Fig. 3: HH CYP3A4-mediated midazolam intrinsic clearance is increased by gefitinib and recapitulated in NADH-HLM but not canonical NADPH-HLM.

A The effect of increasing concentrations of gefitinib on the scaled CL_int,u of midazolam, the prototypic substrate of cytochrome P450 3A4 in HH and how this compares with the scaled CL_int,u in NADPH-HLM. The data represents the mean + SD of three independent replicates. Statistical significance was assessed by the Brown-Forsythe and Welch ANOVA followed by Dunnett’s T3 multiple comparison of the control to the other groups. B Effect of increasing concentrations of gefitinib on formation of the primary metabolite (1´-hydroxy midazolam) and its secondary metabolite (1´-hydroxymidazolam glucuronide) expressed as response ratio (peak area/internal standard). The data represents a single determination. C Schematic view of the entry of midazolam and gefitinib into an intact hepatocyte and effect of gefitinib on the subsequent well-established midazolam sequential metabolism^46,75 – effect of gefitinib occurs at the level of formation of 1´-hydroxymidazolam. D The ability of gefitinib to elicit the same effect on midazolam scaled CL_int,u in HH relative to NADH-HLM or NADPH-HLM. The data are from three independent determinations expressed as a percentage of the control (without gefitinib, set to 100%). At each concentration of gefitinib, statistical significance was assessed by the Brown-Forsythe and Welch ANOVA followed by Dunnett’s T3 multiple comparison of the gefitinib effect on HH midazolam scaled CL_int,u to the NADH-HLM and NADPH-HLM groups.