Abstract
Apparent intrinsic clearance (CLint,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 CLint,app values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CLint,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 (kup) and metabolism (kmet). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [kup/(kmet + kup)]/[1 + (Chep/Cbuffer) × (Vhep/Vbuffer)], where the Chep, Cbuffer, Vhep, and Vbuffer 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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.010793.
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ABBREVIATIONS: CLint, intrinsic clearance; CLint,app, apparent intrinsic clearance; P450, cytochrome P450; LC/MS/MS, liquid chromatography coupled to tandem mass spectrometry; 7-EC, 7-ethoxycoumarin; fu,hep-inc, free fraction in hepatocyte incubations.
- Received April 24, 2006.
- Accepted June 19, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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