Tamiflu and Zanamivir: are they effective in treating Influenza or not ?

A recently published report from the  Cochrane Collaboration suggested that two drugs which are used in the treatment of human Influenza are not as effective as reported in clinical studies, so it is worth to pause a moment and recapitulate how these drugs work and take a closer look at the report before rushing to any judgment.

Currently four drugs are approved for the treatment or prophylaxis of Influenza virus infection (apart from vaccinations), two belonging to the class of the adamantanes (Amantadine and Rimantadine) and two belonging to the class of neuraminidase inhibitors (zanamivir -more commonly known as Relenza- and oseltamivir -commonly known as Tamiflu). Although both are targeting processes within the replication cycle, adamantanes are only effective against Influenza A -the most severe and common form of Influenza - whereas the latter is effective against both Influenza A and B. Furthermore, adamantanes are associated with severe toxic side effects and rapid emergence of drug resistance variants, a feature shared with oseltamivir. These drug resistant virus isolates are genetically stable and can be transmitted to non-infected individuals and shed for prolonged periods by immunocompromised patients taking the drug. In the case of Relenza, no stable resistant virus isolates have been reported so far. The potential side effects limit the use of both adamantanes and oseltamivir, although both drugs have their place within the context of an epidemic or a pandemic. It is commonly accepted however that the use as a prophylactic is contraindicated in most cases, excluding immunocompromised patients perhaps (certainly in a non-pandemic); indeed, otherwise healthy individuals should be vaccinated instead. This does not mean that they can not be used to prevent the spread of Influenza within a community of otherwise healthy individuals, but one should make a careful calculation of balancing the cost and side effects with the gain - and vaccination is not only cheaper but also has less side effects. The situation is obviously different in a situation of a pandemic where mass vaccination campaigns only help to control the spread of Influenza in the long term (it takes about two weeks to develop an immunity against Influenza) or in a situation where a new virus emerges to which there is no vaccine available.

In addition, most times people claim “to have the flu” they actually do not have Influenza but are infected with other common viruses - and neither the adamantanes nor the neuraminidase inhibitors would interfere with those since they are specific for Influenza.

In order to understand how these drugs we have to take a look at the replication cycle of Influenza virus.

                      Replication of Influenza virus (abridged)

The surface of all Influenza viruses contains two glycoproteins -hemagglutinin (HA) and neuraminidase (NA) - the combination of these which defines the particular Influenza strain (e.g. H1N1, H5N1 or H3N7) and the variation of these molecules determines if the virus can enter human cells or not. This variation however also necessitates a new seasonal vaccine each year since the composition of the surface molecules changes each year - often these changes are small enough that antibodies against a previous strain might offer a partial protection, sometimes however a completely new strain emerges and thus having the potential to cause a pandemic. The former process is referred as antigen drift while the latter is referred to as antigenic shift.

Hemagglutinin binds the receptor on the cell surface, sialic acid, and facilitates the entry of the viral particle into the cell and the release of the viral genome into the cytoplasm of the cell in a process akin to other RNA viruses such as the Coronaviridae or Filoviridae.

Following the synthesis and assembly of nascent viral particles, the viral particle needs to be released from the cell surface in a process called budding - a process that involves cleavage of the cellular receptor (sialic acid; SA) by the viral neuraminidase. Neuraminidase deficient viral particles are attached to the cell surface and cannot be released from the cell surface and are limited to one round of replication. Furthermore, the neuraminidase is also required for infection of cells of the upper respiratory system where it cleaves the sialic acid moiety of the mucin thus allowing the virus to infect airway epithelia cells.

Apart from the hemagglutinin and neuraminidase the viral envelope of Influenza A contains other proteins required for viral replication. Of particular interest in this context is the viral M2 protein, which in infected cells it colocalizes with sites of virus budding as well as cholesterol containing lipid raft domains on the cell surface and incorporated into the virion during the budding process. Together with M1, M2 is responsible for the formation of filamentous Influenza virus particles - those that are commonly found during an infection but are largely absent in virus grown in eggs (on a side note, filamentous Influenza virus particles enter cells with macropinocytosis, a receptor independent entry pathway and are the prevalent morphology in patients - spherical particles are common in viruses grown in eggs). The M2 protein not only assists in forming filamentous viral particles, it  is more importantly a proton selective ion channel which is activated by the low pH (4.9) of the endosome (concurrently with HA) thus allowing -following structural changes- the viral genome being released into the cytoplasm and imported into the nucleus of the infected cell.

The adamantanes inhibit these processes, both the formation of spherical and filamentous viral particles as well as the release of the viral genome of the cell, whilst the neuraminidase inhibitors inhibit the cleavage of SA - and thus viral entry as well as the release of newly synthesized viral particles from the cell surface.

                          The Tamiflu and Relenza debate

The debate about the effectiveness started as soon as both drugs were approved by the FDA for the use to treat and prevent Influenza and intensified during the “swine flu”, A/H1N1/2009, epidemic when governments worldwide began to store Tamiflu and Relenza. Early clinical trials of both zanamivir and oseltamivir suggested that both drugs can prevent the onset of Influenza if taken early and reduced symptoms 24 hrs following treatment of infected patients. More importantly, both drugs reduced the onset and complications of secondary bacterial infections - the very same which often might have lead to the death of patients during the Influenza pandemic in 1918/1919 and were the reason that scientist initially suspected bacteria to be the causative agent of the disease in the first place.

These clinical studies also emphasized the importance that both drugs need to be administrated early in the infection in order to reduce symptoms. So what is all the fuzz about? The authors of the recent report reanalyzed the 107 published and unpublished clinical study reports available from European Medicines Agency (EMA) and the manufactures of zanamivir and oseltamivir, GlaxoSmithKline and Roche respectively, as well as comments published by the FDA, EMA and the Japanese regulator. The main objective was to identify the potential benefits and harms associated with these drugs in placebo and non-placebo treated groups by reanalyzing the data from non-published and published studies from both manufactures and regulatory bodies. Criteria for effectiveness included (1) the time from taking the drug  to alleviation of the first symptoms (2) hospitalizations (3) secondary infections as a result of Influenza virus infection (3) side effects of the drug (such as nausea) and (4) prophylaxis - certainly an important factor in an epidemic. Contrary to what is reported in the media, the report acknowledges that in cases of symptomatic Influenza oseltamivir and zanamivir do protect household members of infected individuals - but fail to do so in cases of asymptomatic/non -Influenza disease (the attentive reader might have realize that both drugs were specifically designed against Influenza - so who would expected that they work against other viruses anyway?). Moreover, zanamivir significantly reduces the risk of bronchitis in adults (not in children though). Even the authors concede that both drugs may have benefits. Interestingly the authors published a report in 2009 stating that oseltamivir does not prevent infections of the lower respiratory tract. 
In this context one might speculate that the receptor specificity of Influenza might determine the sensitivity to neuroaminidase inhibitors.
Airway epithelia cells of in lower respiratory tract are known to express α2-3 linked sialic acid -the receptor for avian and avian-like high risk influenza virus (such as H5N1). Cells in the upper respiratory tract however preferentially express α2-6 linked sialic acid - with the exception of children, which preferentially express α2-3 linked sialic acid receptors. Recent results however indicate that the infectivity of recombined (reverse genetically modified) H5N1 which is specific for SAα2-6 is not altered by oseltamivir nor zanamivir, thus implying that other factors might play role in conferring drug resistance (apart from the HA and NA) - however it can not be ruled out that the mutations induced in the HA gene contribute to mutations of the NA. Some data derived from studies using Influenza virus infecting wild aquatic birds suggest that additional N-linked glycans at the top of HA are required for effective binding of Influenza Virus to the host cell receptor (this applies to both H5 and H7 viruses).

So what is the conclusion? Both oseltamivir and zanamivir seem to offer some protection and alleviation of symptoms. The core of the problem seems however that pharmaceutical companies often withhold data from clinical studies - this however is not unique to antiviral drugs. In the end, people may realize that getting vaccinated in the first place might be the best prophylactic. Finally, more antiviral drugs need to be developed. Two recently identified promising candidates include resveratrol and epigallocatechin gallate, both plant derived polyphenols and can be found in green tea as well as red wine. Isoquercetin has been shown to prevent the formation of oseltamivir and amantadine resistant virus particles, thus being another potential antiviral drug. 

Further reading

Moscona, A. (2005). Neuraminidase Inhibitors for Influenza New England Journal of Medicine, 353 (13), 1363-1373 DOI: 10.1056/NEJMra050740 

McKimm-Breschkin JL (2013). Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. Influenza and other respiratory viruses, 7 Suppl 1, 25-36 PMID: 23279894 




























































                                                                                             



Rossman JS, Jing X, Leser GP, Balannik V, Pinto LH, & Lamb RA (2010). Influenza virus m2 ion channel protein is necessary for filamentous virion formation. Journal of virology, 84 (10), 5078-88 PMID: 20219914 

Rossman JS, Leser GP, & Lamb RA (2012). Filamentous influenza virus enters cells via macropinocytosis. Journal of virology, 86 (20), 10950-60 PMID: 22875971 

Fontana, J., Cardone, G., Heymann, J., Winkler, D., & Steven, A. (2012). Structural Changes in Influenza Virus at Low pH Characterized by Cryo-Electron Tomography Journal of Virology, 86 (6), 2919-2929 DOI: 10.1128/JVI.06698-11

Chan MC, Chan RW, Yu WC, Ho CC, Yuen KM, Fong JH, Tang LL, Lai WW, Lo AC, Chui WH, Sihoe AD, Kwong DL, Wong DS, Tsao GS, Poon LL, Guan Y, Nicholls JM, & Peiris JS (2010). Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract. The American journal of pathology, 176 (4), 1828-40 PMID: 20110407 

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Gambaryan, A., Tuzikov, A., Bovin, N., Yamnikova, S., Lvov, D., Webster, R., & Matrosovich, M. (2003). Differences Between Influenza Virus Receptors on Target Cells of Duck and Chicken and Receptor Specificity of the 1997 H5N1 Chicken and Human Influenza Viruses from Hong Kong Avian Diseases, 47 (s3), 1154-1160 DOI: 10.1637/0005-2086-47.s3.1154 

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Doshi P, Jefferson T, & Del Mar C (2012). The imperative to share clinical study reports: recommendations from the Tamiflu experience. PLoS medicine, 9 (4) PMID: 22505850 

Loregian A, Mercorelli B, Nannetti G, Compagnin C, & Palù G (2014). Antiviral strategies against influenza virus: towards new therapeutic approaches. Cellular and molecular life sciences : CMLS PMID: 24699705 

Kim Y, Narayanan S, & Chang KO (2010). Inhibition of influenza virus replication by plant-derived isoquercetin. Antiviral research, 88 (2), 227-35 PMID: 20826184