Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir

The intrinsic metabolic clearance of saquinavir, nelfinavir, and ritona-vir was determined over a range of concentrations (0.02–20 M) in both rat liver microsomes and fresh isolated rat hepatocytes in sus-pension. Clearance valueswere found to be concentration dependent for both systems, and at low concentrations, microsomal clearance wasmuch greater (7–14-fold) than in hepatocytes. Kinetic parameters showed substantially lower microsomal Km values (5–42 nM) com-pared with suspended rat hepatocytes (34–270 nM) but similar scaled Vmax values (2–26 nmol/min/g liver). In the absence of metabolism (achieved by pretreating hepatocytes with amechanism-based inhib-itor of cytochrome P450), saquinavir, nelfinavir, and ritonavir were actively and rapidly taken up into hepatocytes (cell/medium con-centration ratios of 306-3352), and intracellular unbound drug con-centrations between 5- and 12-fold higher than extracellular un-bound concentrations were achieved. Comparison of the rate o