1. MATERIALS and METHODS
  2. 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.