| Assay Method Information | |
| | Inhibition Effect of the Compounds of the Present Invention on the Enzyme Activity of Testosterone Metabolite Site of CYP3A4 in Human Liver Microsomes |
| Description: | Table 4: I. Experimental Materials and Instruments1. Phosphate buffer solution (PBS);2. NADPH (Sigma N-1630);3. Human liver microsome (Corning Gentest);4. ABI QTrap 4000 liquid chromatograph/mass spectrometer (AB Sciex);5. Inertsil C8-3 column, 4.6×50 mm, 5 μm (Dikma Technologies Inc., USA); and6. CYP probe substrate (testosterone/100 μM, SIGMA K1003) and positive control inhibitor (ketoconazole, Dr. Ehrenstorfer GmbH, C17322500).II. Experimental Procedures100 mM PBS buffer was formulated, which was then used to formulate 2.5 mg/ml human microsome solution and 5 mM NADPH solution. The 5× concentration of the compound working solution was diluted with PBS in gradients (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). The 5× concentration of ketoconazole working solution was diluted with PBS in gradients (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM). Dextromethorphan working solution was diluted with PBS to a concentration of 50 μM.20 μl of 2.5 mg/ml microsome solution, 20 μl of 50 μM testosterone working solution, 20 μl of MgCl2 solution and 20 μl of the compound working solution (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM, different reaction systems for each concentration) were mixed well. For the positive control group, the compound was replaced with the same concentration of ketoconazole. The mixture together with 5 mM NADPH solution was pre-incubated at 37° C. for 5 minutes. After 5 minutes, 20 μl of NADPH was added to each well, the reaction was initiated, and the plate was incubated for 30 minutes. All the incubated samples were present in duplicate. After 30 minutes, 250 μl of acetonitrile containing internal standard (100 ng/ml camptothecin) was added to all samples, mixed well, shaken at 800 rpm for 10 minutes, and then centrifuged at 3700 rpm for 10 minutes. 80 μl of the supernatant was taken and analyzed by LC-MS/MS. |
| Affinity data for this assay | |
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