreated mice in their H2N2 infection model using electron spin resonance spectroscopy. This methodology was unavailable to us due to lack of the necessary equipment. Perrone et al.²² used a commercially available kit to measure total lung nitric oxide levels in infected mice, but they did not determine whether L-NMMA treatment reduced those levels. An examination of their data indicated that there was only a 2.5-fold maximal difference of nitric oxide levels between infected and uninfected mice, and there were large standard deviations to the data. This maximal difference only occurred in an avian H5N1 virus infection and was much less with infections using 1918 and Texas 1991 H1N1 viruses. Thus, it seemed unlikely that statistically significant differences in nitric oxide levels between L-NMMA treated and placebo-treated mice could be distinguished by this method. For this reason, we did not pursue this approach either.

In this report, L-NMMA was evaluated alone and in combination with oseltamivir for protection of mice infected intranasally with influenza A H1N1pdm virus. L-NMMA treatment did not confer protection by itself. Oseltamivir was evaluated at doses that were partially protective in order to demonstrate whether an improvement would be rendered by combination treatments. L-NMMA and oseltamivir were synergistically active, resulting in increases in the numbers of survivors over oseltamivir treatment alone. Lung virus titers were not significantly lower than placebo at six days by any treatment, including treatment with oseltamivir. Oseltamivir treatment alone does not diminish lung virus titers dramatically,³² except when used at much higher doses than were used here. Treatment of an influenza A (H2N2) virus infection with L-NMMA alone was previously shown to not diminish lung virus titer.²¹

In a recent review, Fedson¹⁹ pointed out examples of immunomodulators that failed to provide a benefit in mice, and suggested that their lack of benefit may be due to studying them against lethal infections that are difficult to treat. He gave other examples where certain immune-acting compounds protected a significant percentage of mice from death following infections, but in experiments where many of the placebos also survived the infection. Since the present studies employed a lethal influenza virus infection model, the experimental design may not have been optimal. However, we were successful in demonstrating statistically significant differences among treatment groups and showed that drug combination treatment was superior to monotherapy.

Akaike et al.²¹ reported that L-NMMA alone provided some protection from death in their influenza A H2N2 infection model. However, the results reported here did not reproduce those earlier results of protection by monotherapy. This may have been due to differences in the severity of the two infection models (inferring that the H1N1 infection was more severe than the previously-used H2N2 infection), and not necessarily that one type of virus infection is susceptible to treatment and another is not. It might be possible that the H1N1pdm model is sufficiently different from the other influenza models in which an effect of monotherapy with L-NMMA was reported, and perhaps indicates an L-NMMA target that lies outside of the respiratory tract. Mice strains and weights of mice also differed between the two studies, which may have contributed to the differences seen.

Studying drug efficacy in sub-lethal infections seems logical with L-NMMA, but it is fraught with problems in terms of providing statistically interpretable data. Because of lower mortality, the studies lose statistical power unless larger group sizes are used, making the experiments much more expensive. Comparing body weight differences among groups may be useful. However, because of intra-group variability, large weight differences between treatment groups (or larger group sizes) must occur in order to achieve statistical significance. Antiviral drug treatment may not prevent much weight loss during weight decline (during the acute phase of the infection), but may benefit the recovery phase. Body weight recovery measures are complicated because of the differences in numbers of survivors per group and because mice that barely survived the infection may not regain their weight quickly, as seen in the oseltamivir (1 mg/kg/day) group of this report (Figures 1(b) and 2(b)). A new approach that we have used to evaluate the efficacy of antiviral compounds during sub-lethal infections includes plethysmography to measure functional lung parameters. Here, larger group sizes may also be needed due to variability in order to evaluate differences in efficacy of test compounds.

We hypothesized that L-NMMA alone should have exhibited some protective effect against an influenza A/California/04/2009 (H1N1pdm) virus infection in mice, based upon positive benefits of the compound against influenza A (H2N2) and reconstructed 1918 pandemic (H1N1) virus infections in mice.^21,22 However, this was not the case in the 2009 H1N1pdm virus infection. The use of L-NMMA combined with oseltamivir was beneficial in treating the H1N1 pandemic infection, showing a synergistic improvement on survival of mice. L-NMMA may not be an attractive drug candidate for treating uncomplicated influenza because it requires parenteral administration. In addition, a report showed that inhaled L-NMMA did not prevent mortality of influenza-infected mice.³³ L-NMMA might be considered for treatment of severely ill patients who require hospitalization and where intravenous drug administration would be manageable. However, side effects of L-NMMA treatment have been reported in human trials, such as cardiac incidents.^34,35

It is not known whether earlier treatment intervention with L-NMMA will be beneficial or deleterious to the host, since its function is to quell the host’s hyperinflammatory response. In addition, oseltamivir combination treatments with L-NMMA using later antiviral drug start times have not been investigated. Thus, further work using modified treatment regimens may be warranted.

Ethical approval

The animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Utah State University in the AAALAC-accredited Laboratory Animal Research Center.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by contract HHSN272201000039I from the Respiratory Diseases Branch, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA.