INTRODUCTION

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The highly infectious nature of influenza virus has the potential to put whole communities at risk of infection. Particularly vulnerable are closed populations such as elderly nursing home residents (24) and armed forces. Influenza virus infections are primarily controlled by the prophylactic use of vaccines (4, 16). However, the constant antigenic change associated with influenza virus, coupled with the often inadequate protection afforded by vaccines, especially in those with inefficient immune responses (elderly, very young, and immunocompromised), highlights the need for an effective antiviral agent. In addition, the efficacy of available vaccines in the face of the emergence of new antigenic subtypes (as shown by the recent outbreaks of H5N1 [6] and H9N2 infections in humans) would be minimal. The identification of an anti-influenza virus chemotherapeutic agent that could be used prophylactically in high-risk patients with either a poor immune response to vaccines (e.g., the elderly) (3, 24, 29) or as an adjunct to vaccines in an outbreak scenario where cover is required while immunity develops is clearly of great importance (8, 17). In addition, such an agent would be invaluable on the emergence of a new antigenic subtype.

The utility of the currently available anti-influenza drugs, amantadine and rimantadine (7, 29), has been limited by the lack of activity against influenza B viruses (8, 32). Both drugs have been shown to be effective in preventing laboratory infections with influenza A viruses in volunteers (25, 28), and amantadine has proven effective in controlling an outbreak of influenza in a nursing home (1) when given prophylactically. However, the emergence of viruses resistant to these agents is both rapid and significant (14, 18). Thus the concomitant prophylactic and therapeutic use of these agents, in closed or semiclosed communities, may be contraindicated. In addition, these agents have unwanted neurological side effects (11).

The development of influenza virus-specific neuraminidase inhibitors has been a major breakthrough in the treatment of this disease. These may be typified by the inhaled inhibitor zanamivir and the oral inhibitor GS4104 (26, 31), which act by the inhibition of the viral surface glycoprotein neuraminidase (sialidase) and which were developed by a process of rational design (30). Zanamivir (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid; Relenza; GG167) has been shown to be effective for the duration and severity of illness in clinical trials (12, 13, 27). The efficacy data in these studies were predicted by the studies of animal models of influenza virus infection. Thus zanamivir has been shown to be an effective antiviral in both mouse (10, 22) and ferret (23) animal models of influenza virus infection. Early studies, however, showed that when doses of zanamivir were given up to 18 h before infection and for 4 days postinfection, the overall efficacy was enhanced compared to a postinfection regimen alone (22). Similar results were obtained in phase I and phase II clinical trials (12, 13), in which the addition of a single dose 4 h before influenza virus challenge, in conjunction with doses twice or six times daily given therapeutically, gave superior reductions in total symptom score and a nasal virus titer reduction. However, the efficacy of this compound as a prophylactic agent alone has not previously been examined. The experiments reported here investigate the prophylactic efficacy of a single intranasal dose of zanamivir in both mouse and ferret models of influenza A virus infection. These data suggest that the frequency of zanamivir administration could be reduced for prophylactic use compared with the frequency of administration for the treatment of symptomatic influenza. Clinical studies, in which daily administered zanamivir has shown a positive protective effect when given prophylactically, have now been reported (15).

	MATERIALS AND METHODS

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Reagents. Unless stated otherwise, all reagents, media components, tissue culture cell lines and methods, and virus assays by enzyme-linked immunosorbent assay (ELISA), were as described previously (20-22, 33).

Influenza viruses. Influenza viruses A/Singapore/1/57 (H2N2) and A/Mississippi/1/85 (H3N2) were supplied by A. J. Hay and were typed by A. Douglas (National Institute for Medical Research, Mill Hill, London, United Kingdom). Stock virus pools were generated in fertile hens' eggs.

Influenza virus infection in mice. The protocol for infecting mice has been described previously (30). Mice, anesthetized by the inhalation of ether, were inoculated in the external nares with 50 µl of a virus suspension containing 10^4.5 50% tissue culture infective doses (TCID₅₀)/ml. This inoculum had previously been determined to give high titers of virus 24 h postinfection.

Treatment procedure and regimen. A single dose of zanamivir was administered at time points between 3 and 240 h prior to infection. The compound (dissolved in phosphate-buffered saline [PBS]) was given intranasally in a volume of 50 µl to groups of 7 to 10 mice anesthetized by inhalation of ether. Sham-treated control animals received PBS only.

Assay of virus in lung homogenate samples. Individual lung homogenates were prepared from mice culled 24 h postinfection. As described previously (2, 22, 30) the titers of lung virus were assayed by ELISA. Reductions in virus titer were expressed as a percentage of values from PBS-treated control animals. The Kruskal-Wallis rank sum test was used to determine the statistical significance of treatment regimens in reducing lung virus titers.

Autoradiography. ¹⁴C-radiolabelled zanamivir (5 MBq/mg; >99% radiopurity), labelled at the 4-guanidino position, was given intranasally to two anesthetized mice per group, in a volume of 50 µl. At 10-min, 45-min, 90-min, and 24-h time points following dosing, each group of mice was sacrificed and frozen in liquid nitrogen prior to analysis by whole-body autoradiography.

Influenza virus infection in ferrets. While under light anesthesia (isofluorane), groups of four ferrets (female; 0.75 to 1.2 kg) were infected by intranasal instillation of 250 µl of influenza virus A/Mississippi/1/85 containing 10^4.5 TCID₅₀/ml, as described previously (30).

Treatment procedure and regimen. While under light anesthesia, animals received a single intranasal dose of 12.5 mg (0.25 ml/kg of body weight) of zanamivir/kg of body weight 48 h prior to infection. Virus-infected control animals were sham dosed with distilled water. Ferret weights were recorded daily for 8 days. Nasal washings were taken on days 1 to 8 following challenge, as described previously (19), and the washings were used for estimation of virus titer by ELISA and turbidity (19). The area under the curve (AUC) for virus titers for the samples taken on days 1 to 8 for each ferret was calculated from the antilog values obtained from the ELISA log₁₀ values. This value was then converted back to a log₁₀ value, and the log₁₀ geometric mean AUC value for the group of ferrets was calculated. The Duncan multiple-range test was used to determine statistical significance of treatment regimens in reducing lung virus titres. Temperature profiles of ferrets were recorded every 10 min by implanted telemetric transmitters (Dataquest; Data Sciences, St. Paul, Minn.), prior to and up to 8 days following infection (30). AUC values were calculated for the period of pyrexic response (0 to 96 h); AUCs were computed as the area above and below the preinfection mean. Percentage reductions in pyrexia compared with controls were calculated from the AUC values. Pyrexia was also defined as the elevation of core body temperature by two standard deviations (or more) above the preinfection mean temperature for a period of at least 12 h during the postinfection period. Blood samples were taken 5 days preinfection and 21 days postinfection for the determination of neutralizing serum antibody levels to the infecting virus (19).

	RESULTS

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Efficacy of prophylactic regimens of zanamivir in mouse models of influenza A infection. Single intranasal doses of between 12.5 and 1.56 mg of zanamivir/kg given 51 h prior to infection gave statistically significant reductions in lung virus titers (P  0.01), compared with titers in vehicle-treated control animals (Table 1). In addition a good dose response was evident (99.13 to 90.83% reductions compared with control titers). However, single intranasal doses of less than 12.5 mg/kg given 7 days prior to infection did not significantly reduce lung virus titers of mice (Table 2).

View larger version (120K): [in this window] [in a new window]   FIG. 1.   Autoradiography sections of mice dosed intranasally with 14C-labelled zanamivir 90 min and 24 h prior to sacrifice. GI, gastrointestinal.

Efficacy of prophylactic regimens of zanamivir in a ferret model of influenza A infection. In ferrets treated with a single 12.5-mg/kg intranasal dose of zanamivir 48 h prior to infection, nasal wash virus titers were reduced by 60% (AUC between days 1 and 8) compared with those for vehicle-dosed animals (Table 4). However, although this value was not statistically significant (P = 0.051), statistically significant reductions in virus titers (P  0.0005) were obtained on days 1 and 2 postinfection (Table 4). These results are not surprising, as later samples from an animal given a single prophylactic dose prior to infection are less likely to show reductions due to clearance, albeit slow.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Core body temperatures of ferrets given a single intranasal dose of 12.5 mg of zanamivir/kg 48 h prior to infection with influenza virus A/Mississippi/1/85. Solid line, mean; dotted lines, 2 standard deviations.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Core body temperatures of control ferrets infected with influenza virus A/Mississippi/1/85. Solid line, mean; dotted lines, 2 standard deviations. Solid line, mean; dotted lines, 2 standard deviations.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Zanamivir, a potent inhibitor of influenza virus neuraminidase, has been shown in both animal models and clinical trials to be an effective therapeutic agent for influenza. Recent clinical studies of healthy adults have shown that once daily administration of zanamivir is an effective prophylaxis dosing strategy for the prevention of influenza (15). We report here the prophylactic activity of this compound in animal models of influenza virus infection and show that doses as low as 1.56 mg of zanamivir/kg given 51 h prior to infection significantly reduce lung virus titers in infected mice, compared with untreated animals. In addition, in mice, a single intranasal dose of zanamivir given up to 7 days prior to infection with influenza A virus was effective in reducing lung virus titers. This observation was unexpected since in this species the compound is rapidly cleared from plasma (an elimination half-life of 10 min following intravenous administration [22]), suggesting that there may be a substantial "depot" of at least part of the dose in the lung after topical administration. In humans, zanamivir is also rapidly eliminated when given intravenously (half-life = 1.6 h [9]). Following intranasal drops or inhaled administration, maximum serum concentrations were reached within 2 h postdose (9). However, the terminal-phase half-lives (3.4 and 2.9 h, respectively) suggested the presence of either a slow or complex absorption process in humans (9). On the basis of urine excretion data, the bioavailabilities following intranasal drops and inhaled administration were estimated to be approximately 10 and 25%, respectively (5, 9).

The retention of zanamivir in the respiratory tract tissues of mice is supported by whole-body autoradiography studies, in which radiolabelled zanamivir remained detectable in the lungs of mice up to 24 h after a single intranasal dose.

Similarly, in ferrets, a single intranasal dose 48 h prior to infection was able to significantly reduce the clinical signs of infection (pyrexia, nasal wash turbidity) and viral shedding, compared with those for vehicle-treated animals. This provides further evidence of the retention of compound in the respiratory tract, since elimination from the plasma in ferrets is again rapid (43-min elimination half-life after intravenous administration [unpublished in-house data]).

The significant prevention of symptoms in ferrets, however, did not affect the generation of a serum antibody response specific to the infecting virus. This is necessary since a good serum antibody response is important in protection from reinfection with homologous virus and has some protective value against other antigenically related subtypes.

In conclusion, the data suggest that infrequent administration of zanamivir may be a successful strategy for prophylaxis, in the clinical situation.