Abstract

The effect of human serum albumin (Hsa) and human liver cytosol (Hlc) on the in vitro enzyme kinetics of the formation of hydroxytolbutamide (CYP2C9 marker reaction) and the inhibitory effect of gemfibrozil on tolbutamide hydroxylation were examined using human liver microsomes. The addition of Hsa greatly decreased the unbound concentrations of tolbutamide and gemfibrozil in the incubation medium, whereas Hlc only slightly decreased them. The unboundK_m value for tolbutamide hydroxylation was 123 μM without Hsa and Hlc, and 73, 88, and 64 μM in the presence of Hsa (5 mg/ml), Hlc (0.5 mg/ml), and Hsa plus Hlc, respectively. The predicted in vivo hepatic clearance (CL_h) of tolbutamide based on enzyme kinetics without Hsa and Hlc (0.06 ml/min/kg) was 40% of its in vivo clearance (0.15 ml/min/kg) based on published data. Addition of 5 mg/ml Hsa and 0.5 mg/ml Hlc to the incubation medium distinctly improved the prediction, with the coaddition of Hsa and Hlc yielding the most accurate value (0.14 ml/min/kg). TheK_i (6 μM) of gemfibrozil for CYP2C9, calculated using total drug concentrations, was increased by Hlc (8 μM), Hsa (40 μM), or both (72 μM). However, when the unbound substrate and inhibitor concentrations were considered, theK_i (6 μM without Hsa and Hlc) was not markedly altered by Hsa (4 μM), Hlc (8 μM), or both Hsa and Hlc (9 μM). The present findings suggest that the addition of Hsa and Hlc to microsomal incubation media may yield enzyme kinetic estimates more comparable with in vivo results.