peramivir and Orthomyxoviridae-Infections

The recent emergence of the highly pathogenic H5N1 subtype of avian influenza virus (AIV) and of the new type of human influenza A (H1N1) have emphasized the need for the development of effective anti-influenza drugs. Presently, neuraminidase (NA) inhibitors are widely used in the treatment and prophylaxis of human influenza virus infection, and tremendous efforts have been made to develop more potent NA inhibitors to combat resistance and new influenza viruses. In this review, we discuss the structural characteristics of NA catalytic domains and the recent developments of new NA inhibitors using structure-based drug design strategies. These drugs include analogues of zanamivir, analogues of oseltamivir, analogues of peramivir, and analogues of aromatic carboxylic acid and present promising options for therapeutics or leads for further development of NA inhibitors that may be useful in the event of a future influenza pandemic.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Benzoic Acid; Cyclopentanes; Drug Design; Enzyme Inhibitors; Guanidines; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H5N1 Subtype; Influenza, Human; Models, Molecular; Neuraminidase; Orthomyxoviridae; Orthomyxoviridae Infections; Oseltamivir; Zanamivir

Zanamivir and oseltamivir, the currently marketed influenza virus neuraminidase inhibitors (NAIs), are prescribed for the treatment and prophylaxis of influenza and are being stockpiled for pandemic influenza. Oseltamivir resistance has been reported in up to 2% of patients in clinical trials of oseltamivir and in up to 18% of treated children. There are also reports in at least three patients treated with oseltamivir for influenza A (H5N1) infections. At this stage, there are no reports of resistance occurring to zanamivir in immunocompetent patients. Zanamivir and oseltamivir bind differently at the neuraminidase catalytic site and this contributes to different drug resistance profiles. The magnitude and duration of NAI concentrations at the site of infection are also expected to be important factors and are determined by route and timing of drug administration, dose, and pharmacokinetic differences between patients. In addition, the type, strain, and virulence of the influenza strain and the nature of the immune response all appear to play a role in determining the likelihood of drug resistance arising. The clinical significance of a particular NAI-resistant isolate from a patient is often not clear but virus viability and transmissibility are clearly important characteristics. Early initiation of NAI treatment in suspected cases of influenza is important for maximizing efficacy and minimizing the risk of drug resistance. Higher NAI doses and longer periods of treatment may be required for patients with influenza A (H5N1) infections but further work is needed in this area.

Topics: Acids, Carbocyclic; Adult; Animals; Antiviral Agents; Catalytic Domain; Child; Clinical Trials as Topic; Cyclopentanes; Drug Administration Schedule; Enzyme Inhibitors; Guanidines; Humans; Immunocompromised Host; Influenza A Virus, H5N1 Subtype; Influenza, Human; Neuraminidase; Orthomyxoviridae; Orthomyxoviridae Infections; Oseltamivir; Seasons; Sentinel Surveillance; Viral Proteins; Virulence; Zanamivir

Equine influenza A virus (EIV) of the H3N8 subtype is an important pathogen causing acute respiratory disease in horses. Peramivir is a selective inhibitor of the influenza virus neuraminidase (NA). The characteristics of peramivir are not only its capacity for parenteral administration, but also its strong affinity for NA and slow off-rate from the NA-peramivir complex, suggesting that it could lead to a prolonged inhibitory effect and thus allow a lower dosing frequency. The aims of this study were to evaluate the inhibitory efficacy of peramivir against the NA activities of EIV in vitro and the treatment efficacy of a single intravenous dose of peramivir in horses experimentally infected with EIV. Peramivir inhibited the activities of NA from the seven contemporary EIV strains in vitro, with 50% inhibitory concentrations ranging from 0.10 to 0.20 nmol/L. Horses treated with a single IV dose of peramivir (3,000 mg/600 mL/animal, 7.8-9.3mg/kg of bodyweight) showed significantly milder clinical signs (pyrexia, nasal discharge and cough) with a shorter duration than control horses injected with normal saline. Moreover, the mean duration of virus shedding for the horses treated with peramivir was significantly shorter than for the control horses. These findings suggested that a single IV administration of peramivir had good potential for the treatment of equine influenza, and may help to limit the spread of the disease in the horse population.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Chromatography, Liquid; Cyclopentanes; Guanidines; Horse Diseases; Horses; Influenza A Virus, H3N8 Subtype; Injections, Intravenous; Neuraminidase; Orthomyxoviridae Infections; Tandem Mass Spectrometry

Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors (baloxavir marboxil and favipiravir) can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations.

Topics: Acids, Carbocyclic; Amides; Animals; Antiviral Agents; Cell Line; Dibenzothiepins; Dogs; Drug Combinations; Drug Resistance, Viral; Drug Synergism; Guanidines; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Madin Darby Canine Kidney Cells; Morpholines; Neuraminidase; Nucleic Acid Synthesis Inhibitors; Orthomyxoviridae Infections; Oseltamivir; Pyrazines; Pyridones; Ribavirin; Triazines; Viral Proteins; Virus Replication; Zanamivir

Human pandemic H1N1 2009 influenza virus causes significant morbidity and mortality with severe acute lung injury due to the excessive inflammatory reaction, even with neuraminidase inhibitor use. The anti-inflammatory effect of anti-high-mobility group box-1 (HMGB1) monoclonal antibody (mAb) against influenza pneumonia has been reported. In this study, we evaluated the combined effect of anti-HMGB1 mAb and peramivir against pneumonia induced by influenza A (H1N1) virus in mice. Nine-week-old male C57BL/6 mice were inoculated with H1N1 and treated with intramuscularly administered peramivir at 2 and 3 days post-infection (dpi). The anti-HMGB1 mAb or a control mAb was administered at 2, 3, and 4 dpi. Survival rates were assessed, and lung lavage and pathological analyses were conducted at 5 and 7 dpi. The combination of peramivir with the anti-HMGB1 mAb significantly improved survival rate whereas the anti-HMGB1 mAb alone did not affect virus proliferation in the lungs. This combination therapy also significantly ameliorated histopathological changes, neutrophil infiltration, and macrophage aggregation by inhibiting HMGB1, inflammatory cytokines, and oxidative stress. Fluorescence immunostaining showed that the anti-HMGB1 mAb inhibited HMGB1 translocation from type I alveolar epithelial cells. In summary, combining anti-HMGB1 with conventional anti-influenza therapy might be useful against severe influenza virus infection.

Topics: Acids, Carbocyclic; Animals; Antibodies, Monoclonal; Antiviral Agents; Cyclopentanes; Cytokines; Drug Therapy, Combination; Guanidines; HMGB1 Protein; Inflammation; Injections, Intramuscular; Lung; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Orthomyxoviridae Infections; Pneumonia, Viral

Influenza B viruses are important human pathogens that remain inadequately studied, largely because available animal models are poorly defined. Here, we developed an immunocompromised murine models for influenza B virus infection, which we subsequently used to study pathogenicity and to examine antiviral efficacy of the neuraminidase inhibitor peramivir. We studied three influenza B viruses that represent both the Yamagata (B/Massachusetts/2/2012 and B/Phuket/3073/2013) and Victoria (B/Brisbane/60/2008, BR/08) lineages. BR/08 was the most pathogenic in genetically modified immunocompromised mice [BALB scid and non-obese diabetic (NOD) scid strains] causing lethal infection without prior adaptation. The immunocompromised mice demonstrated prolonged virus shedding with modest induction of immune responses compared to BALB/c. Rather than severe virus burden, BR/08 virus-associated disease severity correlated with extensive virus spread and severe pulmonary pathology, stronger and persistent natural killer cell responses, and the extended induction of pro-inflammatory cytokines and chemokines. In contrast to a single-dose treatment (75 mg/kg/day), repeated doses of peramivir rescued BALB scid mice from lethal challenge with BR/08, but did not result in complete virus clearance. In summary, we have established immunocompromised murine models for influenza B virus infection that will facilitate evaluations of the efficacy of currently available and investigational anti-influenza drugs.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Bronchoalveolar Lavage Fluid; Cyclopentanes; Cytokines; Disease Models, Animal; Female; Guanidines; Immunocompromised Host; Inflammation Mediators; Influenza B virus; Mice; Mice, Inbred NOD; Mice, SCID; Morbidity; Mortality; Orthomyxoviridae Infections; Viral Load; Virus Replication

Topics: Acids, Carbocyclic; Amino Acid Substitution; Animals; Antiviral Agents; Catalytic Domain; Computer Simulation; Cyclopentanes; Drug Resistance, Viral; Enzyme Inhibitors; Genetic Fitness; Guanidines; Influenza A Virus, H1N1 Subtype; Inhibitory Concentration 50; Mice; Molecular Dynamics Simulation; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Reverse Genetics

High morbidity and mortality associated with human cases of highly pathogenic avian influenza (HPAI) viruses, including H5N1 influenza virus, have been reported. The purpose of the present study was to evaluate the antiviral effects of peramivir against HPAI viruses. In neuraminidase (NA) inhibition and virus replication inhibition assays, peramivir showed strong inhibitory activity against H5N1, H7N1 and H7N7 HPAI viruses with sub-nanomolar activity in enzyme assays. In H5N1 viruses containing the NA H275Y mutation, the antiviral activity of peramivir against the variant was lower than that against the wild-type. Evaluation of the in vivo antiviral activity showed that a single intravenous treatment of peramivir (10 mg/kg) prevented lethality in mice infected with wild-type H5N1 virus and also following infection with H5N1 virus with the H275Y mutation after a 5 day administration of peramivir (30 mg/kg). Furthermore, mice injected with peramivir showed low viral titers and low levels of proinflammatory cytokines in the lungs. These results suggest that peramivir has therapeutic activity against HPAI viruses even if the virus harbors the NA H275Y mutation.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Cytokines; Disease Models, Animal; Guanidines; Humans; Influenza A Virus, H5N1 Subtype; Influenza A Virus, H7N1 Subtype; Influenza A Virus, H7N7 Subtype; Influenza, Human; Lung; Mice; Mutation; Neuraminidase; Orthomyxoviridae Infections; Viral Load; Virus Replication

China-made Peramivir, an anti-influenza neuraminidase inhibitor drug, is manufactured and widely used in China. Although effective if initiated within 48 h of the onset of symptoms, yet we observed that this drug shows an inconclusive efficacy if treatment is delayed in clinical. Thus we evaluated the efficacy of delayed treatment of China-made Peramivir in a mouse model.. The mouse model of influenza infection was made and Peramivir was administered intravenously for 5 days following infection, and weight loss, lung index, viral shedding and survival rates were monitored.. Peramivir (60 mg/kg · d, repeated intravenous injections, quaque die (QD) × 5 days) enhanced survival rate and suppressed weight loss when treatment was initiated 24, 36, 48, or even 60 h post-infection (p.i.) (p < 0.01), compared with the virus-untreated group, and efficacy was abolished at 72 h p.i.. However the efficacy of delayed treatment was dose dependent, with the highest dose (90 mg/kg · d) even showing efficacy at 72 h p.i.. Furthermore, Peramivir (60 mg/kg · d, repeated intravenous injections, QD × 5 days) also reduced the lung virus titer 24 and 36 h p.i. on day 5, and even at 48 and 60 h p.i. on day 7 after infection, and the lung index was also improved. What is interesting that the concentration of the drug was maintained in blood after infected.. Delayed treatment with China-made Peramivir can reduce the severity of influenza disease, accelerate viral clearance and enhance the survival rate. This drug therefore shows good efficacy and is a promising candidate to control the influenza epidemic in China.

Topics: Acids, Carbocyclic; Administration, Intravenous; Animals; Antiviral Agents; China; Cyclopentanes; Disease Models, Animal; Drug Administration Schedule; Female; Guanidines; Influenza A Virus, H1N1 Subtype; Injections, Intravenous; Lung; Male; Mice; Orthomyxoviridae Infections; Viral Load; Virus Shedding

Pneumococcal pneumonia often occurs secondary to influenza infection and accounts for a large proportion of the morbidity and mortality associated with seasonal and pandemic influenza outbreaks. Peramivir is a novel, intravenous neuraminidase inhibitor that exhibits potent antiviral activity against influenza A and B viruses. We investigated the efficacy of peramivir for modulating the severity of secondary pneumococcal pneumonia.. CBA/JNCrlj mice, infected with influenza virus and superinfected with Streptococcus pneumoniae, were treated with either intravenous peramivir (single or multiple doses of 60 mg/kg/day) or oral oseltamivir at doses of 10 or 40 mg/kg/day in divided doses. The survival rate, viable bacterial count and virus titre in the lungs, as well as cytokine/chemokine concentration and histopathological findings were compared between both groups.. The median duration of survival of coinfected mice was significantly prolonged by treatment with multiple doses of peramivir, relative to mice treated with oseltamivir at either dose. Viable bacterial counts and virus titres in the lungs were significantly reduced by intravenous peramivir treatment compared with no treatment or oral oseltamivir treatment. The production of inflammatory cytokines/chemokines was also significantly suppressed by multiple dosing of peramivir compared with oseltamivir. Increased survival appeared to be mediated by decreased inflammation, manifested as lower levels of inflammatory cells and proinflammatory cytokines in the lungs and less severe histopathological findings. The lungs of mice treated with multiple doses of peramivir showed mild inflammatory changes compared to oseltamivir.. This study demonstrated that a multiple-dose regimen of intravenous peramivir was more efficacious than a single peramivir dose or multiple doses of oseltamivir for improving outcomes in pneumococcal pneumonia following influenza virus infection in mice.

Topics: Acids, Carbocyclic; Administration, Intravenous; Administration, Oral; Animals; Antiviral Agents; Chemokine CXCL2; Chemokines; Coinfection; Cyclopentanes; Guanidines; Influenza A Virus, H1N1 Subtype; Interferon-gamma; Interleukin-6; Lung; Male; Mice; Mice, Inbred CBA; Orthomyxoviridae Infections; Oseltamivir; Pneumonia, Pneumococcal; Streptococcus pneumoniae; Survival Analysis; Treatment Outcome; Tumor Necrosis Factor-alpha

Antiviral drug susceptibility is one of the evaluation criteria of pandemic potential posed by an influenza virus. Influenza A viruses of swine (IAV-S) can play an important role in generating novel variants, yet limited information is available on the drug resistance profiles of IAV-S circulating in the U.S. Phenotypic analysis of the IAV-S isolated in the U.S. (2009-2011) (n=105) revealed normal inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir, zanamivir, and peramivir. Screening NA sequences from IAV-S collected in the U.S. (1930-2014) showed 0.03% (1/3396) sequences with clinically relevant H274Y-NA substitution. Phenotypic analysis of IAV-S isolated in the U.S. (2009-2011) confirmed amantadine resistance caused by the S31N-M2 and revealed an intermediate level of resistance caused by the I27T-M2. The majority (96.7%, 589/609) of IAV-S with the I27T-M2 in the influenza database were isolated from pigs in the U.S. The frequency of amantadine-resistant markers among IAV-S in the U.S. was high (71%), and their distribution was M-lineage dependent. All IAV-S of the Eurasian avian M lineage were amantadine-resistant and possessed either a single S31N-M2 substitution (78%, 585/747) or its combination with the V27A-M2 (22%, 162/747). The I27T-M2 substitution accounted for 43% (429/993) of amantadine resistance in classic swine M lineage. Phylogenetic analysis showed that both S31N-M2 and I27T-M2 emerged stochastically but appeared to be fixed in the U.S. IAV-S population. This study defines a drug-susceptibility profile, identifies the frequency of drug-resistant markers, and establishes a phylogenetic approach for continued antiviral-susceptibility monitoring of IAV-S in the U.S.

Topics: Acids, Carbocyclic; Adamantane; Amino Acid Substitution; Animals; Antiviral Agents; Base Sequence; Cyclopentanes; Dogs; Drug Resistance, Viral; Enzyme Inhibitors; Genotype; Guanidines; Influenza A virus; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H1N2 Subtype; Influenza A Virus, H3N2 Subtype; Madin Darby Canine Kidney Cells; Mutation, Missense; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Phenotype; Phylogeny; Swine; Swine Diseases; Time Factors; United States; Viral Proteins; Zanamivir

Influenza virus infection increases susceptibility to bacterial infection and mortality in humans. Although the efficacy of approved intravenous peramivir, a neuraminidase (NA) inhibitor, against influenza virus infection has been reported, its efficacy against bacterial co-infection, which occurs during the period of viral shedding, was not fully investigated. To further understand the significance of treatment with peramivir, we assessed the efficacy of peramivir against a bacterial co-infection model in mice caused by clinically isolated influenza A(H1N1)pdm09 virus and Streptococcus pneumoniae.. Mice were infected with influenza A(H1N1)pdm09. Peramivir was intravenously administered after the viral infection. At 2days post viral infection, the mice were infected with S. pneumoniae. Peramivir efficacy was measured by the survival rates and viral titers, bacterial titers, or proinflammatory cytokine concentrations in lung homogenates.. Peramivir treatment reduced the mortality of mice infected with influenza virus and S. pneumoniae. The survival rate in the peramivir-treated group was significantly higher than that in the oseltamivir-treated group. Viral titers and proinflammatory cytokine responses in the peramivir-treated group were significantly lower than those in the oseltamivir-treated group until at 2days post viral infection. Bacterial titer was significantly lower in the peramivir-treated group than in the oseltamivir-treated group at 4days post viral infection.. These results demonstrated that peramivir inhibits viral replication, consequently leading to bacterial clearance and prevention of mortality during severe murine bacterial co-infection, which occurs during the period of viral shedding, with the efficacy of peramivir being superior to that of oseltamivir.

Topics: Acids, Carbocyclic; Administration, Intravenous; Animals; Antiviral Agents; Bacterial Load; Coinfection; Cyclopentanes; Cytokines; Disease Models, Animal; Dogs; Guanidines; Influenza A Virus, H1N1 Subtype; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Oseltamivir; Pneumococcal Infections; Streptococcus pneumoniae; Viral Load; Virus Replication

The number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or I219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitors in vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Drug Resistance, Viral; Enzyme Inhibitors; Female; Guanidines; Humans; Influenza A Virus, H5N1 Subtype; Influenza A Virus, H7N9 Subtype; Influenza Vaccines; Influenza, Human; Macaca; Mice; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Primates; Vaccination; Viral Proteins; Virus Replication

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.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Dogs; Drug Administration Schedule; Enzyme Inhibitors; Female; Guanidines; Humans; Influenza A Virus, H7N9 Subtype; Injections, Intramuscular; Lung; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred C57BL; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Survival Analysis; Treatment Outcome; Viral Load; Viral Proteins; Virus Replication

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.

Topics: Acids, Carbocyclic; Administration, Intravenous; Animals; Antiviral Agents; Body Temperature; Body Weight; Chemokine CCL2; Cyclopentanes; Drug Administration Schedule; Female; Guanidines; Influenza A Virus, H5N1 Subtype; Interferon-gamma; Interleukin-12 Subunit p40; Interleukin-6; Macaca fascicularis; Orthomyxoviridae Infections; Time Factors; Treatment Outcome; Tumor Necrosis Factor-alpha; Virulence; Virus Replication

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.

Topics: Acids, Carbocyclic; Animals; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antibodies, Neutralizing; Antibodies, Viral; Antiviral Agents; Cell Line; Cyclopentanes; Dogs; Drug Therapy, Combination; Female; Guanidines; Immunization, Passive; Immunocompromised Host; Influenza A Virus, H5N1 Subtype; Interleukin-6; Lung; Macaca fascicularis; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred BALB C; Models, Animal; Neuraminidase; Orthomyxoviridae Infections; Viral Load

Antiviral drugs are being used for therapeutic purposes against influenza illness in humans. However, antiviral-resistant variants often nullify the effectiveness of antivirals. Combined medications, as seen in the treatment of cancers and other infectious diseases, have been suggested as an option for the control of antiviral-resistant influenza viruses. Here, we evaluated the therapeutic value of combination therapy against oseltamivir-resistant 2009 pandemic influenza H1N1 virus infection in DBA/2 mice. Mice were treated for five days with favipiravir and peramivir starting 4 hours after lethal challenge. Compared with either monotherapy, combination therapy saved more mice from viral lethality and resulted in increased antiviral efficacy in the lungs of infected mice. Furthermore, the synergism between the two antivirals, which was consistent with the survival outcomes of combination therapy, indicated that favipiravir could serve as a critical agent of combination therapy for the control of oseltamivir-resistant strains. Our results provide new insight into the feasibility of favipiravir in combination therapy against oseltamivir-resistant influenza virus infection.

Topics: Acids, Carbocyclic; Amides; Animals; Antiviral Agents; Body Weight; Cyclopentanes; Dogs; Drug Resistance, Viral; Drug Synergism; Drug Therapy, Combination; Female; Guanidines; Influenza A Virus, H1N1 Subtype; Lung; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred DBA; Orthomyxoviridae Infections; Oseltamivir; Pyrazines; Survival Rate

The efficacy of intravenous peramivir against influenza A (H1N1) 2009 virus infection was evaluated in mice in which the immune system was suppressed by cyclophosphamide (CP) treatment. The mortality rate of the vehicle control group was 100%, and the mice lost 20% of their body weight on average by day 13 postinfection (p.i.). Repeated administration of peramivir (40 mg/kg of body weight once a day, given intravenously for 20 days), starting at 1 h p.i., significantly reduced mortality, body weight loss, viral titers, and cytokine production in infected mice compared with results for administration of vehicle (P < 0.01). In addition, repeated administration of peramivir, starting at 24 h, 48 h, or 72 h p.i., also resulted in increases in survival rates and reduction of viral titers in the lungs (P < 0.01). The mean days to death (MDD) of the vehicle group was 14.5 days, while in the groups treated with peramivir starting at 24 h, 48 h, and 72 h p.i., the MDDs were >23.0, 20.9, and 21.8 days, respectively. In comparison, repeated administration of oseltamivir phosphate (5 mg/kg twice a day, given orally for 20 days), starting at 24 h, 48 h, and 72 h p.i., also significantly prevented body weight loss, whereas no significant differences in mortality rates and viral titers in the lungs were observed compared with results for the vehicle group. These data indicated that repeated administration of peramivir was effective in promoting the survival and reducing virus replication in immunosuppressed mice infected with influenza A (H1N1) 2009 virus.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Body Weight; Cyclopentanes; Cyclophosphamide; Drug Administration Schedule; Female; Guanidines; Immunosuppressive Agents; Influenza A Virus, H1N1 Subtype; Injections, Intravenous; Lung; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Oseltamivir; Phosphorous Acids; Survival Analysis; Treatment Outcome

Highly pathogenic H5N1 virus remains a potential threat to humans. Over 289 fatalities have been reported in WHO confirmed human cases since 2003, and lack of effective vaccines and early treatments contribute to increasing numbers of cases and fatalities. H5N1 encephalitis is a recognized cause of death in Vietnamese cases, and brain pathology is described in other human cases and naturally infected animals. However, neither pathogenesis of H5N1 viral infection in human brain nor post-infection effects in survivors have been fully investigated. We report the brain pathology in a ferret model for active infection and 18-day survival stages. This model closely resembles the infection pattern and progression in human cases of influenza A, and our report is the first description of brain pathology for longer term (18-day) survival in ferrets. We analyzed viral replication, type and severity of meningoencephalitis, infected cell types, and cellular responses to infection. We found viral replication to very high titers in ferret brain, closely correlating with severity of meningoencephalitis. Viral antigens were detected predominantly in neurons, correlating with inflammatory lesions, and less frequently in astrocytes and ependymal cells during active infection. Mononuclear cell infiltrates were observed in early stages predominantly in cerebral cortex, brainstem, and leptomeninges, and less commonly in cerebellum and other areas. Astrogliosis was mild at day 4 post-infection, but robust by day 18. Early and continuous treatment with an antiviral agent (peramivir) inhibited virus production to non-detectable levels, reduced severity of brain injury, and promoted higher survival rates.

Topics: Acids, Carbocyclic; Animals; Antigens, Viral; Antiviral Agents; Astrocytes; Brain Diseases; Central Nervous System; Cyclopentanes; Cytokines; Ferrets; Guanidines; Influenza A Virus, H5N1 Subtype; Meningoencephalitis; Neuraminidase; Orthomyxoviridae Infections; Virus Replication

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.

Topics: Acids, Carbocyclic; Amino Acid Substitution; Animals; Antiviral Agents; Cell Line; Cyclopentanes; Dogs; Drug Resistance, Viral; Ferrets; Guanidines; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Inhibitory Concentration 50; Male; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Pandemics; Reverse Genetics; Viral Proteins; Virus Replication; Zanamivir

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Guanidines; Horse Diseases; Influenza A Virus, H3N8 Subtype; Orthomyxoviridae Infections

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.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Drug Resistance, Viral; Female; Guanidines; Influenza A Virus, H1N1 Subtype; Injections, Intramuscular; Mice; Mice, Inbred BALB C; Mutation; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Treatment Outcome; Viral Proteins

New and emerging influenza virus strains, such as the pandemic influenza A (H1N1) virus require constant vigilance for antiviral drug sensitivity and resistance. Efficacy of intramuscularly (IM) administered neuraminidase (NA) inhibitor, peramivir, was evaluated in mice infected with recently isolated pandemic A/California/04/2009 (H1N1, swine origin, mouse adapted) influenza virus. A single IM injection of peramivir (four dose groups), given 1h prior to inoculation, significantly reduced weight loss (p < 0.001) and mortality (p < 0.05) in mice infected with LD90 dose of pandemic A/California/04/2009 (H1N1) influenza virus compared to vehicle group. There was 20% survival in the vehicle-treated group, whereas in the peramivir-treated groups, survival increased in a dose-dependent manner with 60, 60, 90 and 100% survivors for the 1, 3, 10, and 30 mg/kg doses, respectively. Weight loss on day 4 in the vehicle-treated group was 3.4 gm, and in the peramivir-treated groups was 2.1, 1.5, 1.8 and 1.8 g for the 1, 3, 10 and 30 mg/kg dose groups, respectively. In the treatment model, peramivir given 24h after infection as a single IM injection at 50mg/kg dose, showed significant protection against lethality and weight loss. There was 13% survival in the vehicle-treated group while in the peramivir-treated group at 24, 48, and 72 h post infection, survival was 100, 40, and 50%, respectively. Survival in the oseltamivir groups (10 mg/kg/d twice a day, orally for 5 days) was 90, 30 and 20% at 24, 48 and 72 h, respectively. These data demonstrate efficacy of parenterally administered peramivir against the recently isolated pandemic influenza virus in murine infection models.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Body Weight; Cyclopentanes; Disease Models, Animal; Female; Guanidines; Influenza A Virus, H1N1 Subtype; Injections, Intramuscular; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Rodent Diseases; Survival Analysis

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.

Topics: Acids, Carbocyclic; Animals; Cyclopentanes; Ferrets; Guanidines; Influenza B virus; Injections, Intravenous; Macaca; Orthomyxoviridae Infections

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.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cell Line; Cyclopentanes; Dogs; Guanidines; Influenza A Virus, H1N1 Subtype; Injections, Intramuscular; Male; Mice; Mice, Inbred BALB C; Mutation; Neuraminidase; Orthomyxoviridae Infections; Viral Proteins

Oseltamivir and peramivir are being considered for combination treatment of serious influenza virus infections in humans. Both compounds are influenza virus neuraminidase inhibitors, and since peramivir binds tighter to the enzyme than oseltamivir carboxylate (the active form of oseltamivir), the possibility exists that antagonistic interactions might result when using the two compounds together. To study this possibility, combination chemotherapy experiments were conducted in vitro and in mice infected with influenza A/NWS/33 (H1N1) virus. Treatment of infected MDCK cells was performed with combinations of oseltamivir carboxylate and peramivir at 0.32-100μM for 3 days, followed by virus yield determinations. Additive drug interactions with a narrow region of synergy were found using the MacSynergy method. In a viral neuraminidase assay with combinations of inhibitors at 0.01-10nM, no significant antagonistic or synergistic interactions were observed across the range of concentrations. Infected mice were treated twice daily for 5 days starting 2h prior to virus challenge using drug doses of 0.05-0.4mg/kg/day. Consistent and statistically significant increases in the numbers of survivors were seen when twice daily oral oseltamivir (0.4mg/kg/day) was combined with twice daily intramuscular peramivir (0.1 and 0.2mg/kg/day) compared to single drug treatments. The data demonstrate that combinations of oseltamivir and peramivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Treatment with these two compounds should be considered as an option.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cell Line; Cyclopentanes; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Interactions; Drug Therapy, Combination; Guanidines; Humans; Influenza A Virus, H1N1 Subtype; Mice; Mice, Inbred BALB C; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir

Influenza viruses of the N1 neuraminidase (NA) subtype affecting both animals and humans caused the 2009 pandemic. Anti-influenza virus NA inhibitors are crucial early in a pandemic, when specific influenza vaccines are unavailable. Thus, it is urgent to confirm the antiviral susceptibility of the avian viruses, a potential source of a pandemic virus. We evaluated the NA inhibitor susceptibilities of viruses of the N1 subtype isolated from wild waterbirds, swine, and humans. Most avian viruses were highly or moderately susceptible to oseltamivir (50% inhibitory concentration [IC(50)], <5.1 to 50 nM). Of 91 avian isolates, 7 (7.7%) had reduced susceptibility (IC(50), >50 nM) but were sensitive to the NA inhibitors zanamivir and peramivir. Oseltamivir susceptibility ranged more widely among the waterbird viruses (IC(50), 0.5 to 154.43 nM) than among swine and human viruses (IC(50), 0.33 to 2.56 nM). Swine viruses were sensitive to oseltamivir, compared to human seasonal H1N1 isolated before 2007 (mean IC(50), 1.4 nM). Avian viruses from 2007 to 2008 were sensitive to oseltamivir, in contrast to the emergence of resistant H1N1 in humans. Susceptibility remained high to moderate over time among influenza viruses. Sequence analysis of the outliers did not detect molecular markers of drug-resistance (e.g., H275Y NA mutation [N1 numbering]) but revealed mutations outside the NA active site. In particular, V267I, N307D, and V321I residue changes were found, and structural analyses suggest that these mutations distort hydrophobic pockets and affect residues in the NA active site. We determined that natural oseltamivir resistance among swine and wild waterbirds is rare. Minor naturally occurring variants in NA can affect antiviral susceptibility.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Birds; Catalytic Domain; Cyclopentanes; Drug Resistance, Viral; Enzyme Inhibitors; Guanidines; Humans; Influenza A virus; Influenza A Virus, H1N1 Subtype; Influenza in Birds; Influenza, Human; Models, Molecular; Mutation; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Protein Conformation; Species Specificity; Swine; Swine Diseases; Viral Proteins; Zanamivir

Efficacy of combination of the intramuscularly administered neuraminidase (NA) inhibitor, peramivir, and the orally administered M2 ion channel blocker, rimantadine was evaluated in mouse influenza A/Victoria/3/75 (H3N2) model. Mice were challenged with a sub-lethal virus dose (0-40% mortality in placebo group) and changes in body weights were analyzed by three-dimensional effect analysis to assess mode of drug interactions. Compounds were administered in a 5-day treatment course starting 1h before viral inoculation. The peramivir and rimantadine doses ranged from 0.3-3 mg/kg/d and 5-30 mg/kg/d, respectively. The maximum mean weight loss of 5.19 g was observed in the vehicle-infected group on day 10. In the 1 and 3 mg/kg/d peramivir monotherapy groups, the weight losses were 4.3 and 3.55 g, respectively. In the rimantadine monotherapy group, the weight losses were 3.43, 2.1, and 1.64 g for the 5, 10, and 30 mg/kg/d groups, respectively. Combination of 1mg/kg/d peramivir with 5 and 10 mg/kg/d rimantadine produced weight losses of 1.69 and 0.69 (p<0.05 vs. vehicle and individual agent), respectively, whereas the combination of 3.0 mg/kg/d peramivir with 10 and 30 mg/kg/d rimantadine did not show any weight loss (p<0.05 vs. vehicle and individual agent). The three-dimensional analysis of the weight loss for the majority of the drug combinations of peramivir and rimantadine tested demonstrated synergistic antiviral effects.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Administration Schedule; Drug Synergism; Female; Guanidines; Influenza A Virus, H3N2 Subtype; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Rimantadine; Survival Rate; Weight Loss

The post-exposure therapeutic efficacy of injectable peramivir against highly pathogenic avian influenza type A H5N1 was evaluated in mice and in ferrets. Seventy to eighty percent of the H5N1-infected peramivir-treated mice, and 70% in the oseltamivir treated mice survived the 15-day study period, as compared to 36% in control (vehicle) group. Ferrets were infected intranasally with H5N1 followed by treatment with multiple doses of peramivir. In two of three trials, a statistically significant increase in survival over a 16-18 day period resulted from peramivir treatment, with improved survival of 40-64% in comparison to mock-treated or untreated animals. Injected peramivir mitigates virus-induced disease, reduces infectious virus titers in the lungs and brains and promotes survival in ferrets infected intranasally with this highly neurovirulent isolate. A single intramuscular peramivir injection protected mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Ferrets; Guanidines; Influenza A Virus, H5N1 Subtype; Injections, Intramuscular; Lung; Mice; Orthomyxoviridae Infections; Oseltamivir; Survival Analysis; Treatment Outcome

In the event of an influenza outbreak, antivirals including the neuraminidase (NA) inhibitors, peramivir, oseltamivir, and zanamivir may provide valuable benefit when vaccine production is delayed, limited, or cannot be used. Here we demonstrate the efficacy of a single intramuscular injection of peramivir in the mouse influenza model. Peramivir potently inhibits the neuraminidase enzyme N9 from H1N9 virus in vitro with a 50% inhibitory concentration (IC(50)) of 1.3+/-0.4 nM. On-site dissociation studies indicate that peramivir remains tightly bound to N9 NA (t(1/2)>24h), whereas, zanamivir and oseltamivir carboxylate dissociated rapidly from the enzyme (t(1/2)=1.25 h). A single intramuscular injection of peramivir (10mg/kg) significantly reduces weight loss and mortality in mice infected with influenza A/H1N1, while oseltamivir demonstrates no efficacy by the same treatment regimen. This may be due to tight binding of peramivir to the N1 NA enzymes similar to that observed for N9 enzyme. Additional efficacy studies indicate that a single injection of peramivir (2-20mg/kg) was comparable to a q.d.x 5 day course of orally administered oseltamivir (2-20mg/kg/day) in preventing lethality in H3N2 and H1N1 influenza models. A single intramuscular injection of peramivir may successfully treat influenza infections and provide an alternate option to oseltamivir during an influenza outbreak.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Guanidines; Influenza A Virus, H1N1 Subtype; Injections, Intramuscular; Mice; Neuraminidase; Orthomyxoviridae Infections

An inbred murine model (BALB/c) was utilized to assess the protective effect of the immunomodulator methyl inosine 5'-monophosphate (MIMP) against infection with influenza A/PR/8/34 (H1N1) virus. Contrary to the data reported for outbred mice (NMRI) infected with the aerosolized virus (Masihi, Hadden, 2003. J. Int. Immunopharmacol. 3, 1205-1215), there were no improvements in the outcomes of infection in the inbred animals treated with MIMP intranasally 1 day before the challenge and/or orally after the challenge for 5 days (up to 10 mg/kg/day). Nevertheless, complete protection against lethality was afforded by the treatment with the neuraminidase inhibitor peramivir given once daily for 5 days after the challenge (10 mg/kg/day). We speculate that the rapid progression of the disease in inbred mice caused by the intranasal challenge may render the MIMP-treatment ineffective. Our results emphasize the need for careful consideration of murine strains and routes of virus challenge in the design of experiments utilizing lethal influenza virus infection.

Topics: Acids, Carbocyclic; Age Factors; Animals; Antiviral Agents; Body Weight; Cyclopentanes; Disease Models, Animal; Female; Guanidines; Influenza A Virus, H1N1 Subtype; Inosine Monophosphate; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections

Cyclopentane derivatives, designated as BCX-1812, BCX-1827, BCX-1898, and BCX-1923, were tested in parallel with oseltamivir carboxylate and zanamivir for the in vivo activity in mice infected with A/Turkey/Mas/76 X A/Beijing/32/92 (H6N2) influenza virus. The compounds were tested orally and intranasally at different dose levels. BCX-1812, BCX-1827, and BCX-1923 showed more than 50% protection at 1mg/kg/day dose level on oral treatment. The intranasal treatment was 100% effective even at 0.01 mg/kg/day for all four compounds. On comparison with oseltamivir carboxylate and zanamivir, these four cyclopentane derivatives have shown equal or better efficacies. The synthesis of two new compounds, BCX-1898 and BCX-1923, is also described.

Topics: Acetamides; Acids, Carbocyclic; Administration, Intranasal; Administration, Oral; Animals; Antiviral Agents; Cyclopentanes; Dose-Response Relationship, Drug; Guanidines; Mice; Orthomyxoviridae; Orthomyxoviridae Infections; Oseltamivir; Pyrans; Sialic Acids; Structure-Activity Relationship; Survival Rate; Treatment Outcome; Zanamivir

Experiments were run to determine the effect of oral gavage treatment with the cyclopentane influenza virus neuraminidase inhibitor peramivir (BCX-1812, RWJ-270201) in influenza A (H1N1) virus-infected mice that had their immune system suppressed by cyclophosphamide (CP) therapy or in severe combined immune deficient (SCID) mice. Treatment of CP-immunosuppressed mice with peramivir using doses of 100, 10, or 1mg/kg/day was begun 2.5 or 8 days post-virus exposure and continued twice daily for 3 or 5 days. The 5-day therapy was more effective than the 3-day treatment, as seen by significantly increased survivor numbers, lessened decline in arterial oxygen saturation, reduced lung consolidation, and inhibition of lung virus titers. Infected SCID mice were also responsive to peramivir therapy begun 8 days after virus exposure and continued for 5 days, although antiviral effects did not include prevention of death and were dependent upon the viral challenge dose received. These data indicate that peramivir may have potential for treatment of influenza virus-infected immunosuppressed patients.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Cyclophosphamide; Female; Guanidines; Immunocompromised Host; Immunosuppressive Agents; Influenza A virus; Lung; Mice; Mice, SCID; Neuraminidase; Orthomyxoviridae Infections; Oxygen; Survival Rate

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.

Topics: Acetamides; Acids, Carbocyclic; Animals; Antiviral Agents; Area Under Curve; Body Temperature; Cell Count; Cyclopentanes; Enzyme Inhibitors; Ferrets; Guanidines; Half-Life; Hemagglutination Inhibition Tests; Influenza A virus; Lung; Male; Nasal Cavity; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir

Treatment of virus infections with compounds acting by different mechanisms may lead to more potent effects when these agents are used in combination. Under this premise, two known active influenza virus inhibitors, ribavirin and the novel cyclopentane influenza virus neuraminidase inhibitor (1S,2S,3R,4R)-3-[(1S)-(acetylamino)-2-ethylbutyl]-4-[(aminoiminomethyl)amino]-2-hydroxy-cyclopentanecarboxylic acid (RWJ-270201, BCX-1812) were studied. Experiments in cell culture demonstrated that RWJ-270201 plus ribavirin synergistically reduced extracellular influenza A/NWS/33 (H1N1) virus yields at low concentrations of each inhibitor. Mice were treated with ribavirin at 20 and 6.25 mg/kg/day combined with RWJ-270201 at 1, 0.32, or 0.1 mg/kg/day, or used alone. Treatments were twice daily for 5 days starting 4 h before exposure to influenza A/NWS virus. Only RWJ-270201 alone at 1 mg/kg/day significantly prevented mortality. In contrast, most drug combinations increased survival significantly compared to the placebo group. Doses of the two compounds used in combination delayed the mean day of death, and improved arterial oxygen saturation levels, as measured on day 11 of the infection. The combination of the two inhibitors produced additive to synergistic interactions in these mouse experiments with no enhancement of host toxicity. Treatment of influenza infections in the clinical setting may benefit by these two agents in combination.

Topics: Acids, Carbocyclic; Alphainfluenzavirus; Animals; Antiviral Agents; Cell Culture Techniques; Cyclopentanes; Dose-Response Relationship, Drug; Drug Interactions; Female; Guanidines; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Ribavirin

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.

Topics: Acetamides; Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Disease Models, Animal; Female; Guanidines; Influenza A virus; Influenza B virus; Lung; Mice; Mice, Inbred C57BL; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Respiratory Function Tests; Ribavirin; Virus Replication

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.

Topics: Acetamides; Acids, Carbocyclic; Administration, Intranasal; Administration, Oral; Animals; Antiviral Agents; Cyclopentanes; Enzyme Inhibitors; Female; Guanidines; Inhibitory Concentration 50; Mice; Mice, Inbred BALB C; Neuraminidase; Orthomyxoviridae; Orthomyxoviridae Infections; Oseltamivir; Pyrans; Sialic Acids; Species Specificity; Survival Analysis; Time Factors; Zanamivir

The cyclopentane derivative [1S,2S,3R,4R]-3-[(1S)-1-(acetylamino)-2- ethylbutyl]-4-[(aminoiminomethyl)amino]-2-hydroxy-cyclopentanecarboxylic acid (RWJ-270201) has been previously reported to be a potent and selective inhibitor of influenza virus neuraminidase, and to inhibit infections with this virus in vitro, in mice, and in clinical challenge studies. The effect of oral gavage therapy of 100 mg/kg/day of RWJ-270201 administered twice daily for 5 days beginning 16 h prior to virus exposure, on various immune factors of importance in response to primary influenza infection was determined in mice infected with influenza A/Shangdong/09/93 (H3N2) virus. Spleens taken from the mice 2 h after termination of treatment were processed for cytotoxic T lymphocytes (CTL) and natural killer (NK) cell activity and for enumeration of macrophages, T, T-helper, T-suppressor/cytotoxic, and B cells. Saline-treated mice and normal mice were run in parallel. Treatment had no significant effect on any immune parameter. In a second experiment, mice infected with influenza A/NWS/33 (H1N1) were treated similarly with RWJ-270201 beginning 4 h pre-virus exposure. Treatment prevented any deaths from occurring, and markedly lessened arterial oxygen decline, lung consolidation, and lung virus titers. The mice developed mean neutralizing antibody (NA) titers of 1:592, and six of seven rechallenged mice resisted rechallenge with the same virus, indicating the initial virus-inhibitory effect also did not prevent the animals from developing an adequate humoral immune response to the virus.

Topics: Acids, Carbocyclic; Animals; Antibody Formation; Antiviral Agents; Cyclopentanes; Enzyme Inhibitors; Female; Guanidines; Influenza A virus; Lung; Lymphocytes; Mice; Mice, Inbred BALB C; Neuraminidase; Orthomyxoviridae Infections; Oxygen; Phenotype; Spleen

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.

Topics: Acids, Carbocyclic; Animals; Antiviral Agents; Cyclopentanes; Disease Models, Animal; Female; Guanidines; Mice; Mice, Inbred BALB C; Neuraminidase; Orthomyxoviridae Infections; Treatment Outcome

The influenza virus neuraminidase inhibitor RWJ-270201 (cyclopentane carboxylic acid, 3-[cis-1-(acetylamino)-2-ethylbutyl]-4[(aminoiminomethyl)amino]-2-hydroxy-[cis, 2S, 3R, 4R]) was significantly inhibitory to an infection in mice induced by influenza A/NWS/33 (H1N1) virus when oral gavage (p.o.) treatment with 10 mg/kg per day was delayed at least 60 h after virus exposure. Treatment was 5 mg/kg twice daily for 5 days. Viral challenge doses of influenza A/Shangdong/09/93 (H3N2) virus ranging from the LD(70) to the LD(100) did not affect the marked antiviral efficacy of 12.5 mg/kg of RWJ-270201 administered p.o. twice daily for 5 days beginning 4 h pre-virus exposure; infection by an approximate 2 LD(100) dose (10(8) cell culture infectious doses/ml) was only weakly inhibited by the same treatment as seen by significant increase in mean day to death. Murine infections induced by influenza A/Bayern/57/93 (H1N1) and B/Lee/40 viruses were significantly inhibited by 100, 10, and 1 mg/kg per day of RWJ-270201 using the above treatment regimen; influenza A/PR/8/34 (H1N1) virus infections in mice were only moderately inhibited, the antiviral effects using this virus being lessening of arterial oxygen decline, reduced lung consolidation, and inhibition of lung virus titers primarily at the higher dosages.

Topics: Acids, Carbocyclic; Administration, Oral; Animals; Antiviral Agents; Cyclopentanes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Guanidines; Influenza A virus; Influenza B virus; Mice; Mice, Inbred BALB C; Neuraminidase; Orthomyxoviridae Infections

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.

Topics: Acetamides; Acids, Carbocyclic; Animals; Antiviral Agents; Body Weight; Brain; Cyclopentanes; Disease Models, Animal; Dogs; Female; Guanidines; Influenza A virus; Influenza A Virus, H5N1 Subtype; Influenza A Virus, H9N2 Subtype; Lung; Mice; Mice, Inbred BALB C; Neuraminidase; Orthomyxoviridae Infections; Oseltamivir; Pyrans; Sialic Acids; Treatment Outcome; Virus Replication; Zanamivir

Topics: Acids, Carbocyclic; Administration, Oral; Animals; Antiviral Agents; Catalytic Domain; Crystallography, X-Ray; Cyclopentanes; Drug Design; Enzyme Inhibitors; Guanidines; Influenza A virus; Influenza B virus; Mice; Models, Molecular; Neuraminidase; Orthomyxoviridae Infections; Protein Binding; Stereoisomerism; Structure-Activity Relationship