The Use of Sandwich-Cultured Rat Hepatocytes to Determine the Intrinsic Clearance of Compounds With Different Extraction Ratios: 7-Ethoxycoumarin and Warfarin

Abstract

ABSTRACT: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.Predicting the kinetic parameters of compounds in vivo using data from in vitro experiments is a fast developing area of research (Obach, 1999;Haenen et al., 2002;Lau et al., 2002;. Intrinsic clearance, which is the key parameter enabling in vitro-in vivo extrapolation, can be determined from various in vitro models, which include microsomes, liver slices, and isolated hepatocytes (including hepatocyte suspensions and plated hepatocytes) (Cross and Bayliss, 2000). A major disadvantage of these models is their short-term use, because of declining enzyme activities and viability (Berthiaume et al., 1996;Berry et al., 1997).Recently, we have successfully developed the alternative approach of using sandwich-cultured rat hepatocytes in determining the in vitro intrinsic clearance of the slowly metabolized compound tolbutamide (Treijtel et al., 2004). The sandwich culture is a long-term in vitro system allowing for long incubation periods. Besides prolonged cell viability, other advantages of this system are cellular morphology comparable to the in vivo situation, including cellular polarity, physiological levels of protein secretion, development of an extensive bile canalicular network, and expression of drug transporter proteins. Furthermore, the most important phase I enzyme a...

Macitentan Does Not Interfere with Hepatic Bile Salt Transport

“…The hepatocyte volume was estimated from cell diameters measured before plating (23 mm). This value is in good agreement with previously published data, which are 16.2 mm for human hepatocytes (Mateus et al, 2013) and 24 mm for rat hepatocytes (Treijtel et al, 2005). The unbound fraction in the hepatocyte homogenate (f u,homogenate ) was calculated using Eq.…”

Evaluation of Hepatic Disposition of Paroxetine Using Sandwich-Cultured Rat and Human Hepatocytes

“…Other researchers have compared individual metabolic enzyme activity between the two methods independently (Kern et al, 1997;Slaus et al, 2001;Lau et al, 2002;Treijtel et al, 2004). SCH is beneficial for the determination of the metabolic intrinsic clearance of slowly metabolizing compounds, because it allows long-term incubation (Treijtel et al, 2004;Treijtel et al, 2005). The incubation period was up to 24 hours in the present study, whereas Treijtel et al conducted Metabolite profiles of radioactivity were analyzed by radio-HPLC after the incubation of [ metabolic stability studies over 48 hours with SCH for tolbutamide and warfarin and concluded that successful prediction of metabolic intrinsic clearance of these slowly metabolizing drugs was possible.…”

Biokinetic Modeling andin Vitro–in VivoExtrapolations

“…The loss of differentiated functions, e.g., the decline in cytochrome P-450-dependent biotransformation in hepatocyte cultures (Wortelboer et al, 1991), still is an obstacle in the full use of in vitro systems. The development of more sophisticated culture systems, including cells in an improved matrix environment such as hepatocyte sandwich cultures (Treijtel et al, 2005) or tissue slices (Van de Bovenkamp et al, 2007), shows promising results. Another issue is the fact that metabolism may take place in tissues other than the ultimate target for the toxicity of the metabolite, thus making the determination of the parent compound's toxicity in vitro systems solely consisting of the target tissue for the metabolite of little use.…”

“…Here too, kinetic modeling is a useful tool for the interpretation of the data. An example of such an in vitro biokinetic modeling is the study by Treijtel et al (2005).…”