| Assay Method Information | |
| | Inhibition Assay |
| Description: | The potential of the test compound to act as a competitive inhibitor of CYP3A4 was evaluated in in vitro assays, using human liver microsomes and the reference substrate midazolam. The test compound was solved in acetonitrile. Human liver microsomal preparation (pool of HLM) was applied for the assay. A stock solution of the test compound was added to phosphate buffer containing EDTA, NADP, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase. This mixture was sequentially diluted on a Genesis Workstation (Tecan, Crailsheim, FRG). After pre-warming, reaction was initiated by addition of a mixture of probe substrate (midazolam). Finally, the incubation mixtures contained human liver microsomes at protein concentration of 60 ug/mL, NADPH-regenerating system (1 mM NADP, 5.0 mM glucose 6-phosphate, glucose 6-phosphate dehydrogenase (1.5 U/mL), 1.0 mM EDTA, the test compound at 6 different concentrations, 2.5 uM midazolam as probe substrate, and phosphate buffer (50 mM, pH 7.4) in a total volume of 200 uL. Incubations were performed on a Genesis Workstation (Tecan, Crailsheim, FRG) in 96-well plates (Microtiter plate, 96-well plate) at 37° C. Stock solution of probe substrate was prepared in water (midazolam 10 mM). Ketoconazole was used as positive control of a direct-acting inhibitor. The reference samples (substrate, but no inhibitor) were incubated in parallel in sextuple and contained the same amount of solvent as the test incubations. Reactions were stopped by addition of 100 uL acetonitrile containing the internal standard. Precipitated proteins were removed by centrifugation of the well plate, supernatants were analyzed by LC-MS/MS. |
| Affinity data for this assay | |
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