Q172H Replacement Overcomes Effects on the Metabolism of Cyclophosphamide and Efavirenz Caused by CYP2B6 Variant with Arg262

Abstract

ABSTRACT:There are a number of reports indicating that CYP2B6*6 (c.516G>T and c.785A>G) is responsible for decreased clearance of efavirenz (EFV), although increased disposition of cyclophosphamide (CPA) in individuals with this polymorphism was observed. Thus, we hypothesized that the effects of the two single nucleotide polymorphisms (SNPs) of CYP2B6*6 on the metabolism of drugs might be considerably different between these two agents. To clarify this possibility, we expressed two major variants of this enzyme, CYP2B6.6 (Q172H and K262R) and CYP2B6.4 (K262R), and investigated metabolic activities of these variants toward EFV and CPA. Kinetic analyses clearly indicated that CYP2B6.4 possessed enhanced metabolic activity toward EFV compared with that of the wild-type enzyme (CYP2B6.1), whereas CPA was metabolized less efficiently by CYP2B6.4 than by CYP2B6.1. On the other hand, CYP2B6.6 showed a completely opposite character, suggesting that Q172H gives inverse effects on metabolic activities of CYP2B6 affected by K262R. Although it is recognized that effects of amino acid change in cytochrome P450 on the metabolic activity depend on substrates, this study revealed SNPs giving an opposite effect on the metabolism of two clinically important drugs currently used. Furthermore, this study provides the first evidence that Q172H can reverse the direction of the effect caused by K262R in CYP2B6 on the metabolism of certain drugs.

Differences in Methadone Metabolism by CYP2B6 Variants

“…In an insect cell system with coexpressed P450 reductase and b 5 , CYP2B6.6 Cl int for bupropion 4-hydroxylation was lower, whereas efavirenz 8-hydroxylation was less but not significantly different from CYP2B6.1 (Xu et al, 2012). CYP2B6.6 activity in other systems (E. coli, Cos-1, Cos-7) was lower toward bupropion and efavirenz (Zhang et al, 2011), and ketamine (Li et al, 2013), but greater for cyclophosphamide (Xie et al, 2003;Ariyoshi et al, 2011;Raccor et al, 2012) and artemether (Honda et al, 2011), and unchanged for selegiline (Watanabe et al, 2010). The present and previous (Gadel et al, 2013) experiments show that methadone is one of the most catalytically diminished substrates for CYP2B6.6.…”

“…CYP2B6.4 results are even more substrate-dependent. In E. coli, Cos-1, and Cos-7 systems, CYP2B6.4 cyclophosphamide 4-hydroxylation was 25% lower than CYP2B6.1 (Ariyoshi et al, 2011;Raccor et al, 2012), the catalytic efficiency for 7-ethoxy-4-trifluoromethylcoumarin, bupropion, and efavirenz was decreased to half, one-third, and unchanged, respectively (Zhang et al, 2011), and artemether and selegiline metabolism was almost doubled (Watanabe et al, 2010;Honda et al, 2011). Methadone metabolism by CYP2B6.4 in the present investigation was greater than by CYP2B6.1.…”

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

“…This variant has been associated with enhanced cyclophosphamide metabolism in vitro (Xie et al, 2003;Ariyoshi et al, 2011) and in vivo (Nakajima et al, 2007), which seems to be primarily driven by the (Ariyoshi et al, 2011). The CYP2B6*6 allele appears to alter substrate metabolism in two ways: 1) by decreasing (e.g., cyclophosphamide) or increasing (e.g., efavirenz) substrate binding (Ariyoshi et al, 2011;Zhang et al, 2011;present data) probably due to changes in the three-dimensional structures of the protein; and 2) by reducing catalytic efficiency secondary to reduced protein expression (Hofmann et al, 2008). Overall, altered substrate binding and/or catalytic activity as a result of amino acid changes seem to play a critical role in determining the substratedependent functional consequences of the CYP2B6*6 allele.…”

“…However, mounting evidence indicates that reduced protein expression alone may not explain the functional consequences of this allele. For substrates that include cyclophosphamide, this allele is associated with enhanced metabolism despite reduced protein expression (Xie et al, 2003), which appears due to substantially lower K m in the variant versus wild-type protein (Ariyoshi et al, 2011).…”

“…The CYP2B6 gene is highly polymorphic as reflected by 29 associated alleles, many suballeles, and single nucleotide polymorphisms (SNPs) (http://www.imm.ki.se/CYPalleles/cyp2b6.htm). Of the variants identified so far, the CYP2B6*6 haplotype defined by two nonsynonymous SNPs, 516GϾT (Q172H) and 785AϾG (K262R), is clinically important because this allele or the SNP tagging it (G516T) occurs at high frequency in all ethnic populations [14 -62% ] and has been associated with functional consequences in expressed systems (Ariyoshi et al, 2001;Jinno et al, 2003;Bumpus and Hollenberg, 2008;Watanabe et al, 2010;Ariyoshi et al, 2011;Zhang et al, 2011) and in human liver microsomes (HLMs) (Lang et al, 2001;Lamba et al, 2003;Xie et al, 2003;Hesse et al, 2004;Desta et al, 2007). Subsequent to the demonstration that CYP2B6 is the principal clearance mechanism of efavirenz in vitro (Ward et al, 2003), several studies have documented that the CYP2B6*6 allele or its tagging SNP is at increased risk for higher efavirenz exposure and/or adverse effects (Haas et al, 2004;Tsuchiya et al, 2004;Zanger et al, 2007).…”