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Rapid Antiviral Effect of Inhaled Zanamivir in the Treatment of Naturally Occurring Influenza in Otherwise Healthy Adults

  1. Guy Boivin1,
  2. Nathalie Goyette1,
  3. Isabelle Hardy1,
  4. Fred Aoki2,
  5. Anthony Wagner3 and
  6. Sylvie Trottier1
  1. 1Infectious Disease Research Center, Centre Hospitalier Universitaire de Québec, and Division of Microbiology, Laval University, Québec
  2. 2Departments of Medicine, Medical Microbiology, Pharmacology, and Therapeutics, University of Manitoba, Winnipeg
  3. 3Glaxo Wellcome, Mississauga, Canada
  1. Reprints or correspondence: Dr. Guy Boivin, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, RC-709, 2705 Blvd. Laurier, Sainte-Foy, Québec, Canada G1V 4G2 (Guy.Boivin{at}crchul.ulaval.ca).

  1. Presented in part: 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, 26 September 1999 (abstract H021).

 
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Abstract

The antiviral and clinical effects of inhaled zanamivir (10 mg twice daily for 5 days, started within the first or second day of a flulike illness) were evaluated in a randomized, placebo-controlled trial during the 1997–1998 influenza season in Canada. Pharyngeal secretions were collected with swabs every 12 h during 6 days, and symptoms were self-evaluated twice daily during 14 days. After only 12 h of treatment (1 dose), median virus titers decreased by 1.0 log10 TCID50/mL in the zanamivir group (n = 17), compared with a 0.42-log10 increase in the placebo group (n = 10; P = .08). This was associated with a 4.5-day (47.4%) reduction in the median time to alleviation of all significant flu symptoms in the zanamivir recipients (P = .03 after adjusting for the initial virus titer and the time between onset of symptoms and treatment). Resistance to zanamivir was not detected in virus isolates by either phenotypic or genotypic assays.

Amantadine has been available in the United States for the treatment of influenza for >3 decades. Nevertheless, its use is limited by its narrow spectrum of activity (effective against influenza A viruses only), frequent side effects, and rapid and frequent emergence of viral resistance [1]. A new class of anti-influenza agents, the neuraminidase (NA) inhibitors, has recently been developed [2, 3]. Zanamivir is a promising compound of this class of agents, with in vitro activity against influenza A viruses 500–1000-fold greater than that of amantadine, as well as a broader spectrum of activity including influenza B viruses [2, 3]. Recent clinical trials of topically applied (inhaled with or without intranasal) zanamivir have showed clinical efficacy, consisting in a reduction in duration of symptoms of 1.0–2.5 days versus placebo, with greater benefits in more ill (febrile) patients and those who begin treatment earlier [4, 5]. Herein we evaluated the antiviral effects of zanamivir in relationship with clinical efficacy in a subset of adolescents and adults with naturally occurring influenza A virus infections. Drug susceptibility and genotypic studies were also done on serial isolates recovered during treatment for assessing the emergence of viral resistance.

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Subjects and Methods

Study protocol

Subjects enrolled in Québec and Winnipeg as part of Glaxo Wellcome protocol NAIA3002 were selected for detailed virologic studies. This was a double-blind, randomized, placebo-controlled multicenter study conducted during the 1997–1998 winter to investigate the efficacy and safety of inhaled zanamivir (Glaxo Wellcome, Mississauga, Canada) at a dosage of 10 mg twice daily for 5 days in the treatment of symptomatic influenza virus infections [5]. Briefly, healthy persons ⩾12 years old and high-risk subjects (defined as elderly patients or those with chronic respiratory, cardiovascular, or renal diseases) were enrolled if they were able to take the first dose of study medication on the first or second day of their influenza-like symptoms. An influenza-like episode was defined by the presence of fever (temperature ⩾37.8°C or ⩾37.2°C for subjects ⩾65 years old) and at least 2 of the following—headache, myalgia, sore throat, and cough—at a time influenza was circulating in the community. At the screening visit, a physical examination was conducted and a throat swab was collected. Serial swabs were then obtained by a research nurse every 12 h, prior to each dose of zanamivir. Swabs were immediately placed in 2 mL of a virus transportation medium (Cellmatics; Difco, Detroit) and kept at 4°C for a maximum of 36 h before cell inoculation in a central laboratory in Québec. Patients' symptoms and fever were self-assessed on a diary card by use of a 4-point scale 4 times daily during treatment and then twice daily for another 9 days. The primary end point of the clinical trial was the length of time to alleviation of all clinically important symptoms, as defined by no fever and other flu symptoms recorded as absent or mild for at least 24 h.

Laboratory procedures

On receipt, an aliquot (500 μL) of the virus transport medium was diluted in Hanks' balanced salt solution. Eight 10-fold dilutions were inoculated in quadruplicate onto MDCK cells in 24-well plates to determine the TCID50. Another aliquot of 200 μL was inoculated in a vial containing MDCK cells for hemadsorption testing and viral typing by use of monoclonal antibodies (BioWhittaker, Walkersville, MD). A nested multiplex polymerase chain reaction (PCR) assay was done on frozen samples by use of sets of primers for influenza A virus H1, influenza A virus H3, and influenza B virus, as previously reported [6]. Susceptibility to zanamivir, reported as the IC50, was assessed by an NA assay [3] and a plaque reduction (PR) test [7]. The NA and hemagglutinin (HA)-1 genes of selected H3N2 virus isolates were reverse-transcribed and PCR-amplified (C-Therm Polymerase One-Step RT-PCR System; Boehringer, Mannheim, Germany) with NA primers 14/1420 [8] or HA1 primers 7/1184 [9]. PCR products were sequenced by use of a cycle sequencing kit (Big Dye Terminator Cycle Sequencing Ready Reaction; Perkin-Elmer Applied Biosystems, Foster City, CA) and an automated DNA sequencer (ABI Prism 377 DNA Sequencer; Perkin-Elmer).

Statistical analysis

All analyses were done with SAS systems (version 6.12; SAS Institute, Cary, NC) and procedures.

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Results

Clinical efficacy of zanamivir

Thirty-five patients were enrolled in the trial; of these, 27 (77%) had an influenza virus infection laboratory-confirmed by both culture and PCR (100% concordance on day 1). All subjects had influenza A virus H3 infections as demonstrated by multiplex PCR. Of the subjects with laboratory-confirmed infections, 10 (37%) received a placebo and 17 (63%) received zanamivir. Three influenza virus-positive high-risk subjects were enrolled (2 in the placebo group and 1 in the zanamivir group). The median time to alleviation of all flu symptoms for influenza virus-positive subjects was 9.5 days in the placebo group and 5.0 days in the zanamivir group (47.4% difference; P = .35). By use of a regression model (lifereg procedure in SAS systems, SAS Institute, Cary, NC) to adjust for censoring time and 2 important variables, that is, the initial virus titer and the time between onset of symptoms and treatment, the adjusted median times to alleviation of symptoms for the placebo and zanamivir groups were, respectively, 11.6 and 5.3 days, which represents a 54.0% reduction (P = .03).

Antiviral effect of zanamivir

After 12 h of treatment, the median virus load increased by 0.42 log10 TCID50/mL for placebo recipients, compared with a 1.0 log10 decrease for zanamivir patients (P = .08). At 24 h, the median virus load was similar to the baseline value for the placebo group but was still 1 log10 lower than the pretherapy level in the zanamivir group (P = .35). Mean virus titers at serial 12-h time points were also calculated after adjusting for the initial virus titer (which was higher in the zanamivir group) and the time between onset of symptoms and treatment (table 1). At most time points, there was a trend toward a lower virus load in the zanamivir group than in the placebo group, although the difference was significant only at 96 h (P = .04 after adjusting for the 2 variables). Similarly, the mean area under the curve of virus titers for the zanamivir group was lower than that of the placebo group (199.08 vs. 219.82 log10 TCID50 × days/mL), but the difference did not achieve statistical significance (P = .27 after adjusting for the 2 variables). Zanamivir also reduced the median time to the last positive culture by 18 h (96 vs. 78 h) and the median time to the last positive PCR result by 12 h (108 vs. 96 h) compared with placebo (neither analysis was statistically significant). Finally, the median maximum peak titer was 1 log10 lower in the zanamivir group than in the placebo group (3.38 vs. 4.33 log10 TCID50, respectively).

Table 1
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Table 1

Mean influenza virus titers in zanamivir and placebo recipients by time after beginning treatment.

Emergence of zanamivir resistance

By the NA assay, the mean IC50 values of pretherapy and last positive isolates for the zanamivir recipients were 4.15 and 4.76 nM, respectively (table 2). Only 2 subjects (nos. 2 and 11; 11.8%) shed final isolates that showed a ⩾2-fold increase in IC50 (3.8- and 2.4-fold) compared with pretherapy values. These 2 subjects were among the 3 who still had positive cultures on day 5, although their virus titers were low (⩽1 log10 TCID50/mL). By use of the PR assay, pre- and posttreatment mean IC50 values were 0.05 and 0.06 μM, respectively (table 2). Four (23.5%) of the treated subjects (nos. 6, 8, 12, and 17) shed posttreatment isolates with IC50 values ⩾2-fold (range, 2.50–4.16 times) those of the initial strains. For genotypic studies, we elected to sequence the NA genes of the isolates from the 2 subjects (nos. 2 and 11) with a ⩾2-fold change in zanamivir IC50 values by the NA assay. Pretherapy and last positive isolates from these 2 subjects had identical NA sequences. We also sequenced the HA1 genes of paired isolates from all subjects (n = 8) who were still shedding the virus on day 4 or 5, including the 2 subjects (nos. 8 and 17) whose isolates had the highest IC50 values by the PR assay. Again, all paired isolates from those patients had identical HA1 sequences with the exception of patient 15. The last positive isolate of the latter patient contained a single nucleotide mutation resulting in an Arg150 → Lys change. However, both HA1 sequences directly amplified from the initial and last positive original clinical samples were identical and were coded for a Lys at position 150. In summary, therefore, we found no evidence of resistance by use of either phenotypic or genotypic assay.

Table 2
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Table 2

Susceptibility results of initial (a) and of last positive (b) influenza virus isolates from patients who received zanamivir.

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Discussion

We performed a detailed analysis of the virologic effects of zanamivir as part of a clinical trial conducted in Canada during the 1997–1998 influenza season. Although a limited number of subjects were evaluated, 3 important conclusions can be drawn. First, zanamivir produced a rapid antiviral effect following inhalation, and this was noted as early as 12 h after beginning treatment. Second, the decrease in virus load induced by zanamivir correlated with a significant reduction in the median time to alleviation of symptoms. Last, neither phenotypic nor genotypic assays detected any evidence of emergence of zanamivir-resistant strains during therapy.

Because influenza virus titers significantly correlate with fever and systemic symptoms and because virus titers peak early within the first 2 days after experimental human influenza virus infection [10], it appears rational to rapidly inhibit viral replication to obtain significant clinical benefit. In our study, the peak virus load in the placebo group was reached 12–24 h after enrollment (or within 48–60 h of the onset of symptoms), also suggesting that treatment of influenza should be initiated early to obtain maximal clinical benefit. The latter observation was confirmed in a phase II clinical trial in which patients who initiated zanamivir earlier (within 30 h) experienced greater clinical benefits [4]. Inhaled zanamivir allows rapid delivery of the drug in the respiratory tract to the site of influenza virus replication [11], with minimal potential for drug interactions and toxicity [4, 5]. Consistent with the latter fact, we found a reduction in the virus titers after only 12 h of treatment. We also showed a trend to lower mean virus titers at different time points and to shorter viral shedding by culture and PCR in favor of zanamivir, although these differences were not as pronounced as those previously reported [4]. In contrast to the latter study, we quantified virus titers by use of pharyngeal swabs (instead of nasal washings) from subjects who received inhaled zanamivir only (instead of inhaled and intranasal zanamivir). It is possible that swallowing led to rapid clearance of the drug from the throat or that pharyngeal swab samples were diluted by nasal secretions. Nevertheless, the moderate reduction in the virus load induced by zanamivir resulted in a drastic reduction in the median time to alleviation of influenza symptoms, with a 47% difference compared with placebo (before correcting for dependent variables). This difference may be superior to the 25%–30% reduction seen in a previous similar study [5]. Considering the physiopathology of influenza virus infections, we believe that most of the clinical benefit seen in our study is due to the early reduction in virus titers at 12 h, although we cannot exclude a later effect because of the significant reduction found at 96 h.

Zanamivir-resistant influenza viruses selected in the laboratory were shown to contain mutations in the active site of the NA gene and/or in the region of the receptor-binding pocket of the HA gene [7, 12, 13]. Thus far, there has been only 1 clinical report of the recovery of a zanamivir-resistant influenza virus. This was a mutant influenza B virus recovered from an immunocompromised child who received zanamivir for 2 weeks [14]. Interestingly, despite sequential emergence of HA and NA mutations as well as reduction in the NA sensitivity to zanamivir, the child's last isolate remained susceptible in a PR assay on MDCK cells. Thus, phenotypic evaluation of viral susceptibility to NA inhibitors is limited by the fact that no tissue culture system adequately reflects the receptor specificity of human cells of the respiratory tract. Consequently, determination of influenza virus susceptibility should include both phenotypic and genotypic assays and possibly animal experiments to determine growth properties of the mutants.

We found no phenotypic or genotypic evidence for development of resistance to zanamivir during a 5-day treatment course in 17 immunocompetent subjects with naturally occurring influenza A virus (H3N2) infections. This is consistent with the absence of resistance for virus isolates evaluated during phase II studies [15]. The low rate of emergence of viral resistance to zanamivir could be explained by the very conserved catalytic site of the NA and by its essential role in viral replication [2]. In a single isolate (patient 15), we found an amino acid change in the HA gene (Arg150→Lys) not present before zanamivir treatment. However, it is unlikely that this mutation was selected by zanamivir, because no divergent sequences were found between amplified HA sequences from the first and last positive original clinical samples. Larger susceptibility studies on a greater number of isolates should be done, with emphasis on the development of an adequate and sensitive phenotypic assay.

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Footnotes

  • Informed consent was obtained from all patients, according to guidelines in use at the Centre Hospitalier Universitaire de Québec and at the Faculty of Medicine of the University of Manitoba.

  • Financial support: Glaxo Wellcome Canada. G.B. is a fellow of the Medical Research Council of Canada.

  • Received September 24, 1999.
  • Revision received December 9, 1999.
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  1. J Infect Dis. (2000) 181 (4): 1471-1474. doi: 10.1086/315392
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