Drug metabolism by CYP2C8.3 is determined by substrate dependent interactions with cytochrome P450 reductase and cytochrome b5

Abstract

Genetic polymorphisms in CYP2C8 can influence the metabolism of important therapeutic agents and cause interindividual variation in drug response and toxicity. The significance of the variant CYP2C8*3 has been controversial with reports of higher in vivo but lower in vitro activity compared to CYP2C8*1. In this study, the contribution of the redox partners cytochrome P450 reductase (CPR) and cytochrome b5 to the substrate dependent activity of CYP2C8.3 (R139K, K399R) was investigated in human liver microsomes (HLMs) and E. coli expressed recombinant CYP2C8 proteins using amodiaquine, paclitaxel, rosiglitazone and cerivastatin as probe subtrates. For recombinant CYP2C8.3, clearance values were two- to five-fold higher compared to CYP2C8.1. CYP2C8.3’s higher kcat seems to be dominated by a higher, but substrate specific affinity, towards cytochrome b5 and CPR (KD and Km,red) which resulted in increased reaction coupling. A stronger binding affinity of ligands to CYP2C8.3, based on a two site binding model, in conjunction with a five fold increase in amplitude of heme spin change during binding of ligands and redox partners could potentially contribute to a higher kcat. In HLMs, carriers of the CYP2C8*1/*3 genotype were as active as CYP2C8*1/*1 towards the CYP2C8 specific reaction amodiaquine N-deethylation. Large excess of cytochrome b5 compared to CYP2C8 in recombinant systems and HLMs inhibited metabolic clearance, diminishing the difference in kcat between the two enzymes, and provides an explanation for the discrepancy to in vivo data. In silico studies illustrate the genetic differences between wild type and variant on the molecular level.

Interindividual Variability in Hepatic Expression of the Multidrug Resistance-Associated Protein 2 (MRP2/ABCC2): Quantification by Liquid Chromatography/Tandem Mass Spectrometry

“…In summary, this is the first study to investigate MRP2 quantification in a large number of human livers that have been extensively phenotyped for cytochrome P450 activity and expression (Paine et al, 1997;Lin et al, 2002;Lalovic et al, 2004;Naraharisetti et al, 2010;Kaspera et al, 2011). Such MRP2 quantification data combined with MRP2 activity/expression data in cell lines expressing recombinant MRP2 or in hepatocytes will be a tremendous asset toward using physiologically based pharmacokinetic models to predict drug (or metabolite) hepatic exposure and clearance.…”

Activity, Inhibition, and Induction of Cytochrome P450 2J2 in Adult Human Primary Cardiomyocytes

“…Protein expression was performed as previously described (Cheesman et al, 2003;Kaspera et al, 2011) and harvested cells were resuspended in storage buffer and stored in -80°C until purification.…”

“…Frozen pellets were thawed on ice and resuspended in 100 mM potassium phosphate (pH 7.4) containing 20% glycerol and protease inhibitors. Purification was conducted following established procedures (Kaspera et al, 2011).…”

“…Recombinant CYP2J2 was reconstituted with reductase and lipid according to previously established protocols (Kaspera et al, 2011). Briefly, the mixture used was as follows: 1 pmol/ml recombinant CYP2J2 was mixed with 2 pmol/ml rat cytochrome P450 reductase (CPR), 1 pmol/ml cytochrome b 5 , buffer containing 100 mM potassium phosphate (pH 7.4), and 50 mM DLPC on ice for 40 minutes with intermittent mixing.…”

Effects of theCYP2B6*6Allele on Catalytic Properties and Inhibition of CYP2B6 In Vitro: Implication for the Mechanism of Reduced Efavirenz Metabolism and Other CYP2B6 Substrates In Vivo

“…As has been shown for other cytochromes P450 (P450s) such as CYP2C8 (Kaspera et al, 2011) and CYP2C9 (Kumar et al, 2006), several factors inherent to specific enzyme sources that include differences in cytochrome b5 (Cyt b5) contents may influence in vitro kinetic parameters and inhibition constants in a substrate-dependent manner. Cyt b5 has been reported to activate several P450s including CYP2B6 (Reed and Hollenberg, 2003;Jushchyshyn et al, 2005), but its influence on the catalytic properties of CYP2B6.6 protein has not been studied.…”