Dr. Emil Ionita
I. Cantacuzino Institute

Inhibitorii M2 si inhibitorii neuraminidazei (NAIs) reprezinta cele doua clase majore de anitivirale disponibile pentru tratamentul si preventia gripei. Inhibitorii M2 sunt ieftini ca pret, insa ei sunt eficace doar in cazul virusurilor gripale tip A, iar rezistenta se instaleaza rapid. Virusurile gripale A H3N2 si cele pandemice A(H1N1)pdm09 sunt déjà rezistente la inhibitorii M2 asa cum sunt si multe virusuri gripale H5N1. Exista 4 NAI cu licenta in anumite parti ale lumii, precum : zanamivir, oseltamivir, peramivir, si NAI laninamivir cu actiune intarziata.Acest articol reprezinta o trecere inrevista a datelor recente din literature de specialitate asupra rezistentei la NAIs. Resistance poate fi atat NAI-cat si specifica de subtip datorita diferentei structurale a NAI. Aceasta rezulta in profiluri diferite de rezistenta la medicament de exemplu, mutatia H274Y confera rezistenta la oseltamivir si peramivir, dar nu si la zanamivir, si numai in N1 NAs. Mutatiile la E119, D198, I222, R292, si N294 pot de asemenea sa reduca sensibilitatea la NAI .
Cuvinte cheie: inhibitori ai neuraminidazei (NAI) virusului gripal A, inhibitori M2, rezistenta la zanamivir.


The M2 inhibitors and the neuraminidase inhibitors (NAIs) are two major classes of antivirals available for the treatment and prevention of influenza. The M2 inhibitors are cheap, but they are only effective against influenza A viruses, and resistance arises rapidly. The current influenza A H3N2 and pandemic A (H1N1)pdm09 viruses are already resistant to the M2 inhibitors as are many H5N1 viruses. There are four NAIs licensed in some parts of the world, zanamivir, oseltamivir, peramivir, and a long-acting NAI, laninamivir. This is a review of the recent literature data on resistance to the NAIs. The resistance can be both NAI- and subtype specific because of differences in their chemistry and subtle differences in NA structures. This results in different drug resistance profiles, for example, the H274Y mutation confers resistance to oseltamivir and peramivir, but not to zanamivir, and only in N1 NAs. Mutations at E119, D198, I222, R292, and N294 can also reduce NAI sensitivity.
Keywords: Influenza neuraminidase inhibitors, M2 inhibitors, zanamivir resistance.


Research data[1] have confirmed that Influenza viruses have three surface proteins, the hemagglutinin(HA), neuraminidase (NA), and M2 protein. The HA binds to terminal sialic acids on cellular receptors, after which the virus is endocytosed. The low pH (5Æ5–6Æ0) of the endosome activates the M2 proton channel in the influenza A virus membrane to allow acid to enter the virus, prior to HA-mediated fusion, triggering the release of the virus ribonucleoprotein (RNP). After replication, the NA of progeny virions cleaves sialic acids from the cell receptors and from the HA and NA which are also glycosylated, to release progeny virions from the cell surface and prevent self aggregation. There are two major classes of antivirals licensed for the treatment and prevention of influenza, the M2 inhibitors and the NA inhibitors (NAIs). By blocking the M2 proton channel, the M2 inhibitors prevent release of the virus RNP for migration to the nucleus of the cell.
The NA inhibitors (NAIs) prevent release of newly formed virions from the cell surface.
The M2 inhibitors, amantadine and rimantadine, only act on influenza A viruses. Although influenza B viruses have a BM2 protein which is analogous to the M2 protein in influenza A, this is not sensitive to the M2 inhibitors.
The M2 inhibitors have two potential binding sites on the M2 protein: a high-affinity site in the ion channel pore and a second low-affinity site on the lipid face of the pore.[2]
The two most common mutations V27A and S31N are in the ion channel pore, confirming this as the pharmacologically relevant site.
However, although the M2 inhibitors are cheap and have been around for almost 50 years,[3] their use for the treatment of influenza has been limited, in part because resistant viruses emerge rapidly in treated patients, and in a single passage in tissue culture.[4] The pandemic A(H1N1)pdm09 virus was already resistant[5], and the majority of seasonal A(H3N2) viruses have been resistant since the mid-2000s, with an increase in oseltamivir-resistant seasonal H1N1 viruses also observed.[6,7] Many of the H5N1 strains circulating in Southeast Asia, especially in Vietnam and Thailand, are also resistant to M2 inhibitors.[8,9] Due to resistance, the usefulness of these drugs is currently limited. This review therefore focuses on the most recently developed NAIs.

Licensed neuraminidase inhibitors
There are two NAIs licensed globally for the treatment and prevention of influenza. Relenza (zanamivir) was the first in this class[10] followed by Tamiflu (oseltamivir).[11] Zanamivir was developed based on two key findings. Firstly, the transition-state analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) was known to be a weak inhibitor of the NA. Secondly, the structure of the sialic acid substrate in complex with the enzyme active site revealed an empty negatively charged pocket in the region of the C4 on the sugar ring.
This suggested that substitution of the C4-OH with a larger basic residue might lead to higher affinity binding.[12] A single substitution of the C4-OH with a 4-guanidino group enhanced binding more than 10 000- fold over DANA . Zanamivir is administered by oral inhalation as it is not absorbed. Oseltamivir was subsequently designed based on knowledge from zanamivir.
While based on DANA, it has a cyclohexene ring with two substitutions compared with DANA. It has a C4 amino group and a bulky hydrophobic pentyl ether side chain in place of the glycerol side chain. It is administered as the prodrug oseltamivir phosphate and converted by hepatic esterases to the active compound oseltamivir carboxylate.
Peramivir has subsequently been developed[13] and is now licensed in Japan and for emergency use in some other countries while undergoing further clinical trials. It is based on DANA, but has a cyclopentane ring and features of both zanamivir and oseltamivir, the C4-guanidino substitution and a hydrophobic side chain, respectively. It is only effective if administered intravenously. A fourth compound, laninamivir (Inavir), based on zanamivir with a 7-OCH3 substitution, is a long-acting NAI[14] and is administered by oral inhalation as a single 40 mg dose of the laninamivir octanoate prodrug. Laninamivir has been licensed in Japan and is undergoing clinical trials in other countries.
The NAIs prevent release and spread of progeny virions by blocking NA function. The sensitivity of the NA enzyme to the NAIs is evaluated in an in vitro enzyme inhibition
assay, using either a fluorescent[15] or chemiluminescent substrate.
The IC50 is defined as the concentration inhibiting 50% of the enzyme activity compared with the uninhibited control. Decreased sensitivity due to a mutation in the NA is identified by an elevated IC50.[16,17,18] Sensitivities vary in different laboratories due to subtle differences in assay methodology, but in general influenza A(H3N2) viruses are slightly more sensitive to oseltamivir than N1 subtype viruses. Conversely, N1 subtype viruses are slightly more sensitive to zanamivir than to N2 subtype viruses. IC50s are generally <5 nM for both drugs for N1 and N2 subtypes.Influenza B viruses have slightly higher IC50s for zanamivir,but they are still <10 nM.[17,19]. In contrast, influenza B viruses have 10–20-fold higher IC50s for oseltamivir compared with influenza A viruses [17,19].
Oseltamivir is taken orally twice daily, with a dose of 75 mg for adults. The levels of oseltamivir in plasma are estimated to be in the range from 400 to 1200 nM[20,21] and in saliva to be <5% of plasma levels.[22] Thus, levels in the upper respiratory tract may be significantly lower than 100 nM. This may only be 20–50 times the IC50s for influenza A strains and 2–5-fold higher than the IC50s for wild-type influenza B strains.
Zanamivir dosing is 10 mg inhaled twice daily, delivering high levels to the upper respiratory tract, estimated to be up to 10 000 nM.[23,24] This would be up to 5000-fold higher than the average IC50s for influenza A viruses.

Emergence of resistance
In early studies, resistance to oseltamivir emerged both in challenge studies and in naturally acquired infections, with resistant virus isolated from 1 to 4% of oseltamivir-treated adult patients.[25,26]
Due to differences in the chemical structures of the inhibitors, many of the mutations do not confer reduced sensitivity to all the NAIs. Additionally, despite high conservation of residues in the active site, there are mutations which confer resistance in only one subtype, for example,H274Y (H275Y in N1 numbering) confers oseltamivir resistance only in N1, E119V, and R292K confer high-level oseltamivir resistance only in N2.
In early studies, resistant viruses could be isolated from 4 to 8% of oseltamivir-treated pediatric patients, possibly due to prolonged virus shedding in children.[27,28] However, three post-release studies of oseltamivir-treated children have demonstrated much higher frequencies of resistant viruses. The first two studies were conducted in Japan, where weight-based dosing of 2 mg ⁄ kg of oseltamivir is used for children. In one study, in which the viruses were primarily H1N1, oseltamivir-resistant H274Y viruses were isolated from 7 of 43 children (16%).[29]
The H274Y mutation decreases sensitivity to both oseltamivir and peramivir, but not to zanamivir. In the second study, predominantly of H3N2 viruses, resistant viruses with E119V (2), R292K (6), or N294S (1) mutations were isolated from 9 of 50 children (18%).[30] The first two mutations have only been seen clinically in N2 subtype viruses, with E119V and N294S conferring reduced sensitivity specifically to oseltamivir.
The N294S mutation has also been seen in the N1 subtype (N295S in N1 numbering) conferring a mild reduction in zanamivir sensitivity, but a greater reduction in oseltamivir sensitivity.[31,32]
There were concerns that the weight-based dosing used in Japan delivered suboptimal concentrations of oseltamivir, facilitating selection of resistant viruses. Hence, another trial was carried out in the United Kingdom, in which tiered weight-based dosing of oseltamivir was used.[33].
Despite small numbers of patients, significant resistance was seen. Three of 11 patients (27%) infected with H1N1 viruses shed H274Y-resistant virus and one of [34] patients(3%) infected with H3N2 viruses shed an R292K-resistant virus. None of 19 patients infected with influenza B shed a resistant virus.
Due to concerns over the high level of oseltamivir resistance seen in the Japanese pediatric studies, a 3-year study was carried out in Japan to monitor for the emergence of resistant virus after zanamivir therapy. A total of 273 children were enrolled over three influenza seasons from 2006 to 2009.[34]. All children enrolled were <15 years of age, and were influenza positive by a rapid diagnostic assay and culture positive by throat swab. Samples from pre- and posttreatment were tested for resistant virus by RT-PCR and in enzyme assays of cultured virus. Three viruses from two subjects infected with H1N1 viruses showed virus with reduced sensitivity prior to treatment. One virus with a N70S mutation in the NA showed a 46-fold increase in IC50 for zanamivir.
Two viruses from one subject had a Q136K NA mutation, which showed a 300-fold increase in IC50 for zanamivir, but this mutation was only detected after culture and not in the primary sample. Hence, despite more patients than the oseltamivir pediatric trials, no emergence of resistance was seen in 273 zanamivir-treated children.
Seasonal H1N1 Early results demonstrated that mutations in the NA which conferred reduced NAI sensitivity also impacted on the function of the NA such that resistant viruses were compromised in their fitness and unlikely to be transmitted.[35,36] However, in late 2007, several seasonal H1N1 viruses with an H274Y mutation were isolated in Norway.
There was a minimal use of oseltamivir in Norway, and none of the patients had a history of drug exposure. Subsequent testing revealed 183 of 272 isolates (67%) bore this mutation. This virus was clearly fit, and transmissible and within weeks resistant viruses were detected in North America, Europe, and Asia.[37,38] The resistant viruses continued to spread to the southern hemisphere,[39] ultimately displacing the sensitive virus. More than 90% of H1N1 isolates were resistant by 2008–2009, with IC50s in the fluorescent assay generally in the 500–1000 nM range.
It appears that permissive mutations had evolved that enabled the NA to tolerate the H274Y mutation, maintaining fitness of the enzyme.[40]. The substitutions include R193G, R221Q, V233M, and D343N (R194G, R222Q, V234M, and D344N in N1 numbering). While H274Y is the primary mutation seen in N1 viruses, a seasonal H1N1 virus with an I222V mutation, conferring reduced susceptibility to oseltamivir,was also detected in surveillance of community isolates from untreated patients.[41].
Pandemic influenza A(H1N1)pdm09The sudden emergence and spread of the swine-derived influenza virus from Mexico led to the displacement of the oseltamivir-resistant seasonal H1N1 virus by the new A(H1N1)pdm09 virus. However, given the awareness of oseltamivir resistance, much closer monitoring has been carried out, by both phenotypic testing by enzyme assay and sequencing.


Resistance to the NAIs can be both drug and virus type or subtype specific. However, while resistance is often defined as greater than a 10-fold change in IC50 compared with the wild type, because some viruses have a higher base line IC50, for example, influenza B and clade 2 H5N1 strains, such viruses may be clinically resistant with only a few fold increase in IC50. It may be more appropriate to define resistance in terms of the IC50 and the drug concentrations delivered to the upper respiratory tract. There is also an issue of how the IC50 is measured, because values from the chemiluminescent assay are often lower than those from the fluorescent assay, and IC50 kinetics experiments [1].demonstrate how the IC50 can change with incubation times in the NAI assays.
Hence, there is currently no consensus on a definition of resistance, as it can really only be demonstrated by the lack of response to treatment.
Development of new inhibitors with different modes of action should also be a priority.


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