Utility of Hepatocytes in Predicting Drug Metabolism: Comparison of Hepatic Intrinsic Clearance in Rats and Humans in Vivo and in Vitro

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:We investigated hepatic in vitro intrinsic clearance (CL int,in vitro ) in freshly isolated or cryopreserved hepatocytes and compared with CL int,in vivo by using nine model compounds, FK1052, FK480, diazepam, diltiazem, troglitazone, quinotolast, FK079, zidovudine, and acetaminophen, in rats and humans. The compounds showed a broad range of in vivo hepatic extraction ratios (rat, 0.05-0.93; humans, 0.03-0.76) and were metabolized by hepatic P450, UDPglucuronosyltransferase, sulfotransferase, and/or esterase. CL int,in vitro was determined from substrate disappearance rate at 1 M in hepatocytes. CL int,in vivo was calculated from in vivo pharmacokinetic data using two frequently used mathematical models (the well stirred and dispersion models). When estimating rat CL int,in vitro in freshly isolated hepatocytes, the rat scaling factor values (CL int,in vivo /CL int,in vitro ) showed marked difference among the model compounds (0.2-73.1-fold). The rat CL int,in vitro values in freshly isolated hepatocytes were in good agreement with these in cryopreserved hepatocytes. Human CL int,in vitro were determined by use of cryopreserved hepatocytes. When human CL int,in vitro was regarded as the predicted CL int,in vivo , the observed and predicted CL int,in vivo for FK1052, FK480, troglitazone, and FK079 differed markedly (12.4-199.0-fold). In contrast, using human CL int,in vitro corrected with the rat scaling factors yielded better predictions of CL int,in vivo that were mostly within 5-fold of the actual values. These results make the evaluation using hepatocytes more useful and provide a basis for predicting hepatic clearance using hepatocytes.Recently, pharmacokinetic investigation has played an increasingly important role in drug discovery. In particular, it is very important to predict human hepatic metabolic clearance because most drugs are eliminated from the body predominantly by hepatic metabolism. For predicting hepatic clearance, theoretical aspects of in vitro/in vivo scaling based on a physiological model and clearance concepts have been developed (Roberts and Rowland, 1986). Application of this method has been successful in predicting in vivo hepatic clearance in rats for many drugs metabolized by P450 1 from in vitro metabolism data using rat liver microsomes and isolated hepatocytes (Sugiyama et al., 1988;Houston and Carlile, 1997). Because human liver samples have become more readily available, it would also be very useful to predict in vivo outcomes from in vitro data in humans. However, there has been relatively limited application of this approach (Hoener, 1994), and there have been some failed attempts at the prediction of human hepatic clearance. For example, Iwatsubo et al. (1997) and Houston and Carlile (1997) reported that CL int,in vitro generally exhibited a positive correlation with CL int,in vivo , but in some cases animal or human clearance values were not well predicted from in vitro studies. To improve the predict...

A Unified Model for Predicting Human Hepatic, Metabolic Clearance From in Vitro Intrinsic Clearance Data in Hepatocytes and Microsomes

“…CL int, in vivo data were provided in several reports (Shibata et al, 2002;Naritomi et al, 2003). Relationships obtained using the parallel tube model to compute CL int, in vivo were very similar to those reported (data not shown).…”

“…CL int, in vitro data derived from hepatocyte (Lave et al, 1997a,b;Lau et al, 2002;Shibata et al, 2002;Naritomi et al, 2003) or microsomal incubations (Carlile et al, 1999;Obach, 1999;Naritomi et al, 2001;Andersson et al, 2004) were generated in the authors' laboratory and collated from several published studies (Tables 1-3). Incubation conditions for data generated in the authors' laboratory have been detailed previously (Austin et al, 2002;McGinnity et al, 2004).…”

“…For the remaining datasets, the unbound fraction in the incubation (fu inc ) was applied either as reported by the authors (Naritomi et al, 2003) or predicted from a consideration of chemical class and either log D 7.4 or log P: for microsomes, log(1 Ϫ fu/fu) ϭ 0.53 log P/D Ϫ1.42 (Austin et al, 2002); and for hepatocytes, log(1 Ϫ fu/fu) ϭ 0.40 log P/D Ϫ1.38 (Austin et al, 2005).…”

“…The hepatocyte data were compiled from several sources: incubations conducted with more simple cell suspensions (Lau et al, 2002;Naritomi et al, 2003;authors' laboratory), and cells cultured in the presence of exogenous protein. Additional protein was either autologous (human) serum (Shibata et al, 2002) or 10% fetal calf serum (FCS) for incubations conducted with cultured cells for up to 72 h (Lave et al, 1997a,b;Schneider et al, 1999).…”

Human microdose evaluation of the novel EP1 receptor antagonist GSK269984A

“…A similar approach can be undertaken for human CL predictions, although for certain classes of compound there is a lack of correlation between the CLi value derived in vitro using human microsomes or hepatocytes and that Human microdose study of GSK269984A calculated in vivo [43]. Ostensibly, the present data would lend support to other studies that have shown how the prediction of human CL in vivo can be improved by the appropriate incorporation of in vitro data.For example, predictions of human CL based on allometric scaling from pre-clinical in vivo data can be improved by first normalizing in vivo CL values using the respective in vitro CLi values [44], while other investigators have proposed the initial correction of in vitro human CLi using a scaling factor derived from animal studies [45,46]. However, in this current study, the values obtained for GSK269984A CLi in microsomes (rat, dog, primate and human), S9 fractions (rat, dog, primate and human) and hepatocytes (rat, primate and human) were essentially equal across all species [16].…”