Comparison of Intrinsic Clearance in Liver Microsomes and Hepatocytes from Rats and Humans: Evaluation of Free Fraction and Uptake in Hepatocytes

Abstract

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 (f u ). The f u in hepatocyte incubations (f u,hep-inc ) was influenced not only by the free fraction of compounds in the incubation buffer (f u,buffer ) but also by the rate constants of uptake (k up ) and metabolism (k met ). This report provides a new derivation for f u,hep-inc , which can be expressed as, 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 f u,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.The determination of in vitro intrinsic clearance (CL int ) for drug candidates in the early discovery stage is a common practice in the pharmaceutical industry (Houston, 1994;Lave et al., 1997;Obach et al., 1997). The CL int values of drug candidates can help to confirm whether metabolism is the main clearance pathway when it is compared with the total body clearance in vivo. It is also helpful in rank-ordering drug candidates based on their metabolic stabilities, assessing species and gender differences in metabolic clearance, and projecting the metabolic clearance of drug candidates in humans. The in vitro CL int may be derived from enzyme kinetic data such as V max /K m (Lin et al., 1996;Tan and Pang, 2001;Griffin and Houston, 2004) or from the in vitro t 1/2 values where subK m substrate concentrations are used (Lave et al., 1997, Obach, 1999Lau et al., 2002;Jones and Houston, 2004). The CL int can be calculated from the experimental apparent intrinsic clearance, CL int,app , by correcting for free fraction of test compounds in the incubations. To further predict the in vivo hepatic clearance from the in vitro intrinsic clearance, a well stirred model is often used (Naritomi et al., 2001;Ito and Houston, 2004). A survey of literature revealed that in hepatocyte incubations, the free fraction of test compound has not been well defined. Simply assuming a steady state where the intracellular free concentration equals the extracellular free concentration may allow one to roughly estimate CL int for some compounds. However, clearance, after a dose in vitro or in vivo, is actually a dynamic system such that at any given time the amount of compound getting into a cell typically equals the amount of compound leaving the cell by diffus...

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