Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction

Abstract

ABSTRACT:The hepatic uptake of quinine, fluvoxamine, and fluoxetine (0.1-10 M) was investigated with freshly isolated rat hepatocytes. The cell-to-medium concentration ratios (K p ) were concentration-dependent: the mean maximum K p values (at 0.1 M) were 150 (quinine), 500 (fluvoxamine), and 2000 (fluoxetine). There was also a large capacity site that was not saturable over the concentration range used (possibly partition into the phospholipid component of membranes); representing this site, the mean minimum K p values (at 10 M) were 30 (quinine), 200 (fluvoxamine), and 500 (fluoxetine). To eliminate concomitant metabolism, cells were pretreated with the irreversible P450 inhibitor, aminobenzotriazole. The saturable uptake was substantially eliminated after exposure to carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (ATP inhibitor). The difference between the maximum and minimum K p for these three amine drugs, as well as for dextromethorphan, propranolol, and imipramine, was within a limited range of 3-fold, indicating a common magnitude of saturable uptake. Basic, permeable drugs are expected to be sequestered into lysosomes, which actively maintain their low internal pH (ϳ5) using ATP, and this process is predictable from the combined effects of pH-driven ion accumulation and unsaturable binding representing partition into membranes. The resultant predicted maximum K p correlated strongly with the observed maximum K p . Thus, at low substrate concentrations, the fraction of drug unbound in the hepatocyte incubation (critical for assessing drug clearance and drug-drug interaction potential) may be dependent upon saturable as well as unsaturable binding, and for lipophilic, basic drugs, this can be readily estimated assuming a common degree of uptake into lysosomes.

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

“…After appropriate binding corrections, more potent hepatocyte inhibition (as evident by lower K i values) would suggest the involvement of hepatic transporters because a higher concentration of unbound drug available for enzyme inhibition can be achieved compared with a similar incubation concentration in microsomes (Ito et al, 1998). Alternatively, similar values would indicate that any hepatic accumulation results from intracellular binding or lysosomal trapping (Grime and Riley, 2006;Hallifax and Houston, 2007). We recently demonstrated good concordance between K i values (range 0.05-30 M) determined in rat hepatic microsomes and freshly isolated hepatocytes using seven P450 inhibitors (fluconazole, fluoxetine, fluvoxamine, ketoconazole, miconazole, omeprazole, and quinine) with a range of uptake properties (cell/medium concentration ratios 4 -6000).…”

Hepatocellular Binding of Drugs: Correction for Unbound Fraction in Hepatocyte Incubations Using Microsomal Binding or Drug Lipophilicity Data

“…where K a represents microsomal protein binding affinity, P the microsomal protein concentration (mg/ml), K p the hepatocyte/medium concentration ratio, and V R a V cell /V inc ratio, where V cell is the cell volume and V inc the incubation volume (Brown et al, 2007b;Hallifax and Houston, 2007). V R is 0.005 at the cell concentration of 10 6 cells/ml (Brown et al, 2007b).…”

“…A database of 39 drugs and their corresponding fu mic and fu hep values was collated either from in house data or from the literature (Austin et al, 2002(Austin et al, , 2005Brown et al, 2007a;Hallifax and Houston, 2007). In the aforementioned studies, different methods were used to determine the drug binding in hepatocyte incubations, namely oil centrifugation (using live cells), dialysis (using dead cells), and ultrafiltration (using dead cells).…”

“…The authors proposed a linear 1:1 relationship between microsomal and hepatocyte binding for incubations of 1 mg/ml and 10 6 cells/ml, respectively, indicating that the fu hep can be extrapolated from microsomal studies. However, the applicability of this correlation has been questioned because of the small number of compounds investigated (Hallifax and Houston, 2007).…”

“…The authors proposed a linear 1:1 relationship between microsomal and hepatocyte binding for incubations of 1 mg/ml and 10 6 cells/ml, respectively, indicating that the fu hep can be extrapolated from microsomal studies. However, the applicability of this correlation has been questioned because of the small number of compounds investigated (Hallifax and Houston, 2007).To further explore the relationship between binding in microsomal and hepatocyte systems, a detailed analysis was carried out involving 39 drugs. A nonlinear empirical equation is proposed as an alternative to the linear relationship to relate binding between the two systems.…”