2.5 Enzyme phenotype of M1 production and determination of
inhibition kinetics by gefitinib.
The in vitro metabolism experiments showed that M1 was mainly
formed in liver microsomes. Therefore, the enzyme phenotype of M1
formation was evaluated using CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4A11
and CYP4F2 in our previous work. The CYP1A1, CYP1B1, CYP2C9 and CYP3A4
could catalyze the metabolism of ningetinib into M1. The gefitinib
inhibition on the formation of M1 was explored in CYP1A1, CYP1B1,
CYP2C9, CYP3A4 and HLMs incubations. After optimisation of the enzyme
concentration and incubation time, the incubation conditions in the
linear range were selected. The reaction mixture containing 100 mM PBS
(pH 7.4) supplemented with CYPs (5, 10, 5 or 10
pmol·mL-1 of CYP1A1, CYP1B1, CYP2C9 or CYP3A4,
respectively) or HLMs (0.05 mg·protein·mL-1) was
incubated with 0.01–30 μM gefitinib. In addition, different
concentrations of ningetinib were added to the CYP1A1 (0.1, 0.3, 0.9,
2.7 or 8 μM), CYP1B1 (0.05, 0.1, 0.2, 0.5 or 1 μM), CYP2C9 (3, 6, 12 or
30 μM), CYP3A4 (3, 5, 15, 30 or 60 μM) and HLMs reaction mixtures (1, 3,
6, 10 or 30 μM). The reactions were initiated by the addition of NADPH
(2 mM). The volume of each reaction mixture was 100 μL. An equal volume
of ice-cold acetonitrile was added to terminate the reaction at 5, 15,
60, 30 and 60 min after the reaction’s initiation. All incubation
samples were triplicated and stored at -20 °C before LC-MS/MS analysis.