Development of a method to predict in vivo drug metabolism and excretion from in vitro data with human hepatic tissues and/or recombinant proteins

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The content of the present study can be classified into two parts. Each part is described separately :1. Quantitative prediction of in vivo metabolism from in vitro data with human microsomes/P-450 expressing systemBy kinetically analyzing the previously reported data, we determined the metabolic activity determined with human microsomes in vitro and that determined in humans in vivo. We found a nice 1 : 1 correlation between the two parameters, suggesting that in vivo disposition can be extrapolated from in vitro data. In order to comfirm this conclusion, we also performed experiments with YM796, which is metabolized by CYP3A4 enzyme. Kinetic parameters (Km and Vmax) were determined in vitro with microsomes. These parameter values were further used in predicting the drug disposition in vivo after oral administration. In this prediction, dispersion model was used, in which the nonlinear metabolism was considered. By analyzing the disposition of YM796 after oral administration, we found … More that the in vivo disposition of this drug can be extrapolated from in vitro data. Moreover, we examined the metabolism of YM796 by using the microsomes from leukoblastoma transfected with CYP3A4 cDNA.It was indicated that the metabolic data with human microsomes can be predicted from the kineticparameters determined with the recombinant enzymes after correcting the amount of the isozymes in the human microsomes. These results suggest that the disposition in humans can be predicted from the data determined with the recombinant enzymes.2. Determination of transport activity using the mammalian cells transfected with cDNA for transportersBy comparing the transport activity determined in isolated hepatocytes along with that determined in the cells transfected with the cloned cDNA for transporters, we determined the contribution of each transporter to the hepatic uptake of ligands. Moreover, we performed the genetic analysis of atransporter (canalicular multispecific organic anion transporter ; cMOAT) responsible for the excretion of organic anions into the bile. We had also examined the function of cloned cMOAT cDNA by preparing the stable transfectant. ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione, atypical substrate for cMOAT,into membrane vesicles isolated from NIH/3T3 cells was stimulated by transfection of rat cMOAT cDNA These results suggest that the methodology employed in the present study may be useful in the quantitative prediction of transport from the activity determined with the cloned cDNA products. Less