Title
Modeling the in vitro intrinsic clearance of the slowly metabolized compound tolbutamide determined in sandwich-cultured rat hepatocytes
Author
Treijtel, N.
Barendregt, A.
Freidig, A.P.
Blaauboer, B.J.
van Eijkeren, J.C.H.
TNO Voeding
Publication year
2004
Abstract
An alternative approach is introduced in determining the in vitro intrinsic clearance of slowly metabolized compounds. The long-term sandwich rat hepatocyte culture was exploited, allowing for sufficient substrate depletion to obtain a reliable clearance estimation; in its physiology, it resembles the in vivo liver, thus allowing in vivo extrapolation of the in vitro clearance value. Substrate depletion of tolbutamide and the formation of its metabolites hydroxytolbutamide and carboxytolbutamide were measured in the medium and sandwich layer. Depletion data from the medium were fitted to a mathematical model incorporating system-dependent parameters (diffusion, protein binding, and partitioning) to calculate the hepatocytes' intrinsic clearance. Based on the decrease of the parent compound in the medium, a specific intrinsic clearance value, i.e., clearance per unit of volume of hepatocytes, of 0.085 min -1 was fitted. This value was in accordance with in vivo and in vitro values from the literature. The model was verified with substrate depletion data from the sandwich layer. Data on metabolite formation showed an incomplete mass balance. A radiochemical experiment revealed the presence of three additional metabolites. These metabolites were analyzed by liquid chromatography-mass spectometry. One was identified as p-tolysulfonylurea. The structure of the other two needs to be elucidated. After the addition of these compounds to the metabolic pattern, the mass balance was completed. The in vitro clearance value was incorporated in a physiologically based pharmacokinetic literature model of tolbutamide that accurately describes the plasma concentration. The approach used in this study successfully predicts the intrinsic clearance of tolbutamide. In addition, the sandwich rat hepatocyte culture also proves to be useful in the identification of metabolic pathways.
Subject
Biology
Physiological Sciences
4 tolylsulfonylurea
carboxytolbutamide
hydroxytolbutamide
sulfonylurea derivative
tolbutamide
unclassified drug
addition reaction
animal cell
article
cell culture
controlled study
drug blood level
drug clearance
in vitro study
in vivo study
liquid chromatography
liver cell
mass spectrometry
mathematical model
nonhuman
priority journal
radiochemistry
rat
Animals
Cells, Cultured
Hepatocytes
Male
Metabolic Clearance Rate
Models, Biological
Rats
Rats, Wistar
Tolbutamide
Animalia
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DOI
https://doi.org/10.1124/dmd.32.8.884
TNO identifier
237913
ISSN
0090-9556
Source
Drug Metabolism and Disposition, 32 (8), 884-891
Document type
article