In-vivo and In-vitro Metabolic Clearance of Midazolam, a Cytochrome P450 3A Substrate, by the Liver under Normal and Increased Enzyme Activity in Rats

Abstract

Abstract The metabolic clearance of midazolam, a cytochrome P450 (CYP) 3A substrate, by the liver under normal and increased enzyme activity in rats was determined in-vivo and in-vitro to elucidate the reproducibility of the in-vivo hepatic extraction ratio of midazolam from the in-vitro study. The hepatic enzyme activity was modified by pretreating rats with a CYP inducer such as dexamethasone and clotrimazole. The in-vivo hepatic extraction ratio (ERh,obs) of midazolam under a steady-state plasma concentration (approx. 3 nmol mL−1) in untreated (control) rats was 0.864. This value increased to 0.984 in dexamethasone-pretreated rats and to 0.964 in clotrimazole-pretreated rats. The in-vitro hepatic intrinsic clearance (CLint,in-vitro), expressed as mL min−1 (mg microsomal protein)−1, of midazolam was estimated as Vmax (Km)−1 by in-vitro metabolism studies using liver microsomes. The CLint,in-vitro value was converted to the CLint,cal value, expressed as mL min−1 kg−1, by considering the microsomal protein content (g liver)−1 and the microsomal protein content (g liver)−1 kg−1. The estimated CLint,cal value was then converted to the ERh value (ERh,cal) according to the well-stirred, the parallel-tube and the dispersion models. The ERh,cal values obtained by the parallel-tube model were in good agreement with corresponding in-vivo ERh,obs values. In conclusion, it was demonstrated that high hepatic clearances of midazolam under normal and increased CYP3A activity were reasonably predicted from in-vitro metabolism studies using liver microsomes.