| Assay Method Information | |
| | Reversible CYP Inhibition Assay |
| Description: | A seven-point semi-log dilution of each test compound (up to 30 NM) or positive control inhibitors for CYP2C9 (up to 1000 nM sulphenazole), CYP2D6 (up to 500 nM quinidine) and CYP3A4 (up to 250 nM ketoconazole) were dissolved in DMSO and added to the incubation plate using a Tecan 300 (normalized for 0.3% DMSO content). Final reaction conditions were: human liver microsomes (0.1 mg/mL), potassium phosphate buffer 100 mM pH 7.4, 1 mM magnesium chloride, 5 NM diclofenac, 5 NM dextromethorphan and 2.5 μM midazolam. After a pre-incubation at 37 00. The reaction was started with the addition of NADPH solution at the final concentration of 1 mM and carried out at 37 00 for 5 minutes. The reaction was stopped by the addition of 1 volume of cold acetonitrile with internal standard (labetalol) to the incubation plate. The incubation plate was centrifuged at 3000 rpm for 10 minutes to precipitate proteins, and the supernatant was analyzed by LC-MS/MS. Metabolite area ratio (4-OH-diclofenac, dextrorphan and 1-OH-midazolam) versus ISTD area ratio was used as the quantitative signal. Data were analyzed using the plot log of concentration (×-axis) versus the percentage of inhibition (y axis) and the IC50, Hill Slope and R2 were determined. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |