M2 Ion Channel Inhibitors Research Articles

Preparing for the Next Influenza Season: Monitoring the Emergence and Spread of Antiviral Resistance.

The relaxation of pandemic restrictions in 2022 has led to a reemergence of respiratory virus circulation worldwide and anticipation of substantial influenza waves for the 2022/2023 Northern Hemisphere winter. Therefore, the antiviral susceptibility profiles of human influenza viruses circulating in Germany were characterized. Between October 2019 (week 40/2019) and March 2022 (week 12/2022), nasal swabs from untreated patients with acute respiratory symptoms were collected in the national German influenza surveillance system. A total of 598 influenza viruses were isolated and analyzed for susceptibility to oseltamivir, zanamivir and peramivir, using a neuraminidase (NA) inhibition assay. In addition, next-generation sequencing was applied to assess molecular markers of resistance to NA, cap-dependent endonuclease (PA) and M2 ion channel inhibitors (NAI, PAI, M2I) in 367 primary clinical samples. Furthermore, a genotyping assay based on RT-PCR and pyrosequencing to rapidly assess the molecular resistance marker PA-I38X in PA genes was designed and established. While NAI resistance in the strict sense, defined by a ≥ 10-fold (influenza A) or ≥5-fold (influenza B) increase of NAI IC50, was not detected, a subtype A(H1N1)pdm09 isolate displayed 2.3- to 7.5-fold IC50 increase for all three NAI. This isolate carried the NA-S247N substitution, which is known to enhance NAI resistance induced by NA-H275Y. All sequenced influenza A viruses carried the M2-S31N substitution, which confers resistance to M2I. Of note, one A(H3N2) virus displayed the PA-I38M substitution, which is associated with reduced susceptibility to the PAI baloxavir marboxil. Pyrosequencing analysis confirmed these findings in the original clinical specimen and in cultured virus isolate, suggesting sufficient replicative fitness of this virus mutant. Over the last three influenza seasons, the vast majority of influenza viruses in this national-level sentinel were susceptible to NAIs and PAIs. These findings support the use of antivirals in the upcoming influenza season.

Insights into the direct anti-influenza virus mode of action of Rhodiola rosea

Background: The anti-influenza A virus activities and contents of previously isolated most active flavonoids (rhodiosin and tricin) from a standardized hydro-ethanolic R. rosea root and rhizome extract (SHR-5®), did not fully explain the efficacy of SHR-5®. Moreover, the mode of antiviral action of SHR-5® is unknown.Purpose: To determine the anti-influenza viral principle of SHR-5® we evaluated i) the combined anti-influenza virus effect of rhodiosin and tricin, ii) the impact of its tannin-enriched fraction (TE), iii) its antiviral spectrum and mode of action, and iv) its propensity for resistance development in vitro.Methods: The combined anti-influenza virus effect of rhodiosin and tricin and the impact of TE were investigated with cytopathic effect (CPE)-inhibition assays in MDCK cells. A tannin-depleted fraction (TD) and TE were prepared by polyamide column chromatography and dereplicated by LC-MS. Plaque-reduction assays provided insights into the anti-influenza virus profile, the mode of action, and the propensity for resistance development of SHR-5®.Results: Our results i) did not reveal synergistic anti-influenza A virus effects of rhodiosin and tricin, but ii) proved a strong impact of TE mainly composed of prodelphinidin gallate oligomers. iii) TE inhibited the plaque-production of influenza virus A(H1N1)pdm09, A(H3N2), and B (Victoria and Yamagata) isolates (including isolates resistant to neuraminidase and/or M2 ion channel inhibitors) with 50% inhibitory concentration values between 0.12 - 0.53 µg/ml similar to SHR-5®. Mechanistic studies proved a virucidal activity, inhibition of viral adsorption, viral neuraminidase activity, and virus spread by SHR-5® and TE. iv) No resistance development was observed in vitro.Conclusion: For the first time a comprehensive analysis of the anti-influenza virus profile of a hydro-ethanolic R. rosea extract (SHR-5®) was assessed in vitro. The results demonstrating broad-spectrum multiple direct anti-influenza virus activities, and a lack of resistance development to SHR-5® together with its known augmentation of host defense, support its potential role as an adaptogen against influenza virus infection.

Identification of entry inhibitors with 4-aminopiperidine scaffold targeting group 1 influenza A virus

Influenza A viruses (IAVs) cause seasonal flu and occasionally pandemics. The current therapeutics against IAVs target two viral proteins - neuraminidase (NA) and M2 ion-channel protein. However, M2 ion channel inhibitors (amantadine and rimantadine) are no longer recommended by CDC for use due to the emergence of high level of antiviral resistance among the circulating influenza viruses, and resistant strains to NA inhibitors (oseltamivir and zanamivir) have also been reported. Therefore, development of novel anti-influenza therapies is urgently needed. As one of the viral surface glycoproteins, hemagglutinin (HA) mediates critical virus entry steps including virus binding to host cells and virus-host membrane fusion, which makes it a potential target for anti-influenza drug development. In this study, we report the identification of compound CBS1116 with a 4-aminopiperidine scaffold from a chemical library screen as an entry inhibitor specifically targeting two group 1 influenza A viruses, A/Puerto Rico/8/34 (H1N1) and recombinant low pathogenic avian H5N1 virus (A/Vietnam/1203/04, VN04Low). Mechanism of action studies show that CBS1116 interferes with the HA-mediated fusion process. Further structure activity relationship study generated a more potent compound CBS1117 which has a 50% inhibitory concentration of 70 nM and a selectivity index of ~4000 against A/Puerto Rico/8/34 (H1N1) infection in human lung epithelial cell line (A549).

Efficacy, Mechanism and Antiviral Resistance of Neuraminidase Inhibitors and Adamantane against Avian Influenza

Vaccination and antiviral drug are often used to control influenza. However, the effectiveness of vaccine was impaired due to the emergence of new variant of virus strain. Antiviral drug consists of prophylactic and curative substances, namely M2 ion channel inhibitors (adamantane; amantadine and rimantadine) and neuraminidase (NA) inhibitors (NAIs; oseltamivir, zanamivir, peramivir, laninamivir). The synthesis and modification of antiviral neuraminidase (NA) inhibitors (NAIs) and adamantanes increased the antiviral effectiveness. The mechanism of the neuraminidase inhibitor is to prevent influenza infection by inhibiting the release of the virus from internal cells. Adamantane is antiviral drug that selectively inhibits the flow of H+ ions through M2 protein to prevent the uncoating virus particles getting into the endosome. The substitution of (H275Y, S247N, I223L, K150N, R292K, I222T, R152K, R118K, E119V) on NA protein caused resistance of avian influenza virus against the neuraminidase inhibitor. The combination of mutations (S247N, I223L, K150N) increased the resistance of influenza A (H5N1) virus. The diffusion of adamantane resistance varies among HA subtypes, the species of host, the period of isolation, and region. Mutations at residues of 26, 27, 30, 31 or 34 transmembrane M2 protein caused adamantane resistance. The unique substitution (V27I) of M2 protein of clade 2.3.2 H5N1 subtype isolated in Indonesia in 2016 has been contributed to the amantadine resistance. Antiviral combination of M2 ion channel inhibitors and neuraminidase (NA) inhibitors is effective treatments for the resistance.

Aryl and Arylalkyl Substituted 3-Hydroxypyridin-2(1H)-ones: Synthesis and Evaluation as Inhibitors of Influenza A Endonuclease.

Seasonal influenza infections are associated with an estimated 250-500 000 deaths annually. Resistance to the antiviral M2 ion-channel inhibitors has largely invalidated their clinical utility. Resistance to neuraminidase inhibitors has also been observed in several influenza A virus (IAV) strains. These data have prompted research on inhibitors that target the cap-snatching endonuclease activity of the polymerase acidic protein (PA). Baloxavir marboxil (Xofluza®), recently approved for clinical use, inhibits cap-snatching endonuclease. Resistance to Xofluza® has been reported in both in vitro systems and in the clinic. An X-ray crystallographic screening campaign of a fragment library targeting IAV endonuclease identified 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating agent at the active site. We have reported the structure-activity relationships for 3-hydroxypyridin-2(1H)-ones and 3-hydroxyquinolin-2(1H)-ones as endonuclease inhibitors. These studies identified two distinct binding modes associated with inhibition of this enzyme that are influenced by the presence of substituents at the 5- and 6-positions of 3-hydroxypyridin-2(1H)-ones. Herein we report the structure-activity relationships associated with various para-substituted 5-phenyl derivatives of 6-(p-fluorophenyl)-3-hydroxypyridin-2(1H)-ones and the effect of using naphthyl, benzyl, and naphthylmethyl groups as alternatives to the p-fluorophenyl substituent on their activity as endonuclease inhibitors.

Influenza Virus: Small Molecule Therapeutics and Mechanisms of Antiviral Resistance.

Influenza viruses cause severe upper respiratory illness in children and the elderly during seasonal epidemics. Influenza viruses from zoonotic reservoirs can also cause pandemics with significant loss of life in all age groups. Although vaccination is one of the most effective methods to protect against seasonal epidemics, seasonal vaccines vary in efficacy, can be ineffective in the elderly population, and do not provide protection against novel strains. Small molecule therapeutics are a critical part of our antiviral strategies to control influenza virus epidemics and pandemics as well as to ameliorate disease in elderly and immunocompromised individuals. This review aims to summarize the existing antiviral strategies for combating influenza viruses, the mechanisms of antiviral resistance for available drugs, and novel therapeutics currently in development. We systematically evaluated and synthesized the published scientific literature for mechanistic detail into therapeutic strategies against influenza viruses. Current IAV strains have developed resistance to neuraminidase inhibitors and nearly complete resistance to M2 ion channel inhibitors, exacerbated by sub-therapeutic dosing used for treatment and chemoprophylaxis. New tactics include novel therapeutics targeting host components and combination therapy, which show potential for fighting influenza virus disease while minimizing viral resistance. Antiviral drugs are crucial for controlling influenza virus disease burden, but their efficacy is limited by human misuse and the capacity of influenza viruses to circumvent antiviral barriers. To relieve the public health hardship of influenza virus, emerging therapies must be selected for their capacity to impede not only influenza virus disease, but also the development of antiviral resistance.

Baloxavir: game-changer or much ado about nothing?

Baloxavir: game-changer or much ado about nothing?

Stimulation of alpha2-adrenergic receptors impairs influenza virus infection

Influenza A viruses cause seasonal epidemics and occasional pandemics. The emergence of viruses resistant to neuraminidase (NA) inhibitors and M2 ion channel inhibitors underlines the need for alternate anti-influenza drugs with novel mechanisms of action. Here, we report the discovery of a host factor as a potential target of anti-influenza drugs. By using cell-based virus replication screening of a chemical library and several additional assays, we identified clonidine as a new anti-influenza agent in vitro. We found that clonidine, which is an agonist of the alpha2-adrenergic receptor (α2-AR), has an inhibitory effect on the replication of various influenza virus strains. α2-AR is a Gi-type G protein-coupled receptor that reduces intracellular cyclic AMP (cAMP) levels. In-depth analysis showed that stimulation of α2-ARs leads to impairment of influenza virus replication and that α2-AR agonists inhibit the virus assembly step, likely via a cAMP-mediated pathway. Although clonidine administration did not reduce lung virus titers or prevent body weight loss, it did suppress lung edema and improve survival in a murine lethal infection model. Clonidine may thus protect against lung damage caused by influenza virus infection. Our results identify α2-AR-mediated signaling as a key pathway to exploit in the development of anti-influenza agents.

Identification of camphor derivatives as novel M2 ion channel inhibitors of influenza A virus

Amantadine derivatives have been the only drugs marketed as M2 inhibitors of influenza A for decades.

Conjugation to Polymeric Chains of Influenza Drugs Targeting M2 Ion Channels Partially Restores Inhibition of Drug-Resistant Mutants

By attaching multiple copies of the influenza M2 ion channel inhibitors amantadine (1) and rimantadine (2) to polymeric chains, we endeavored to recover their potency in inhibiting drug-resistant influenza viruses. Depending on loading densities, as well as the nature of the drug, the polymer, and the spacer arm, polymer-conjugated drugs were up to 30-fold more potent inhibitors of drug-resistant strains than their monomeric parents. In particular, a 20% loading density and a short linker group on the negatively charged poly-l-glutamate resulted in one of the most potent inhibitors for 2's conjugates against drug-resistant influenza strains. Although full recovery of the inhibitory action against drug-resistant strains was not achieved, this study may be a step toward salvaging anti-influenza drugs that are no longer effective.

Novel hemagglutinin-based influenza virus inhibitors.

Influenza virus has caused seasonal epidemics and worldwide pandemics, which caused tremendous loss of human lives and socioeconomics. Nowadays, only two classes of anti-influenza drugs, M2 ion channel inhibitors and neuraminidase inhibitors respectively, are used for prophylaxis and treatment of influenza virus infection. Unfortunately, influenza virus strains resistant to one or all of those drugs emerge frequently. Hemagglutinin (HA), the glycoprotein in influenza virus envelope, plays a critical role in viral binding, fusion and entry processes. Therefore, HA is a promising target for developing anti-influenza drugs, which block the initial entry step of viral life cycle. Here we reviewed recent understanding of conformational changes of HA in protein folding and fusion processes, and the discovery of HA-based influenza entry inhibitors, which may provide more choices for preventing and controlling potential pandemics caused by multi-resistant influenza viruses.

New Strategy for High Throughput Screening of Anti-Influenza Virus M2 Ion Channel Inhibitors

Since the recent emergence of swine and avian flu, there has been a sense of urgency to discover new anti-influenza drugs. In this study, a stable cell line with M2 ion channel/ enhanced Green fluorescent protein (EGFP) co-expression was established in order to develop an EGFP-based high-throughput assay for screening M2 ion channel inhibitors. This assay directly monitors the proton conductivity of the M2 ion channel by measuring the fluorescence intensity of EGFP, which is dependent on and sensitive to pH. The ability of amantadine to inhibit the M2 ion channel was detected by this novel EGFP-based assay and then confirmed by a patch clamp recording assay. With this assay, (1S,2S,3S,5R)-(+)-3-Isopinocampheylamine and (1R,2R,3R,5S)-(-)-3-Isopinocampheylamine were identified to inhibit M2 ion channel with an antiviral profile similar to that of amantadine. This new technique will facilitate the discovery of pharmaceutical candidates and will aid in the development of new, potent, and safe anti-influenza agents.

Antiviral Activity of the Plant Extracts from Thuja orientalis, Aster spathulifolius, and Pinus thunbergii Against Influenza Virus A/PR/8/34

Influenza viruses cause significant morbidity and mortality in humans through epidemics or pandemics. Currently, two classes of anti-influenza virus drugs, M2 ion-channel inhibitors (amantadin and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir), have been used for the treatment of the influenza virus infection. Since the resistance to these drugs has been reported, the development of a new antiviral agent is necessary. In this study, we examined the antiviral efficacy of the plant extracts against the influenza A/PR/8/34 infection. In vitro, the antiviral activities of the plant extracts were investigated using the cell-based screening. Three plant extracts, Thuja orientalis, Aster spathulifolius, and Pinus thunbergii, were shown to induce a high cell viability rate after the infection with the influenza A/PR/8/34 virus. The antiviral activity of the plant extracts also increased as a function of the concentration of the extracts and these extracts significantly reduced the visible cytopathic effect caused by virus infections. Furthermore, the treatment with T. orientalis was shown to have a stronger inhibitory effect than that with A. spathulifolius or P. thunbergii. These results may suggest that T. orientalis has anti-influenza A/PR/8/34 activity.

Design and synthesis of pinanamine derivatives as anti-influenza A M2 ion channel inhibitors

The adamantanes are a class of anti-influenza drugs that inhibit the M2 ion channel of the influenza A virus. However recently, the clinical effectiveness of these drugs has been called into question due to the emergence of adamantane-insensitive A/M2 mutants. Although we previously reported (1R,2R,3R,5S)-3-pinanamine 3 as a novel inhibitor of the wild type influenza A virus M2 protein (WT A/M2), limited inhibition was found for adamantane-resistant M2 mutants. In this study, we explored whether newly synthesized pinanamine derivatives were capable of inhibiting WT A/M2 and selected adamantane-resistant M2 mutants. Several imidazole and guanazole derivatives of pinanamine were found to inhibit WT A/M2 to a comparable degree as amantadine and one of these compounds 12 exhibits weak inhibition of A/M2-S31N mutant and it is marginally more effective in inhibiting S31NM2 than amantadine. This study provides a new insight into the structural nature of drugs required to inhibit WT A/M2 and its mutants.

Genotypic and phenotypic resistance of pandemic A/H1N1 influenza viruses circulating in Germany

In response to the rapid global spread of an antigenically novel A/H1N1 influenza virus in 2009, the World Heath Organization (WHO) recommended surveillance and monitoring for antiviral resistance of influenza viruses. We designed and evaluated pyrosequencing (PSQ)-based genotypic assays for high-throughput analysis of the susceptibility of pandemic A/H1N1 influenza viruses to neuraminidase (NA) inhibitors. A total of 1570 samples circulating in Germany between April 2009 and April 2010 were tested for determination of molecular markers of resistance to the NA inhibitors oseltamivir and zanamivir, and 635 of them were evaluated by phenotypic fluorescence-based assay with MUNANA substrate. Eight (0.5%) viruses were resistant to oseltamivir due to the H274Y NA substitution (N2 numbering). Six of these oseltamivir-resistant cases were treatment-related; four of them were selected in immunocompromised patients, two in patients suffered from chronic diseases. The two remaining oseltamivir-resistant viruses seem to have evolved in the absence of drug treatment and were isolated from immunocompetent healthy patients. All tested A/H1N1 pandemic viruses were sensitive to zanamivir. In addition, analysis of 1011 pandemic A/H1N1 virus samples by a PSQ-based assay according to the WHO protocol revealed the presence of mutation S31N in the M2 protein that conferred resistance to M2 ion channel inhibitors. Our data demonstrate a low incidence of oseltamivir-resistant pandemic A/H1N1 influenza variants isolated under drug selection pressure as well as community-acquired or naturally evolving viruses.

Maternal and Neonatal Outcomes After Antepartum Treatment of Influenza With Antiviral Medications

Pregnant women are at increased risk of severe morbidity and mortality, secondary to influenza infection. Vaccination programs are the cornerstone of influenza preventive efforts and when they fail, 2 classes of antiviral drugs are important for both postexposure prophylaxis and treatment. The first class, the M2 ion channel inhibitors, includes amantadine and rimantadine, and is effective for the prophylaxis and treatment of influenza A. The second class, the neuraminidase inhibitors, includes oseltamivir, which is effective against both influenza A (including novel H1N1) and B. Because of increasing resistance to oseltamivir, this drug has been used with M2 ion channel inhibitors as 2-drug therapy for influenza A. Little data are not available on the safety of these 2 drug classes when used for treatment of influenza in pregnant women. This retrospective cohort study compared the maternal and neonatal outcomes of pregnant women treated antepartum for influenza with M2 ion channel inhibitors (amantadine, rimantadine), oseltamivir, or a control group. The study was conducted during 5 influenza seasons at a hospital in Texas. The comparative data showed that antepartum treatment with M2 ion channel inhibitors (n = 104), oseltamivir (n = 135), and a control group (n = 82,097) were not associated with increased rates of the following outcomes: gestational diabetes (P = 0.388), preterm birth (P = 0.190), premature rupture of the membranes (P = 0.154), or preeclampsia (P = 0.209). No differences were found among the 3 groups regarding the duration of hospital stay for the mother or the infant, the incidence of fever in labor, stillbirth, or major or minor malformations. Moreover, there were no significant differences among singleton live-born neonates without major malformations in fetal weight (P = 0.186), number of neonates who required intubation (P = 0.552) or intensive care nursery admission (P = 0.418), or the incidence of hyperbilirubinemia (P = 0.282). There were no neonatal deaths among live-born singletons and none had grade 3 or 4 intraventricular hemorrhages or seizures. Two preterm neonates exposed to antivirals in the second trimester developed necrotizing enterocolitis. One was exposed to amantadine and the other to oseltamivir. These findings show that antepartum-antiviral exposure does not increase the risk of adverse maternal or neonatal outcomes.

Drugs in Development for Influenza

The emergence and global spread of the 2009 pandemic H1N1 influenza virus reminds us that we are limited in the strategies available to control influenza infection. Vaccines are the best option for the prophylaxis and control of a pandemic; however, the lag time between virus identification and vaccine distribution exceeds 6 months and concerns regarding vaccine safety are a growing issue leading to vaccination refusal. In the short-term, antiviral therapy is vital to control the spread of influenza. However, we are currently limited to four licensed anti-influenza drugs: the neuraminidase inhibitors oseltamivir and zanamivir, and the M2 ion-channel inhibitors amantadine and rimantadine. The value of neuraminidase inhibitors was clearly established during the initial phases of the 2009 pandemic when vaccines were not available, i.e. stockpiles of antivirals are valuable. Unfortunately, as drug-resistant variants continue to emerge naturally and through selective pressure applied by use of antiviral drugs, the efficacy of these drugs declines. Because we cannot predict the strain of influenza virus that will cause the next epidemic or pandemic, it is important that we develop novel anti-influenza drugs with broad reactivity against all strains and subtypes, and consider moving to multiple drug therapy in the future. In this article we review the experimental data on investigational antiviral agents undergoing clinical trials (parenteral zanamivir and peramivir, long-acting neuraminidase inhibitors and the polymerase inhibitor favipiravir [T-705]) and experimental antiviral agents that target either the virus (the haemagglutinin inhibitor cyanovirin-N and thiazolides) or the host (fusion protein inhibitors [DAS181], cyclo-oxygenase-2 inhibitors and peroxisome proliferator-activated receptor agonists).

Maternal and Neonatal Outcomes After Antepartum Treatment of Influenza With Antiviral Medications

To review the maternal and neonatal outcomes after antepartum exposure to M2 ion channel inhibitors or oseltamivir to provide some guidance on the risk, if any, of antiviral medication during pregnancy. This was a retrospective cohort study examining maternal and neonatal outcomes after antepartum exposure to antiviral therapy for influenza. We evaluated maternal characteristics, pregnancy outcomes, and fetal outcomes and compared them with our overall obstetric population. Exposure to antiviral therapies (M2 ion channel inhibitors [n=104] compared with oseltamivir [n=135] compared with the control group [n=82,097]) during pregnancy was not associated with increased rates of preterm birth (7% compared with 10% compared with 6%, P=.190), premature rupture of membranes (23% compared with 16% compared with 22%, P=.154), gestational diabetes (4% compared with 8% compared with 6%, P=.388), or preeclampsia (6% compared with 1% compared with 4%, P=.209). Exposure was not associated with increased duration of hospital stay for mother or neonate. There were no differences in the incidence of minor malformations (19% compared with 15% compared with 22%, P=.101). Liveborn singletons without major malformations did not have differences in fetal weight (3,238+/-586 g compared with 3,281+/-642 g compared with 3,336+/-571 g, P=.186), need for intubation (2% compared with 0.8% compared with 1%, P=.552), intensive care nursery admission (3% compared with 3% compared with 2%, P=.418), or hyperbilirubinemia (12% compared with 9% compared with 8%, P=.282). Liveborn singletons had no grade 3 or 4 intraventricular hemorrhages, seizures, or neonatal deaths. Two preterm neonates exposed to different classes of medications had necrotizing enterocolitis (1.0% compared with 0.8% compared with 0.02%, P<.001). We found no evidence of an association between antepartum antiviral exposure and adverse outcomes. II.

A Measure of Resistance: Detecting Tamiflu Metabolite in Sewage Discharge and River Water

During the flu season each year, combating the different strains of the influenza virus becomes a public health priority, with treatment dependent upon two groups of antiviral drugs: neuraminidase inhibitors and M2 ion channel inhibitors. Oseltamivir phosphate, marketed as Tamiflu, is a popular neuraminidase inhibitor widely used to treat flu symptoms. Oseltamivir carboxylate (OC), Tamiflu’s active metabolite, is known to withstand activated sludge treatment at sewage treatment plants (STPs), but less is known about how much OC may make its way into waterways that receive STP effluent. Now a new study conducted in Kyoto City, Japan, during the 2008–2009 flu season reports some of the first measurements of OP occurrences in STP discharge and in river water [EHP 118:103–107; Ghosh et al.]. According to the World Health Organization, between 250,000 and 500,000 people die each year from influenza, and each year, millions of people take Tamiflu to battle flu symptoms. After the sewage treatment process, the excreted active metabolite remains in STP effluent and travels to waterways where effluent is discharged. The investigators in the current study collected samples from STP effluent and from river water on three different occasions: at the beginning of the flu season, during the peak period, and 2 weeks after the peak period. Using solid-phase extraction followed by liquid chromatography–tandem mass spectrometry, they measured the highest concentration of OC, 293.3 ng/L, in an STP discharge sample collected during the peak of the flu season. Concentration amounts were higher in effluent from STPs that used traditional activated sludge treatment; in contrast, effluent from plants that used advanced ozonation as tertiary treatment contained significantly lower OC levels (37.9 ng/L). River water samples showed a range of OC levels from 6.6 to 190 ng/L during the peak of the flu season. Previous research indicates OC concentrations ranging from 80 to 230 ng/L will disable 50% of the influenza virus. This level of exposure is most likely to kill virus particles that are particularly susceptible to OC, while selecting for viruses that are resistant to the drug’s effects. The authors note, “During a common flu season, waterfowl can ingest large quantities of OC with virus. . . . Exposing waterfowl infected with influenza A virus to elevated levels of OC in open waterways could trigger the development of Tamiflu-resistant viral strains.” The authors suggest ozonation as tertiary treatment that may reduce OC load in STP effluent. They also recommend conducting further investigations to determine the fate of antiviral drugs at every interval of the STP process.

A Measure of Resistance: Detecting Tamiflu Metabolite in Sewage Discharge and River Water

During the flu season each year, combating the different strains of the influenza virus becomes a public health priority, with treatment dependent upon two groups of antiviral drugs: neuraminidase inhibitors and M2 ion channel inhibitors. Oseltamivir phosphate, marketed as Tamiflu, is a popular neuraminidase inhibitor widely used to treat flu symptoms. Oseltamivir carboxylate (OC), Tamiflu’s active metabolite, is known to withstand activated sludge treatment at sewage treatment plants (STPs), but less is known about how much OC may make its way into waterways that receive STP effluent. Now a new study conducted in Kyoto City, Japan, during the 2008–2009 flu season reports some of the first measurements of OP occurrences in STP discharge and in river water [EHP 118:103–107; Ghosh et al.]. According to the World Health Organization, between 250,000 and 500,000 people die each year from influenza, and each year, millions of people take Tamiflu to battle flu symptoms. After the sewage treatment process, the excreted active metabolite remains in STP effluent and travels to waterways where effluent is discharged. The investigators in the current study collected samples from STP effluent and from river water on three different occasions: at the beginning of the flu season, during the peak period, and 2 weeks after the peak period. Using solid-phase extraction followed by liquid chromatography–tandem mass spectrometry, they measured the highest concentration of OC, 293.3 ng/L, in an STP discharge sample collected during the peak of the flu season. Concentration amounts were higher in effluent from STPs that used traditional activated sludge treatment; in contrast, effluent from plants that used advanced ozonation as tertiary treatment contained significantly lower OC levels (37.9 ng/L). River water samples showed a range of OC levels from 6.6 to 190 ng/L during the peak of the flu season. Previous research indicates OC concentrations ranging from 80 to 230 ng/L will disable 50% of the influenza virus. This level of exposure is most likely to kill virus particles that are particularly susceptible to OC, while selecting for viruses that are resistant to the drug’s effects. The authors note, “During a common flu season, waterfowl can ingest large quantities of OC with virus. . . . Exposing waterfowl infected with influenza A virus to elevated levels of OC in open waterways could trigger the development of Tamiflu-resistant viral strains.” The authors suggest ozonation as tertiary treatment that may reduce OC load in STP effluent. They also recommend conducting further investigations to determine the fate of antiviral drugs at every interval of the STP process.