Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans

Abstract

Aims To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CL int ) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. Methods V max , K m and CL int (defined as V max /K m ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Results Microsomal CL int values differed by over two orders of magnitude, with the means ranging from 0.18 ( phenytoin) to 40.70 (diclofenac) ml minmicrosomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CL int tended to be underpredicted. While the in vivo unbound K m for phenytoin, 1-5 mm is substantially lower than the value determined in microsomes based on total concentrations (56 mm), correction for the in vitro binding reduces this value to 20 mm and 6 mm in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide K m . Conclusions An appreciation of the utility of in vitro prediction can be best achieved when the range of CL int values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CL int values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent. Keywords: CYP2C9, human microsomes, intrinsic clearance, in vitro predictionsin vitro and in vivo clearances of 25 drugs and were able Introduction to demonstrate a significant correlation despite the numerous sources of these data and the low sample A general strategy to quantitatively predict in vivo drug clearance from CL int values estimated from in vitro drug numbers upon which the parameters were based (see later). There remains a need for more indepth examinmetabolising systems has recently been explored and evaluated in the rat using a data base of 35 drugs which ations of the utility of human in vitro kinetic data for the prediction of the in vivo situation. show a range of turnovers covering four orders of magnitude [1][2][3]. The ultimate application of such aAs there is a shortage of good quality human liver material from which hepatocytes can be successfully prediction strategy is with human tissue, and although there have been many studies with human in vitro tissue, isolated, the vast majority of researchers employ the hepatic microsomal fraction as their enzyme source due often sufficient methodological detail is lacking from the original report to allow a systematic analysis. Iwatsubo to its ease of preparation from tissue of varying quality and i...

Effect of Albumin on Phenytoin and Tolbutamide Metabolism in Human Liver Microsomes: An Impact More Than Protein Binding

“…To minimize nonspecific binding of the substrates to microsomal proteins, lower enzyme concentrations were used in the present study (250 and 125 g/ml for PHT and TLB, respectively), compared with previous reports (Ludden et al, 1997;Carlile et al, 1999). Less than 10% PHT (5 M) and TLB (50 M) was bound to microsomal protein, and the value for TLB was negligible when its concentration reached 1000 M (data not shown).…”

“…For a better understanding of the effect of protein binding on the estimation of in vitro kinetic parameters, we examined phenytoin (PHT) and tolbutamide (TLB) metabolism in human liver microsomes. These two CYP2C9 substrates are known to differ in terms of protein binding and turnover rates (Bajpai et al, 1996;Carlile et al, 1999). …”

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

“…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).…”

“…Incubation conditions for data generated in the authors' laboratory have been detailed previously (Austin et al, 2002;McGinnity et al, 2004). Data from microsomal studies reflected a variety of methods including formal Michaelis-Menten kinetic analysis [CL int ϭ V max /K m for specific metabolite(s) formation (Carlile et al, 1999;Andersson et al, 2004)] and substrate depletion at low substrate concentrations (Obach, 1999;Naritomi et al, 2001), which was used for all hepatocyte data. Datasets were compiled with several key objectives in mind: to expand existing databases substantially, to provide some assessment of interlaboratory variability, and to complement external datasets in terms of representation from different chemical classes covering a range of physicochemical properties.…”

Metabolism of fibrates by cytochrome P450s and UDP-glycosyltransferases in rat and human liver microsomes

“…This may be partly due to the fact that the contributions of the major metabolic pathways to fibrate clearance have not been systematically investigated. Previous in vitro studies generally focused on a specific pathway by adding either NADPH or UDPGA to initiate oxidative or conjugated reactions [22,29]. Such strategies may hence underestimate the true metabolic rate of fibrates when using in vitro-in vivo extrapolation studies because the contribution of both pathways should not be ignored.…”