Determination of Activity of Cytochrome P4503A in the Rat Intestinal Microsomes Using Midazolam as a Probe and Optimization of Incubation Conditions in Vitro

OBJECTIVE To determine the activity of CYP3A and optimize the incubation conditions in the rat intestinal microsomes.METHODS The formation of 1′-hydroxymidazolam determined by RP-HPLC was used to indicate the activity of CYP3A.Single effect experimental design was used to optimize the incubation conditions and the enzyme kinetics was evaluated by graphical analysis with Lineweaver-Burk double reciprocal plots (1/velocity vs.1/[substrate]).RESULTS The calibration curves of 1′-hydroxymidazolam were linear in the range of 9.10-910.00 ng·mL-1 ( r2= 0.999 9).The optimal incubation conditions were 10 μmol·L-1,4 μg and 20 min for midazolam concentration,intestinal microsomes and incubation time,respectively.The parameters of CYP3A in the intestinal microsomes for Km and Vmax were 7.61 μmol·L-1 and 1.26 nmol·min-1·mg-1,respectively.CONCLUSION The HPLC method is simple,accurate,rapid,and suitable for the determination of 1′-hydroxymidazolam in the intestinal microsomes.The incubation conditions of CYP3A and its kinetics can be applied for study on the interaction between drugs metabolized by CYP3A.