The “Albumin Effect” and in Vitro-in Vivo Extrapolation: Sequestration of Long-Chain Unsaturated Fatty Acids Enhances Phenytoin Hydroxylation by Human Liver Microsomal and Recombinant Cytochrome P450 2C9

Abstract

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...

Prediction of Drug Clearance by Glucuronidation from in Vitro Data: Use of Combined Cytochrome P450 and UDP-Glucuronosyltransferase Cofactors in Alamethicin-Activated Human Liver Microsomes

“…The improved prediction accuracy with the use of the parallel tube liver model was in good agreement with the observations by Ito and Houston (2005). It has recently been reported that the addition of BSA to microsomal incubation decreases the K m and consequently increases the clearance estimates for UGT2B7 and UGT1A9 substrates (Rowland et al, 2007(Rowland et al, , 2008b; an analogous effect was observed on CYP2C9 (Carlile et al, 1999;Rowland et al, 2008a). The rationale is that BSA sequesters the inhibitory effect of unsaturated long-chain fatty acids released during the incubation on certain UGTs.…”

“…The -fold change in CL int is in good agreement with a 5-fold increase in phenytoin CL int observed in the presence of 2% BSA (Rowland et al, 2008b). Diclofenac and phenytoin are reported to be metabolized at the same CYP2C9 binding site (Kumar et al, 2006); therefore, the effects on diclofenac are probably caused by a decrease in the CYP2C9 K m value in a manner similar to that of phenytoin (Rowland et al, 2008a), resulting in an increased CL int .…”

“…The addition of BSA to incubations sequesters the fatty acids, resulting in a 9-to 10-fold increase in the intrinsic clearance (CL int ) of propofol (Rowland et al, 2008b) and zidovudine (Rowland et al, 2007). A comparable "albumin effect" on the CYP2C9 substrate phenytoin was also observed (Rowland et al, 2008a).…”

The “Albumin Effect” and Drug Glucuronidation: Bovine Serum Albumin and Fatty Acid-Free Human Serum Albumin Enhance the Glucuronidation of UDP-Glucuronosyltransferase (UGT) 1A9 Substrates but Not UGT1A1 and UGT1A6 Activities

“…We have reported previously that BSA and HSAFAF but not HSA decrease the K m values for zidovudine glucuronidation by UGT2B7 and HLM and for phenytoin hydroxylation by CYP2C9 and HLM. The decrease in K m values, approximately 1 order of magnitude, arises from sequestration of long-chain unsaturated fatty acids released from microsomes or cell lysate during the course of an incubation (Rowland et al, 2007(Rowland et al, , 2008. Long-chain unsaturated fatty acids, particularly arachidonic, linoleic, and oleic, were shown to be potent competitive inhibitors of UGT2B7 and CYP2C9 (Tsoutsikos et al, 2004;Rowland et al, 2007Rowland et al, , 2008.…”

“…The decrease in K m values, approximately 1 order of magnitude, arises from sequestration of long-chain unsaturated fatty acids released from microsomes or cell lysate during the course of an incubation (Rowland et al, 2007(Rowland et al, , 2008. Long-chain unsaturated fatty acids, particularly arachidonic, linoleic, and oleic, were shown to be potent competitive inhibitors of UGT2B7 and CYP2C9 (Tsoutsikos et al, 2004;Rowland et al, 2007Rowland et al, , 2008. The present work indicates that the same mechanism is responsible for the enhanced glucuronidation of UGT1A9 substrates observed in the presence of BSA and HSAFAF.…”