Review of the clinical effectiveness of the neuraminidase inhibitors against influenza B viruses

Influenza A and B viruses cause significant morbidity and mortality worldwide each year. The neuraminidase inhibitors (NAIs) are the most commonly used class of influenza antiviral drugs for the treatment of infected patients. In vitro studies have shown that influenza B viruses are significantly less susceptible to oseltamivir and other neuraminidase inhibitors compared with influenza A viruses. Following analysis of published clinical studies, we show that oseltamivir does appear to have lower effectiveness in patients infected with influenza B virus compared with influenza A infected patients, but due to insufficient studies on zanamivir, laninamivir or peramivir, it was not possible to conclude the relative effectiveness of these drugs against influenza A virus compared with B virus.     

Use of neuraminidase inhibitors to combat pandemic influenza

Since the last influenza pandemic in 1968, neuraminidase (NA) inhibitors have been licensed for the treatment and prophylaxis of seasonal influenza. Continuing outbreaks of highly pathogenic avian influenza H5N1 since 2004 have focused attention on the timing of the next pandemic and preparedness plans. Although immunization is the principal means of influenza prophylaxis, a well-matched efficacious vaccine is unlikely to be widely available for several months following the emergence of the pandemic strain. NA inhibitors could be used to contain and eliminate an emerging pandemic virus at source. If unsuccessful, they could still play a crucial role in reducing the medical impact of pandemic influenza as it spreads through countries. Accordingly, many authorities are creating stockpiles of NA inhibitors. However, the use of stockpiled drugs for treatment or prophylaxis, the rapid delivery to newly diagnosed cases and unknown characteristics of an emergent pandemic strain pose significant challenges to determining optimal use of stockpiles.     

Detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use

Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) develops at a low level following drug treatment, and person-to-person transmission of resistant virus has not been recognized to date. The Neuraminidase Inhibitor Susceptibility Network (NISN) was established to follow susceptibility of isolates and occurrence of NAI resistance at a population level in various parts of the world. Isolates from the WHO influenza collaborating centers were screened for susceptibilities to oseltamivir and zanamivir by a chemiluminescent enzyme inhibition assay, and those considered potentially resistant were analyzed by sequence analysis of the neuraminidase genes. During the first 3 years of NAI use (1999 to 2002), 2,287 isolates were tested. Among them, eight (0.33%) viruses had a >10-fold decrease in susceptibility to oseltamivir, one (0.22%) in 1999 to 2000, three (0.36%) in 2000 to 2001, and four (0.41%) in 2001 to 2002. Six had unique changes in the neuraminidase gene compared to neuraminidases of the same subtype in the influenza sequence database. Although only one of the mutations had previously been recognized in persons receiving NAIs, none were from patients who were known to have received the drugs. During the 3 years preceding NAI use, no resistant variants were detected among 1,054 viruses. Drug use was relatively stable during the period, except for an approximate 10-fold increase in oseltamivir use in Japan during the third year. The frequency of variants with decreased sensitivity to the NAIs did not increase significantly during this period, but continued surveillance is required, especially in regions with higher NAI use.     

Selective solubilization of hemagglutinin and neuraminidase from influenza viruses.

A novel approach is described for the isolation of hemagglutinin and neuraminidase from influenza viruses. In contrast to published procedures, the two glycoproteins are selectively solubilized, leaving an intact viral subparticle which contains lipid, RNA and non-glycoproteins. The pronounced differences in size allow a simple separation of the solubilized proteins from the virus 'core'.     

Novel alpha- and beta-amino acid inhibitors of influenza virus neuraminidase

In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 alpha- and beta-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited K(i) values in the 50 microM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the alpha-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the beta-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the alpha- or beta-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.     

Impact of neuraminidase mutations conferring influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic backgrounds

Subtype-specific neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) have been reported during in vitro passages and in clinic. In this study, we evaluated the impact of various NA mutations (E119A/G/V, H274Y, R292K and N294S) on the susceptibility profiles to different NAIs (oseltamivir, zanamivir and peramivir) using recombinant NA proteins of influenza A/WSN/33 (H1N1) and A/Sydney/5/97-like (H3N2) viruses. In the Nl subtype, the E119V mutation conferred cross-resistance to oseltamivir, zanamivir and peramivir [1,727-2,144 and 5,050-fold increase in IC50 values compared with wild-type (WT)] whereas only oseltamivir-resistance (1,028-fold increase in IC50) was conferred by the same mutation in the N2 subtype. The N294S mutation conferred resistance to oseltamivir in both the NI and N2 subtypes (197- and 1,879-fold increase in IC50 values, respectively) whereas the H274Y mutation conferred resistance to oseltamivir (754-fold increase) and peramivir (260-fold increase) in the N1 subtype only. The virulence of reverse genetics-rescued A/WSN/33 viruses harbouring H274Y and N294S NA mutations was investigated in Balb/c mice. The WT and H274Y recombinants had identical LD50 values (103 PFUs) and generated similar viral lung titres, whereas a higher LD50 (10 PFUs) and a 1-log decrease in viral lung titres were obtained with the N294S mutant. This study shows that some NA mutations at framework residues may confer resistance to one or three NAIs depending on the viral subtype. It suggests that certain drug-resistant NA mutants may still be virulent although additional studies using clinical isolates are needed to confirm our results.     

Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations

OBJECTIVES AND METHODS: An influenza B virus plasmid-based rescue system was used to introduce site-specific mutations, previously observed in neuraminidase (NA) inhibitor-resistant viruses, into the NA protein of six recombinant viruses. Three mutations observed only among in vitro selected zanamivir-resistant influenza A mutants were introduced into the B/Beijing/1/87 virus NA protein, to change residue E116 to glycine, alanine or aspartic acid. Residue E116 was also mutated to valine, a mutation found in the clinic among oseltamivir-resistant viruses. An arginine to lysine change at position 291 (292 N2 numbering) mimicked that seen frequently in influenza A N2 clinical isolates resistant to oseltamivir. Similarly, an arginine to lysine change at position 149 (152 in N2 numbering) was made to reproduce the change found in the only reported zanamivir-resistant clinical isolate of influenza B virus. In vitro selection and prolonged treatment in the clinic leads to resistance pathways that require compensatory mutations in the haemagglutinin gene, but these appear not to be important for mutants isolated from immunocompetent patients. The reverse genetics system was therefore used to generate mutants containing only the NA mutation. RESULTS AND CONCLUSIONS: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.     

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.     

Susceptibilities of simian immunodeficiency virus to protease inhibitors

We used a focal infectivity assay with HeLa H1-JC.37 cells to directly compare susceptibilities of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) to protease inhibitors. SIVmac239 was inhibited by indinavir, saquinavir, and ritonavir, with 50% effective concentrations (means +/- standard deviations) of 39 +/- 8, 55 +/- 3, and 13 +/- 5 nM, respectively. The corresponding values for inhibition of HIV-1 were 66 +/- 4, 47 +/- 10, and 25 +/- 14 nM, respectively.     

New millennium antivirals against pandemic and epidemic influenza: the neuraminidase inhibitors

The mushroom shaped outer spike protein of influenza, neuraminidase, was first discovered nearly 60 years ago. Its importance in viral replication was soon recognised both at the point of viral release from the cell and also enabling passage of virus through nasal fluid to reach the cell. The enzyme active site was identified by x-ray crystallography, allowing an atomic study of interaction of enzyme with the sialic acid substrate. Analogues could then be identified and synthesized and became a focused target for antivirals. With the current threat of bioterrorism and the potential for the emergence of a new pandemic strain in the near future, efforts are underway to develop more potent second-generation anti-neuraminidase inhibitors with enhanced protective and therapeutic effects. Here we review older and newer neuraminidase inhibitors and the role that they will play in the fight against influenza in its epidemic and pandemic face.     

Development of novel entry inhibitors targeting emerging viruses

Emerging viral diseases pose a unique risk to public health, and thus there is a need to develop therapies. A current focus of funding agencies, and hence research, is the development of broad-spectrum antivirals, and in particular, those targeting common cellular pathways. The scope of this article is to review screening strategies and recent advances in this area, with a particular emphasis on antivirals targeting the step of viral entry for emerging lipid-enveloped viruses such as Ebola virus and SARS-coronavirus.     

Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008

The surveillance of seasonal influenza virus susceptibility to neuraminidase (NA) inhibitors was conducted using an NA inhibition assay. The 50% inhibitory concentration values (IC₅₀s) of 4,570 viruses collected globally from October 2004 to March 2008 were determined. Based on mean IC₅₀s, A(H3N2) viruses (0.44 nM) were more sensitive to oseltamivir than A(H1N1) viruses (0.91 nM). The opposite trend was observed with zanamivir: 1.06 nM for A(H1N1) and 2.54 nM for A(H3N2). Influenza B viruses exhibited the least susceptibility to oseltamivir (3.42 nM) and to zanamivir (3.87 nM). To identify potentially resistant viruses (outliers), a threshold of a mean IC₅₀ value + 3 standard deviations was defined for type/subtype and drug. Sequence analysis of outliers was performed to identify NA changes that might be associated with reduced susceptibility. Molecular markers of oseltamivir resistance were found in six A(H1N1) viruses (H274Y) and one A(H3N2) virus (E119V) collected between 2004 and 2007. Some outliers contained previously reported mutations (e.g., I222T in the B viruses), while other mutations [e.g., R371K and H274Y in B viruses and H274N in A(H3N2) viruses) were novel. The R371K B virus outlier exhibited high levels of resistance to both inhibitors (>100 nM). A substantial variance at residue D151 was observed among A(H3N2) zanamivir-resistant outliers. The clinical relevance of newly identified NA mutations is unknown. A rise in the incidence of oseltamivir resistance in A(H1N1) viruses carrying the H274Y mutation was detected in the United States and in other countries in the ongoing 2007 to 2008 season. As of March 2008, the frequency of resistance among A(H1N1) viruses in the United States was 8.6% (50/579 isolates). The recent increase in oseltamivir resistance among A(H1N1) viruses isolated from untreated patients raises public health concerns and necessitates close monitoring of resistance to NA inhibitors.     

Recent progress in chemical approaches for the development of novel neuraminidase inhibitors

Influenza virus is the main cause of an infectious disease called influenza affecting the respiratory system including the throat, nose and lungs. Neuraminidase inhibitors are reagents used to block the enzyme called neuraminidase to prevent the influenza infection from spreading. Neuraminidase inhibitors are widely used in the treatment of influenza infection, but still there is a need to develop more potent agents for the more effective treatment of influenza. Complications of the influenza disease lead to death, and one of these complications is drug resistance; hence, there is an urgent need to develop more effective agents. This review focuses on the recent advances in chemical synthesis pathways used for the development of new neuraminidase agents along with the medicinal aspects of chemically modified molecules, including the structure–activity relationship, which provides further rational designs of more active small molecules.

Influenza virus is the main cause of an infectious disease called influenza affecting the respiratory system including the throat, nose and lungs.     

Administration of neuraminidase inhibitors for the treatment of Japanese patients infected with the novel influenza A (H1N1)

The Centers for Disease Control and Prevention in the United States recommend the use of neuraminidase inhibitors for treating the novel influenza A (H1N1) in patients requiring desirable antiviral agents. However, the efficacy and side effects of neuraminidase inhibitors in Japanese patients infected with the novel influenza A (H1N1) are not well known. It is worth reporting on the results of the administration of neuraminidase inhibitors in Japanese patients infected with the novel influenza A (H1N1), as it is believed the number of Japanese patients will increase in the future. We treated seven Japanese patients (five adults and two juveniles) infected with the novel influenza A (H1N1) with oseltamivir and zanamivir, and good clinical courses were obtained. Our study indicates that oseltamivir and zanamivir may indeed be useful in Japanese patients infected with the novel influenza A (H1N1) in cases where antiviral therapy is required.     

A reverse genetics study of resistance to neuraminidase inhibitors in an influenza A/H1N1 virus

A system of reverse genetics was used to generate influenza A/H1N1 viruses harbouring neuraminidase (NA) mutations previously associated with resistance to NA inhibitors in various viral subtypes. The His274Tyr and Glu119Gln mutants were rescued whereas the Arg292Lys and Glu1l9 --> Gly, Val, Ala or Asp mutants could not be generated. In NA inhibition assays, the His274Tyr mutant was resistant to oseltamivir (430-fold over wild-type) and BCX-1812 (50-fold) but was sensitive to zanamivir. A similar trend was seen when the mutant was evaluated by plaque reduction assay (PRA). The Glu119Gln mutant expressed a low level of resistance to oseltamivir (nine-fold) and zanamivir (fourfold) in NA inhibition assay but was only marginally resistant to oseltamivir (fourfold) in PRA. The replication capacity of both mutants, in particular that of the His274Tyr virus, was impaired when compared with the wild-type virus in vitro.     

广东流感H3N2亚型神经氨酸酶基因变异和耐药分析

目的 揭示广东地区2007～2010年甲型H3N2毒株神经氨酸酶(NA)基因特征、变异及对奥司他韦和扎那米韦的药物敏感性.方法 采用时空抽样方法选取毒株,检测广东2007～2010年甲型H3N2毒株NA基因核苷酸序列,同时检索全球NA基因序列作为参照,采用Mega 5.05和BioEdit 7.0.1软件对NA基因核苷酸序列进行比对和分析;分析毒株变异进化速度;采用NA酶活性阻断试验检测病毒的奥司他韦和扎那米韦药物敏感性.结果 广东2007～2010年H3N2毒株NA基因同义进化(Ks)和错义进化(Ka)速度分别为1.69×10-3～1.84×10-3核苷酸/年和1.07×10-3～1.15×10-3核苷酸/年,Ks值约为Ka值的1.5倍,揭示NA基因受自然选择压力较大.疫苗株A/Perth/10/2009与2010年和2011年广东毒株NA基因同源性分别为97.7%～98.2%、97.6%～97.7%.广东毒株NA基因有6个抗原表位变异,尤其是367位和369位氨基酸的变异频率较高.毒株A/Guangdong/548/2008发生耐药相关位点D151V,出现对奥司他韦敏感性降低;其余分离毒株均对奥司他韦和扎那米韦敏感;各年份毒株药物敏感性差异没有统计学意义.结论 广东2007～2010年甲型H3N2毒株NA基因的6个抗原表位有变异,抗原变异的积累可能出现流感暴发;D151V变异可能降低H3N2病毒对奥司他韦敏感性,但广东大多数H3N2毒株对奥司他韦和扎那米韦仍敏感.     

Resistance to anti-influenza drugs: adamantanes and neuraminidase inhibitors

Development of effective drugs for the treatment or prevention of epidemic and pandemic influenza is important in order to reduce its impact. Adamantanes and neuraminidase inhibitors are two classes of anti-influenza drugs available for influenza therapy currently. However, emergence of resistance to these drugs has been detected, which raises concerns regarding their widespread use. In this review, resistance to the adamantanes and neuraminidase inhibitors will be discussed in relation to both epidemic and pandemic influenza viruses.     

Comparative effectiveness of neuraminidase inhibitors in patients with influenza: A systematic review and network meta-analysis

The aim of this study was to use a network meta-analysis (NWA) to evaluate the relative efficacy and safety of various neuraminidase inhibitors (NAIs) in reducing the duration of influenza symptoms, and thereby, informing the selection of suitable therapeutic regimens for patients with influenza. We conducted a systematic review of randomized controlled trials comparing the clinical effects of four NAIs administered to patients with influenza and placebo. Relevant studies were found in the PubMed and Cochrane databases. Unpublished studies were collected from the ClinicalTrials.gov registry and through hand searching. We carried out NWA to compare the different regimens with each other and across subgroups of age and medical status (high-risk patients). A total of 58 two-arm studies were identified. Five regimens were efficacious in reducing the time to alleviation of influenza symptoms in all populations; this efficacy was comparable. No significant improvements were seen in combination therapy groups. The mean difference in the time to alleviation of symptoms ranged from 12.78 to 19.51 h. According to the summarized mean difference and surface under the cumulative ranking curve (SUCRA), peramivir (SUCRA = 82.6%), zanamivir (SUCRA = 64%), and oseltamivir (SUCRA = 55.1%) were the three top-ranking drugs for treating influenza. Zanamivir and peramivir were the preferred pharmacologic intervention among all investigated interventions based on the calculated “value preference of SUCRA.” This study is a network meta-analysis to explore the therapeutic effects of NAIs in patients with influenza. Peramivir might be the best choice for reducing the time to alleviation of symptoms.     

Laninamivir and its prodrug, CS-8958: long-acting neuraminidase inhibitors for the treatment of influenza

Oseltamivir and zanamivir are currently licensed worldwide for influenza treatment and chemoprophylaxis. Both drugs require twice-daily administration for 5 days for treatment. A new influenza drug, laninamivir (code name R-125489), and its prodrug form, CS-8958 (laninamivir octanoate or laninamivir prodrug), which are long-acting neuraminidase inhibitors, are introduced in this review. Laninamivir potently inhibited the neuraminidase activities of various influenza A and B viruses, including subtypes N1-N9, pandemic (2009) H1N1 virus, highly pathogenic avian influenza (HPAI) H5N1 viruses and oseltamivir-resistant viruses. Because of the long retention of laninamivir in mouse lungs after an intranasal administration of CS-8958, therapeutic administration of a single dose of CS-8958 showed superior efficacy to repeated administrations of zanamivir or oseltamivir in animal infection models for influenza A and B viruses. These include pandemic (2009) H1N1 virus and HPAI H5N1 virus. Prophylactic single administration of CS-8958, as early as 7 days prior to infection, also showed superior efficacy. Finally, the potential of a single inhalation of CS-8958 for influenza patients was demonstrated by clinical studies, and CS-8958 has been approved and is commercially available as Inavir(?) (Daiichi Sankyo Co., Ltd, Tokyo) in Japan.