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THE USE OF SANDWICH-CULTURED RAT HEPATOCYTES TO DETERMINE THE INTRINSIC CLEARANCE OF COMPOUNDS WITH DIFFERENT EXTRACTION RATIOS: 7-ETHOXYCOUMARIN AND WARFARIN

Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands (N.T., H.v.H., A.B., B.J.B.); and National Institute for Public Health and the Environment, Bilthoven, the Netherlands (J.C.H.v.E.)

The application of sandwich-cultured rat hepatocytes for the identification of the hepatic intrinsic clearance of compounds with widely varying extraction ratios was investigated. We previously showed the applicability of this in vitro system, in combination with a model describing molecular diffusion, hepatocyte/medium partition, and nonsaturated metabolism, which resulted in a successful identification of this parameter for tolbutamide. This approach is further validated using the compounds 7-ethoxycoumarin and warfarin, covering a 100-fold range of extraction ratios. Clearance of these two substrates could be reliably determined, but only if the depletion of the parent compound in medium as well as in the hepatocyte sandwich was measured. Sensitivity analyses showed that the time course of depletion of the parent compound in medium, especially for warfarin, is insensitive to the partition and diffusion parameter values, whereas depletion in the hepatocyte sandwich was far more sensitive. When varying the volumes of collagen in the sandwich culture, it appears that the most reliable kinetic parameters could be obtained by fitting the data with the smaller collagen volume and that these parameters obtained from fitting to data of the larger volumes generally cannot be verified satisfactorily with the data of the smaller volumes. The values of hepatic clearance that were obtained after extrapolation of the intrinsic clearance to the hepatic clearance from blood were comparable within a factor of 2 to hepatic clearance data in the literature. This indicates that this sandwich culture and modeling system can be applied for the identification of the hepatic intrinsic clearance rate of the total range from low to high clearance compounds.