ABSTRACT:This study characterized the mechanism by which bovine serum albumin (BSA) reduces the K m for phenytoin (PHY) hydroxylation and the implications of the "albumin effect" for in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates. BSA and essentially fatty acid-free human serum albumin (HSA- The use of in vitro kinetic data to predict drug metabolic stability in vivo has attracted widespread interest, particularly in preclinical drug development. Most commonly, intrinsic clearance (CL int ) determined in vitro is scaled to an in vivo CL int and hepatic clearance (CL H ) using human liver microsomes (HLMs) or hepatocytes as the enzyme source (Houston 1994;Iwatsubo et al., 1997;Obach et al., 1997;McGinnity et al., 2004). Although in vitro models and scaling approaches have been progressively refined in recent years, in vitro-in vivo extrapolation (IV-IVE) typically results in underprediction of in vivo CL int and CL H (and hence hepatic extraction ratio, E H ). With HLMs as the enzyme source, in vivo CL int is underpredicted by approximately an order of magnitude for drugs eliminated by either cytochrome P450-or UDP-glucuronosyltransferase-catalyzed biotransformation (Boase and Miners, 2002;Ito and Houston, 2005). Prediction bias is improved using hepatocytes (Soars et al., 2002;Riley et al., 2005), but there remains an approximate 4-fold underprediction of the in vivo CL int Brown et al., 2007).FAFThe reason for the underprediction of in vivo CL int (and CL H ) based on human liver microsomal kinetic data remains unclear. Nonspecific microsomal binding, variability in experimental conditions and physiological scaling factors, and inappropriate kinetic modeling all affect the reliability of in vitro-in vivo extrapolation , but significant underprediction remains even when these factors are taken into account. Similarly, involvement of hepatic uptake transporters potentially accounts for the improved predictivity of kinetic data generated with hepatocytes, although recent data suggest that hepatic uptake is not rate-limiting in the clearance of lipophilic drugs (Doherty et al., 2006;Halifax and Houston, 2006).The addition of bovine serum albumin (BSA) to incubations of HLMs has been reported to increase the rate of metabolism of CYP2C9 substrates (Ludden et al., 1997;Carlile et al., 1999;Tang et al., 2002;Zhou et al., 2004) and microsomal CL int values of UGT2B7 substrates (Uchaipichat et al., 2006;Rowland et al., 2007 19885/3327892 DMD 36:870-877, 2008 Printed in U.S.A.
870at ASPET Journals on May 10, 2018 dmd.aspetjournals.org
Downloaded fromtion by HLMs in incubations supplemented with 2% BSA were 6-to 20-fold lower compared with K m values generated in the absence of BSA. Maximal velocity was generally unaffected by BSA, although Carlile et al. (1999) reported a 2-fold reduction in the V max for tolbutamide tolymethylhydroxylation. Consistent with this observation, long-chain unsaturated fatty acids, including oleic (C18:1n-9), linoleic (C18:2n-6), and arachidonic (C20:4n-6) acids, are...