Determination of the rate-limiting step in the hepatic elimination of YM796 by isolated rat hepatocytes.

Purpose: The membrane permeability clearance and intrinsic metabolic clearance of a drug in the liver were estimated using isolated rat hepatocytes, and the rate-limiting step in the overall intrinsic clearance of the drug in vivo was investigated. For this purpose, an anti-dementia drug, (S)-(-)-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro [4,5] decane-L-tartarate monohydrate (YM796) was used as a model drug.

Methods: The parent drug and its metabolites in both medium and cells were separated by thin-layer chromatography (TLC). The total amount of drug taken up by hepatocytes and the total amount of metabolites were plotted against the AUC of YM796 in the medium or cells to obtain the kinetic parameters.

Results: While the influx clearance (PS(inf)) through the sinusoidal membrane defined in terms of YM796 concentration in the medium was almost constant, irrespective of the concentration of YM796 in the medium, the intrinsic metabolic clearance (CL(int)) and the efflux clearance (PS(eff)) defined in terms of the total concentration of YM796 in the cells markedly decreased and increased, respectively, as the concentration of YM796 increased. The overall intrinsic metabolic clearance (CL(int,all), defined in terms of the YM796 concentration in the medium, corresponding to the hepatic intrinsic clearance obtained from the in vivo pharmacokinetic data on the drug, was comparable with PS(inf) at low concentrations of YM796. As the YM796 concentration increased, however, CL(int,all) fell markedly approaching CL(int).

Conclusions: While, at low concentrations of YM796, CL(int,all) was predominantly affected by membrane permeability clearance, at high concentrations it was completely rate-determined by the intrinsic metabolic clearance because of the marked reduction in CL(int) resulting from the saturation of YM796 metabolism.