| Assay Method Information | |
| | Inhibition of Cytochrome P450 Isoenzymes |
| Description: | First, the test compound (10.0 mM) was diluted to prepare a working solution (100×the final concentration) with a concentration of 1.00 mM. Simultaneously, working solutions were respectively prepared for positive inhibitors of the P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 (with midazolam as a probe substrate), and CYP3A4 (with testosterone as a probe substrate)) and specific probe substrates thereof. Human liver microsomes, stored in a refrigerator at a temperature lower than −60° C., were thawed on ice, and once fully dissolved, were diluted with PB to prepare a working solution of a specific concentration (0.127 mg/mL). 20.0 μL of probe substrate was added to the reaction plate (20.0 μL of PB was added to the Blank wells), followed by the addition of 158 μL of human liver microsomal working solution to the reaction plate. The reaction plate was then placed on ice for later use. Then, 2.00 μL of the test compound (N=1) and specific inhibitors (N=2) were added to the corresponding wells. In groups without inhibitors (either with test compound or positive inhibitors), the corresponding organic solvent was added. The organic phases for the test compound control samples and positive control samples were DMSO and MeOH at a ratio of 1 to 1, and DMSO and MeOH at a ratio of 1 to 9, respectively. After pre-incubating in a 37° C. water bath for 10 minutes, 20.0 μL of the cofactor (NADPH) solution was added to the reaction plate. For the CYP3A4 metabolic reaction using midazolam as the probe substrate, the reaction lasted 3 minutes; for the CYP2C19 reaction using (S)-mephenytoin as the probe substrate, and the CYP2D6 reaction using dextromethorphan as the probe substrate, the reaction lasted 20 minutes; for all other reactions, the reaction lasted 10 minutes. The reaction was then terminated by adding 400 μL of pre-cooled acetonitrile solution (containing 200 ng/ml of internal standards Tolbutamide and Labetalol). Placed on a shaker, the reaction plate was shaken and evenly mixed for 10 minutes, then centrifuged at 4° C. and 4000 rpm for 20 minutes. 200 μL of the supernatant was taken and added to 100 pL of water for sample dilution. Lastly, the plate was sealed, shaken to ensure the content was thoroughly mixed, and analyzed by LC/MS/MS. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |