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John F. Enders Lecture 2006: Antivirals for Influenza Adrian K. Ong1,3 and Frederick G. Hayden2,4 1

Communicable Diseases Cluster and 2Global Influenza Program, World Health Organization, Geneva, Switzerland; 3Department of Infectious Diseases, Tan Tock Seng Hospital, Singapore; 4Departments of Medicine and Pathology, University of Virginia School of Medicine, Charlottesville

The long history of influenza drug development has both contributed practical advances in antiviral chemotherapy and improved the understanding of influenza pathogenesis and epidemiology. The role played by these antivirals continues to grow with the dual threats of seasonal and pandemic influenza. The neuraminidase inhibitors are proven effective for the chemoprophylaxis and treatment of influenza A and B, although early therapy is essential for disease mitigation. Studies of topically applied zanamivir have demonstrated the importance of viral replication in the lower respiratory tract, even in uncomplicated influenza. Antiviral resistance, especially to the M2 ion channel inhibitors, sometimes limits clinical utility. Oseltamivir-resistant variants may emerge during treatment but have not yet circulated widely and are usually less fit than wild-type virus; most retain susceptibility to zanamivir. The transmission fitness cost of these resistant variants is drug-, neuraminidase subtype–, and mutation-specific. Continued vigilance in drug resistance surveillance is imperative, as is research into the development of new agents that will provide the potential for alternative and combination antiviral therapy.

Influenza drug development has been restricted in scope and relatively slow, compared with that for other viral infections, in part because of the lack of commercial incentives [1]. Despite the paucity of available agents, antivirals provide a key pharmacologic measure in responding to seasonal influenza and to infections by novel influenza viruses. Recently, greater impetus has been placed on both stockpiling of existing antivirals and research for new ones because of their central role in pandemic influenza preparedness [2, 3]. The present review provides personal perspectives on the development of influenza antiviral therapy by the senior author

Received 25 January 2007; accepted 20 February 2007; electronically published 7 June 2007. Potential conflicts of interest: none reported. Presented in part: John F. Enders Lecture, 44th Annual Meeting of the Infectious Diseases Society of America, Toronto, Canada, 13 November 2006. Correspondence: Dr. Frederick G. Hayden, World Health Organization, 20 Ave. Appia, CH-1211 Geneva, Switzerland ([email protected]). The Journal of Infectious Diseases 2007; 196:181–90  2007 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2007/19602-0004$15.00 DOI: 10.1086/518939

(F.G.H.), who initiated his studies of influenza during his training at the Division of Infectious Diseases, University of Rochester, Rochester, New York, 3 decades ago. The review selectively focuses on studies conducted by F.G.H. while at the University of Virginia and by his collaborators and includes updates on studies of topical antiviral administration, antiviral drug resistance, and combination therapy that were started during his fellowship with Dr. R. Gordon Douglas, Jr. (University of Rochester). A key lesson has been that the development of influenza antivirals has not only led to valuable pharmacologic interventions for influenza management but also provided important insights into mechanisms of viral replication and the pathogenesis and epidemiology of influenza disease in humans [1]. NEURAMINIDASE INHIBITOR (NAI) DEVELOPMENT Cell-surface receptors bearing terminal sialic acid residues serve as the initial attachment site for influenza A and B viruses. These sialic acid residues are

subsequently cleaved by influenza viral neuraminidase (also known as sialidase) to promote the efficient release of progeny virus particles from infected cells and facilitate virus spread within the respiratory tract [4]. The elucidation of these viral replication mechanisms was the culmination of decades of investigation and ultimately led to the development of specific inhibitors [1]. The work of George Hirst at the Rockefeller Institute in the early 1940s described the hemagglutination of erythrocytes by influenza viruses and the loss of erythrocyte receptors for virus under certain conditions. Hirst first postulated the existence of a viral enzyme that altered cellular receptors on red blood cells [5]. This research was further developed by F. MacFarlane Burnet et al. in Melbourne, who conducted a series of studies and first described “receptor-destroying enzyme” obtained from the filtrates of Vibrio cholerae cultures [6, 7]. This receptor-destroying enzyme (RDE) rendered erythrocytes nonagglutinable by influenza viruses and topical application of RDE in mice lungs reduced influenza infection [8]. These PERSPECTIVE • JID 2007:196 (15 July) • 181

findings were corroborated in experiments performed much later by other groups; MDCK or EAC cells that were briefly treated with V. cholerae sialidase (the equivalent of RDE) were rendered less susceptible to infection by influenza viruses [9, 10]. Furthermore, Burnet’s group showed that respiratory mucus (mucoproteins containing sialic acid residues) competitively inhibited influenza hemagglutination and infectivity and that this was reversed by either RDE treatment or the addition of live virus containing the fresh enzyme [7]. In 1948, Burnet’s group [8] wrote the following 2 prescient observations: (1) “Theoretically there are several possible ways in which a prophylactic or therapeutic effect could be developed on the basis of these conceptions. Cell receptors in the respiratory tract might be removed by the regular inhalation of RDE or other destructive agent” and (2) “an effective ‘competitive poison’ for the virus enzyme might be similarly administered which, when deposited on the mucus film lining the respiratory tract, would render this an effective barrier against infection.” However, at the time they concluded that “neither of these approaches seems even remotely practicable” (p. 410) [8]. Nearly 60 years later, the first of these hypothesized modalities is about to enter clinical testing. A novel molecule consisting a fusion construct of an Actinomycoses viscosus sialidase and a human epithelial anchor domain effectively cleaves terminal sialic acid residues from receptors recognized preferentially by either human (a2,6 linkages) or avian (a2,3 linkages) influenza viruses [11]. The molecule, designated “DAS181,” is inhibitory for a range of influenza A and B viruses in cell culture and provides prolonged antiviral and cell protective effects in vitro. When topically applied to airway epithelium in mouse and ferret models of influenza, DAS181 shows antiviral and disease benefits [11]. Preclinical safety studies are currently being completed, and the molecule is expected to start phase 1 testing in 2007. 182 • JID 2007:196 (15 July) • PERSPECTIVE

In 1999, 150 years after Burnet’s observation, the previously impracticable use of a “competitive poison” became a clinical reality when zanamivir, a sialic acid analogue, was approved for clinical use. Decades before in Australia, Gottschalk [12] had first established that the target of the viral enzyme (neuraminidase) was sialic acid bearing receptors. In the mid1970s, Palese and Compans [13] and Palese et al. [14] showed that the inhibition of viral neuraminidase, including the use of relatively impotent sialic acid analogues, was associated with the inhibition of viral release and spread from infected cells. A major breakthrough came in 1983 with the solution of the crystal structure of the viral neuraminidase by Peter Colman et al. in Melbourne [15]. The structural data allowed rational drug design to proceed and led to the description of zanamivir 10 years later [16, 17]. This was closely followed by the development of the first orally active NAI, oseltamivir, by Kim and colleagues [18, 19]. The NAIs entered into clinical practice in 1999, and, subsequently, oral oseltamivir has become one of the principal drugs of choice for managing seasonal influenza and pandemic stockpiling [2, 20, 21]. CLINICAL APPLICATIONS OF NAIS Zanamivir. The effectiveness of topical application to the respiratory tract of inhaled antivirals was demonstrated initially in animal models using amantadine and rimantadine, M2 ion channel inhibitors [22]. As an infectious diseases fellow, F.G.H. conducted the initial human studies of the safety and pharmacologic characteristics of nebulized amantadine [23] A subsequent placebo-controlled study of amantadine demonstrated significant, albeit modest, antiviral effects and clinical benefits in uncomplicated influenza [24]. Inhaled amantadine was associated with dose-limiting airway irritation, so this strategy needed to wait for an alternative drug; the experience, though, helped lay

the foundation for subsequent work with topical zanamivir. In the early proof-of-concept studies with zanamivir, it was demonstrated that intranasal drug, administered even once daily, was highly protective against intranasal viral challenge in experimentally exposed volunteers [25, 26]. However, naturally acquired influenza illness commonly involves the lower respiratory tract, with tracheobronchitis and small-airway dysfunction present in apparently uncomplicated disease. When zanamivir progressed to testing in natural influenza, there was legitimate uncertainty about where in the respiratory tract (nose, throat, tracheobronchial tree, distal airways, and/ or alveoli) a topical antiviral needed to be delivered to be effective for the prevention or treatment of natural influenza. Pharmacologic studies had established that orally inhaled zanamivir was deposited principally in the pharynx and, to a lesser extent, lower airways [27]. Consequently, one of the first field studies with zanamivir was a 4-way randomization to compare intranasal and inhaled administration for postexposure prophylaxis (PEP) in household contacts [28]. The influenza illness attack rate in the intranasal group was no different from that in the placebo group. This was consistent with earlier studies showing that intranasal interferon (IFN) did not protect against natural influenza [29–31]. By contrast, orally inhaled zanamivir alone or when combined with intranasal drug reduced illness rates by half [28]. Subsequent studies of orally inhaled zanamivir showed high levels of protection against influenza illness (efficacies of 80%–85%) during seasonal and PEP [32–34] (table 1) and ultimately led to its approval for prophylaxis in the United States and many other countries. Such intervention studies have demonstrated that antiviral administration to the nose alone is insufficient to protect against natural influenza and support the hypothesis that many influenza infections are acquired by droplets that are

Table 1. Laboratory-confirmed influenza illness prevention in household contacts with antiviral postexposure prophylaxis with or without index case treatment.

Treatment, antiviral [ref.] Postexposure prophylaxis without index treatment Amantadine [35]

Season (virus)

Age range studied

Reduction in secondary influenza illness, %

Adult prophylaxis regimen/comments

1967–68 (A/H2N2)

Adults/children 12 years old

100

1988–89 (A/not stated) 2000–01 (A/H3N2, B)

Adults/children Adults/children 15 years old

70

200 mg daily for 10 days

82

10 mg inhaled daily for 10 days

1998–99 (A/H3N2, B)

Adults/children 112 years old

89

75 mg daily for 7 days

Amantadine [35]

1968–69 (A/H3N2)

Adults/children 12 years old

6

Rimantadine [38]

1987–89 (A/H3N2, A/H1N1) Adults/children

Zanamivir [32]

1998–99 (A/H3N2, B)

Adults/children 15 years old

82

Oseltamivir [39]

2000–01 (A/H3N2, B)

Adults/children 11 year old

85

Rimantadine [36] Zanamivir [33] Oseltamivir [37] Combined index treatment and postexposure prophylaxis

a b

100 mg twice/day for 10 days

a

100 mg every 12 h for 10 days (resistance transmission not studied) 100 mg twice/day id for 10 days (resistance transmission found)

3b

10 mg inhaled daily for 10 days (no resistance transmission noted)

b

75 mg daily for 10 days (no resistance transmission noted)

Clinical influenza illness in contacts. Excludes contacts positive for influenza before prophylaxis.

deposited in the tracheobronchial tree or, perhaps, the pharynx. The same uncertainty existed regarding delivery of zanamivir for treatment of influenza. In an initial placebo-controlled study comparing orally inhaled zanamivir to a combination of inhaled and intranasal, early treatment (within 30 h of illness onset) resulted in both groups experiencing a 3-day reduction in illness duration [40]. The addition of intranasal dosing reduced nasal viral titers and nasal symptoms but did not speed overall recovery, compared with inhaled delivery alone. In a meta-analysis, inhaled zanamivir also reduced lower respiratory tract complications leading to antibiotic use but did not significantly reduce those in the upper respiratory tract [41]. Taken together, these observations highlight the importance of inhibiting viral replication in the lower respiratory tract, even in uncomplicated influenza. Oseltamivir. As with zanamivir, oseltamivir has been shown to have high levels of protective efficacy when used either in postcontact or seasonal prophylaxis [37,

39, 42, 43]. In household members exposed to influenza, the efficacy of PEP was 68%–89% (table 1) with no evidence of the emergence or transmission of oseltamivir-resistant variants in one study [37, 39]. The high protection levels observed with NAI prophylaxis have led to plans for possible use of mass targeted chemoprophylaxis in efforts to contain an emerging influenza pandemic [44, 45, 46]. The household-based studies of PEP with NAIs also confirmed the substantial risks of influenza transmission within families [47] and the epidemiologic importance of school-aged children as introducers of virus. In addition, further analysis of data from these studies has suggested that early oseltamivir treatment of ill index cases may be associated with some reduction in the risk of virus transmission to household contacts [48]. Studies in seasonal influenza have shown that early oseltamivir treatment can reduce lower respiratory tract complications, including pneumonia, and reduce hospitalizations from all causes in both previously healthy and at-risk ambulatory

adults [49, 50]. One controlled study of ambulatory children with influenza documented decreases in viral replication, illness measures, and complications, particularly new episodes of otitis media, leading to antibiotic use [51]. In high-risk nursing home patients, a retrospective evaluation found that the receipt of oseltamivir within 48 h of symptom onset was associated with a lower likelihood of being prescribed antibiotics, being hospitalized, or dying [52]. In more recent studies, treatment with oseltamivir, even when administered beyond 48 h of symptom onset in hospitalized patients, was associated with reduced mortality and shortened hospitalization [53, 54]. Studies of oseltamivir conducted in collaboration with Stephen Straus and colleagues at the National Institutes of Health have also helped elucidate the role of the host immune response in symptom pathogenesis of human influenza infection. In experimentally infected volunteers, the nasal levels of several mediators (e.g., interleukin-6, IFN-a, IFN-g, and tumor necrosis factor–a) increased after infection PERSPECTIVE • JID 2007:196 (15 July) • 183

and correlated with both measures of illness and of viral replication [55, 56]. Oseltamivir administration initiated at 28 h after viral challenge was associated with a significant reduction in the magnitude and duration of viral replication, as well as with concomitant reductions in symptom scores and modulation of nasal proinflammatory mediator responses [42]. Even greater reductions in these measures were noted in studies involving intravenous administration of zanamivir before viral inoculation [56, 57]. Taken together, these results suggest a direct linkage among viral replication, cytokine and chemokine responses, and symptom pathogenesis. Such observations are especially pertinent in light of the findings of cytokine dysregulation, so-called cytokine storm, in avian influenza H5N1 human infections [58, 59]. De Jong et al. [60] have shown that prolonged, high-level viral replication is central to excessive mediator responses, disease pathogenesis, and mortality risk in H5N1 disease. Specifically, this study demonstrated strong positive correlations between H5N1 RNA levels in the pharynx at time of hospital admission and elevated plasma levels of multiple cytokines and chemokines. It is likely that active viral replication is driving these exaggerated host responses, such that early control of replication is essential to improve outcomes. In this regard, the published experience with oseltamivir for the treatment of patients with illness caused by H5N1 virus is limited, with no clear reductions in mortality [59, 61]. The apparent lack of benefit may be due to a variety of reasons, particularly late initiation of therapy after the occurrence of pulmonary injury, given that most of these patients presented at the end of the first week of illness with progressive viral pneumonia [61]. Recent findings suggest that early treatment reduces mortality [62]. Other possible contributory factors to poor responses include prolonged replication of virus in a immunologically naive host, extrapulmonary viral dissemination in some patients, inadequate dosing and/ 184 • JID 2007:196 (15 July) • PERSPECTIVE

or oseltamivir absorption because of gastrointestinal dysfunction in patients who were critically ill or had diarrhea, and the emergence of oseltamivir resistance during therapy [63]. In animal models of H5N1 infection, higher oseltamivir doses are often required for the control of viral replication [64, 65] A controlled study of higher dose oseltamivir treatment for avian or severe human influenza is one of the first studies planned by the Southeast Asia Influenza Clinical Research Network (see below). ANTIVIRAL DRUG RESISTANCE The high mutation rate of RNA viruses, such as influenza virus, allows the selection of viruses in the laboratory and in clinical practice that are resistant to currently used antiviral drugs. Resistance in the laboratory to amantadine and rimantadine (adamantanes) was recognized shortly after their discovery as antiinfluenza agents in the early 1960s [66]. One project of F.G.H. during his infectious disease fellowship was the development and application of a simple plaque-inhibition assay in MDCK cell monolayers [67] to detect adamantane resistance in clinical isolates. Despite its limitations and the development of other phenotypic and genotypic assays [68], this method remains in use today. The study of resistant variants by Alan Hay et al. [69] was key to understanding the mechanisms of antiviral action of the adamantanes and the role of the M2 pro-

tein in viral replication. This tetrameric protein forms an ion channel that mediates an influx of hydrogen ions into the virion during early replication that promotes dissociation of viral RNA segments and allows them to move to the nucleus for subsequent replication [73]. It also plays a role in later stages of replication. Adamantanes block this channel function, but single amino acid changes at one of 4–5 sites in the transmembrane domain lead to high-level resistance to the entire class. The most common change in clinical isolates is a substitution of a bulkier asparagine for serine at position 31. Clinical studies in the 1980s found that resistant variants emerged rapidly in rimantadine-treated children and adults [74, 75]. Resistant variants have been detected at high frequencies (table 2) among isolates from patients who have been treated with adamantanes, and householdand institution-based studies have demonstrated the potential of these variants to be transmitted to close contacts and to cause failures of chemoprophylaxis (table 1) [38, 76]. Higher frequencies of resistance emergence have been observed in hospitalized children when sensitive techniques are used [80] and in immunocompromised hosts [72 77, 78]. In comparison, frequencies are lower but not inconsequential with oseltamivir (table 2) [70, 71, 79]. Importantly, in immunocompromised hosts, dual resistance to oseltamivir and M2 ion channel inhibitors has emerged during sequential therapy of in-

Table 2. Detection of drug-resistant influenza during or shortly after antiviral treatment. Frequency of resistance, % Study population [ref.] Outpatient adults Outpatient children Inpatient children Immunocompromised patients

Oseltamivir

M2 inhibitor

0.4 5.5 16–18

∼30 ∼30 80

Yes

133

NOTE. Data taken from refs. [70–72, 74, 75, 77, 79, and 80].

fluenza A infections—an approach that should be avoided [72]. The frequency of resistance emergence is an important variable, but transmission fitness is the key to whether such variants might become a public health problem. This potential was elucidated initially in randomized, controlled household-based studies of chemoprophylaxis to prevent influenza transmission. Adamantanes and NAIs are effective when used for PEP in households, when the index case is not concurrently treated and the circulating strain is susceptible (table 1) [33, 35, 36, 37]. By contrast, 2 studies, in which ill index cases received the same drug (amantadine or rimantadine) as that used for PEP in healthy contacts, found very little protection (table 1) [38, 81]. A multicenter study that included households in Charlottesville, Virginia, documented that transmission of resistant variants from the treated index case caused failures of prophylaxis in contacts, who experienced typical influenza illness [38]. By contrast, oral oseltamivir and inhaled zanamivir provided high levels of protection and no resistance transmission in subsequent studies [32, 39]. Such findings indicate important differences in these drug classes with respect to the risk of resistance emergence and transmission. Another approach to testing the transmission risk of resistant variants is use of animal models. Ferrets are susceptible to nonadapted human influenza viruses and continue to be used for studies of influenza pathogenesis, transmission, and antiviral efficacy. Sweet et al. [82] compared the replication of an adamantane-resistant H3N2 variant harboring the serine to asparagine mutation at position 31 (S31N) with its susceptible parental virus in ferrets and determined that these viruses grew comparably and caused the same degree of febrile and nasal inflammatory cell responses. This variant and other adamantane-resistant ones were fully replication competent and pathogenic. Earlier studies by Bean et al. [83] in chickens had similar findings. These observations suggested

that such variants could compete with wild-type strains in the absence of selective drug pressure and circulate in the community. Of note, recent studies with oseltamivir-resistant influenza A viruses in ferrets have found that some neuraminidase mutations lead to loss of replication competence and transmissibility, whereas others can be transmitted from one ferret to another [84]. Adamantane resistance was not observed to a significant extent in community isolates until 2003, when global surveillance of H3N2 subtype viruses by the US Centers for Disease Control and Prevention detected a dramatic surge in the frequency of resistance, from a background rate of 1%–3% to 170% in China [73, 85]. This was followed by increases in the frequency of detection of resistant variants, all of which harbored the same S31N mutation, in multiple countries. In the United States the frequency jumped from ∼15% in early 2005 to 190% in late 2005 and early 2006 [68]. The frequency of resistance due to this same mutation has also increased in H1N1 isolates [86]. These surveillance results show the potential for rapid global circulation of adamantane-resistant influenza A viruses in the absence of continuing significant selective drug pressure. Furthermore, the widespread circulation of adamantane inhibitor–resistant human influenza (especially among H3N2 and, less often, H1N1) and many H5N1 strains makes this class of drugs unreliable in current clinical practice [68, 85]. Given these findings, concern has emerged regarding the development of resistance to NAIs, in particular oseltamivir. No NAI resistance was found before clinical use [87]. To date the oseltamivir-resistance mutations that have emerged during treatment predominantly occur at 3 sites (R292K in N2, E119V in N2, and H274Y in N1) [2]. The H274Y mutation in N1 viruses, first described by Gubareva et al. [88] in the context of experimental human influenza, is of particular concern. Two (4%) of 50 adult volunteers inocu-

lated with an H1N1 virus and given oseltamivir shed these resistant variants, in both instances associated with upswings in viral loads in nasal lavages, although not symptoms [88]. This mutation confers high-level resistance to oseltamivir with a 1400-fold reduction in susceptibility. The frequency of development of this mutation emerging during treatment is reportedly as high as 16% in children infected with H1N1 infection [79]. More ominously, de Jong et al. [63] reported detection of the H274Y resistant variant during or shortly after a standard course of oseltamivir in 2 (25%) of 8 human H5N1 infections, in whom the detection of resistance emergence appeared to be associated with fatal outcome. However, the H274Y mutation appears to come at a biological cost to the virus. Replication efficiency is reduced in cell culture, and infectiousness is reduced in relevant animal models [89, 90]. However, this oseltamivir-resistant variant is transmissible between experimentally infected ferrets [84] and has been detected in community isolates of H1N1 viruses [91, 95]. The clinical importance of a second mutation (N294S) associated with reduced oseltamivir susceptibility (10–30-fold) in H5N1 virus remains to be determined [90]. In general, these neuraminidase mutations conferring resistance to NAIs appear to alter the fitness of influenza virus and their transmissibility, which results in different public health implications than those for M2 ion channel inhibitors [92, 93]. To evaluate changes in susceptibility to current NAIs over time in community isolates, the Neuraminidase Inhibitor Susceptibility Network has been conducting prospective surveillance in collaboration with the World Health Organization Global Influenza Surveillance Network [94]. The frequency of resistance to oseltamivir or zanamivir was !0.5% during the first 3 years of monitoring conducted after approval of the drugs in 1999 [95]. Japan has had the highest per capita use of oseltamivir in the world. During a single influenza season in 2003–2004, ∼6 million PERSPECTIVE • JID 2007:196 (15 July) • 185

treatment courses of oseltamivir were given, sufficient to treat ∼5% of the population [96]. Of nearly 1200 community H3N2 isolates collected across Japan, only 3 variants (0.3%) were found to have phenotypic resistance and to harbor resistance mutations (2 E119V and 1 R292K) [96]. This very low level of detectable resistance in the community despite substantial drug use is reassuring. However, recent community surveys in Japan have suggested somewhat higher levels of transmission of resistant influenza A and B viruses [91, 97], so that ongoing surveillance of NAI resistance in both human and animal influenza viruses is essential. The current and investigational NAIs are sialic acid analogues that differ somewhat in chemical structure. One result is that differing patterns of drug interaction with conserved residues in the active enzyme translate into different patterns of cross-resistance. As an example, the histidine to tyrosine mutation at position 274 in N1 neuraminidase (H274Y) confers 1400-fold reduced susceptibility for oseltamivir but is fully susceptible to zanamivir and A-315675, a novel NAI [98, 99]. Similarly peramivir, another investigational NAI, loses substantial activity in the presence of the H274Y mutation, but the observed inhibitory levels are well below those that can be achieved by parenteral administration. The recent solution of the crystal structure of N1 neuraminidase from an H5N1 virus has provided an understanding of the structural basis for the differences in drug interactions with the active enzyme site and pointed to the possibility for designing even more potent inhibitors [100]. ANTIVIRAL COMBINATIONS AND NEW THERAPIES Combination therapy is now standard practice for several viral infections but represented a novel concept in the mid-1970s, especially with regard to influenza. One of the earliest references on this subject came from Russian investigators in the late 1960s, who reported that a combination 186 • JID 2007:196 (15 July) • PERSPECTIVE

of amantadine and interferon exerted increased antiviral effects in vitro [101]. Further in vitro studies by F.G.H. during his fellowship showed that combinations of adamantanes and ribavirin [102] and, later, a triple regimen of adamantanes, ribavirin, and IFN-a [103] demonstrated enhanced activity against influenza A. This combination would still be a relevant consideration for clinical study in infections by amantadine-susceptible strains. Other groups have studied various combinations of therapy in animal models [22, 104–106]. The sole controlled clinical trial was conducted through the National Institute of Allergy and Infectious Disease (NIAID)–sponsored Collaborative Antiviral Study Group [107]. This was a prospective, randomized, saline aerosol–controlled comparison of the effects of oral rimantadine and nebulized zanamivir versus rimantadine monotherapy in adults hospitalized with serious lower respiratory tract disease caused by influenza virus. The

trial was underenrolled, in part because of the availability of an alternative oral treatment (oseltamivir) during the study, but the limited virological results suggested a trend toward a shorter duration of viral detection in the combination group. This group also had less protracted cough, a finding that supported the conclusion that zanamivir administered by nebulizer was generally well tolerated in this high-risk population. Of particular interest, the only adamantane-resistant variants were observed in the monotherapy group. The finding of reduced resistance emergence with a combination of M2 ion channel and NAIs has been confirmed recently in preclinical studies [105]. However, this combination would not be beneficial against viruses already possessing adamantane resistance. Given the prevalence of antiviral resistance, alternative therapies and agents for influenza are being sought (table 3). A number of new compounds—including

Table 3. Investigational antiinfluenza agents that have shown activity in animal models or are of clinical investigative interest and their routes of administration.

Agent

Route of administration [ref.]

NA inhibitors Peramivir Zanamivir A-32278

IV/IM [114] IV [57] Oral [115]

Long-acting NA inhibitors CS8958/R-118958

Topical

Zanamivir dimmers Conjugated sialidase DAS181

Topical [116] Topical [11]

Hemagglutinin inhibitors Cyanovirin-N Sialylglycopolymer Entry blocker Polymerase inhibitors Ribavirin Viramidine siRNA T-705 Protease inhibitor aprotinin Antibodies Interferons

Topical Topical Topical [109] Topical, IV, oral Oral Topical, IV [111] Oral [110] Topical IV, IM [108, 112, 113] IM, SC

NOTE. IM, intramuscular, IV, intravenous; NA, neuraminidase; SC, subcutaneous; siRNA, small interfering RNA.

conjugated sialidase [11], hemagglutinin inhibitors [109], small interfering RNA [111], polymerase inhibitors [110], and protease inhibitors—have shown activity in animal models, and several of these, principally parenteral or topically applied long-acting NAIs, are in clinical trials. There are several agents with novel mechanisms of action that are scheduled to enter clinical trials soon, and recent reports on the possible value of serotherapy, including the activities of polyclonal and monoclonal antibodies in animal models of H5N1 infection, are encouraging [108, 112, 113]. At present, there is no injectable antiinfluenza agent that is yet approved for use, although both intravenous zanamivir [57] and parenteral peramivir [114] are in active development. Parenteral administration is a strategy to reliably deliver high drug concentrations to seriously ill patients. In initial proof-of-principle studies, intravenous zanamivir was found to be highly active in experimentally infected volunteers, with virtually complete protection against measures of infection and illness [57]. Of note, the intravenous regimen used provides peak plasma concentrations that are ∼100-fold higher than those observed with standard oral doses of oseltamivir [27]. It remains to be determined whether such high drug exposure will translate into more immediate control of replication and clinical benefit in severe influenza. To take influenza treatment research forward, a new collaborative network called the Southeast Asia Influenza Clinical Research Network [117] has recently been established. This multicenter collaborative group currently includes health care institutions in Vietnam, Thailand, and Indonesia, as well as the National Institutes of Health Clinical Center and several international partners, including NIAID, Oxford University, Wellcome Trust, and the World Health Organization. It will initiate studies of the pathogenesis and management of both avian and severe human influenza. This network builds on existing infrastructure and will be a part-

nership of major research institutions in target countries. Such international teamwork is especially important given the current need for better management approaches for seasonal and avian influenza, in addition to fostering influenza pandemic preparedness [118].

Acknowledgments We thank the many volunteers and patients who participated and who continue to participate in the clinical trials that have allowed progress in this field. We also thank the large number of academic, governmental, and pharmaceutical industry colleagues who have been key collaborators with the senior author on the studies described in this review, including Stephen Straus, Scott Fritz, Elizabeth Higgs, Michael Polis, Henry Masur, John Beigel, and Clifford Lane (National Institute of Allergy and Infectious Diseases [NIAID], Bethesda, MD); Richard Whitley and John Gnann (NIAID Collaborative Antiviral Study Group, University of Alabama–Birmingham); Maria Zambon (Health Protection Agency, London, UK); Alan Perelson and Niko Stilianakis (Los Alamos National Laboratory, Los Alamos, NM); Jeremy Farrar and Menno de Jong (Oxford Research Unit, Ho Chi Minh City, Vietnam); Laurent Kaiser (University of Geneva, Geneva, Switzerland); Fred Aoki (University of Manitoba, Winnipeg, Canada); Arnold Monto (University of Michigan, Ann Arbor); R. Gordon Douglas, Jr., and John Treanor (University of Rochester, Rochester, NY); and Larisa Gubareva, Monica Lobo, Birgit Winther, Jack Gwaltney, Jr., Robert Powers, Rebecca Tominack, Carol Sable, Steven Sperber, Eurico Arruda, David Calfee, and Michael Ison (University of Virginia, Charlottesville). We also acknowledge the essential contributions of staff in the Respiratory Disease Study Unit at the University of Virginia over many years, particularly Gloria Hipskind, Carolyn Crump, and Diane Ramm.

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