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The disposition of atorvastatin, cerivastatin, and indomethacin, established substrates of rat hepatic basolateral uptake transporters, has been evaluated in suspended rat hepatocytes. Cell and media concentration-time data were simultaneously fitted to a model incorporating active uptake, permeation, binding, and metabolism. Use of the model to estimate the ratio of intracellular to extracellular steady-state free drug concentrations demonstrated the strong influence of active uptake on the kinetics of atorvastatin (18:1) and cerivastatin (8:1), in comparison with indomethacin (3.5:1). Indomethacin, however, was shown to have a higher uptake clearance (599 ± 101 μl/min/106cells) than atorvastatin (375 ± 45 μl/min/106cells) and cerivastatin (413 ± 47 μl/min/106cells). The high passive permeability of indomethacin (237 ± 63 μl/min/106cells) clearly negated the effect of the active transport on the overall disposition. An analogous physiological model was constructed that allowed prediction of the in vivo pharmacokinetics, including the free intracellular concentration in liver. Hepatic clearance was well predicted by the model, in contrast to predictions based on standard methods. Volume of distribution was well predicted for indomethacin and predicted reasonably for atorvastatin and cerivastatin and higher than might be expected for an acid compound. Furthermore, the terminal half-life predictions for all three compounds were within 2-fold of the observed values. The ability to estimate the free-intracellular hepatic concentration of uptake substrates has major benefits in terms of predicting pharmacokinetics, potential CYP-mediated drug-drug interactions, and efficacy of hepatically targeted therapeutics.

A fully automated assay to determine the enzymology of drug oxidation by the major human hepatic cytochrome P450s (CYPs; CYP1A2, -2C9, -2C19, -2D6, and -3A4) coexpressed functionally in Escherichia coliwith human NADPH-P450 reductase has been developed and validated. Ten prototypic substrates were chosen for which clearance was primarily CYP-dependent, and the activities of these five major CYPs were represented. A range of intrinsic clearance (CLint) values were obtained for substrates in both pooled human liver microsomes (HLM; 1–380 μl · min−1mg−1) and recombinant CYPs (0.03–7 μl · min−1pmol−1) and thus the percentage contribution of individual CYPs toward their oxidative metabolism could be estimated. All the assignments were consistent with the available literature data. Tolbutamide was metabolized by CYP2C9 (70%) and CYP2C19 (30%), diazepam by CYP2C19 (100%), ibuprofen by CYP2C9 (90%) and CYP2C19 (10%), and omeprazole by CYP2C19 (68%) and CYP3A4 (32%). Metoprolol and dextromethorphan were primarily CYP2D6 substrates and propranolol was metabolized by CYP2D6 (59%), CYP1A2 (26%), and CYP2C19 (15%). Diltiazem, testosterone, and verapamil were metabolized predominantly by CYP3A4. In addition, the metabolite profile for the CYP-dependent clearance of several markers determined by mass spectroscopy was as predicted from the literature. There was a good correlation between the sum of individual CYP CLintand HLM CLint(r2= 0.8, P< .001) for the substrates indicating that recombinant CYPs may be used to predict HLM CLintdata. This report demonstrates that recombinant human CYPs may be useful as an approach for the prediction of the enzymology of human CYP metabolism early in the drug discovery process.