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

Abstract

ABSTRACT:The intrinsic metabolic clearance of saquinavir, nelfinavir, and ritonavir was determined over a range of concentrations (0.02-20 M) 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.

Comparative Use of Isolated Hepatocytes and Hepatic Microsomes for Cytochrome P450 Inhibition Studies: Transporter-Enzyme Interplay

“…This behavior is consistent with the differences reported here in terms of K i values and the resulting Kp u, app . Interestingly, the ratio of microsomal to hepatocyte K m values (Parker and Houston, 2008) is similar to the corresponding ratio of K i values (Table 5).…”

“…In contrast, the hepatic clearance of the protease inhibitors is very high (Shibata et al, 2000;Parker and Houston, 2008). We have studied this in some detail in vitro and have dmd.aspetjournals.org demonstrated that in hepatocytes the metabolic clearance is transporter rate-limited (Parker and Houston, 2008).…”

“…The activity of members of the solute carrier and ATP-binding cassette families of transporter proteins may provide a mechanistic explanation for the limited success achieved for some drugs using standard (hepatic microsomal) in vitro experiments to predict pharmacokinetic aspects of clearance and drug-drug interaction (DDI) potential (Lam et al, 2006;Soars et al, 2007;Parker and Houston, 2008). Although various theoretical scenarios involving hepatic transporters can be proposed, specific examples with experimental confirmation of an unequivocal nature have been largely restricted to the statins (Lau et al, 2006;Shitara et al, 2006;Paine et al, 2008).…”

“…Four drugs were identified for study (CLAR, ENX, NFV, and SQV), all of which have known transporter involvement and show substantial hepatocellular accumulation (Sasabe et al, 1997;Yamano et al, 1999;Su et al, 2004;Maeda et al, 2007;Seithel et al, 2007;Parker and Houston, 2008;Giacomini et al, 2010). These drugs also show a range of metabolic clearance values (Table 1), allowing enzymetransporter interplay to be assessed.…”

Clearance Prediction of HIV Protease Inhibitors in Man: Role of Hepatic Uptake

“…10 Moreover, in addition to a carrier-mediated process, passive transmembrane permeation significantly contributed to the hepatic uptake of saquinavir and ritonavir. 11 In general, previous results imply that a carrier-mediated process is involved in the hepatic uptake of HIV PI.…”