- MATERIALS and METHODS
- Clinical trials
A multi-center, randomized, double-blind, controlled phase 2 study was
conducted during the 2015-16 influenza season with BXM in Japanese
adults aged 20–64 years with uncomplicated influenza (Japic
CTI-153090).23 In the subsequent 2016-17 influenza
season, an open-label study was conducted with BXM in otherwise healthy
pediatric patients aged 6 months to <12 years with
uncomplicated influenza (Japic CTI-163417).24 The
CAPSTONE-1 study (ClinicalTrials.gov NCT02954354) was conducted in the
United States and Japan as a double-blind, placebo- and
oseltamivir-controlled, randomized trial that enrolled outpatients 12 to
64 years of age with influenza-like illness in
2016-17.7 The CAPSTONE-2 study (ClinicalTrials.gov
NCT02949011) was a double-blind, placebo- and oseltamivir-controlled
trial involving outpatients aged ≥12 years in 551 sites in 17 countries
and territories, and eligible patients had clinically diagnosed
influenza-like illness, at least one risk factor for influenza-related
complications (eg, age >65 years), and a symptom duration
of < 48 hours.8 Written informed
consent was obtained from all the patients in clinical trials, and all
methods related to clinical samples were derived according to standard
operating procedures in accordance with the protocol approved by the
institutional review board (IRB), all applicable regulatory
requirements, and the current Good Clinical Practice (GCP) guidelines.
In the clinical trials, baseline variant monitoring was conducted to
evaluate BXA susceptibility of the viruses in the baseline samples from
nasopharyngeal/pharyngeal swabs. In addition, genotypic analysis was
performed using paired pre- and post-treatment swab samples from BXM
treated patients to identify treatment-emergent AA substitutions that
were associated with reduced susceptibility to BXA.
Compounds, cells and viruses
Baloxavir acid (S-033447; BXA) was synthesized at Shionogi & Co., Ltd.,
Osaka, Japan, and favipiravir was purchased from PharmaBlock Sciences,
Inc., Nanjing, China. MDCK, RPMI2650 and 293T cells were cultured as
described previously.10 For generation of recombinant
viruses by reverse genetics, the plasmid set of rgA/WSN/33 (H1N1),
rgA/Victoria/3/75 (H3N2) and rgB/Maryland/1/59 were used as described
previously.10
Phenotypic analyses of variant viruses
The plaque reduction assay was conducted as described
previously.10 A series of mutant influenza viruses was
generated by Virapur (SanDiego, CA, USA) using reverse genetics to
determine drug sensitivity to BXA using Virapur’s ViraDot Assay. The
assay is a modification of the HINT assay developed by Gubareva et
al.19, and is based on a single round of replication
of influenza virus in MDCK cells. Briefly, 3x104 MDCK
cells/well were plated in 96-well plates 1 day prior to infection. Cells
were infected (500 Dot-forming units/well) and BXA serially dilutions
were added. Plates were incubated overnight at 37°C (A viruses) or 34°C
(B virus) before the cells were fixed and permeabilized with ice-cold
100% methanol. Cells were probed with a mouse monoclonal anti-A/NP
antibody (Millipore Sigma MAB8251) against Influenza A and anti-B/NP
(Millipore Sigma MAB8661) against Influenza B for 1 hour at 37°C and
washed three times with PBS before anti-mouse IgG peroxidase labeled
secondary polyclonal antibody (Sigma #A3682) was added and incubated at
37°C for 1 hour. Cells were washed three times with DPBS and
virus-infected cells were detected using TrueBlue substrate (KPL, Cat#
5510-0050/-0030) and the CTL ImmunoSpot System with the BioSpot software
module BioSpot 7.0.23.2 Professional. EC50 values were
determined from dose-response curves using GraphPad Prism.
Evaluation of virus replicative capacity was previously
described.10 Briefly, 2x105cells/well MDCK or 1x106 cells/well RPMI2650 cells
were seeded on 24-well plates 1 day prior to infection. MDCK and
RPMI2650 cells were infected with 10 and 100 TCID50/well
of the viruses, respectively. The infected cells were incubated at 37°C
in a 5% CO2 incubator for 1 hour, followed by
exchanging the inoculum to MEM containing 3 μg/mL trypsin and incubation
at 37°C in the 5% CO2 incubator. The culture
supernatants were collected at the indicated time points, and viral
titers (log10TCID50/mL) were determined
on MDCK cells.