Radioligand binding assay for the human CB1 receptor

The radioligand binding assay on the human CB1 receptor was performed by Eurofins Discovery (St Charles, MO). Briefly, human recombinant Chem-1 cells were used, and cells were incubated with 2 nM [3H] SR141716A, 2.4 nM [3H] WIN-55,212–2, or 0.5 nM [3H] Estradiol for 90 min at 37 °C. Non-specific binding was estimated in the presence of 10 μM CP-55,940, 10 μM R(+)- WIN-55,212–2, or 1 μM diethylstilbestrol. Test compounds were prepared in assay buffer (20 mM HEPES, pH 7.0, 0.5% BSA) with serial dilution. Membranes were filtered and washed four times, and the filters were counted to determine specifically bound radioligand. IC₅₀ values were determined by a non-linear, least squares regression analysis using the MathIQTM statistical software (ID Business Solutions Ltd., UK). The inhibition constants (Ki) values were calculated by the Cheng-Prussoff equation using the observed IC₅₀ of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the dissociation constants of the ligand.