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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 microl/min/10(6) cells) than atorvastatin (375 +/- 45 microl/min/10(6) cells) and cerivastatin (413 +/- 47 microl/min/10(6) cells). The high passive permeability of indomethacin (237 +/- 63 microl/min/10(6) cells) 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.

The intrinsic metabolic clearance of saquinavir, nelfinavir, and ritonavir was determined over a range of concentrations (0.02-20 microM) in both rat liver microsomes and fresh isolated rat hepatocytes in suspension. Clearance values were found to be concentration dependent for both systems, and at low concentrations, microsomal clearance was much greater (7-14-fold) than in hepatocytes. Kinetic parameters showed substantially lower microsomal K(m) values (5-42 nM) compared with suspended rat hepatocytes (34-270 nM) but similar scaled V(max) values (2-26 nmol/min/g liver). In the absence of metabolism (achieved by pretreating hepatocytes with a mechanism-based inhibitor of cytochrome P450), saquinavir, nelfinavir, and ritonavir were actively and rapidly taken up into hepatocytes (cell/medium concentration ratios of 306-3352), and intracellular unbound drug concentrations between 5- and 12-fold higher than extracellular unbound concentrations were achieved. Comparison of the rate of uptake into hepatocytes with the rate of metabolism in hepatocytes and microsomes indicates that the former is the rate-limiting step at low concentrations. The rate of metabolism saturates at lower concentrations (100-400-fold) than the rate of uptake; hence, at the high concentrations metabolic rate-limited clearance occurs. In conclusion, the clearance of saquinavir, nelfinavir, and ritonavir is extremely rapid, and it is proposed that in the case of hepatocytes and by inference in vivo, the rate of uptake limits the metabolic clearance of these three drugs.