Avicel rc 581

For the preparation of carriers in the form of pellets, menthol racemic, camphor racemic and ammonium bicarbonate (Dr. Kulich Pharma, Hradec Králové, Czech Republic) were used as the pore-forming substances. Neusilin^® US2 (magnesium aluminometasilicate, Fuji Chemical Industry, Toyama, Japan) was used as a substance with large SSA and sorption capacity, while Avicel^® PH-101 (microcrystalline cellulose, FMC Biopolymer, Wallingstown, Ireland) and Avicel^® RC-581 (microcrystalline cellulose and sodium carboxymethylcellulose, FMC Biopolymer, Brussel, Belgium) were used as fillers and spheronization agents. Purified water (Ph. Eur. 10) was used as a wetting agent. All materials were of European Pharmacopoeia grade.

The antiviral activity of baloxavir was also determined by using a focus reduction assay (Tilmanis et al., 2017 (link)) under Avicel overlays (Matrosovich et al., 2006 (link)). Confluent monolayers of MDCK cells in 96-well plates were inoculated with 100 μl of 1000 focus-forming unit (FFU)/well of viruses. Virus adsorption was carried out for 1 h at 37°C and then 100 μl of 1.2% Avicel RC-581 (FMC BioPolymer) in culture medium containing serial dilutions (0.025–2500 nM) of baloxavir acid was added to each well in triplicate. The cells were incubated for 24 h and then fixed with formalin. After the formalin was removed, the cells were immunostained with a mouse monoclonal antibody against influenza A or B virus nucleoprotein (Merck; MAB8251, MAB8661), followed by a horseradish peroxidase-labeled goat anti-mouse immunoglobulin (SeraCare) as previously described (Tilmanis et al., 2017 (link)). The infected cells were stained with TrueBlue Substrate (SeraCare) and then washed with distilled water. After drying, the number of FFU was quantified by using an ImmunoSpot S6 Analyzer, ImmunoCapture software, and BioSpot software (CTL) as previously described (Tilmanis et al., 2017 (link); van Baalen et al., 2017 (link)). The IC₅₀ values were calculated by using GraphPad Prism.

The hemagglutination inhibition assay (HI) was performed in 96-well plates (U-bottom) by a standard method, essentially as described previously [16 ] using 0.5% chicken red blood cells suspended in PBS (pH 7.2). For the plaque reduction neutralization assay, virus was titrated to approximately 250 pfu/ml and incubated with concentrations of mAb from 20 to 0.032 μg/ml for 1 hour at room temperature before infection of MDCK cells. After 1.5 hour adsorption, virus inoculum was removed and the cells overlaid with a 50:50 mixture of 2X EMEM plaque media (BioWhittaker, Walkersville, MD) and 2.4% Avicel RC-581 (FMC BioPolymer, Philadelphia, PA) [17 (link)] with TPCK-trypsin (Sigma-Aldrich Corp. St. Louis, MO) at a final concentration of 2 μg/ml. At 96 hours, the plaque media was removed and the cells fixed and stained with 0.3% crystal violet/20% methanol. Percent neutralization was calculated for each mAb concentration versus no mAb controls.

PT cells (6.5 × 10⁴) were seeded in 24-well plates and infected with BCoV (MOI 0.00005). After 1 h post-infection (hpi) the inoculum was replaced by 2 mL of 2.4% (w/v) Avicel RC-581 (FMC BioPolymer, Belgium) containing 2 × MEM, 5% FBS and various concentrations of sHA3 (0.0, 0.1, 0.3, 0.4, 0.6, 0.9 µM) or sCS3 (0.0, 0.4, 0.9, 1.0, 2.0, 3.0 µM). After incubation for four days at 37 °C, the cells were fixed with 6% formalin in PBS pH 7.4 prior to staining with 0.2% (w/v) crystal violet in 20% EtOH for 15 min at room temperature. Plaques were counted by using a light microscope (Axiovert 10, Carl Zeiss Microscopy, Jena, Germany) and compound effects were calculated by comparing compound-treated cells versus untreated cells.

An in vitro virus micro-neutralisation assay, based on the concept of plaque reduction, was conducted. Briefly, after 50 μl of twofold serial dilutions of the ferret antisera in DMEM containing 200 nM oseltamivir was added to a monolayer of MDCK-SIAT1 cells in a 96-well plate, 1,000 focus-forming units of the indicated virus (50 µl) was added to each well. After a 1 hour incubation, 1.6% Avicel RC-581 (FMC BioPolymer) in DMEM was overlaid. Oseltamivir was used to prevent receptor binding via NA, and trypsin was not added to avoid virus multiplication. At 18‒20 hours after infection, the cells were stained with mouse anti-nucleoprotein (NP) monoclonal antibodies (MAB8257 and MAB8258; Millipore, Consett, US), followed by an anti-mouse IgG antibody conjugated with horseradish peroxidase. NP-positive cells were visualised with KPL TrueBlue substrate (Kirkegaard and Perry Laboratories) to count foci. The reciprocal number of the minimum dilution of sera needed to achieve 50% focus reduction was used as the neutralisation titre.

HuH7 cells seeded in 6-well clusters were infected with recombinant MERS-CoV at low MOI (30 PFU/well) for 1 h at 37°C. Subsequently, the inoculum was replaced with 2 ml of a 1.2% suspension of Avicel (RC-581; FMC Biopolymer) (101 (link)) in Dulbecco’s minimal essential medium (DMEM) (containing 2% FCS and antibiotics) and serial dilutions of 5-FU (F6627; Sigma-Aldrich) or ribavirin (R9644; Sigma-Aldrich) ranging from 0 to 400 μM. Cells were incubated at 37°C for 72 h and fixed with 7.4% formaldehyde, and plaques were visualized using crystal violet staining.
To compare the effect of 5-FU treatment on the progeny titers of wt and nsp14-E191D rMERS-CoV, confluent monolayers of HuH7 were incubated for 30 min at 37°C with solvent or a range of 5-FU concentrations. The drug was then removed and cells were infected at an MOI of 0.1 for 1 h at 37°C. After removal of the inoculum, EMEM containing 2% FCS and solvent or a matching concentration of 5-FU was added to the wells. Supernatants were collected after 30 h, and rMERS-CoV progeny titers were determined by plaque assay. All drug-treated samples were normalized to the untreated vehicle control, and values were expressed as fold change compared to untreated-virus titers.