| Assay Method Information | |
| | CYP Inhibition Assay |
| Description: | Human liver microsomes (HLM) are stored at −80° C. Before the study, microsomes were thawed in a cold water bath, and then were put on ice immediately. Test compounds and P450 3A4 specific inhibitor ketoconazole were dissolved in DMSO to yield a stock solution of 10 mM. The stock solution was diluted with 50% acetonitrile to get a working solution at the concentration of 1.5 mM. The working solution was further diluted with 0.1 M potassium phosphate buffer to get a series of working solution at concentrations of 150, 50, 15, 5, 1.5, 0.5, 0.15, and 0.05 μM. Incubation mixtures in duplicate contain pooled human liver microsome (0.1 mg/mL), 3.3 mM MgCl2, CYP 3A4 probe substrate testosterone (50 μM), specific inhibitor or test compounds (30, 10, 3, 0.1, 0.03, 0.01, 0.003, 0.01 μM) in 0.1 M potassium phosphate buffer (total volume 0.1 mL). Negative control contains 0.1 M phosphate buffer instead of a specific inhibitor or test compound. The final concentrations of DMSO and acetonitrile were equal to or less than 0.1%. The mixtures are pre-incubated for 10 min at 37° C. Then, 1 mM NADPH is added to initiate a reaction. Following a 10-min incubation at 37° C., the reactions are terminated by the addition of 300 μL acetonitrile containing an internal standard. The formation of the corresponding products is detected by LC/MS/MS. |
| Affinity data for this assay | |
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