Tissue slices revisited: evaluation and development of a short-term incubation for integrated drug metabolism.
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-microm 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 degrees 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 microM) 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.
Thohan
S
S
AstraZeneca R&D Boston, Drug Metabolism and Pharmacokinetics, Waltham, Massachusetts 02451, USA. sanjeev.thohan@AstraZeneca.com
Zurich
M C
MC
Chung
H
H
Weiner
M
M
Kane
A S
AS
Rosen
G M
GM
D000818:Animals; D003374:Coumarins / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D008297:Male; D008722:Methods; D008954:Models, Biological; D051381:Rats; D017207:Rats, Sprague-Dawley; D014468:Umbelliferones / Q000378:metabolism*
D003374:Coumarins; D014468:Umbelliferones; C017299:7-ethoxycoumarin; C031477:7-hydroxycoumarin