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

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 nmolmL(-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 (CL(int,in-vitro)), expressed as mLmin(-1) (mg microsomal protein)(-1), of midazolam was estimated as Vmax (Km)(-1) by in-vitro metabolism studies using liver microsomes. The CL(int,in-vitro) value was converted to the CL(int,cal) value, expressed as mLmin(-1)kg(-1), by considering the microsomal protein content (g liver)(-1) and the microsomal protein content (g liver)(-1)kg(-1). The estimated CL(int,cal) value was then converted to the ERh value (ER(h,cal)) according to the well-stirred, the parallel-tube and the dispersion models. The ERh(h,cal) values obtained by the parallel-tube model were in good agreement with corresponding in-vivo ERh(h,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.