| Assay Method Information | |
| | Time-Dependent Inhibitory Assay |
| Description: | The time-dependent inhibitory effect of the test compound on the activity of human liver microsomal cytochrome P450 isoenzyme CYP2C19 was determined.Experimental MethodThe experiment was divided into two groups. In the first group, human liver microsomes (HLM) were used as the incubation system. The test articles with a series of concentrations were added to the incubation system, followed by the addition of a coenzyme factor (NADPH) solution. The preincubation was carried out at 37° C. for 30 minutes. After preincubation, a probe substrate solution was added thereto. After a certain period of incubation, the reaction was terminated. The amount of probe substrate metabolites generated in the incubation solution was determined, and the enzyme activity was calculated. In the second group, human liver microsomes (HLM) were used as the incubation system. The test articles with a series of concentrations were added to the incubation system, followed by the addition of potassium phosphate buffer. The preincubation was carried out at 37° C. for 30 minutes. After preincubation, a mixture of NADPH and the probe substrate was added thereto. After a certain period of incubation, the reaction was terminated. The amount of probe substrate metabolites generated in the incubation solution was determined, and the enzyme activity was calculated.[0370]First, the test compound (10.0 mM) was diluted by gradient to prepare a working solution (100× the final concentration) with concentrations of 5.00, 1.65, 0.500, 0.165, 0.0500, 0.0165, and 0.00500 mM, respectively. Simultaneously, working solutions were respectively prepared for positive inhibitors of the P450 isoenzyme CYP2C19, probe substrate, and NADPH. Human liver microsomes, stored in a refrigerator at a temperature below −60° C., were thawed on ice, and once fully dissolved, were diluted with potassium phosphate buffer (PB) to prepare a working solution of a specific concentration (0.169 mg/mL).Then, 147.5 μL of human liver microsomal working solution was added to the reaction plate, and the reaction plate was placed on ice for use; at this time, 2.50 μL of the test compound (N=1) and the working solution (N=2) of the positive control inhibitor at various concentrations were added to the corresponding wells, and the corresponding organic solvent was added to the group without inhibitors (test compound or positive inhibitor); the reaction plate was incubated for 10 minutes at 37° C., and then 50.0 μL of NADPH solution or potassium phosphate buffer was added to reaction wells of the first or second group, respectively, to start the reaction; the reaction plate was pre-incubated at 37° C. for 30 minutes; 50.0 μL of the substrate solution or a mixture of NADPH and the substrate was added to the reaction wells of the first or second group, respectively, to start the reaction. |
| Affinity data for this assay | |
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