4.10. CYP450 Inhibition Study

The inhibition of cytochrome P450 3A4 isoform by Si306 and pro-Si306 was measured by determining its activity on the substrate testosterone.

The activity of CYP3A4 was evaluated by measuring the 6β-hydroxylated testosterone formation (CYP3A4-specific reaction). The amount of 6β-hydroxylated metabolite was assessed in the absence and presence of Si306, pro-Si306 and ketoconazole (testosterone concentration of 100 μM in phosphate buffer 0.025 M, pH 7.4). The compounds were solubilized in DMSO and added in a test tube to give final concentrations 1, 10, 25, 50 and 0.01, 0.05, 0.5, 5 μM, (DMSO did not exceed 2%). Ketoconazole was used as standard selective inhibitor of CYP3A4 isoenzyme [55]. Incubations were carried out in a mix containing human liver microsomes (1 mg/mL of protein) in phosphate buffer 0.025 M, pH 7.4, NADPH (200 μM) previously solubilized in a MgCl₂ 48 mM solution. The final incubation volume was 0.25 mL. After a 40 min-incubation period at 37 °C, the reaction was stopped by adding 1 mL of acetonitrile in the presence of corticosterone 2 μM as internal standard. The samples were centrifuged for 20 min at 5000 rpm. Concentrations of testosterone and its metabolite 6β-hydroxytestosterone were assessed by the HPLC method with UV detection. Calibration curve for quantitative analysis of 6β-hydroxytestosterone in the samples was obtained using commercial 6β-hydroxytestosterone purchased from Sigma Aldrich. For the statistical analysis, GraphPad InStat 3.0 (GraphPad Software, San Diego, CA, USA) was used.