Issues in pharmacotherapy of 2009 H1N1 influenza infectionYK Gupta, BM Padhy
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.70945
Source of Support: None, Conflict of Interest: None
The pandemic caused by the 2009 H1N1 influenza A virus has been a cause of great concern for healthcare professionals and the scientific community worldwide. Due to the widespread resistance of the virus to adamantanes, pharmacotherapy is currently limited to neuraminidase inhibitors, oseltamivir and zanamivir. The use of neuraminidase inhibitors in India is primarily associated with issues of patient and physician awareness, variability in disease management guidelines, safety and efficacy in the Indian population, need for active drug safety monitoring, and development of resistance due to possible misuse. In addition, other issues like availability of the drugs in retail and stockpiling by the public health authorities need careful introspection. The development of influenza vaccines in India and its adequate availability to the country's populace also poses significant challenges in the management of the pandemic. In light of the limited therapeutic options available for the management of the disease, research on novel targets and pharmacological agents would also be beneficial in addressing the challenges of future outbreaks.
Keywords: Neuraminidase, influenza A virus, oseltamivir, vaccines
The influenza virus, known to be a circulating pathogen in the human population since the 16th century is notable for its unique ability to cause recurrent epidemics and global pandemics. The previous century saw three such pandemics. In 1918, the first pandemic (Spanish flu) caused by influenza A (H1N1) led to the death of around 30-100 million people worldwide (including 7 million in India) and loss of around 16% in the global GDP. The other two pandemics in 1957 and 1968 were relatively milder but still killed nearly 1 million people.  Influenza A viruses are classified into subtypes based on the antigenicity of hemagglutinin (HA) and neuraminidase (NA) molecules.  The ability of this virus to undergo genetic reassortments causes unpredictable changes in its antigens and the consequent immune response leads to recurrent epidemics of febrile respiratory disease every 1-3 years. 
The current millennium saw the appearance of a new subtype of influenza A (H5N1) in 2003 known as avian flu. As the world was preparing for a pandemic of avian flu, another novel influenza A (H1N1) virus made a dramatic appearance in Mexico in March 2009. The novel influenza A (H1N1) virus has the genetic structure resulting from the reassortment of genes from four influenza viruses, i.e., North American swine influenza, Asia/Europe swine influenza, human influenza, and avian influenza (non-H5).  Its common name "swine flu" is a misnomer as this virus is not firmly established to cause flu in swines.  In early June 2009, swine flu, which is now called 2009 H1N1 influenza A was declared a pandemic, affecting many countries worldwide for which the World Health Organization (WHO) noted a level VI precaution, the highest level of precaution possible.  As on May 16, 2010, 31,866 laboratory-confirmed cases have been reported in India of which 1,517 have been fatal. 
Luckily, the 2009 H1N1 virus is not highly pathogenic, and the number of deaths and severe disease has been lower than previous pandemics. However, the current pandemic has forced the scientific community and health policy makers onto a common platform to critically assess and address key issues related to the adoption of treatment guidelines, pricing, procurement, stockpiling and rational use of anti-influenza drugs, pharmacovigilance, and indigenous development of vaccines and other novel pharmacological anti-influenza agents. This article attempts to discuss some of the aforementioned issues primarily from an Indian perspective.
Presently, two classes of antiviral drugs have been approved by the US Food and Drug Administration (FDA) for use in treating or preventing influenza virus infections: M2 ion channel blockers (adamantanes) and neuraminidase inhibitors (NAIs). The M2 blockers, amantidine and rimantidine, are effective against influenza A viruses, but not influenza B viruses, which lack the M2 protein. However, use of the M2 blockers has been associated with the rapid emergence of drug-resistance mutations of the M2 protein among human influenza A viruses of H3N2 subtype and H1N1 subtypes circulating in certain geographic areas. A recent study by the United States Centre for Disease Control (CDC) has reported 100% resistance of 2009 H1N1 to the adamantanes rendering them almost inept for treatment or prophylaxis. 
Two NAIs, oseltamivir (Tamiflu [Hoffman-La Roche, Ltd]) and zanamivir (Relenza [GlaxoSmithKline]) are approved by US FDA for use against type A and type B influenza infections. The NAIs target the active site of the NA protein, inhibiting its sialidase activity that is essential for virus release. Although zanamivir and oseltamivir are similar in their mode of action, the drugs have different structural and biochemical properties, which influence their route of administration. Both the drugs require twice daily administrations for treatment. Zanamivir is administered by inhalation with a dry powder inhaler. The bioavailability of the drug is 10-20% by inhalation, compared with 2% by oral administration. About 90% of the absorbed dose is excreted unchanged in the urine. On the other hand, oseltamivir is administered orally (75 mg/dose) as a prodrug and 75% is metabolized to its active form, oseltamivir carboxylate, and reaches the systemic circulation. Oseltamivir carboxylate is almost entirely eliminated in urine and dose adjustment is required in the case of severe renal impairment. ,, Further, a third NAI, peramivir, formulated for intravenous administration is undergoing clinical trials and is expected to be marketed soon.  Two other NAIs, laninamivir (Daiichi Sankyo) and A-315675 (Abbott Laboratories) are at the preclinical stage. 
The NAIs, oseltamivir and zanamivir, are also approved for the management of H1N1 influenza by the Indian drug regulatory authority and are available for restricted retail sale in the country. However, various issues associated with the use of NAIs often arise and answers are sought from the scientific community and health policy makers.
Guidelines for therapeutic use
The US CDC recommends treatment with oseltamivir or zanamivir for all hospitalized patients with confirmed, probable, or suspected 2009 H1N1 or seasonal influenza. Some groups appear to be at increased risk of influenza-related complications and can be started on early empiric therapy with NAIs. Those at high risk include children younger than 2 years of age, adults 65 years of age or older, pregnant women and women up to 2 weeks postpartum, and persons with chronic pulmonary, cardiovascular (except hypertension), renal, hepatic, hematological, or metabolic disorders. In addition, the immunocompromised and those younger than 19 years of age, receiving long-term aspirin therapy should be treated with NAIs.  Emergency use authorization of intravenous (IV) peramivir, has been allowed by US FDA for the treatment of adult patients who have not responded to either oral or inhaled antiviral therapy or when drug delivery by a route other than IV is not expected to be dependable or is not feasible. 
However, in India, the patients have been categorized in categories A, B, and C based on the severity of their symptoms and presence of risk factors. While category A patients do not require treatment with oseltamivir, category B and C patients do.  It is proposed that zanamivir should be reserved for the treatment of cases of 2009 H1N1 resistant to oseltamivir.
Evidence for benefits from antiviral treatment in studies of uncomplicated seasonal influenza is strongest when treatment is started within 48 h of illness onset. Initiating treatment as soon as possible after illness onset is also likely to reduce the risk of severe outcomes including severe illness or death. The recommended duration of treatment is 5 days. Hospitalized patients with severe infections (such as those with prolonged infection or who require intensive unit care admission) require longer treatment courses. 
Guidelines for prophylactic use
Infected persons may shed influenza virus, and potentially be infectious to others, beginning 1 day before they develop symptoms to up to 7 days after they become ill. The CDC recommends that postexposure antiviral chemoprophylaxis with either oseltamivir or zanamivir can be considered for persons who are at higher risk for complications of influenza and are a close contact of a confirmed, probable, or suspected case of 2009 H1N1 during the infectious period. It is also indicated for healthcare personnel or public health workers, who have had a recognized, unprotected close-contact exposure with a confirmed, probable, or suspected case of 2009 H1N1 during the infectious period.  The drug of choice for chemoprophylaxis is oseltamivir and the dose is half that of the therapeutic dose, to be administered for 10 days after the last exposure. Therapeutic and prophylactic dosing schedules for children are similar (2 mg/kg twice a day for 5 days for treatment, and 2 mg/kg once a day for 10 days for prophylaxis).  A nearly similar guideline regarding chemoprophylaxis has been issued by the Health Ministry of India.  Recommendations notwithstanding, the role of prophylactic antiviral therapy for 2009 H1N1 is yet to be firmly established, especially in children. Subtle differences in therapeutic and prophylactic guidelines therefore do exist between various countries, yet most recommend a restricted use of NAIs only in the high-risk population.
Although the guidelines are available in public domain and can be downloaded from the CDC, WHO and Health Ministry's H1N1 pandemic influenza websites, awareness about them is still lacking among the healthcare professionals. Therefore, awareness programs to educate the physicians as well as the patients are imperative to ensure that the guidelines are accessed and properly adhered to.
In September 2009, the Indian Union Health and Family Welfare Ministry issued a notification under Section 26 E of the Drugs and Cosmetics Act, 1940, allowing a regulated retail sale of zanamivir and oseltamivir through designated pharmacies. Until then, NAIs were only available in designated hospitals identified by the government for treating and quarantining H1N1 cases.  Both the drugs have been labeled under Schedule X of Drugs and Cosmetics Act, and the patient will have to produce two prescriptions issued by the same doctor at the time of purchase. The chemist will have to keep one copy of the prescription for 2 years. In India, only 60 distributors and about 300 chemists have this license that allows them to sell drugs falling under Schedule X.  In addition, five Indian pharmaceutical companies, namely, Daiichi Ranbaxy, Cipla, Metco, Hetero, and Strides Acrolabs Ltd., have been permitted to manufacture oseltamivir and provide it for retail sale as well as stockpiling by public health authorities. This authorization led to widespread debates within the medical fraternity about the risks and benefits of availability of the drug in retail. The risk of adverse reactions especially due to self-medication, panic hoarding in households, and development of resistance in the virus was pointed out if the drug was made available in retail. However, it was also argued that retail sale would ease the burden on the public health system and prevent black marketing and unauthorized internet-based sale of fake versions of the drugs. ,, The Indian manufacturers have priced the Indian version of oseltamivir around USD 20 as opposed to USD 60 for Tamiflu making it more affordable and accessible to the larger public. 
The innovator of oseltamivir, Roche, has provided more than 270 million doses of Tamiflu to 96 governments and orders continue to grow. Developed countries like Australia, Canada, UK, and USA have sizeable stockpiles of oseltamivir to cover 25-50% of their population. , The Indian government has built up a stockpile of 30 million doses (3 million courses) of oseltamivir. Around 7.2 million doses have already been decentralized, with every district having stock to treat 1,000 patients and 35 metros having stock to treat 1,000 patients.  Daiichi Ranbaxy Laboratories has got orders to supply 900,000 doses of oseltamivir. Strides Arcolab also has a contract order from the Ministry of Health for a supply of 740,000 doses of oseltamivir. In addition, the Indian government has procured around 9 million doses of oseltamivir from Hetero, the only domestic company that has a manufacturing agreement with Roche to make the low-cost version of the patented version. The government is procuring oseltamivir at around INR 280 for a pack of 10 tablets. The Health Ministry is also considering stockpiling of zanamivir. 
How large should the stockpile of antivirals be, has been a matter of debate. Modeling studies are not only useful for providing insights into the severity of influenza epidemics and pandemics but also for estimating the required size of the antiviral stockpile. The first mathematical model used to describe an influenza epidemic, known as the susceptible-infectious-recovered (SIR) model was developed early in the 20 th century by Kermack and McKendrick. , The SIR model has been used as a basis for all subsequent influenza models. The rate of spread of an infection is represented by the basic reproductive number R0, defined as the average number of new infections that one case generates, in an entirely susceptible population, during the time of infection. The feasibility of controlling an epidemic depends on the value of the R0. The more severe the epidemic (i.e., the greater the value of R0) the more intensive the interventions must be to significantly reduce the number of infections and deaths. 
Various studies have evaluated the level of interventions needed to contain influenza epidemics of varying severities. A metapopulation stochastic model-based study of a hypothetical avian influenza pandemic capable of spreading through 3,100 urban areas in 220 countries reported that when R0 is less than 1.9, the impact of the disease can be significantly reduced if there were enough antivirals to treat ~2-6% of the population. However, in the case of a very severe epidemic (R0 of 2.3) even if ~20% of the population were treated with antiviral drugs, 30-50% of the population would still be infected. , A recent modeling study based on initial outbreak data of 2009 H1N1 in Mexico has estimated the R0 to be between 1.4 and 1.6, which could be controlled by an effective use of antiviral drugs and vaccination strategies. , Therefore, with the current 2009 H1N1 influenza strain, the government stockpile of 30 million doses (3 million courses) of oseltamivir, used appropriately, would be sufficient for approximately 0.3% of India's population of 1.15 billion individuals.  However, if R0 of 2009 H1N1 were to be equivalent to the 1918 H1N1 influenza strain (R0 between 2 and 3), health agencies would require much larger stockpiles of antiviral drugs to cover at least 20% of the population. 
The requirement of drugs or vaccines necessary for control of infections would be lower if interventions are specifically targeted toward susceptible populations. Model-based studies on the emerging influenza epidemic in South East Asia have reported that targeted antiviral prophylaxis along with pre-exposure vaccination, and quarantine could effectively contain even moderately severe or severe epidemics. ,, A recent study from the UK has reported that prompt treatment with oseltamivir along with a widespread use of prophylactic measures significantly reduced the transmission of 2009 H1N1 virus. The study also reported a 71% reduction in laboratory-confirmed cases of 2009 H1N1 in those contacts who received oseltamivir within 3 days of the onset of symptoms in the index case. 
Nevertheless, stockpiling strategies cannot solely rely on model-based assumptions as they may not be encountered in real-life pandemics. Other factors such as adoption of containment strategies, social distancing, migration and travel patterns, vaccination coverage, availability of funds, drug supply, distribution and storage facilities, resistance patterns, and extensibility of the shelf life of the drugs are taken into account.  In addition, the population growth rate, the urban-rural distribution, the proportion of high-risk individuals, the possibility of using antiviral combinations, and the amount of drug required for pre-exposure prophylaxis of front-line healthcare workers and emergency care providers are also considered while determining the most appropriate stockpile size for the country. ,
The commonest adverse effect reported in the literature on oseltamivir is dose-related nausea which occurs twice as frequently as placebo when used for prophylaxis. In controlled clinical trials, approximately 10% of patients had nausea without vomiting, and an additional 10% experienced vomiting. Cases of insomnia have also been reported with the use of oseltamivir. In recent years, there have also been a number of postmarketing case reports (mainly from Japan) of neuropsychiatric events (such as delirium, hallucinations, confusion, and abnormal behavior leading to injury, convulsions, and encephalitis), particularly in children younger than 16 years.  The most common adverse events reported in more than 1.5% of all patients treated with zanamivir are sinusitis and dizziness. In addition, there have been reports of fatal respiratory complications like bronchospasm, following the inhalation of zanamivir. Neuropsychiatric events particularly in the pediatric age group have also been reported. 
A review of the available information on the safety of oseltamivir in pediatric patients by US FDA suggested that the increased reports of neuropsychiatric events in Japanese children are most likely related to an increased awareness of influenza-associated encephalopathy, increased access to oseltamivir in that population, and a coincident period of intensive monitoring of adverse events. This prompted the addition of precautions to the US product label for oseltamivir. An internet-based cross-sectional survey carried out on children in three schools in London also revealed that those administered oseltamivir developed neuropsychiatric symptoms, most frequently difficulty in sleeping, bad dreams/nightmares, and poor concentration.  However, a recent metanalysis has failed to demonstrate any increase in neuropsychiatric events in patients administered oseltamivir over those who were not. Further, the study reported no genetic predilection for the development of neuropsychiatric adverse events in the Japanese populace.  The plethora of case reports of neuropsychiatric events, prompted researchers to investigate the inhibitory effect of NAIs on human sialidase enzyme that is present in the brain. The study concluded that human sialidases were not significantly inhibited with conventional doses of oseltamivir or zanamivir. However, this study was carried out in vitro using recombinant human sialidases and inhibition of silalidases in vivo cannot entirely be ruled out. 
Data about the frequency, type, and severity of adverse events related to the use of NAIs in the Indian population are still lacking. However, due to restricted availability of oseltamivir and zanamivir through selected healthcare facilities and chemists, the incidence of adverse events can be accurately estimated by means of cohort event monitoring. This method can provide the total number of adverse events encountered as well as the number of patients exposed to the drugs. As quality of data collected will be higher, causality assessment would be simpler in this method as opposed to spontaneous reports. Such a system of pharmacovigilance has been pilot tested for artemisinin-based combination therapy (ACT) for uncomplicated Falciparum malaria and its operational procedures could be modified for monitoring of adverse events of NAIs. The project carried out by department of pharmacology, All India Institute of Medical Sciences and National Institute of Malaria Research, involved conduct of sensitization cum training workshops for medical officers in malaria endemic zones to identify and report adverse events encountered during ACT, using specially designed reporting forms. In just a month, over 50 filled forms were received from Orissa and Karnataka (unpublished data). However, this method is resource intensive and requires proactive involvement of all the stakeholders involved in drug safety evaluation. In addition to cohort event monitoring, spontaneous reports of NAI-related adverse events would also help in understanding the safety profile of the drugs. In cognizance of the active pharmacovigilance required, the recently launched Pharmacovigilance Programme for India by Central Drugs Standard Control Organisation, Government of India, has also envisaged focused monitoring of NAI-related adverse events at selected ADR monitoring centers.
In 2006, it was believed that influenza virus was unlikely to develop resistance to NAIs as they were specifically designed to target viral neuraminidase enzyme, and any mutation in the neuraminidase would compromise the virulence. The available evidence at that time also supported this notion as H274Y (H275Y in N1 numbering) mutation in neuraminidase that confers resistance to oseltamivir significantly reduced the replication of H1N1 strains and their virulence in experimental models of influenza. Furthermore, the strains with this mutation were sensitive to zanamivir. The incidence of oseltamivir resistance was considered rare until 2006-2007. However, in December, 2008, the CDC reported that nearly all cases of influenza A (H1N1), the predominant circulating strain for the seasonal influenza, had developed resistance to oseltamivir. Recent studies have dispelled the notion of attenuated virulence in mutated strains of H1N1 and found no discernible differences in the predisposing factors, clinical symptoms, or complications between oseltamivir-resistant cases and matched oseltamivir-susceptible cases. 
2009 H1N1 influenza viruses are currently sensitive to oseltamivir and zanamivir, but are resistant to the adamantanes. However, oseltamivir-resistant 2009 H1N1 viruses have been identified, typically among persons who develop illness while receiving oseltamivir for chemoprophylaxis or in immunocompromised patients with influenza who are being treated. As on May 20, 2010, the CDC has reported 54 cases of oseltamivir-resistant 2009 H1N1 in the USA. , In India, the resistance of 2009 H1N1 to oseltamivir has not been reported.
It is interesting to note that isolates from two oseltamivir-resistant 2009 H1N1 cases which developed during prophylactic treatment have revealed the presence of I223V mutation in neuraminidase besides the H275Y mutation. , However, the frequency of the development of resistance in typical H1N1 viruses do not correlate well with the extent of oseltamivir usage and selective drug pressure may not always lead to the development of resistance. For example, 67.3% oseltamivir resistance is observed among typical H1N1 strains in Norway, where oseltamivir is rarely used, and obtained only on prescription as opposed to only 3% in Japan, the country with the highest per capita oseltamivir use. 
Significant resistance to zanamivir has not yet been reported in influenza A viruses. There is only one report of zanamivir resistance in an immunocompromized child infected with influenza B virus, yet concerns about the development of zanamivir resistance in H1N1 are high following its increased use in the last 2 years. 
The milieu in which the influenza virus develops resistance to NAIs is complex, from the time since licensure of oseltamivir and zanamivir in late 1990s until recently, and resistance to these agents have remained at a low level, even in the countries where it is used the most.  Nevertheless, it is likely that prudent use of the NAIs in a rational manner may prevent the development of resistance in the virus as well as reduce the incidence of adverse events. 
Influenza vaccination is considered the most effective method for preventing influenza and influenza-related complications. Limited data from serologic studies of persons who received vaccination with seasonal influenza vaccines suggest that they do not confer protection against 2009 H1N1 influenza A virus.  More than a dozen multinational companies including Sanofi-Aventis, GlaxoSmithKline, Novartis, Baxter, CSL, and AstraZeneca have invested huge sums of money in developing vaccines against 2009 H1N1 virus.  Inactivated and live attenuated monovalent vaccines containing the A/California/7/2009 H1N1 strain have been recently approved by the US FDA, European Medicines Agency (EMEA), and Australian Therapeutic Goods Administration (TGA). 
Indian biopharmaceutical companies like Cadila, Panacea Biotech, Bharat Biotech, and the Serum Institute of India are also actively engaged in the development of the influenza vaccine in the country.  While Cadila recently received marketing approval for its vaccine from the Drugs Controller General (India), clinical development programs of the vaccines from Panacea Biotech and Serum Institute of India are nearing completion.  The Health Ministry also has recently imported 1.5 million doses of a split virus, inactivated, nonadjuvanted monovalent vaccine from Sanofi-Aventis in order to vaccinate high-risk individuals. The healthcare workers in the community health centers will be vaccinated in the first phase followed by subsequent vaccination of emergency care providers and other healthcare workers.  However, it has to be remembered that due to the large size of the population, there will be a heavy demand for the vaccines in India. Further, as most vaccines would expire in 1-2 years, periodic replacements of vaccine stockpiles would be required and may trigger heated political and public debates on perceived "wastage."  In addition, as these vaccines are being developed on a fast-track basis, issues related to quality and safety that may not have been addressed during the development phase may pose challenges later on.
Although most of the current focus is on the development of drugs which target the activity of the NA protein, agents that target the HA are also effective in the laboratory setting.
Drugs targeting the HA and showing promise as potential anti-influenza agents include nitazoxanide (NTZ), a thiazolide anti-infective licensed in the USA for treating enteritis caused by Cryptosporidium parvum and Giardia lamblia, its active metabolite tizoxanide, and the second-generation thiazolides.  Antibodies against the HA protein that can neutralize virus infection can be potentially developed. In mice challenged with influenza A virus, neutralizing antibodies to the HA glycoprotein have been shown to be effective both as a prophylactic and a therapeutic agent. , Small interfering RNAs (siRNAs) designed against conserved sequences in the influenza A virus nucleoprotein, acidic polymerase and matrix genes, suppress virus replication in tissue culture, and have significantly reduced virus yields in tissue culture, and in the lungs of infected mice. The major advantage of RNA inhibitor-based therapeutics in a pandemic influenza situation is that the design of specific siRNAs only requires knowledge of the gene sequence, and the siRNA synthesis can be achieved within a short period of time and at relatively low cost. However, before a siRNA approach can be used in the clinical setting, it will have to be carefully evaluated to examine its specificity in silencing virus gene expression.  The efficacy of chloroquine as an antiviral has also been evaluated. Treatment of cells with chloroquine elevates the endosomal pH, and previous studies have demonstrated its inhibitory effects on influenza virus replication. Moreover, recent studies have reexamined its anti-influenza activity in tissue culture, and the available data are encouraging. ,, Experimental studies have shown that during influenza virus replication, cholesterol-rich lipid-rafts play an important role in the virus maturation process. Statins are able to disrupt lipid-raft membranes by removing membrane-bound cholesterol, and in doing so are able to inhibit the assembly process of influenza virus in tissue culture. ,,
In addition, certain plant-based anti-influenza therapies containing Tinospora cordifolia and Ocimum sanctum have been claimed to be effective in the treatment of 2009 H1N1 infection by Indian traditional healers. The Department of Biotechnology, Government of India, is also undertaking projects to screen various plant products like Japanese plum juice and green tea for their anti-influenza activity. ,
Lessons learnt from the devastating Spanish Flu of 1918, and subsequent pandemics due to mutant influenza viruses make it imperative that we become more cautious and prepared to handle any such future eventuality. The development of global pandemic preparedness strategies and their integration with national/regional pandemic plans is of paramount importance. Increasing public awareness, adoption of preventive measures, deployment of rapid response teams, provision of surveillance and logistic support systems, building adequate drug stockpiles, and monitoring for the development of resistance to NAIs are also essential components of pandemic preparedness measures. ,, In addition, rational, judicious, and optimal use of existing drugs and research and development of novel target-based preventive and curative anti-influenza drugs and vaccines will be critical for mitigating the impact of any future pandemic.