Injectable peramivir mitigates disease and promotes survival in ferrets and mice infected with the highly virulent influenza virus, A/Vietnam/1203/04 (H5N1)

Abstract

The post-exposure therapeutic efficacy of injectable peramivir against highly pathogenic avian influenza type A H5N1 was evaluated in mice and in ferrets. Seventy to eighty percent of the H5N1-infected peramivir-treated mice, and 70% in the oseltamivir treated mice survived the 15-day study period, as compared to 36% in control (vehicle) group. Ferrets were infected intranasally with H5N1 followed by treatment with multiple doses of peramivir. In two of three trials, a statistically significant increase in survival over a 16-18 day period resulted from peramivir treatment, with improved survival of 40-64% in comparison to mock-treated or untreated animals. Injected peramivir mitigates virus-induced disease, reduces infectious virus titers in the lungs and brains and promotes survival in ferrets infected intranasally with this highly neurovirulent isolate. A single intramuscular peramivir injection protected mice against severe disease outcomes following infection with highly pathogenic avian influenza and multi-dose treatment was efficacious in ferrets.

Fig. 1. Effects of post-exposure peramivir treatment on survival and disease development in mice infected with influenza A H5N1. A) Pharmacokinetic (PK) analysis of peramivir in mice

Mice (3 per group) received a single i.m. injection of 10 mg/kg of peramivir. 20 mg/kg and 30 mg/kg doses were modeled using WinNonlin® 5.0.1. Values are presented as log/linear plot. B-D) Post-exposure efficacy of peramivir in ten-week-old mice. All mice were infected intranasally with a dose of 6 × 10² TCID₅₀ of influenza A H5N1 (A/Vietnam/1203/04). Mice (10-11 per group, N) were treated via intramuscular (i.m.) injection with single (+1 h) dose (30 mg/kg) or multiple (+1 h, daily on day +1 through day +4) doses of peramivir (30 mg/kg) or with control (vehicle, +1 h, daily on day +1 through day +4). For comparison, mice were treated with oseltamivir (10 mg/kg/day) per os (p.o., +1 h, daily on day +1 through day +4). Animals were monitored daily for a period of 15 days for disease and death. B) Survival curves. C-D) Severe disease development in post-exposure efficacy trial in ten week-old-mice. C) The percentage of mice in each group that developed paralysis by day 15 is shown. D) The percentage change in body mass from day 1 was calculated for each indicated day (data shown in Supplemental Fig. 1). Severe disease was defined as the loss of ≥20% of their initial body mass.

Fig. 2. Dose-dependent survival and disease development in ferrets infected with influenza A H5N1. A) Cell-grown stock

Seven-week-old ferrets (N=4 per group) were infected intranasally with influenza A H5N1 (A/Vietnam/1203/04) using a range of doses from 1 × 10^-1 to 1 × 10³ TCID₅₀ per animal. Prior to infection, blood was drawn for hematological evaluation. Following infection, animals were monitored daily for a period of 19 days following infection for death (A1) and disease development (A2). B) Egg-grown stock. Six- to eight-week-old ferrets (4-7 per group, N) were infected intranasally with influenza A H5N1 (A/Vietnam/1203/04) using a dose of 1.89 × 10⁴ (N=7), 1.89 × 10³ (N=7), or 1.89 × 10² (N=4) TCID₅₀ per animal or as control, with uninfected egg stock (N=2, indicated by dash). Following infection, animals were monitored daily for a period of 16 days following infection for death (B1) and disease development (B2). Animals that developed paralysis were euthanized.

Fig. 3. Pharmacokinetic (PK) analysis in ferrets

Ferrets (3 per group) received a single i.m. injection of 1, 3 or 9 mg/kg of peramivir. Blood samples were collected at the indicated pre- and post-inoculation time points and analyzed via liquid chromatography with tandem mass spectrophotometry (LS/MS/MS). The average log/linear plasma concentration is shown.

Fig. 4. Effects of post-exposure peramivir treatment on survival in six- to eight-week-old ferrets infected with influenza A H5N1. Survival curves are shown for animals tested in three independent post-exposure efficacy trials: A) trial 1, B) trial 2, and C) trial 3

All ferrets were infected intranasally with influenza A H5N1 (A/Vietnam/1203/04) using the challenge dose indicated in the figure legend. Prior to infection, blood was drawn for hematological evaluation. One group of ferrets was treated with multiple doses of peramivir (30 mg/kg). As controls, one group of ferrets either was treated with vehicle (drug diluent) at +1 h post-infection, and daily on day +1 through day +4 post-infection (trial 1 and trial 3) or was untreated (trial 2). Following H5N1 infection, animals were monitored daily for a period of 16-18 days for death and disease development (see Fig 5). Logrank test comparing the survival for peramivir treated and control groups was performed and the p-value is reported. D) The median survival time for the three peramivir trials is summarized. Asterisk (*) indicates that the p-value obtained comparing the treatment and control (vehicle or untreated) group in the respective trial via logrank test was statistically significant.

Fig. 5. Effects of post-exposure peramivir treatment on levels of infectious virus in the lungs, brain and nasal washes in six- to eight-week-old ferrets infected with influenza A H5N1

Ferrets were infected intranasally with influenza A H5N1 (A/Vietnam/1203/04) using the challenge dose and peramivir treatment regimen described for Trial 3 in Fig 4. Randomly pre-selected ferrets were euthanized at day 4 and day 6 post-infection (N=5/group at each time point). In addition, ferrets that developed paralysis or those that survived to the end of the study (day 18) were euthanized. Organs from euthanized animals were harvested for viral titration using TCID₅₀ assay. The limit of detection, indicated by a dashed line, was 1 × 10⁴ gram (g) of tissue for lungs/brain and 1 × 10³ TCID₅₀ per ml for nasal washes.