Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter‐system extrapolation factors

Abstract

The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

Direct and quantitative evaluation of the human CYP3A4 contribution (fm) to drug clearance using the in vitro SILENSOMES model

“…For example, for CYP1A2, Venkatakrishnan and colleagues reported a 4-fold difference in the RAF when comparing methoxyresorufin or phenacetin (Lipscomb & Poet, 2008;Venkatakrishnan et al, 2000). The same phenomenon was identified for CYP2C9 using diclofenac, tolbutamine and Swarfarin (Crewe et al, 2011;Kumar et al, 2006;Locuson et al, 2007) and with CYP3A4, a 2-fold difference between nifedipine and testosterone (Emoto & Iwasaki, 2007;Patki et al, 2003) is evident. In our study, a 5-fold difference was observed (RAF of 9.8 for testosterone and 49 for nifedipine).…”

Simultaneous Quantification of the Abundance of Several Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzymes in Human Liver Microsomes Using Multiplexed Targeted Proteomics

“…Alternatively, abundance may not reflect the activity when various CYP2C9 genotypes are assessed together. In addition, differences in expression of cytochrome b5 and NADPH P450 reductase in different individuals have been reported to lead to disparities in CYP2C9 enzyme activity (Crewe et al, 2011). These were not known for individual samples under study.…”

Application of Physiologically Based Pharmacokinetic Modeling to Predict Acetaminophen Metabolism and Pharmacokinetics in Children

“…41 Because the in vitro enzyme kinetic data used relative activity to express CYP-mediated NAPQI formation, 44 both in vitro and in vivo values and systemic information was combined to convert this unit to the absolute amount of NAPQI. The unbound intrinsic clearance (CL u,int(enzyme) ) of each CYP and UGT enzyme and the total intrinsic clearance of sulfotransferases were normalized to pmol enzyme (CYPs) or microsomal protein (UGTs and total sulfotransferases) levels by incorporating system-specific information including enzyme abundance, microsomal protein expression level, liver weight, intersystem extrapolation factor for scaling of recombinant CYP in vitro kinetic data, 48 which are provided by the ADME simulator, and intrinsic clearance derived from in vivo study. The maximum velocity for each phase I and phase II pathway or enzymes (V max(enzyme) ) was derived from the calculated CL u,int(enzyme) values using the following equation:…”