In vitro and in vivo activities of T-705 and oseltamivir against influenza virus

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has a potent and selective inhibitory activity against influenza virus. We studied the effects of an infectious dose on the anti-influenza virus activities of T-705 and oseltamivir, a commercially available neuraminidase inhibitor, both in vitro and in vivo. Plaque formation of influenza A/PR/8/34 virus was completely inhibited by 10 microg/ml of T-705 after 72 h incubation, whereas visible plaque formation was detected in the plate treated with GS 4071, the active form of oseltamivir (10 microg/ml). The antiviral activity of T-705 was not influenced by an increase in multiplicity of infection (MOI) from 0.0001 to 1, but that of GS 4071 was influenced in a yield reduction assay. No increase in viral yield was seen in either culture supernatant or cells after removal of T-705 (10 microg/ml) but, in contrast, productive infection recurred in culture supernatant and in cells after removal of GS 4071. In mice infected with a high challenge dose of influenza A/PR/8/34 virus, orally administered T-705 (200 and 400 mg/kg/day) completely prevented the death of mice and the survival rates of mice were significantly higher than those in mice treated with oseltamivir (P<0.01). When the treatment was delayed at 1, 13 and 25 h post infection, oral administration of 200 mg/kg of T-705 significantly prevented the death of mice (P<0.01), and the survival rates of mice treated with T-705 were comparable to those of mice treated with oseltamivir. These results suggest that T-705 has the potential to be a potent inhibitor of human influenza virus infections.     

Utilization of alpha-1-acid glycoprotein levels in the serum as a parameter for in vivo assay of influenza virus inhibitors

Alpha-1-acid glycoprotein (AGP), an acute phase protein in serum assayed by single radial immunodiffusion using a commercially available kit, was found to significantly increase in mice infected with influenza A and B viruses. Experiments were run to determine the rate of increase of serum AGP and its relation to other influenza disease parameters, including lung consolidation, development of lung virus titres, decline in arterial oxygen saturation (SaO2), histopathological changes in the lung, and death of the animal. Maximal AGP levels occurred by day 3 in the animals, at about the same time lung virus titres reached their peak and inflammatory effects were evident in the lung. Serum levels of AGP were then compared with other disease parameters in the evaluation of the anti-influenza A and B virus efficacy of oseltamivir and ribavirin in mice. Treatment was by oral gavage twice daily for 5 days, beginning 4 h before virus exposure using doses of 100, 10, and 1 mg/kg per day of oseltamivir and 75 mg/kg per day of ribavirin. Against the influenza A infection, significant inhibition of death, SaO2 decline, and lung consolidation was seen at all doses of each compound; day-6 AGP levels were reduced in a dose-responsive manner. Lung virus titres were lessened at this time, but to a significant degree only at the high dose of oseltamivir and by ribavirin. The influenza B virus infection, which appeared more severe than the influenza A infection, was also significantly inhibited by both compounds, but to a lesser extent. The serum AGP levels were again lessened by therapy with both compounds. The influence of challenge dose of influenza A virus on AGP level and on the antiviral activity of 20 mg/kg per day of oseltamivir, administered by oral gavage, was determined in mice. The AGP level was in proportion to the viral challenge dose; oseltamivir significantly inhibited AGP levels and all other disease parameters regardless of size of viral inoculum. These data indicate murine AGP levels to be markedly stimulated by infection with influenza A and B viruses, and the level of the protein to be an additional measure of antiviral efficacy.     

Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine

Mannan, a polysaccharide preparation from Saccharomyces cerevisiae, has previously been shown to enhance influenza virus replication in mice by inhibiting host defense collectins. The use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in rodent infection models. When mannan was co-administered with influenza B/Sichuan/379/99 virus to mice, the animals died from the infection, whereas mice infected with only virus survived. Three types of influenza A (H1N1) and another influenza B (Hong Kong/330/01) virus infection were also enhanced by mannan, but not four types of influenza A (H3N2) viruses. Mannan was used at 0.16 or 0.5 mg/mouse for optimal disease-enhancing activity using influenza B/Sichuan/379/99 virus. Using this model, influenza B/Sichuan/379/99 infections were treated with oseltamivir, ribavirin or viramidine (the carboxamidine derivative of ribavirin). When oral gavage treatments started 4 h before virus and mannan challenge, oseltamivir was effective at 2.5, 5 and 10 mg/kg/day. Ribavirin was active at 20, 40 and 80 mg/kg/day. Viramidine was effective at 80 and 160 mg/kg/day but not at 40 mg/kg/day. Active drug doses improved lung consolidation scores and lung weights, with decreases in lung virus titres also noted. Arterial oxygen saturation values in treated groups were significantly better than those of the placebo group on days 7-11 of the infection. Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination to determine how late after infection they could be beneficially administered. Ribavirin alone was very effective (90-100% survival of mice) when treatments started as late as 3 days after infection. Forty percent survival was evident even when treatments started 4 days post-infection. Oseltamivir was active starting treatments 1 day after virus exposure, but lost considerable efficacy when treatments began after that time. The combination of ribavirin and oseltamivir appeared to be no better than ribavirin alone, due to the stronger beneficial effect of ribavirin in this model. The overall results demonstrate that mannan can be used to enhance certain non-lethal influenza virus infections sufficiently to allow antiviral studies.     

Effect of oral gavage treatment with ZnAL42 and other metallo-ion formulations on influenza A H5N1 and H1N1 virus infections in mice

Avian influenza H5N1 infections can cause severe, lethal human infections. Whether influenza A virus treatments effectively ameliorate avian influenza H5N1 human infections is uncertain. The research objective was to evaluate the efficacy of novel zinc and other metallo-ion formulations in two influenza A mouse models. Mice infected with influenza A/Duck/MN/1525/81 (H5N1) virus were treated orally 48 h before virus exposure and then twice daily for 13 days with ZnAL42. The optimal dosing regimen for ZnAL42 was achieved at 17.28 mg/kg 48 h prior to virus exposure, twice daily for 7 days. The survival rate was 80% compared with 10% in the untreated control group and a 100% survival rate with ribavirin (75 mg/kg/day, twice a day for 5 days, beginning 4 h before virus exposure). ZnAL42 treatment significantly lessened the decline in arterial oxygen saturation (SaO2; P < 0.001). This regimen was also well tolerated by the mice. Manganese and selenium formulations were not inhibitory to virus replication when given therapeutically. Mice were also infected with influenza A/NWS/33 (H1N1) virus and were treated 48 h before virus exposure with three dosages of ZnAL42 (8.64, 1.46 or 0.24 mg/kg/day). Treatment was by oral gavage twice daily for 13 days. The highest dose of ZnAL42 was significantly inhibitory to the virus infection as seen by prevention of deaths and lessening of decline in SaO2. The data suggest that the prophylactic use of ZnAL42 is effective against avian influenza H5N1 or H1N1 virus infection in mice and should be further explored as an option for treating human influenza virus infections.     

Efficacy of orally administered T-705 on lethal avian influenza A (H5N1) virus infections in mice

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H5N1 influenza virus in MDCK cells, with the 90% effective concentrations ranging from 1.3 to 7.7 microM, as determined by a virus yield reduction assay. The efficacy was less than that exerted by oseltamivir carboxylate or zanamivir but was greater than that exerted by ribavirin. Experiments with mice lethally infected with influenza A/Duck/MN/1525/81 (H5N1) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h after virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg of body weight/day were well tolerated; each prevented death, lessened the decline of arterial oxygen saturation (SaO(2)), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented the death of the mice. Oseltamivir (20 mg/kg/day), administered per os twice daily for 5 days, was tested in parallel in two experiments; it was only weakly effective against the infection. The four-times-daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented death and the decline of SaO(2). Characterization of the pathogenesis of the duck influenza H5N1 virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H5N1 virus. These data indicate that T-705 may be useful for the treatment of avian influenza virus infections.     

In vitro and in vivo protection against the highly pathogenic H5N1 influenza virus by an antisense phosphorothioate oligonucleotide

BACKGROUND: Current vaccination strategies and antiviral drugs only provide limited protection against influenza virus infection. In this study, we investigated the use of a novel antisense oligonucleotide (named IV-AS), which is specific for the 5'-terminal conserved sequence found in all eight viral RNA segments of influenza A virus. METHODS: The activity of IV-AS was monitored both in vitro, in Madin-Darby canine kidney (MDCK) cells, and in vivo using a mouse model. IV-AS was given intranasally to H5N1-infected mice once daily for 6 days starting 6 h after infection. A three-base mismatch of IV-AS was used as a control. RESULTS: IV-AS inhibited influenza virus A induced cytopathic effects in MDCK cells with the 50% effective concentration (EC50) ranging from 2.2 to 4.4 microM. IV-AS was effective against H5N1 virus in preventing death, lessening weight reduction, inhibiting lung consolidation and reducing lung virus titres. Dosages of 40 and 60 mg/kg/day provided 40% and 60% survival rates and prolonged mean survival days in comparison with the infected control group (P<0.05). The lung index in mice treated with IV-AS, at a dose of 20, 40 or 60 mg/kg/day, had been inhibited on day 4 or 6 (P<0.05 or P<0.01); virus titres in lung had declined to 2.42, 1.51 and 1.54 log10 TCID50/g of lung, respectively, whereas the yields in the infected control mice were 6.00 log10 TCID50/g of lung. CONCLUSIONS: Our results suggest that the 5'-terminal conserved region of influenza A virus RNA segments can be targeted using antisense technology; therefore, IV-AS is a potential drug for prophylaxis and control of influenza virus infections.     

Prophylactic and therapeutic efficacies of poly(IC.LC) against respiratory influenza A virus infection in mice.

Polyriboinosinic-polyribocytidylic acid [poly(IC.LC)] was evaluated for its prophylactic and therapeutic efficacies against respiratory influenza A virus infection in mice. Two doses of poly(IC.LC) (1 mg/kg of body weight per dose) administered intranasally within 12 days prior to infection with 10 50% lethal doses of mouse-adapted influenza A/PR/8 virus fully protected the mice against the infection. Determination of virus titers by hemagglutination and plaque assays showed more than a 2-log10 decrease in virus titers in lung homogenates of pretreated mice compared with those in the lungs of the nonpretreated group. Treatment of infected mice with poly(IC.LC) resulted in a modest (40%) survival rate. These results suggest that poly(IC.LC) provides a highly effective prophylaxis against respiratory influenza A virus infection in mice.     

Rimantadine and oseltamivir demonstrate synergistic combination effect in an experimental infection with type A (H3N2) influenza virus in mice

We studied the combination effect of rimantadine hydrochloride and oseltamivir phosphate on mice infected with influenza A/Aichi/2/68 (H3N2) virus. Compounds were simultaneously administered in a 5-day-treatment course, starting 4 h before intranasal infection with 10 or 20 viral 50% mouse lethal doses. Initially, we tested combinations of oseltamivir (0.05, 0.1 and 0.2 mg/kg/day) and rimantadine (2.5, 5.0 and 7.5 mg/kg/day). Significant differences were recorded between combination-treated groups, and groups with separately applied compounds and the placebo group, such as: protection index of oseltamivir with 5.0 or 7.5 mg/kg rimantadine varied between 34-41% and 43-87%, respectively, whereas the individual effects of oseltamivir, 5 mg/kg of rimantadine and 7.5 mg/kg of rimantadine were 0-10%, 0% and 18.7-29.6%, respectively; mean survival time in combination-treated groups was lengthened by 3.1-6.9 days, in oseltamivir groups by 0-1.9 days, and in rimantadine groups by 0.8-1.3 days at 5 mg/kg and 2.6-3.2 days at 7.5 mg/kg. The three-dimensional method of Prichard and Shipman characterized the combination effect as synergistic. Further, we studied the activity of 0.05 mg/kg/day of oseltamivir combined with 5 mg/kg of rimantadine. Lung virus titre in Madin Darby canine kidney cells, lung index and consolidation score proved the high effectiveness of the combination. When compared with the placebo group, a 2.8 log10 lower titre of 50% cell culture infectious dose (CCID50) was recorded in the combination-treated group at 48-60 h post-infection (the peak of lung virus growth). This is in contrast to the 0.1-1.0 log10 and 1.1-1.4 log10 reduction in CCID50 titre observed in the oseltamivir and rimantadine groups, respectively. These data emphasize the high anti-influenza A potential of the combination.     

Activities of oseltamivir and ribavirin used alone and in combination against infections in mice with recent isolates of influenza A (H1N1) and B viruses

Mouse models have been widely used for evaluating potential influenza virus inhibitors. However, the viral strains traditionally used in these models are fairly old and do not represent currently circulating viruses in nature. We developed two new lethal infection models in mice using mouse-adapted influenza A/New Caledonia/20/99 (H1N1) and influenza B/Sichuan/379/99 viruses. Both virus infections were used to study oral treatment with oseltamivir and ribavirin, both alone and in combination. Oral treatments were given twice daily for 5 days starting 4 h before infection in initial studies. Against influenza A, oseltamivir was active at 10, 20, and 40 mg/kg/day, protected 80-100% of mice from death and reduced lung consolidation - ribavirin was similarly effective at 20, 40, and 80 mg/kg/day. When treatments were initiated after virus challenge, delaying treatment with oseltamivir even 1 day caused it to be ineffective. Ribavirin prevented mortality by 50-80% when treatments were delayed 1-4 days after infection. The combination of the two drugs (oseltamivir at 20 mg/kg/day and ribavirin at 40 mg/kg/day) was no better than ribavirin alone. In contrast to what we observed with influenza A virus infections, oseltamivir and ribavirin showed similar dose-related antiviral activities against influenza B virus infections. The compounds both significantly increased survival when treatments started up to 4 days after infection, but ribavirin was more active than oseltamivir (50-80% survival compared to 30-40% survival, respectively, when starting treatments on days 2-4 after infection). By varying the doses of each drug that were used in combination (oseltamivir at 1.25, 2.5 and 5 mg/kg/day; ribavirin at 5, 10 and 20 mg/kg/day) certain dosage combinations were superior to either compound used alone as assessed by decreased mortality, lung virus titre, lung score and lung weight parameters. These activities differed from published results with older, more established virus strains as oseltamivir was less effective and ribavirin was more active than previously reported.     

Rimantadine Therapy of Influenza A Infection in Mice

Eighty per cent of mice infected with a mouse-adapted strain of influenza virus died within 3 to 8 days after infection. Rimantadine given at maximum protective doses reduced mortality to 10%. This protection is dose related and can be demonstrated with doses from 4.5 to 24 mg per kg per day. Significant survival rates are shown by delaying treatment as long as 48 hr after infection. Lungs of treated mice have significantly less virus than those of controls at 24, 48, and 72 hr after infection. Antibody production as measured by hemagglutination inhibition is not different in treated and controlled mice. These results indicate that rimantadine is an effective prophylactic and therapeutic agent and that its activity is associated with decreased viral titers.     

Early augmentation of interleukin (IL)-12 level in the airway of mice administered orally with clarithromycin or intranasally with IL-12 results in alleviation of influenza infection.

The protective role of interleukin (IL)-12 against influenza infection was assessed by analyzing the efficacies of orally administered clarithromycin (CAM) as an immunomodulator and intranasal administration of recombinant IL-12 in intranasally influenza virus-infected mice. In infected mice, CAM at 20 mg/mouse/day significantly elevated the levels of IL-12 and interferon-gamma on days 2 and 3, respectively, after infection in the bronchoalveolar lavage fluid (BALF), but the levels in the sera were not affected. The levels of IL-4, -6, and -10 were not significantly affected in the sera and BALF. Corresponding with the local elevation of IL-12 level, CAM reduced virus yield and the number of infiltrated cells in the BALF, the severity of pneumonia, and mortality of the treated mice. The potential activity of CAM as an experimental immunomodulator was verified at a dose of 20 mg/mouse/day. Intranasal administration of the optimal dose (20 ng/mouse) of IL-12 on day 2 significantly reduced virus yield in the BALF after infection. The loss of body weight was significantly suppressed by IL-12 administration. The local elevation of IL-12 level at the optimal dose and timing in influenza infection was confirmed to be effective in alleviating the influenza infection in mice treated with the two different ways. Thus, the augmentation of IL-12 production or administration of supplementary IL-12 in the respiratory tract was essential in reducing virus yield in the early phase of influenza and may be crucial for recovery from influenza infection.     

Glycyrrhizin, an active component of licorice roots, reduces morbidity and mortality of mice infected with lethal doses of influenza virus.

The antiviral effect of glycyrrhizin (GR), an active component of licorice roots, was investigated in mice infected with influenza virus A2 (H2N2). When mice that had been exposed to 10 50% lethal doses of the virus were treated intraperitoneally with 10 mg of GR per kg of body weight 1 day before infection and 1 and 4 days postinfection, all of the mice survived over the 21-day experimental period. At the end of this period, the mean survival time (in days) for control mice treated with saline was 10.5 days, and there were no survivors. The grade of pulmonary consolidations and the virus titers in the lung tissues of infected mice treated with GR were significantly lower than those in the lung tissues of infected mice treated with saline. GR did not show any effects on the viability or replication of influenza virus A2 in vitro. When splenic T cells from GR-treated mice were adoptively transferred to mice exposed to influenza virus, 100% of the recipients survived, compared to 0% survival for recipient mice inoculated with naive T cells or splenic B cells and macrophages from GR-treated mice. In addition, the antiviral activities of GR on influenza virus infection in mice were not demonstrated when it was administered to infected mice in combination with anti-gamma interferon (anti-IFN-gamma) monoclonal antibody. These results suggest that GR may protect mice exposed to a lethal amount of influenza virus through the stimulation of IFN-gamma production by T cells, because T cells have been shown to be producer cells of IFN-gamma stimulated with the compound.     

Efficacy of single intravenous injection of peramivir against influenza B virus infection in ferrets and cynomolgus macaques

We evaluated the efficacy of a single intravenous dose peramivir for treatment of influenza B virus infection in ferrets and cynomolgus macaques in the present study. A single dose of peramivir (60 mg/kg of body weight) given to ferrets on 1 day postinfection with influenza B virus significantly reduced median area under the curve (AUC) virus titers (peramivir, 8.3 log(10) 50% tissue culture infective doses [TCID(50)s] · day/ml; control, 10.7 log(10) TCID(50)s · day/ml; P < 0.0001). Furthermore, nasal virus titers on day 2 postinfection in ferrets receiving a single injection of peramivir (30 mg/kg) and AUCs of the body temperature increase in ferrets receiving a single injection of peramivir (30 and 60 mg/kg) were lower than those in ferrets administered oral oseltamivir phosphate (30 and 60 mg/kg/day twice daily for 3 days). In macaques infected with influenza B virus, viral titers in the nasal swab fluid on days 2 and 3 postinfection and body temperature after a single injection of peramivir (30 mg/kg) were lower than those after oral administration of oseltamivir phosphate (30 mg/kg/day for 5 days). The two animal models used in the present study demonstrated that inhibition of viral replication at the early time point after infection was critical in reduction of AUCs of virus titers and interleukin-6 production, resulting in amelioration of symptoms. Our results shown in animal models suggest that the early treatment with a single intravenous injection of peramivir is clinically recommended to reduce symptoms effectively in influenza B virus infection.     

Safety and Immunogenicity of H5N1 Influenza Vaccine Based on Baculovirus Surface Display System of Bombyx mori

Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed. A recombinant Bombyx mori baculovirus, Bmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. The HA protein accounted for approximately 3% of the total viral proteins in silkworm pupae infected with the recombinant virus. Using a series of separation and purification methods, pure Bmgp64HA virus was isolated from these silkworm pupae bioreactors. Aluminum hydroxide adjuvant was used for an H5N1 influenza vaccine. Immunization with this vaccine at doses of 2 mg/kg and 0.67 mg/kg was carried out to induce the production of neutralizing antibodies, which protected monkeys against influenza virus infection. At these doses, the vaccine induced 1:40 antibody titers in 50% and 67% of the monkeys, respectively. The results of safety evaluation indicated that the vaccine did not cause any toxicity at the dosage as large as 3.2 mg/kg in cynomolgus monkeys and 1.6 mg/kg in mice. The results of dose safety evaluation of vaccine indicated that the safe dose of the vaccine were higher than 0.375 mg/kg in rats and 3.2 mg/kg in cynomolgus monkeys. Our work showed the vaccine may be a candidate for a highly effective, cheap, and safe influenza vaccine for use in humans.     

Efficacy of 2'-deoxy-2'-fluororibosides against influenza A and B viruses in ferrets.

Single-dose treatments (5 to 40 mg/kg of body weight given intraperitoneally) of ferrets with 2'-deoxy-2'-fluoroguanosine or its prodrug, 2,6-diamino-purine-2'-fluororiboside, 1 h after infection with influenza A virus significantly inhibited replication of virus in the upper respiratory tract, resulting in amelioration of fever and nasal inflammation. Replication of virus in the lower respiratory tract was also reduced > 100-fold, but three doses were required to prevent replication in the lungs. In ferrets infected with influenza B virus, single-dose treatment (40 mg/kg given intraperitoneally) produced a similar but reduced response in comparison with that in ferrets infected with influenza A virus, indicating that dosing was not optimal for this virus.     

Pharmacodynamic Evaluation of RWJ-270201, a Novel Neuraminidase Inhibitor, in a Lethal Murine Model of Influenza Predicts Efficacy for Once-Daily Dosing

We examined RWJ-270201 in a lethal model of influenza in BALB/c mice. The aim was to delineate the pharmacodynamically linked variable for the drug. Challenge was performed with influenza virus A/Shongdong/09/93 (H3N2). Treatment was administered by gavage. Five doses (1 to 10 mg/kg of body weight) and three schedules (every 24, 12, and 8 h) were evaluated with 10 mice per group. There were 39 placebo-treated mice. Drug exposure was evaluated for infected mice. Exposures were calculated after population modeling of all the plasma concentration-time data simultaneously using the NPEM3 program. Evaluation of dose and schedule with Kaplan-Meier analysis and Cox proportional hazards modeling demonstrated that schedule offered no explanatory power relative to dose alone. Evaluation of peak concentration, trough concentration, and area under the concentration-time curve (AUC) by the same methods revealed that AUC was the dynamically linked variable. Again, schedule offered no further explanatory power when included in the model with AUC. This indicates that AUC is the linked variable and that the anti-influenza effect of RWJ-270201 is independent of schedule. We conclude that once-daily dosing of RWJ-270201 should be evaluated in clinical trials of influenza therapy.     

Amantadine-oseltamivir combination therapy for H5N1 influenza virus infection in mice

BACKGROUND: The clinical management of H5N1 influenza virus infection in humans remains unclear. Combination chemotherapy with drugs that target different viral proteins might be more effective than monotherapy. METHODS: BALB/c mice were treated by oral gavage for 5 days with amantadine (1.5, 15 or 30 mg/kg/day) and oseltamivir (1 or 10 mg/kg/day) separately or in combination. Mice were challenged 24 h after initiation of treatment with 10 mouse 50% lethal doses of either amantadine-sensitive (having S31 in the M2 protein) or amantadine-resistant (having N31 in the M2 protein) recombinant A/Vietnam/1203/04 (H5N1) virus. RESULTS: Combination treatment with amantadine (15 or 30 mg/kg/day) and oseltamivir (10 mg/kg/day) provided greater protection (60% and 90%, respectively) against lethal infection with amantadine-sensitive H5N1 virus than did monotherapy. Moreover, spread of the virus to the brain was prevented by both combination regimens. The efficacy of the drug combinations against amantadine-resistant H5N1 virus was comparable to that of oseltamivir alone. Oseltamivir produced a dose-dependent effect against both recombinant H5N1 viruses (P < 0.05) but did not provide complete protection against lethal infection. Importantly, no mutations in the HA, NA and M2 proteins were detected when the two drugs were used in combination. CONCLUSIONS: Combination chemotherapy provided a survival advantage over single-agent treatment of mice inoculated with neurotropic H5N1 influenza virus. This strategy might be an option for the control of pandemic influenza viruses that are sensitive to amantadine. Combinations that include other drugs should be explored.     

Inhibition of influenza virus replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is consistent with extracellular activity of viral neuraminidase (sialidase).

We demonstrate the potent antiviral activity of a novel viral neuraminidase (sialidase) inhibitor, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GG167), administered by the intranasal route in comparison with those of amantadine and ribavirin in experimental respiratory tract infections induced with influenza A and B viruses. In an extended study in which mice were infected (day 0) with influenza A/Singapore/1/57 virus, with treatments given prophylactically plus twice daily over days 0 to 3 and with mice observed to day 10, we show that intranasally administered GG167 at 0.4 and 0.01 mg/kg of body weight per dose reduced mortality, lung consolidation, and virus titers in the lung, with no virus growing back following the cessation of treatment. In other studies with influenza B/Victoria/102/85 virus in which infected mice were culled after the cessation of treatment, the calculated intranasal dose required to reduce virus titers in the lungs of treated animals to 10% of that seen in untreated controls (EDAUC10 [where AUC is area under the virus titer days curve]) was 0.085 mg/kg per dose. GG167 was inactive against influenza viruses A and B when given by the intraperitoneal or oral route (EDAUC10, > 100 mg/kg per dose). GG167 was metabolically stable, with an elimination half-life of 10 min following intravenous administration. While readily bioavailable by systemic routes, it was poorly bioavailable by the oral route. Its potent efficacy by the intranasal route but lack of efficacy by other routes, relative to those of amantadine and ribavirin, was explicable in terms of its in vitro activity, bioavailability, and pharmacokinetic properties and with the extracellular activity of viral sialidase.     

The Flavonoid Isoliquiritigenin Reduces Lung Inflammation and Mouse Morbidity during Influenza Virus Infection

The host response to influenza virus infection is characterized by an acute lung inflammatory response in which intense inflammatory cell recruitment, hypercytokinemia, and a high level of oxidative stress are present. The sum of these events contributes to the virus-induced lung damage that leads to high a level of morbidity and mortality in susceptible infected patients. In this context, we identified compounds that can simultaneously reduce the excessive inflammatory response and the viral replication as a strategy to treat influenza virus infection. We investigated the anti-inflammatory and antiviral potential activities of isoliquiritigenin (ILG). Interestingly, we demonstrated that ILG is a potent inhibitor of influenza virus replication in human bronchial epithelial cells (50% effective concentration [EC₅₀] = 24.7 μM). In addition, our results showed that this molecule inhibits the expression of inflammatory cytokines induced after the infection of cells with influenza virus. We demonstrated that the anti-inflammatory activity of ILG in the context of influenza virus infection is dependent on the activation of the peroxisome proliferator-activated receptor gamma pathway. Interestingly, ILG phosphate (ILG-p)-treated mice displayed decreased lung inflammation as depicted by reduced cytokine gene expression and inflammatory cell recruitment. We also demonstrated that influenza virus-specific CD8⁺ effector T cell recruitment was reduced up to 60% in the lungs of mice treated with ILG-p (10 mg/kg) compared to that in saline-treated mice. Finally, we showed that administration of ILG-p reduced lung viral titers and morbidity of mice infected with the PR8/H1N1 virus.     

In vitro and in vivo activities of anti-influenza virus compound T-705

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has been found to have potent and selective inhibitory activity against influenza virus. In an in vitro plaque reduction assay, T-705 showed potent inhibitory activity against influenza A, B, and C viruses, with 50% inhibitory concentrations (IC(50)s) of 0.013 to 0.48 microg/ml, while it showed no cytotoxicity at concentrations up to 1,000 microg/ml in Madin-Darby canine kidney cells. The selectivity index for influenza virus was more than 2,000. It was also active against a neuraminidase inhibitor-resistant virus and some amantadine-resistant viruses. T-705 showed weak activity against non-influenza virus RNA viruses, with the IC(50)s being higher for non-influenza virus RNA viruses than for influenza virus, and it had no activity against DNA viruses. Orally administered T-705 at 100 mg/kg of body weight/day (four times a day) for 5 days significantly reduced the mean pulmonary virus yields and the rate of mortality in mice infected with influenza virus A/PR/8/34 (3 x 10(2) PFU). These results suggest that T-705 may be a compound that is useful and highly selective against influenza virus infections and that has a mode of action different from those of commercially available drugs, such as amantadine, rimantadine, and neuraminidase inhibitors.