2.4 In vitro metabolic stability experiments.
To investigate the metabolic stability of ningetinib and the formation of M1, primary human hepatocytes were incubated at 37 °C in WME at 1.0×106 cells/mL containing 3 µM ningetinib. The HLMs, HIMs, HKMs or HLUMs were incubated at 0.5 mg/mL in 100 mM phosphate buffer containing 3.2 mM MgCl2 (PBS, pH 7.4) and ningetinib (3 μM) in the presence of NADPH (2 mM). The volume of each incubation mixture was 100 μL. The reactions were terminated by adding equal volume of ice-cold acetonitrile at 0, 5, 15, 30, 60 and 180 min after the initiation of the reactions. All the incubation samples were triplicated and stored at -20 °C for LC-MS/MS analysis.