| Assay Method Information | |
| | Inhibitory activity curve of compounds on CYP3A4/5 enzymes |
| Description: | Liver microsomes were thawed in a 37° C. water bath prior to use. Liver microsome working solution (final system concentration of 0.5 mg/mL) was prepared. 5 mM NADPH working solution was prepared with phosphate buffered saline. The above stock solution was prepared into a 2 mM working solution with DMSO. The above stock solution was diluted with acetonitrile (4-fold dilution, 6 non-zero concentrations). 238.5 μL of liver microsome working solution was added to a 1.1 mL tube. 1.5 μL of test compound/control working solution/DMSO was added and mixed well by pipetting several times. The mixture was pre-incubated in water bath with shaking at 37° C. for 5 minutes. After the pre-incubation, 60 μL of NADPH working solution was added and mixed well by pipetting several times. The mixture was incubated in water bath with shaking at 37° C. for 10 minutes. Immediately after incubation, 500 μL of quenching solution was added and vortexed for 1 minute. All samples were centrifuged at 4,000 rpm for 15 minutes at 4° C. 300 μL of the above supernatants were aliquoted for further LC-MS/MS analysis. |
| Affinity data for this assay | |
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