2.1. Study 1. Cytopathic Effect (CPE) Assay

In Study 1, the cytopathic effects of indomethacin, ketotifen, and naproxen were compared with remdesivir (positive control).

Vero E6 cells were seeded into 96-well plates at 2 × 10⁴ cells/well in 100 μL seeding medium (MEM supplemented with 1% (w/v) l-glutamine, 2% FBS). Plates were incubated overnight at 37 °C, 5% CO₂. Test compounds were prepared fresh on the day of testing, vortexed and visually inspected to confirm complete dissolution.

The positive control compound remdesivir was prepared as a 10 mM stock in dimethyl sulfoxide (DMSO) and stored at −20 °C. Compound dilutions were prepared on the day of experimentation. A 3-fold, 8 point, DMSO dilution series of each of the four test compounds was performed initially, ranging 100 mM to 0.045 mM. An intermediate dilution series in virus growth medium (MEM supplemented with 1% (w/v) l-glutamine, 2% FBS, 8 μg/mL Tosyl Phenylalanyl Chloromethyl Ketone (TPCK)-Trypsin was generated ranging 800 µM–0.37 µM. A 50 μL volume from each compound intermediate dilution series was added to triplicate wells of the assay plate pre-seeded with Vero E6 cells. The DMSO concentration was maintained at 0.2% in the assay plate. One assay plate per compound was generated.

Similarly, remdesivir was subjected to an initial DMSO dilution series, intermediate dilution series to reduce the DMSO concentration to 0.2% in the assay plate. Remdesivir was tested at a starting concentration of 20 µM.

A 50 μL volume of SARS-CoV-2 diluted in virus growth medium to generate a multiplicity of infection (moi) of 0.05, was added to the assay plates. This moi was previously determined to provide 100% cytopathic effect (CPE) in 4 days. Virus was added to triplicate rows to assess antiviral activity and virus growth medium without virus was added to triplicate rows to assess cytotoxicity.

The percent cell protection achieved by the positive control (remdesivir) and test articles in virus-infected cells was calculated by the formula of Pauwels et al. [24] as shown below:

where:

[ODt]virus = the optical density measured in a well examining the effect of a given concentration of test article or positive control on virus-infected cells.

[ODc]virus = the optical density measured in a well examining the effect of the negative control on virus-infected cells.

[ODc]mock = the optical density measured in a well examining the effect of the negative control on mock-infected cells.

The EC₅₀ values were calculated from the percent cell protection results by non-linear regression analysis using the Hill (sigmoid Emax) formula:

where:

X = test or control article concentration;

Y = percent cell protection;

Min = minimum;

Max = maximum;

D = slope coefficient.