title: Comparison of intrinsic clearance in liver microsomes and hepatocytes from rats and humans: evaluation of free fraction and uptake in hepatocytes.
abstract: Apparent intrinsic clearance (CL(int,app)) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CL(int,app) values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CL(int,app). This could partly be attributed to the differences in the free fraction (fu). The fu in hepatocyte incubations (fu,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (fu,buffer) but also by the rate constants of uptake (k(up)) and metabolism (k(met)). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [k(up)/(k(met) + k(up))]/[1 + (C(hep)/C(buffer)) x (V(hep)/V(buffer))], where the C(hep), C(buffer), V(hep), and V(buffer) represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the fu,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.
authors.affiliation: Drug Metabolism and Pharmacokinetics, Drug Safety and Disposition, Millennium Pharmaceuticals, Inc., MA 02139, USA. chuang.lu@mpi.com
authors.forename: Chuang
authors.initials: C
authors.lastname: Lu
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Richard
authors.initials: R
authors.lastname: Gallegos
authors.forename: Vinita
authors.initials: V
authors.lastname: Uttamsingh
authors.forename: Cindy Q
authors.initials: CQ
authors.lastname: Xia
authors.forename: Gerald T
authors.initials: GT
authors.lastname: Miwa
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Balani
authors.forename: Liang-Shang
authors.initials: LS
authors.lastname: Gan
mesh_terms: D000818:Animals; D001692:Biological Transport; D003374:Coumarins / Q000378:metabolism; D015925:Cryopreservation; D004058:Diffusion; D004353:Drug Evaluation, Preclinical / Q000379:methods; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008657:Metabolic Clearance Rate; D008862:Microsomes, Liver / Q000378:metabolism*; D008874:Midazolam / Q000378:metabolism; D010615:Phenacetin / Q000378:metabolism; D011433:Propranolol / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results
chemical_list: D003374:Coumarins; C017299:7-ethoxycoumarin; D011433:Propranolol; D010615:Phenacetin; D008874:Midazolam