KINETICS OF DIAZEPAM METABOLISM IN RAT HEPATIC MICROSOMES AND HEPATOCYTES AND THEIR USE IN PREDICTING IN-VIVO HEPATIC-CLEARANCE

1. The rates of diazepam (DZ) metabolism to the primary metabolites 3- hydroxydiazepam, 4'-hydroxydiazepam and nordiazepam were studied in vi tro using rat hepatic microsomes and hepatocytes. 4'-hydroxydiazepam h ad the largest intrinsic clearance (V-max/K-m ratio, CL(int)) in both microsomes and hepatocytes representing 49 and 70% of total metabolism respectively. Whereas the contribution of 3-hydroxydiazepam was simil ar in both systems (21-24%), the N-demethylation pathway was greater i n microsomes (27%) than hepatocytes (9%). 2. The pharmacokinetics of D Z were determined in vivo using the intraportal route to avoid blood f low limitations due to the high clearance of DZ. No dose dependency wa s observed in either clearance or steady state volume of distribution, which were estimated to be 38 ml/min/SRW (where SRW is a standard rat weight of 250 g) and 1.3 L/SRW respectively. Blood binding of DZ was concentration independent, the unbound fraction being 0.22. 3. Scaling factors were used to relate the in vitro CL(int) to the in vivo unbou nd clearance. Hepatocytes (123 ml/min/SRW) produced a more realistic p rediction for the in vivo value (174 ml/min/SRW) than microsomes (41 m l/min/SRW). This situation is believed to arise from the quantitative differences in the three metabolic pathways in the two in vitro system s. It is speculated that end product inhibition is responsible for red uced total metabolism in microsomes whereas hepatocytes operate kineti cally in a manner close to in vivo.