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Vol. 302, Issue 1, 43-49, July 2002

Effect of Albumin and Cytosol on Enzyme Kinetics of Tolbutamide Hydroxylation and on Inhibition of CYP2C9 by Gemfibrozil in Human Liver Microsomes

Jun-Sheng Wang1, Xia Wen1, Janne T. Backman and Pertti J. Neuvonen

Department of Clinical Pharmacology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland

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 unbound Km 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 (CLh) 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). The Ki (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, the Ki (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.

1 These authors contributed equally to this study.

0022-3565/02/3021-0043$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics


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