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.