Effects of genetic variants of human P450 oxidoreductase on catalysis by CYP2D6 in vitro

Department of Pediatrics, University of California, San Francisco, California 94143-0978, USA.
Pharmacogenetics and Genomics (Impact Factor: 3.48). 11/2010; 20(11):677-86. DOI: 10.1097/FPC.0b013e32833f4f9b
Source: PubMed

ABSTRACT Cytochrome P450 (P450) oxidoreductase (POR) donates electrons to all microsomal cytochrome P450s, including drug-metabolizing and steroidogenic enzymes. Severe POR mutations cause skeletal malformations and disordered steroidogenesis. The POR polymorphism A503V is found on approximately 28% of human alleles and decreases activities of CYP3A4 and steroidogenic CYP17, but not the activities of steroidogenic CYP21 or drug-metabolizing CYP1A2 and CYP2C19. CYP2D6 metabolizes about 25% of clinically used drugs; we assessed the capacity of POR variants to support the activities of human CYP2D6.
N-27 forms of wildtype (WT), Q153R, A287P, R457H and A503V POR, and WT CYP2D6 were expressed in Escherichia coli. POR proteins in bacterial membranes were reconstituted with purified CYP2D6. Support of CYP2D6 was measured by metabolism of EOMCC (2H-1-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)), dextromethorphan and bufuralol. Michaelis constant (K(m)) and maximum velocity (V(max)) were determined in three triplicate experiments for each reaction; catalytic efficiency is expressed as V(max)/K(m).
Compared with WT POR, disease-causing POR mutants A287P and R457H supported no detectable CYP2D6 activity with EOMCC, but A287P supported approximately 25% activity with dextromethorphan and bufuralol. Q153R had increased function with CYP2D6 (128% with EOMCC, 198% with dextromethorphan, 153% with bufuralol). A503V supported decreased CYP2D6 activity: 85% with EOMCC, 62% with dextromethorphan and 53% with bufuralol.
POR variants have different effects depending on the substrate metabolized. Disease-causing POR mutations R457H and A287P had poor activities, suggesting that diminished drug metabolism should be considered in affected patients. The common A503V polymorphism impaired CYP2D6 activities with two commonly used drugs by 40-50%, potentially explaining some genetic variation in drug metabolism.

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    • "POR, P450 oxidoreductase. Data were compiled from previous reports (Flück et al., 2004, 2010, 2011a; Pandey et al., 2004, 2007, 2010; Huang et al., 2005, 2008; Agrawal et al., 2008, 2010; Nicolo et al., 2010; Sandee et al., 2010). # DBSNP id not available, these were identified in a sequencing study by Bioventures Inc. (Solus et al., 2004) "
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    ABSTRACT: Cytochrome P450 oxidoreductase (POR) supports reactions of microsomal cytochrome P450 which metabolize drugs and steroid hormones. Mutations in POR cause disorders of sexual development. P450 oxidoreductase deficiency (PORD) was initially identified in patients with Antley-Bixler syndrome but now it has been established as a separate disorder of sexual development (DSD). Here we are summarizing the work on variations in POR related to metabolism of drugs and xenobiotics. We have compiled mutation data on reported cases of PORD from clinical studies. Mutations found in patients with defective steroid profiles impact metabolism of steroid hormones as well as drugs. Some trends are emerging that establish certain founder mutations in distinct populations with Japanese (R457H), Caucasian (A287P) and Turkish (399-401) populations showing repeated findings of similar mutations. Most other mutations are found as single occurrences. A large number of different variants in POR gene with more than 130 amino acid changes are now listed in databases. Among the polymorphisms, the A503V allele is found in about 30% of all alleles but there are some differences across different population groups.
    Frontiers in Pharmacology 04/2014; 5(103). DOI:10.3389/fphar.2014.00103 · 3.80 Impact Factor
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    • "In particular the A503V variation (POR * 28) is common with frequencies ranging from 19 to 37% in all major ethnicities. In recombinant systems the variant retained >50% of the wild type activity toward several CYPs (Huang et al., 2008b; Sandee et al., 2010) and CYP3A4 activity was influenced in a substratedependent way (Agrawal et al., 2010). In an in vivo study it was found that POR * 28 TT genotype was associated with a 1.6-fold increase in CYP3A midazolam 1 -hydroxylase activity compared with POR * 28 C carriers, a finding that could be replicated in an "
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    ABSTRACT: CYP3A4 is the most important drug metabolizing enzyme in adult humans because of its prominent expression in liver and gut and because of its broad substrate specificity, which includes drugs from most therapeutic categories and many endogenous substances. Expression and function of CYP3A4 vary extensively both intra- and interindividually thus contributing to unpredictable drug response and toxicity. A multitude of environmental, genetic and physiological factors are known to influence CYP3A4 expression and activity. Among the best predictable sources of variation are drug-drug interactions, which are either caused by PXR-, CAR-mediated gene induction, or by inhibition through coadministered drugs or other chemicals, including also plant and food ingredients. Among physiological and pathophysiological factors are hormonal status, age, and gender, the latter of which was shown to result in higher levels in females compared to males, as well as inflammatory processes that downregulate CYP3A4 transcription. Despite the influence of these nongenetic factors, the genetic influence on CYP3A4 activity was estimated in previous twin studies and using information on repeated drug administration to account for 66% up to 88% of the interindividual variation. Although many single nucleotide polymorphisms (SNPs) within the CYP3A locus have been identified, genetic association studies have so far failed to explain a major part of the phenotypic variability. The term “missing heritability” has been used to denominate the gap between expected and known genetic contribution, e.g. for complex diseases, and is also used here in analogy. In this review we summarize CYP3A4 pharmacogenetics/genomics from the early inheritance estimations up to the most recent genetic and clinical studies, including new findings about SNPs in CYP3A4 (*22) and other genes (POR, PPARA) with possible contribution to CYP3A4 variable expression.
    Frontiers in Genetics 02/2013; 4:12. DOI:10.3389/fgene.2013.00012
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    • "Interindividual variations in POR activity (at least 4-to 5-fold differences) have been found in human liver microsomes (Kaminsky et al., 1984; Hart et al., 2008). The notion that variations in POR expression or activity influence the rates of P450- mediated drug metabolism in patients is supported by several lines of data, including the impact of Por gene knockout on drug clearance in mouse models (Gu et al., 2003; Henderson et al., 2003; Zhang et al., 2009), positive correlations between POR activity and P450-mediated drug metabolism activities in human liver microsomes (Kaminsky et al., 1984; Hart et al., 2008), and the impact of many POR single nucleotide polymorphisms (SNPs) on P450 activities toward drugs and other xenobiotics in reconstituted enzyme systems (Agrawal et al., 2010; Flück et al., 2010; Marohnic et al., 2010; Nicolo et al., 2010; Sandee et al., 2010; Miller et al., 2011). "
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    ABSTRACT: Cytochrome P450 oxidoreductase (POR) is required for drug metabolism by all microsomal cytochrome P450 enzymes. The aim of this study was to investigate whether any of the common single nucleotide polymorphisms (SNPs) in the POR gene and its flanking intergenic sequences correlate with interindividual variations in the warfarin maintenance dose (which is determined partly by rates of warfarin metabolism) in patients undergoing anticoagulation therapy. Warfarin dose and patients' demographic and clinical information were collected from 124 patients, who had attained a stable warfarin dose while receiving treatment at the Stratton VA Medical Center. Genomic DNAs were isolated from blood samples and were genotyped for 15 SNPs (including 10 SNPs on the POR gene). Association analysis was performed on 122 male patients by linear regression. Simple regression analysis revealed that vitamin K epoxide reductase complex subunit 1 (VKORC1) -1639A>G, CYP2C9*2, CYP2C9*3, age, and chronic aspirin therapy were significantly associated with warfarin dose. In contrast, multiple regression analysis revealed that, in addition to several known factors contributing to the variations in warfarin maintenance dose (VKORC1 -1639A>G, CYP2C9*2, CYP2C9*3, CYP4F2 rs2108622, and chronic aspirin therapy), three common POR SNPs (-173C>A, -208C>T, and rs2868177) were also significantly associated with variations in warfarin maintenance dose. These results indicate, for the first time, that three common SNPs in the POR gene may contribute to the interindividual variability in warfarin maintenance dose. Further studies on functional characterization of the POR SNPs identified, including their impact on the in vivo metabolism of additional drugs, are needed.
    Drug metabolism and disposition: the biological fate of chemicals 05/2011; 39(8):1433-9. DOI:10.1124/dmd.111.038836 · 3.25 Impact Factor
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