Efficacy of Repeated Intravenous Administration of Peramivir against Highly Pathogenic Avian Influenza A (H5N1) Virus in Cynomolgus Macaques

Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor α, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.     

Therapeutic activity of intramuscular peramivir in mice infected with a recombinant influenza A/WSN/33 (H1N1) virus containing the H275Y neuraminidase mutation

The therapeutic activity of intramuscular (IM) peramivir was evaluated in mice infected with a recombinant influenza A/WSN/33 virus containing the H275Y neuraminidase (NA) mutation known to confer oseltamivir resistance. Regimens consisted of single (90 mg/kg of body weight) or multiple (45 mg/kg daily for 5 days) IM peramivir doses that were initiated 24 h or 48 h postinfection (p.i.). An oral oseltamivir regimen (1 or 10 mg/kg daily for 5 days) was used for comparison. Untreated animals had a mortality rate of 75% and showed a mean weight loss of 16.9% on day 5 p.i. When started at 24 h p.i., both peramivir regimens prevented mortality and significantly reduced weight loss (P < 0.001) and lung viral titers (LVT) (P < 0.001). A high dose (10 mg/kg) of oseltamivir initiated at 24 h p.i. also prevented mortality and significantly decreased weight loss (P < 0.05) and LVT (P < 0.001) compared to the untreated group results. In contrast, a low dose (1 mg/kg) of oseltamivir did not show any benefits. When started at 48 h p.i., both peramivir regimens prevented mortality and significantly reduced weight loss (P < 0.01) and LVT (P < 0.001) whereas low-dose or high-dose oseltamivir regimens had no effect on mortality rates, body weight loss, and LVT. Our results show that single-dose and multiple-dose IM peramivir regimens retain clinical and virological activities against the A/H1N1 H275Y variant despite some reduction in susceptibility when assessed in vitro using enzymatic assays. IM peramivir could constitute an alternative for treatment of oseltamivir-resistant A/H1N1 infections, although additional studies are warranted to support such a recommendation.     

Phase III randomized, double-blind study comparing single-dose intravenous peramivir with oral oseltamivir in patients with seasonal influenza virus infection

Antiviral medications with activity against influenza viruses are important in controlling influenza. We compared intravenous peramivir, a potent neuraminidase inhibitor, with oseltamivir in patients with seasonal influenza virus infection. In a multinational, multicenter, double-blind, double-dummy randomized controlled study, patients aged ≥ 20 years with influenza A or B virus infection were randomly assigned to receive either a single intravenous infusion of peramivir (300 or 600 mg) or oral administration of oseltamivir (75 mg twice a day [b.i.d.] for 5 days). To demonstrate the noninferiority of peramivir in reducing the time to alleviation of influenza symptoms with hazard model analysis and a noninferiority margin of 0.170, we planned to recruit 1,050 patients in South Korea, Japan, and Taiwan. A total of 1,091 patients (364 receiving 300 mg and 362 receiving 600 mg of peramivir; 365 receiving oseltamivir) were included in the intent-to-treat infected population. The median durations of influenza symptoms were 78.0, 81.0, and 81.8 h in the groups treated with 300 mg of peramivir, 600 mg of peramivir, and oseltamivir, respectively. The hazard ratios of the 300- and 600-mg-peramivir groups compared to the oseltamivir group were 0.946 (97.5% confidence interval [CI], 0.793, 1.129) and 0.970 (97.5% CI, 0.814, 1.157), respectively. Both peramivir groups were noninferior to the oseltamivir group (97.5% CI, <1.170). The overall incidence of adverse drug reactions was significantly lower in the 300-mg-peramivir group, but the incidence of severe reactions in either peramivir group was not different from that in the oseltamivir group. Thus, a single intravenous dose of peramivir may be an alternative to a 5-day oral dose of oseltamivir for patients with seasonal influenza virus infection.     

Susceptibilities of antiviral-resistant influenza viruses to novel neuraminidase inhibitors

The susceptibilities of five zanamivir-resistant and six oseltamivir-resistant influenza viruses were assessed against four neuraminidase (NA) inhibitors, including peramivir and A-315675, by a fluorometric NA activity inhibition assay. The enzyme activity of a majority of the variants was effectively inhibited by either A-315675 or both peramivir and A-315675 (50% inhibitory concentration, <10 nM). A novel oseltamivir-resistant influenza virus B variant carrying substitution at residue 198 (Asp-->Asn) (N2 numbering) retained susceptibility to peramivir and A-315675. In vivo, the Asn198 variant showed no apparent fitness impairment as judged by its recovery on day 5 from the nasal washes of ferrets coinfected with equal doses of the wild-type virus and the Asn198 variant. Based on the sequence analysis of the virus in the nasal washes, oseltamivir treatment (5 mg/kg twice daily for 5 days) did not provide growth advantage to the Asn198 variant. Nevertheless, treatment with A-315675 (prodrug A-322278) reduced the number of the animals (two of seven) shedding the Asn198 variant. These studies indicate that different patterns of susceptibility and cross-resistance between NA inhibitors may prove important if antiviral resistance to zanamivir and oseltamivir were to emerge.     

Assessment of Antiviral Properties of Peramivir against H7N9 Avian Influenza Virus in an Experimental Mouse Model

The H7N9 influenza virus causes a severe form of disease in humans. Neuraminidase inhibitors, including oral oseltamivir and injectable peramivir, are the first choices of antiviral treatment for such cases; however, the clinical efficacy of these drugs is questionable. Animal experimental models are essential for understanding the viral replication kinetics under the selective pressure of antiviral agents. This study demonstrates the antiviral activity of peramivir in a mouse model of H7N9 avian influenza virus infection. The data show that repeated administration of peramivir at 30 mg/kg of body weight successfully eradicated the virus from the respiratory tract and extrapulmonary tissues during the acute response, prevented clinical signs of the disease, including neuropathy, and eventually protected mice against lethal H7N9 influenza virus infection. Early treatment with peramivir was found to be associated with better disease outcomes.     

Efficacy of oseltamivir therapy in ferrets inoculated with different clades of H5N1 influenza virus

Highly pathogenic H5N1 influenza viruses have infected an increasing number of humans in Asia, with high mortality rates and the emergence of multiple distinguishable clades. It is not known whether antiviral drugs that are effective against contemporary human influenza viruses will be effective against systemically replicating viruses, such as these pathogens. Therefore, we evaluated the use of the neuraminidase (NA) inhibitor oseltamivir for early postexposure prophylaxis and for treatment in ferrets exposed to representatives of two clades of H5N1 virus with markedly different pathogenicities in ferrets. Ferrets were protected from lethal infection with the A/Vietnam/1203/04 (H5N1) virus by oseltamivir (5 mg/kg of body weight/day) given 4 h after virus inoculation, but higher daily doses (25 mg/kg) were required for treatment when it was initiated 24 h after virus inoculation. For the treatment of ferrets inoculated with the less pathogenic A/Turkey/15/06 (H5N1) virus, 10 mg/kg/day of oseltamivir was sufficient to reduce the lethargy of the animals, significantly inhibit inflammation in the upper respiratory tract, and block virus spread to the internal organs. Importantly, all ferrets that survived the initial infection were rechallenged with homologous virus after 21 days and were completely protected from infection. Direct sequencing of the NA or HA1 gene segments in viruses isolated from ferret after treatment showed no amino acid substitutions known to cause drug resistance in conserved residues. Thus, early oseltamivir treatment is crucial for protection against highly pathogenic H5N1 viruses and the higher dose may be needed for the treatment of more virulent viruses.     

Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.     

Prophylactic Activity of Intramuscular Peramivir in Mice Infected with a Recombinant Influenza A/WSN/33 (H1N1) Virus Containing the H274Y Neuraminidase Mutation

Peramivir is a neuraminidase (NA) inhibitor (NAI) under development that must be administered by the systemic route. The prophylactic activity of intramuscular (IM) peramivir was evaluated with mice infected with wild-type (WT) and oseltamivir-resistant (H274Y NA mutant) recombinant influenza A/WSN/33 (H1N1) viruses. Treatment regimens consisted of IM injections starting 1 h before viral challenge that were single (45 mg/kg or 90 mg/kg) or multiple (45 mg/kg daily for 5 days). All peramivir regimens prevented mortality and weight loss while significantly reducing lung viral titers (LVT) in mice infected with the WT virus. For animals infected with the H274Y mutant, the multiple-dose regimen completely prevented mortality and was associated with significant reduction in weight loss and LVT compared to untreated animals. In contrast, both single-treatment regimens reduced mortality and weight loss but did not significantly reduce LVT. Although further experiments using different influenza A/H1N1 virus strains and other animal models are needed, our results suggest that 5-day IM peramivir therapy may be considered a prophylactic alternative to control influenza infections caused by oseltamivir-resistant viruses with the H274Y mutation.Neuraminidase (NA) inhibitors (NAIs) constitute one of the most valuable options for the control of influenza epidemics and pandemics. Two NAIs, inhaled zanamivir and oral oseltamivir, have been approved for the treatment and prevention of influenza infections in many countries (16). In addition, other NAIs are at different stages of development. Peramivir, which is a cyclopentane analogue compound, has shown potent in vitro activity against influenza A and B viruses (4). By the use of NAI assays, we previously demonstrated that peramivir 50% inhibitory concentration (IC₅₀) values for Canadian clinical influenza A/H3N2, A/H1N1, and B viruses were lower than those of zanamivir and oseltamivir (10). In other studies, mean IC₅₀ values of clinical influenza A/H1N1 viruses from untreated individuals against peramivir were also lower than those against oseltamivir and zanamivir (14, 15). Furthermore, on-site dissociation studies demonstrated that peramivir remained tightly bound to the NA enzyme with a half-time for the substrate conversion of >24 h compared to 1.25 h for both zanamivir and oseltamivir (5).In controlled trials of prophylaxis and treatment, oral peramivir was associated with reduced viral titers but no significant decrease in time to relief of symptoms, a feature that could be attributed to a low oral bioavailability in humans (6). The bioavailability of peramivir may be improved by using intravenous (IV) or intramuscular (IM) injections. Indeed, comparison of single IM versus oral peramivir with the same dose (10 mg/kg), administered 4 h prior to a lethal influenza A/WSN/33 (H1N1) virus challenge, demonstrated that the IM route was associated with a higher survival rate in mice than that of the oral route (100% versus 50%) (5). Also, a single IV injection of 3 mg/kg of peramivir provided a significant therapeutic effect that was superior to that of oral oseltamivir in a lethal mouse model of influenza A and B virus infections (18). The emergence and rapid dissemination of the seasonal A/Brisbane/59/2007 (H1N1) virus containing the NA mutation H274Y in N2 numbering (H275Y in N1 numbering), which is associated with a high level of resistance to oseltamivir and moderate cross-resistance to peramivir in vitro (9), are a major clinical concern. The aim of the present study was to evaluate the prophylactic efficacy of IM injections of peramivir in mice infected with a recombinant influenza A/WSN/33 (H1N1) virus containing or not containing the H274Y NA mutation, which has been associated with 427- and 48-fold increases in oseltamivir and peramivir IC₅₀ values, respectively, in NAI assays (1).     

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.     

Oral oseltamivir in human experimental influenza B infection

Oseltamivir is the prodrug of Ro64-0802 (GS4071), a potent and selective inhibitor of influenza A and B virus neuraminidases. Three randomized, double-blind, placebo-controlled, parallel-group studies evaluated oral oseltamivir for early treatment (75 or 150 mg twice daily for 5 days) or prevention (75 mg once or twice daily for 7 days) of experimental influenza B virus infection in healthy susceptible adults. Treatment study A (n=60) demonstrated similar trends to treatment study B (n=117), in which 75 mg doses of oseltamivir introduced 24 h after inoculation reduced median area under curve (AUC) virus titre (oseltamivir, 22.7; placebo, 131.1 log10 TCID50 x h/ml; P=0.002) and duration of viral shedding (oseltamivir, 23.9 h; placebo, 95.8 h; P=0.0005). In prevention study C (n=58), oseltamivir did not reduce infection rates (85 versus 84%) but significantly reduced median AUC virus titre (10.0 versus 66.9 log10 TCID50 x h/ml; P=0.03) and duration of viral shedding (36 versus 84 h; P=0.03) compared with placebo. Oseltamivir was well tolerated. No emergence of drug-resistant variants was detected by testing last-day isolates (n=112) in neuraminidase inhibition assays. These results indicate that oseltamivir has significant antiviral activity in experimental human influenza B virus infection when used for prophylaxis or early treatment.     

Oral administration of cyclopentane neuraminidase inhibitors protects ferrets against influenza virus infection

Several cyclopentane inhibitors of influenza virus neuraminidase that have inhibitory activities in tissue culture similar to those of zanamivir and oseltamivir have recently been described. These new inhibitors have been examined for efficacy against a virulent H3N2 influenza virus when administered orally to infected ferrets. Preliminary studies indicated that oral administration of BCX-1923, BCX-1827, or BCX-1812 (RWJ-270201) at a dose of 5 or 25 mg/kg of body weight was active in ferrets in reducing respiratory and constitutional signs and symptoms, but these antivirals affected virus titers in the upper and lower respiratory tracts only marginally. Of the three compounds, BCX-1812 seemed to be the most efficacious and was examined further at higher doses of 30 and 100 mg/kg. These doses significantly reduced peak virus titers in nasal washes and total virus shedding as measured by areas under the curve. Virus titers in lung homogenates were also reduced compared to those in controls, but the difference was not statistically significant. As was observed with BCX-1812 at lower doses, the nasal inflammatory cellular response, fever, and nasal signs were reduced while ferret activity was not, with activity remaining similar to uninfected animals.     

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.     

Oral Administration of a Prodrug of the Influenza Virus Neuraminidase Inhibitor GS 4071 Protects Mice and Ferrets against Influenza Infection

We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.     

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.     

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.     

Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus

Amino acid substitutions at residue I223 of the neuraminidase (NA) protein have been identified in 2009 pandemic influenza (pH1N1) variants with altered susceptibilities to NA inhibitors (NAIs). We used reverse genetics and site-directed mutagenesis to generate the recombinant A/Québec/144147/09 pH1N1 wild-type virus (WT) and five (I223R, I223V, H275Y, I223V-H275Y, and I223R-H275Y) NA mutants. A fluorimetry-based assay was used to determine 50% inhibitory concentrations (IC(50)s) of oseltamivir, zanamivir, and peramivir. Replicative capacity was analyzed by viral yield assays in ST6GalI-MDCK cells. Infectivity and transmission of the WT, H275Y, and I223V-H275Y recombinant viruses were evaluated in ferrets. As expected, the H275Y mutation conferred resistance to oseltamivir (982-fold) and peramivir (661-fold) compared to the drug-susceptible recombinant WT. The single I223R mutant was associated with reduced susceptibility to oseltamivir (53-fold), zanamivir (7-fold) and peramivir (10-fold), whereas the I223V virus had reduced susceptibility to oseltamivir (6-fold) only. Interestingly, enhanced levels of resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir (1,647-, 17,347-, and 16-fold increases in IC(50)s, respectively) were observed for the I223R-H275Y recombinant, while the I223V-H275Y mutant exhibited 1,733-, 2,707-, and 2-fold increases in respective IC(50)s. The I223R and I223V changes were associated with equivalent or higher viral titers in vitro compared to the recombinant WT. Infectivity and transmissibility in ferrets were comparable between the recombinant WT and the H275Y or I223V-H275Y recombinants. In conclusion, amino acid changes at residue I223 may alter the NAI susceptibilities of pH1N1 variants without compromising fitness. Consequently, I223R and I223V mutations, alone or with H275Y, need to be thoroughly monitored.     

Comparison of efficacies of RWJ-270201, zanamivir, and oseltamivir against H5N1, H9N2, and other avian influenza viruses

The orally administered neuraminidase (NA) inhibitor RWJ-270201 was tested in parallel with zanamivir and oseltamivir against a panel of avian influenza viruses for inhibition of NA activity and replication in tissue culture. The agents were then tested for protection of mice against lethal H5N1 and H9N2 virus infection. In vitro, RWJ-270201 was highly effective against all nine NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was superior to that by zanamivir and oseltamivir carboxylate. RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (50% effective concentration, 0.5 to 11.8 microM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death. These results suggest that RWJ-270201 is at least as effective as either zanamivir or oseltamivir against avian influenza viruses and may be of potential clinical use for treatment of emerging influenza viruses that may be transmitted from birds to humans.     

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.     

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.     

In vivo influenza virus-inhibitory effects of the cyclopentane neuraminidase inhibitor RJW-270201

The cyclopentane influenza virus neuraminidase inhibitor RWJ-270201 was evaluated against influenza A/NWS/33 (H1N1), A/Shangdong/09/93 (H3N2), A/Victoria/3/75 (H3N2), and B/Hong Kong/05/72 virus infections in mice. Treatment was by oral gavage twice daily for 5 days beginning 4 h pre-virus exposure. The influenza virus inhibitor oseltamivir was run in parallel, and ribavirin was included in studies with the A/Shangdong and B/Hong Kong viruses. RWJ-270201 was inhibitory to all infections using doses as low as 1 mg/kg/day. Oseltamivir was generally up to 10-fold less effective than RWJ-270201. Ribavirin was also inhibitory but was less tolerated by the mice at the 75-mg/kg/day dose used. Disease-inhibitory effects included prevention of death, lessening of decline of arterial oxygen saturation, inhibition of lung consolidation, and reduction in lung virus titers. RWJ-270201 and oseltamivir, at doses of 10 and 1 mg/kg/day each, were compared with regard to their effects on daily lung parameters in influenza A/Shangdong/09/93 virus-infected mice. Maximum virus titer inhibition was seen on day 1, with RWJ-270201 exhibiting the greater inhibitory effect, a titer reduction of >10(4) cell culture 50% infective doses (CCID(50))/g. By day 8, the lung virus titers in mice treated with RWJ-270201 had declined to 10(1.2) CCID(50)/g, whereas titers from oseltamivir-treated animals were >10(3) CCID(50)/g. Mean lung consolidation was also higher in the oseltamivir-treated animals on day 8. Both neuraminidase inhibitors were well tolerated by the mice. RWJ-270201 was nontoxic at doses as high as 1,000 mg/kg/day. These data indicate potential for the oral use of RWJ-270201 in the treatment of influenza virus infections in humans.