Vol. 29, Issue 10, 1337-1342, October 2001

Tissue Slices Revisited: Evaluation and Development of a Short-Term Incubation for Integrated Drug Metabolism

Sanjeev Thohan, MaryLynn C. Zurich, Ho Chung, Myron Weiner, Andrew S. Kane, and Gerald M. Rosen

AstraZeneca R&D Boston, Drug Metabolism and Pharmacokinetics (S.T.), Waltham, Massachusetts; Zurich Toxicology Consulting (ML.C.Z.), Gainesville, Florida; Division of Experimental Therapeutics, Walter Reed Army Institute of Research (H.C.), Washington, D.C.; Department of Pharmaceutical Sciences (M.W., G.M.R.), University of Maryland, Baltimore, Maryland; and Aquatic Pathobiology Center, Department of Veterinary Medicine (A.S.K.), University of Maryland, College Park, Maryland

This work details the development of a model for the rapid evaluation of drug metabolism in an integrated fashion using in situ architecture of the liver. A Krumdieck tissue slicer was used to generate slices from 10-mm cores of rat liver (approximately 250-µm thick). Initial unsuccessful efforts with 6-well plate-based incubation were overcome with the use of a dynamic (rotating) incubation in 23-ml liquid scintillation vials containing titanium mesh supports for the slice. Incubation of 1 slice/5 ml of a Krebs-Henseleit solution buffered with HEPES showed a <2% increase over the initial 25% release of lactate dehydrogenase over 2 h of incubation at 37°C under ambient oxygen conditions. Coupled O-dealkylase and conjugative metabolism of alkoxycoumarin derivatives was shown to be linear for both 7-methoxy- and 7-ethoxycoumarin (100 µM) with a low amount of nonconjugated 7-hydroxycoumarin (7-HC) at all time points. Metabolic profiles for 7-methoxy- and 7-ethoxycoumarin were compared between slice and microsomal incubations generated from the same tissue. The use of 7-HC as a primary substrate not only provided an assessment of the capacity-based differences in oxidative versus conjugative metabolism but also capacity-based differences in glucuronidation and sulfation. These studies underscore the physiological fact that phase I metabolism has a lower capacity for substrate metabolism than phase II metabolism. Additionally, this technique provides a model for examination of pharmacodynamic and pharmacokinetic influences in the context of maintenance of the in situ architecture of the liver.