Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance

Abstract

Cytochrome P450 2B6 (CYP2B6) belongs to the minor drug metabolizing P450s in human liver. Expression is highly variable both between individuals and within individuals, owing to non-genetic factors, genetic polymorphisms, inducibility, and irreversible inhibition by many compounds. Drugs metabolized mainly by CYP2B6 include artemisinin, bupropion, cyclophosphamide, efavirenz, ketamine, and methadone. CYP2B6 is one of the most polymorphic CYP genes in humans and variants have been shown to affect transcriptional regulation, splicing, mRNA and protein expression, and catalytic activity. Some variants appear to affect several functional levels simultaneously, thus, combined in haplotypes, leading to complex interactions between substrate-dependent and -independent mechanisms. The most common functionally deficient allele is CYP2B6*6 [Q172H, K262R], which occurs at frequencies of 15 to over 60% in different populations. The allele leads to lower expression in liver due to erroneous splicing. Recent investigations suggest that the amino acid changes contribute complex substrate-dependent effects at the activity level, although data from recombinant systems used by different researchers are not well in agreement with each other. Another important variant, CYP2B6*18 [I328T], occurs predominantly in Africans (4–12%) and does not express functional protein. A large number of uncharacterized variants are currently emerging from different ethnicities in the course of the 1000 Genomes Project. The CYP2B6 polymorphism is clinically relevant for HIV-infected patients treated with the reverse transcriptase inhibitor efavirenz, but it is increasingly being recognized for other drug substrates. This review summarizes recent advances on the functional and clinical significance of CYP2B6 and its genetic polymorphism, with particular emphasis on the comparison of kinetic data obtained with different substrates for variants expressed in different recombinant expression systems.

Full text

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REVIEW ARTICLE
published: 05 March 2013
doi: 10.3389/fgene.2013.00024
Pharmacogenetics of cytochrome P450 2B6 (CYP2B6):
advances on polymorphisms, mechanisms, and clinical
relevance
Ulrich M. Zanger1,2* and Kathrin Klein1,2
1Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
2The University of Tuebingen,Tuebingen, Germany
Edited by:
José A. Agúndez, University of
Extremadura, Spain
Reviewed by:
Chin Eap, University of Lausanne
Medical School, Switzerland
Rajeev K. Mehlotra, Case Western
Reserve University, USA
*Correspondence:
Ulrich M. Zanger, Dr. Margarete
Fischer Bosch Institute of Clinical
Pharmacology, Auerbachstrasse 112,
70376 Stuttgart, Germany.
e-mail: uli.zanger@ikp-stuttgart.de
Cytochrome P450 2B6 (CYP2B6) belongs to the minor drug metabolizing P450s in human
liver. Expression is highly variable both between individuals and within individuals, owing
to non-genetic factors, genetic polymorphisms, inducibility, and irreversible inhibition by
many compounds. Drugs metabolized mainly by CYP2B6 include artemisinin, bupropion,
cyclophosphamide, efavirenz, ketamine, and methadone. CYP2B6 is one of the most
polymorphic CYP genes in humans and variants have been shown to affect transcriptional
regulation, splicing, mRNA and protein expression, and catalytic activity. Some variants
appear to affect several functional levels simultaneously, thus, combined in haplotypes,
leading to complex interactions between substrate-dependent and -independent mech-
anisms. The most common functionally deficient allele is CYP2B6*6 [Q172H, K262R],
which occurs at frequencies of 15 to over 60% in different populations. The allele leads
to lower expression in liver due to erroneous splicing. Recent investigations suggest that
the amino acid changes contribute complex substrate-dependent effects at the activity
level, although data from recombinant systems used by different researchers are not
well in agreement with each other. Another important variant, CYP2B6*18 [I328T], occurs
predominantly in Africans (4–12%) and does not express functional protein. A large number
of uncharacterized variants are currently emerging from different ethnicities in the course of
the 1000 Genomes Project.The CYP2B6 polymorphism is clinically relevant for HIV-infected
patients treated with the reverse transcriptase inhibitor efavirenz, but it is increasingly
being recognized for other drug substrates.This review summarizes recent advances on the
functional and clinical significance of CYP2B6 and its genetic polymorphism, with particular
emphasis on the comparison of kinetic data obtained with different substrates for variants
expressed in different recombinant expression systems.
Keywords: bupropion, cyclophosphamide, cytochrome P450, drug metabolism, drug–drug interaction, efavirenz,
pharmacogenetics, pharmacogenomics
INTRODUCTION
The cytochrome P450 (CYP) enzyme CYP2B6 is one of about a
dozen human CYPs that are primarily involved in the biotrans-
formation of drugs and other xenobiotics. The CYP2B6 gene and
its closely related pseudogene, CYP2B7, are located in a tandem
head-to-tail arrangement within a large CYP2 gene cluster on the
long arm of chromosome 19 (Hoffman et al., 2001;Figure 1). The
orthologous genes in dog, mouse, and rat are termed CYP2B11,
Cyp2b10, and CYP2B1, respectively, but in contrast to other mam-
malian species, CYP2B6 is the only functional isozyme of its
subfamily in humans (Nelson et al., 2004). Owing to the existence
of extensive genetic polymorphism as well as strong inhibitors and
inducers, its activity is highly variable in the population. For some
clinically used drugs including the antiretroviral agents efavirenz
and nevirapine, CYP2B6 single nucleotide polymorphisms have
been shown to be useful predictors of pharmacokinetics and drug
response (reviewed in Zanger et al., 2007;Telenti and Zanger,2008;
Rakhmanina and van den Anker,2010 ). However,recent data indi-
cate that pharmacogenetic mechanisms are complex, appear on
several levels of gene expression from the initial mRNA transcript
to splice variants (pre-mRNA splicing and mRNA expression) to
altered proteins, and affect function in various ways including
substrate-dependent and substrate-independent effects. Several
previous reviews are available that cover the biochemical pharma-
cology, molecular genetics, and pharmacogenetics of this enzyme
at various degrees of detail (Ekins and Wrighton, 1999;Turpeinen
et al., 2006;Hodgson and Rose, 2007;Zanger et al., 2007;Wang
and Tompkins, 2008;Mo et al., 2009;Turpeinen and Zanger, 2012).
The purpose of this review is to summarize recent advances in areas
that have an impact on variable expression of CYP2B6 and the
mechanisms and impact of CYP2B6 polymorphism, as observed
by various in vitro approaches as well as in in vivo studies, and to
discuss their functional and clinical implications.
VARIABILITY OF EXPRESSION AND TRANSCRIPTIONAL
REGULATION
Cytochrome P450 2B6 is primarily expressed in the liver where its
contribution to the total microsomal P450 pool has been estimated
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Zanger and Klein Pharmacogenetics of CYP2B6
FIGURE 1 |Structure of genomic regions on chromosome 19.
Schematic representation of the CYP2ABFGST-cluster on chromosome
19 (A) and of the CYP2B6 gene (B). Boxes in the (A) represent genes,
with direction of transcription indicated by arrows.The (B) represents
CYP2B6 exons and introns and shows the most important SNPs as
triangles.
to be within a range of about 1–10%, with a large inter-individual
variability at protein level of roughly 100-fold (see Zanger et al.,
2007 for review and references therein). Although some earlier
studies reported expression in only a fraction of human liv-
ers, newer studies with better antibodies found CYP2B6 to be
present in all investigated human adult liver samples (Hofmann
et al., 2008) while up to one-third of pediatric samples contained
no detectable protein (Croom et al., 2009). In the latter study,
ontogenic differences were studied in liver microsomes from 217
pediatric liver donors. Hepatic median CYP2B6 protein levels were
about twofold higher in the period between birth and 30 days post-
natal compared to fetal samples, and protein levels varied already
over 25-fold in both of these age groups (Croom et al., 2009). Mat-
uration effects may further depend on genotype, as suggested in
a study on HIV-infected children treated with efavirenz (Sueyoshi
et al., 1999;Wang et al., 2003;Faucette et al., 2004,2007).
One of the most important factors contributing to intra- as
well as inter-individual variability is enzyme induction, i.e., de
novo protein synthesis following exposure to certain chemicals.
Regulation of CYP2B gene expression represents the archetypal
example of enzyme induction (Remmer et al., 1973). Human
CYP2B6 is strongly inducible by several drugs including “classi-
cal” inducers such as rifampicin, phenytoin, and phenobarbital
involving a so-called phenobarbital-responsive enhancer module
(PBREM) at −1.7 kb of the CYP2B6 gene promoter, and a dis-
tal xenobiotics-responsive enhancer module (XREM, −8.5 kb),
to which pregnane X receptor (PXR, NR1I2) and/or constitutive
androstane receptor (CAR,NR1I3) bind to mediate increased tran-
scription (Sueyoshi et al., 1999;Wang et al., 2003;Faucette et al.,
2004,2007). Since other CYPs are regulated by overlapping sets
of nuclear receptors, CYP2B6 is often co-induced with CYP2C
enzymes and CYP3A4. CYP2B6 inducers identified to date include
cyclophosphamide (Gervot et al., 1999), hyperforin (Goodwin
et al., 2001), artemisinin antimalarials (Simonsson et al., 2003;
Burk et al., 2005), carbamazepine (Oscarson et al., 2006;Desta
et al., 2007), metamizole (Saussele et al., 2007;Qin et al., 2012),
ritonavir (Kharasch et al., 2008), the insect repellent N,N-diethyl-
m-toluamide (DEET; Das et al., 2008), statins (Feidt et al., 2010),
efavirenz (Ngaimisi et al., 2010;Habtewold et al., 2011). Interest-
ingly, in the latter study, gender influenced the inducibility of
efavirenz 8-hydroxylation, which was higher in women than in the
men (Ngaimisi et al., 2010). In addition to therapeutic drugs, pes-
ticides were found to be powerful inducers of CYP2B6 and other
CYPs through interaction with both PXR and CAR (Das et al.,
2008). Induction of CYP2B6 and other cytochromes P450 and its
clinical consequences has been reviewed by others (Pelkonen et al.,
2008;Mo et al., 2009).
Sex differences in liver expression have been observed in a
number of studies. Females liver donors had higher amounts of
CYP2B6 mRNA (3.9-fold), protein (1.7-fold), and enzyme activity
(1.6-fold) compared to male subjects in a study of 80 ethnically
mixed samples (Lamba et al., 2003). In a study with 235 Cau-
casian liver donors, female samples had 1.6-fold higher expression
level of CYP2B6 mRNA, however, this difference did not trans-
late into higher protein and activity levels and no sex difference
was found when only liver donors without presurgical drug expo-
sure were considered (Hofmann et al., 2008). Discrepant effects
of sex on pharmacokinetics of CYP2B6 substrates, which may be
due to other confounders such as age or smoking status, were
also found in vivo. Higher bupropion hydroxylation rates were
found in adolescent females compared to males (Stewart et al.,
2001) but not in adults (Hsyu et al., 1997). For efavirenz, sev-
eral studies reported elevated plasma concentrations in female
compared to male patients, which is in contrast to the above-
mentioned in vitro findings and may be explained by other factors
such as differences in body fat content and distribution (Burger
et al., 2006;Nyakutira et al., 2008;Mukonzo et al., 2009). The influ-
ence of age on CYP2B6 expression may also depend on sex, as only
males showed a significant increase of liver CYP2B6 at higher age
(Yang et al., 2010).
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Besides liver, CYP2B6 is also consistently expressed in different
parts of respiratory and gastrointestinal tracts, including lung and
nasal mucosa, and also in skin and the kidneys (Choudhary et al.,
2003;Dutheil et al., 2008;Thelen and Dressman, 2009;Leclerc
et al., 2010). The significance of CYP2B6 in these extrahepatic tis-
sues is currently unknown, but it should be remembered that the
enzyme is probably the most important one for many environ-
mental toxins such as pesticides, and its presence in tissues with
barrier function may thus contribute substantially to protection
against these chemicals. In addition, the presence of CYP2B6 in
brain has been demonstrated in human and primate brain tissue
samples and smoking, alcohol consumption, and genetic poly-
morphism have been suggested to contribute to its variability in
this organ (Miksys et al., 2003). In general, CYP levels in extra-
hepatic tissues are far below those of liver, but the localization
to specific regions in the brain may contribute to the activation
or inactivation of centrally acting drugs and to neurological side
effects of certain medications or abused drugs, e.g., “ecstasy” [1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), see below].
This may also explain why efficacy for some centrally acting drugs
is not well correlated to their plasma levels. The potential role of
brain-expressed CYPs including CYP2B6 in the biotransformation
of centrally acting drugs has been reviewed by others (Meyer et al.,
2007;Ferguson and Tyndale, 2011).
THE CHEMICAL INTERACTION PROFILE OF CYP2B6
Recent studies have revealed crystal structures of the CYP2B6
wild-type and K262R variant in complex with various inhibitors
at providing first views into its active site and its plasticity to
adopt different conformations when binding different ligands
(Gay et al., 2010;Shah et al., 2011;Wilderman and Halpert,
2012). Substrates of CYP2B6 are usually fairly lipophilic, neu-
tral or weakly basic non-planar molecules with one or two
hydrogen bond acceptors (Lewis et al., 1999,2004). The CYP2B6
substrate selectivity comprises many diverse chemicals, includ-
ing not only clinically used drugs but also many environmental
chemicals such as pesticides (Turpeinen et al., 2006;Hodgson
and Rose, 2007;Turpeinen and Zanger, 2012). Therapeutically
important drugs metabolized primarily by CYP2B6 include the
prodrug cyclophosphamide, which is converted to the direct
precursor of the cytotoxic metabolites, phosphoramide mus-
tard and acrolein, by 4-hydroxylation (Huang et al., 2000;Roy
et al., 2005), the non-nucleoside reverse transcriptase inhibitor
(NNRTI), efavirenz, which is 8-hydroxylated to become phar-
macologically inactive (Ward et al., 2003;Desta et al., 2007), the
atypical antidepressant and smoking cessation agent bupropion,
which is converted to pharmacologically active hydroxybupro-
pion (Faucette et al., 2000;Hesse et al., 2000;Turpeinen et al.,
2005b), the anesthetics propofol (Court et al., 2001;Oda et al.,
2001) and ketamine (Desta et al., 2012), the analgesic pethidine
(meperidine; Ramírez et al., 2004); the μ-opioid receptor ago-
nist, methadone (Totah et al., 2008), the antimalarial artemisinin
(Svensson and Ashton, 1999;Asimus and Ashton, 2009), among
numerous additional metabolic pathways of other drugs, to which
CYP2B6 contributes in part, such as the antiretroviral, nevirapine
(Erickson et al., 1999), and many others (Turpeinen and Zanger,
2012). Metabolic pathways suitable as probe for CYP2B6 activity
include S-mephenytoin N-demethylation (Ko et al., 1998), bupro-
pion hydroxylation (Faucette et al., 2000;Fuhr et al., 2007) and
efavirenz, based on in vitro investigations (Ward et al., 2003;Desta
et al., 2007).
Endogenous substances metabolized by the enzyme include
arachidonic acid, lauric acid, 17beta-estradiol, estrone, ethiny-
lestradiol, and testosterone 16α- and 16β-hydroxylation (Ekins
et al., 1998).
Cytochrome P450 2B6 furthermore participates in the
biotransformation of the abused drug “ecstasy” (N-methyl-
3,4-methylenedioxymethamphetamine, MDMA), which is N-
demethylated leading to potentially neurotoxic metabolites (Kreth
et al., 2000). It also plays a minor role in nicotine metabolism
(Yamazaki et al., 1999;Yamanaka et al., 2005). CYP2B6 has fur ther-
more been found to be of importance in the metabolism of pesti-
cides and other environmental chemicals and pollutants (Hodgson
and Rose, 2007). In particular the bioactivating oxidation of the
organophosphorus insecticides chlorpyrifos (Crane et al., 2012)
and methyl parathion (Ellison et al., 2012a) to their more toxic
oxon metabolites is mainly catalyzed by CYP2B6, a public health
concern due to their worldwide use and documented human
exposures (Ellison et al., 2012b). Further environmental sub-
strates are the insecticide and endocrine disruptor methoxychlor,
the extensively used insect repellent N,N-diethyl-m-toluamide
(Das et al., 2008), profenofos and other pesticides (Abass and
Pelkonen, 2012), as well as the tobacco-specific nitrosamine,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; Smith
et al., 2003), aflatoxin B1 (Code et al., 1997), and others (Hodgson
and Rose, 2007;Abass and Pelkonen, 2012).
Several structurally unrelated drugs have been shown to inhibit
CYP2B6 and many of them do that in a mechanism-based, irre-
versible manner (Turpeinen et al., 2006;Turpeinen and Zanger,
2012). The thienopyridine derivatives clopidogrel and ticlopi-
dine are prodrugs that selectively inhibit platelet aggregation and
have been in clinical use for the prevention of atherothrombotic
events for several years. Both of them are potent mechanism-
based inhibitors of CYP2B6 (Richter et al., 2004;Zhang et al.,
2011a). The established anticancer agent, thioTEPA (N,N,N-
triethylenethiophosphoramide) was also found to be a highly
selective and mechanism-based CYP2B6 inhibitor (Rae et al.,
2002;Harleton et al., 2004;Richter et al., 2005). A compar-
ison of several selective inhibitors revealed that 2-phenyl-2-
(1-piperidinyl)propane is probably the most selective CYP2B6
inhibitor in vitro (Walsky and Obach, 2007). Recent in vitro obser-
vations identified the progesterone receptor antagonist, mifepris-
tone (RU486; Lin et al., 2009); the anti-Parkinsonian agent selegi-
line (the R-enantiomer of deprenyl; Sridar et al., 2012), methadone
(Amunugama et al., 2012), and tamoxifen (Sridar et al., 2012)
as potent mechanism-based inhibitors. In vivo drug–drug inter-
actions have been reported, for example, between thioTEPA
and cyclophosphamide (Huitema et al., 2000), clopidogrel and
bupropion (Turpeinen et al., 2005a), voriconazole and efavirenz
(Liu et al., 2008;Jeong et al., 2009), clopidogrel and efavirenz
(Jiang et al., 2012), and between ticlopidine and ketamine (Pel-
toniemi et al., 2011). Furthermore, certain non-pharmaceutical
compounds like particular benzylpyridine derivatives have been
characterized as very potent inhibitors of CYP2B6 (Korhonen
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Zanger and Klein Pharmacogenetics of CYP2B6
et al., 2007) and have been utilized for structural modeling
experiments (Gay et al., 2010).
PHARMACOGENETICS OF CYP2B6
The CYPalleles website1currently lists 37 distinct star-alleles, i.e.,
gene haplotypes with a distinct variant amino acid sequence or
with demonstrated functional effect (last accessed: February 21st,
2013). More than 30 amino acid-changing single-nucleotide poly-
morphisms (SNPs) occur in different combinations and together
with additional non-coding variants and many more SNPs not
yet assigned to particular haplotypes. The worldwide variations
in SNP frequencies have been reviewed recently (Li et al., 2012).
Tab l e 1 lists the most important variants in terms of frequency
and functional impact and summarizes updated structural, func-
tional, and frequency information for different ethnicities. In
addition to the CYPallele website, further valuable information
about CYP2B6 SNPs and pharmacogenetics are available on the
websites of The Pharmacogenomics Knowledgebase2, the NCBI por-
tal for short genetic variations, dbSNP3, the 1000 Genomes Catalog of
Human Genetic Variation4, as well as the NHLBI exome sequencing
project5.
1http://www.cypalleles.ki.se/cyp2b6.htm
2http://www.pharmgkb.org/
3http://www.ncbi.nlm.nih.gov/projects/SNP/
4http://www.1000genomes.org/
5http://EVS.gs.washington.edu
CYP2B6*6 AND EFAVIRENZ: IN VIVO,EX VIVO,IN VITRO
The most common variant allele in all populations studied to
date harbors two amino acid changes, Q172H and K262R, and
is termed CYP2B6*6. This haplotype occurs in about 15 to over
60% of individuals, depending on ethnicity (Ta bl e 1 ). Although
additional variants occur in the promoter and in introns, their
functional impact appears to be of limited relevance and will not
be further discussed here (Lamba et al., 2003;Hesse et al., 2004;
Hofmann et al., 2008).
Since the discovery that CYP2B6 is the major enzyme for
efavirenz 8-hydroxylation (Ward et al., 2003), pharmacogenetic
studies have linked the Q172H variant to elevated plasma concen-
trations of efavirenz, indicating decreased enzyme function in vivo.
This finding has been reproduced manifold in different ethnicities
throughout the world (summarized by Telenti and Zanger, 2008;
Rakhmanina and van den Anker, 2010). Three CYP2B6 polymor-
phisms, 15631G>T, 21011T>C, and an intron 3 SNP rs4803419,
were also shown to be associated with efavirenz pharmacokinetics
at genome wide significance (Holzinger et al., 2012).
The potent first-generation NNRTI of HIV-1 is recommended
as initial therapy with two NRTIs in highly active antiretrovi-
ral therapy (HAART) regimes, but patients with subtherapeutic
plasma concentrations can develop resistance and treatment fail-
ure, whereas those with too high plasma levels are at increased
risk of central nervous system (CNS) side effects, which can
lead to treatment discontinuation in a fraction of patients (King
and Aberg, 2008). Q172H variant was furthermore associated
Table 1 |Summary data on selected genetic polymorphisms of CYP2B6.
CYP allele
designationa
Key mutation(s)b
rs number
Location, protein effect Allele frequenciescFunctional effect
CYP2B6*4 g.18053(c.516) A>G
rs2279343
K262R (isolated) 0.00 AA, Af
0.04 Ca
0.05–0.12 As
↑Expression, moderate substrate-
dependent effects
CYP2B6*5 g.25505(c.1459) C>T
rs3211371
R487C 0.01–0.04AA, Af
0.09–0.12 Ca
0.05–0.12 Hs
0.01–0.04 As
↓Expression, in part compensated
by ↑specific activity
CYP2B6*6 g.15631(c.516) G>T
rs3745274 and
g.18053(c.785)A>G
rs2279343
Q172HK262R 0.33–0.5 AA, Af
0.10–0.21 As
0.14–0.27 Ca
0.62 PNG
↓Expression; ↓activity with
efavirenz in vivo; some other
substrates show ↑activity
CYP2B6*18 g.21011(c.983)T>C
rs28399499
I328T 0.04–0.08 AA
0.05–0.12, Af
0.01 HS
0.00 As, Ca, PNG
↓Expression and activity
CYP2B6*22 g.-82T>C
rs34223104
promoter (TATA-box) 0.00–0.025 AA, Af, As
0.024 Ca, Hs
↑Expression and activity
↑Inducibility in vitro
aAccording to CYPallele nomenclature homepage http://www.cypalleles.ki.se.
bGenomic (g.) and cDNA (c.) positions are given in bp.
cSelected frequencies of individual ethnicities (AA, African American; Af African; As Asian; Ca Caucasian; Hs, Hispanic; PNG, Papua New Guineans) compiled from
dbSNP http://www.ncbi.nlm.nih.gov/SNP and from the literature cited in the text.
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with increased neurotoxicity and other CNS side effects (Haas
et al., 2004;King and Aberg, 2008;Lubomirov et al., 2010;Rib-
audo et al., 2010;Maimbo et al., 2011) with HAART-induced
liver injury (Yimer et al., 2011), and with efavirenz treatment
discontinuation and the associated risk of developing drug
resistance (Ribaudo et al., 2006;Lubomirov et al., 2011;Wye n
et al., 2011). Importantly, compound heterozygotes of 516T and
another low activity allele (e.g., *11,*18,*27,*28) also pre-
dict high efavirenz plasma levels (Rotger et al., 2007;Ribaudo
et al., 2010). In prospective, genotype-based dose adjustment
studies the therapeutic dose of efavirenz could be successfully
reduced and CNS-related side effects decreased (Gatanaga et al.,
2007;Gatanaga and Oka, 2009). Using pharmacokinetic modeling
and simulation it was suggested that a priori dose reduction in
homozygous CYP2B6*6 patients would maintain drug exposure
within the therapeutic range in this group of patients (Nyakutira
et al., 2008).
The in vitro data that have accumulated over the years on the
CYP2B6*6 allele draw a more complex picture with functional
consequences on various levels including pre-mRNA splicing, pro-
tein expression, as well as substrate-dependent changes in enzyme
activity and different sensitivity toward irreversible inhibition.
While early studies using recombinantly expressed enzyme vari-
ants found higher 7-ethoxycoumarin O-deethylase activity for
the Q172H variant (Ariyoshi et al., 2001;Jinno et al., 2003), in
genotyped human livers (ex vivo), the *6 allele has been associ-
ated with approximately 50–75% decreased protein levels (Lang
et al., 2001;Desta et al., 2007;Hofmann et al., 2008). An expla-
nation for decreased protein expression was provided based on
the observation that the c.516G>T SNP coding for Q172H in
exon 4 (rs3745274, Tab le 1 ) was correlated to increased amounts
of a hepatic splice variant that lacked exons 4–6, and concur-
rently to decreased amounts of the normal functional transcript.
Recombinant expression of minigene constructs in mammalian
cells proved that the c.516G>T variant was causally involved in
erroneous splicing and lower expression of functional mRNA and
protein (Hofmann et al., 2008). It has been hypothesized that bind-
ing of splice factors to an exonic splicing enhancer(s) located in
exon 4 could be affected by the variant (Zanger and Hofmann,
2008;Sadee et al., 2011). Although reduced expression in liver
appears to satisfactorily explain increased efavirenz plasma con-
centrations in individuals with *6/*6 genotype (Desta et al., 2007),
recent in vitro data of expressed variants seem to indicate that
the amino acid substitutions contribute to changes in catalytic
activity of the enzyme. Structurally this is not easy to compre-
hend, because the Q172H and Lys262Arg amino acid changes
occur in regions of the protein that are not directly located at the
active site or that have been identified as substrate recognition sites
(Figure 2).
Concerning efavirenz and also other substrates, the avail-
able in vitro data are however, not well in agreement with each
other. Tab l e 2 summarizes kinetic parameters for bupropion and
efavirenz for CYP2B6 enzyme variants obtained from different
recombinant expression systems. Using Escherichia coli expression
system, Zhang et al. (2011b) purified six N-terminally truncated
expressed variants to homogeneity and reconstituted them with
NADPH:cytochrome 450 reductase (POR) at a molar ratio of 1:2
FIGURE 2 |CYP2B6 structural model. Selected variant residues are
marked in green (Q172H, K262R, I328T, R487C); substrate recognition sites
(SRS; Nguyen etal., 20 08) and active site residues (ASR; Niu et al., 2011)
are highlighted in blue; the prosthetic heme molecule is shown in red. PDB
file 3IBD was visualized using VMD visual molecular dynamics viewer
http://www.ks.uiuc.edu/Research/vmd/ (Humphrey etal., 1996). The protein
sequence used for determining the crystal structure contains R at position
262 instead of the reference residue K. (A) view along x-axis (light red); (B)
view along y-axis (light green); (C) view along z-axis (light blue)
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Table 2 |Kinetic properties of recombinantly expressed CYP2B6 protein variants with bupropion and efavirenz.
Variant System Bupropion hydroxylation Efavirenz 8-hydroxylation Reference
Km(μM) Vmax (%) CLint (%) Km(μM) Vmax (%) CLint (%)
2B6.1 COS-1 87 100 100 2.07 100 100 Radloff etal. (2013)
E. coli 95 100 100 7.3 100 100 Zhang etal. (2011b)
Sf9 7.7 100 100 Ariyoshi etal. (2011)
Sf9 64 100 100 3.2 100 100 Xu et al. (2012)
2B6.6 COS-1 72 81 98 1.21 107 183 Radloff et al. (2013)
E. coli 380 175 43 198 563 20 Zhang et al. (2011b)
Sf9 12.4 81 50 Ariyoshi etal. (2011)
Sf9 63 139 143 8.8 133 49 Xu et al. (2012)
2B6.4 E. coli 162 60 35 5.5 73 96 Zhang et al. (2011b)
Sf9 9.16 169 142 Ariyoshi etal. (2011)
2B6.5 COS-1 65 44 59 1.15 46 83 Radloff etal. (2013)
E. coli 134 66 47 53 100 5 138 Zhang et al. (2011b)
Only studies which determined kinetic parameters (Km,V
max,orK
cat) were included.
and measured efavirenz and bupropion kinetics. Using Sf9 insect
cell cotransfection, CYP2B6.1, 2B6.4 and 2B6.6 were expressed in
the presence of 10-fold excess of POR, i.e., under saturating condi-
tions, to measure efavirenz kinetics (Ariyoshi et al., 2011). Another
study determined both bupropion and efavirenz kinetics in protein
preparations also derived from insect cells in the presence or
absence of cytochrome b5 (CYB5) but at somewhat more vari-
able ratios in regard to POR (Xu et al., 2012). Radloff et al. (2013)
used the COS-1 expression system, where P450 monooxygenase
activity is supported by endogenously expressed POR, to deter-
mine bupropion and efavirenz kinetics for several novel CYP2B6
variants in comparison to the known variants 2B6.1, 2B6.5, and
2B6.6.
The compilation of data in Ta b l e 2 shows that differences
between the variants were masked by differences between the
expression systems. For example, efavirenz Kmwas moderately
decreased (58%) for COS-1 cell-expressed 2B6.6 compared to
2B6.1 but moderately larger for both insect cell-expressed proteins.
The E. coli-expressed variant showed, however, 27-fold increased
Km. While the COS-1 proteins had almost identical Vmax ,oneof
the insect cellproteins had decreased Vmax (81%), while the other
had increased activity (133%). Again, the E. coli-expressed variant
showed the biggest difference of almost sixfold higher activity for
the variant. Similar discrepancies, albeit less dramatic, were found
with bupropion as substrate (Ta b l e 2).
This data-comparison illustrates the problems that still exist
with recombinant P450 expression systems, and particularly for
CYP2B6, which appears to be an enzyme that sensibly reacts
with activity changes to expression conditions. It is difficult to
pin down the reasons for these differences exactly. Reconstitution
of recombinant or even purified P450 with POR and CYB5 is a
non-trivial problem especially if different protein variants shall
be compared for quantitative kinetic parameters. Reconstitution
under saturating conditions with respect to electron donators, e.g.,
at a POR:P450 ratio of 10, is a straightforward practical way,
but in hepatocytes, POR is stoichiometrically underrepresented
(ratio about 1:10) and may be limiting for monooxygenase activity
(Gomes et al., 2009). Enzyme variants may interact differently with
the electron donors and catalytic differences could thus depend on
reconstitution conditions. In addition, N-terminal modifications
required to achieve high expression in E. coli may interact with
the DNA-polymorphisms to be analyzed. In the COS-cell system,
on the other hand, the POR:P450 ratio can neither be controlled
nor quantified because expression of P450 is too low for spectral
quantitation.
Taken together, the data from expression systems indicated that
catalytic differences may exist between CYP2B6.6 and CYP2B6.1.
However, except for the E. coli study, the differences were rather
modest and at present it cannot be concluded with certainty
whether the CYP2B6.6 variant is catalytically more or less active
compared to the wild-type, atleast for bupropion and efavirenz.
Taken all evidence together, the decrease in hepatic expression due
to erroneous splicing caused by the c.516G>T SNP (Desta et al.,
2007;Hofmann et al., 2008) most plausibly explains most of the
phenotypic in vivo activity differences observed with efavirenz and
bupropion.
CYP2B6*6 SNP-related functional differences were also
observed with inhibitors. In contrast to the wild-type enzyme
the recombinantly expressed K262R variant was not inactivated
by efavirenz, but both enzymes were irreversibly inhibited by 8-
hydroxyefavirenz (Bumpus et al., 2006;Bumpus and Hollenberg,
2008). Lower susceptibility to inhibition of the K262R variant and
the CYP2B6.6 double variant compared to CYP2B6.1 was also
found with respect to sertraline and clopidogrel, as well as sev-
eral other potent drug inhibitors of CYP2B6 (Talakad et al., 2009).
These data indicate a role of genetic polymorphisms in drug–drug
interaction sensitivity of CYP2B6, a finding that warrants further
investigation in vivo.
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Zanger and Klein Pharmacogenetics of CYP2B6
OTHER CYP2B6 VARIANTS AND OTHER SUBSTRATES –
IN VITRO STUDIES
The two amino acid changes that together constitute the *6 allele
also occur in isolation, although at much lower frequencies (Klein
et al., 2005;Zanger et al., 2007;Tab l e 1 ). In most pharmacoge-
netic studies they are not being determined and functional data
is therefore rare, especially concerning *9 (Klein et al., 2005).
Data from recombinant systems as well as liver data suggest
that the K262R variant possesses catalytic activities similar to
the wild-type enzyme, although with different substrates mod-
erately increased or decreased activity was observed (Tables 2 and
3;Desta et al., 2007;Hofmann et al., 2008). The allele with amino
acid change [R487C] in exon 9 (*5 variant; Tab le 1 ) expresses
very low levels of protein which does not translate into simi-
larly reduced activities as measured with bupropion as well as
efavirenz (Lang et al., 2001;Desta et al., 2007;Hofmann et al.,
2008). The variant was shown to have higher specific activity
compared to wild-type (Radloff et al., 2013). In human liver, this
leads to partial compensation of low expression, finally resulting
in a phenotype with moderately decreased activity with bupropion
and efavirenz in vivo. This explains why CYP2B6*5 was not asso-
ciated with efavirenz pharmacokinetics in HIV patients (Burger
et al., 2006).
The second most important functionally deficient allele is
CYP2B6*18 (c.983C>T [I328T]), which occurs predominantly
in African subjects with allele frequencies of 4–11% (Mehlo-
tra et al., 2007;Li et al., 2012). The I328T variant expressed
no detectable protein or activity toward bupropion, 7-ethoxy-4-
trifluoromethylcoumarin (7-EFC), selegiline and artemether in
COS-1 cells whereas a partially defective protein was expressed
in insect cells (Klein et al., 2005;Watanabe et al., 2010;Honda
et al., 2011). This demonstrates another example for expression
system-dependent differences. Most likely the 2B6.18 variant is
temperature-sensitive and thus able to be expressed at the lower
temperature (27◦C) of insect cell culture but not at 37◦C. Inter-
estingly, the I328T+Q172H double variant expressed partially
functional protein in HEK293 cells and in yeast (Wang et al.,
2006), indicating that Q172H can stabilize the I328T variant.
The *18 allele is thus phenotypically a null allele, at least in vitro
with some substrates. This is supported by many in vivo studies
(see below).
At least 12 additional null or low-activity alleles have been
described and analyzed with various substrates (Lang et al., 2004;
Klein et al., 2005;Rotger et al., 2007;Watanabe et al., 2010;Honda
et al., 2011). Although they are rather rare in all investigated pop-
ulations they may have profound effects on drug metabolism if
present in compound heterozygous genotypes, e.g., in combina-
tion with *6 or *18 (Rotger et al., 2007). The CYP2B6*22 allele
is a gain-of-function variant associated with increased transcrip-
tion in vitro (Zukunft et al., 2005) and with increased activity in
vivo (Rotger et al., 2007). It was shown that a -82T>C exchange
alters the TATA-box into a functional CCAAT/enhancer-binding
protein binding site that causes increased transcription from an
alternative downstream initiation site (Zukunft et al., 2005). Inter-
estingly, the -82T>C polymorphism also confers synergistically
enhanced CYP2B6 inducibility by the PXR ligand rifampicin in
human primary hepatocytes (Li et al., 2010).
New variants are discoveredpreferentially in previously unchar-
acterized ethnic groups. Restrepo et al. (2011) described two novel
combinations of known amino acid variants in a Colombian
population. Structural variants including a novel CYP2B6/2B7P1
duplicated fusion allele (CYP2B6*30) were found when individ-
uals from various ethnicities were screened for copy number
variations (Martis et al., 2012). Furthermore, three novel and
five previously uncharacterized amino acid variants in different
combinations (CYP2B6*33 to *37) were identified by resequenc-
ing the CYP2B6 gene in a Rwandese cohort of HIV-1-infected
patients (Radloff et al., 2013). The variants were then function-
ally studied by COS-1 cell expression and by in silico prediction
tools. At least four of the variants were shown to result in com-
plete or almost complete loss of function with bupropion and
efavirenz as substrates. The detailed comparison of in vitro func-
tionality of the variants with in silico prediction tools including a
thorough substrate docking simulation analysis points at the chal-
lenge to deal with the hundreds of new variants that exist in all
populations as currently uncovered by next generation sequenc-
ing approaches and large scale population projects (see links
above).
Table 3 |Properties of recombinantly expressed CYP2B6 protein variants with other clinical substrates.
Artemether1
COS-7 cells
(Honda et al., 2011)
Selegiline2
COS-7 cells
(Watanabe et al., 2010)
Chlorpyrifos3
COS-1 cells
(Crane et al., 2012)
Cyclophosphamide4
Sf9 cells
(Ariyoshi et al., 2011)
Cyclophosphamide4
E. coli
(Raccor et al., 2012)
Variant Km(μM) Vmax(%) Km(μM) Vmax (%) Km(μM) Vmax(%) Km(mM) Vmax (%) Km(mM) Vmax (%)
2B6.1 3.1 100 48.2 100 1.84 100 2.68 100 3.6 100
2B6.6 6.72 416 56.6 169 1.97 254 1.62 99 4.0 155.2
2B6.4 2.73 196 45.8 147 1.09 1094 2.75 74 3.5 67.1
2B6.5 6.87 55 70.1 85 0.80 441 5.1 72.4
1O-Demethylation.
2N-Demethylation (mean values were calculated for several expressions of CYP2B6.1).
3Desulfation.
44-Hydroxylation.
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Zanger and Klein Pharmacogenetics of CYP2B6
CLINICAL STUDIES WITH DIFFERENT DRUGS
The widely used anticancer and immunosuppressant pro-
drug cyclophosphamide depends on bioactivation to 4-
hydroxycyclophosphamide for cytotoxic activity. Bioactivation is
highly variable in cancer patients and has been attributed mainly
to CYP2B6 in vitro and in vivo with contributions from CYP2C19
and CYP3A4 (Chang et al., 1993;Raccor et al., 2012). The case
of cyclophosphamide 4-hydroxylation deserves particular atten-
tion, as it exemplifies substrate-dependent effects of CYP2B6
pharmacogenetics. Cyclophosphamide 4-hydroxylation was ini-
tially reported to be enhanced in livers genotyped CYP2B6*6/*6
(Xie et al., 2003), which was confirmed in several later in vivo
studies (Xie et al., 2006;Nakajima et al., 2007;Torimoto and
Kohgo, 2008). However, other in vivo studies analyzing phar-
macokinetics or clinical outcome also presented contradictory or
negative results (Singh et al., 2007;Ekhart et al., 2008;Melanson
et al., 2010;Yao et al., 2010;Raccor et al., 2012). In vitro, insect
cell-expressed recombinant CYP2B6.4 [K262R] had lower activ-
ity for cyclophosphamide 4-hydroxylation (Ariyoshi et al., 2011;
Raccor et al., 2012). The CYP2B6.4 and CYP2B6.6 variants thus
display mirror-inverted catalytic activities toward efavirenz and
cyclophosphamide, in that the former variant is the catalytically
more active one with efavirenz, whereas the opposite is true for the
latter variant (Tab l e 3). A direct comparison of catalytic properties
of the two variants with the reference enzyme expressed in insect
cells supports this inverse behavior of the two variants toward these
two substrates (Ariyoshi et al., 2011). Interestingly, several studies
associated other variants including CYP2B6*4, *5, *8, and *9 with
lower 4-OH cyclophosphamide formation in vivo or with worse
outcome (Takada et al., 2004;Bray et al., 2010;Helsby et al., 2010;
Joy et al., 2012). Taken together, the data concerning cyclophos-
phamide from both in vivo and in vitro indicate that CYP2B6
polymorphism plays a role, although the studies are so far not yet
conclusive. This may be explained by different study size design
as well as lack of consistency in allele definition and genotype
information among studies (Helsby and Tingle, 2011).
In addition to efavirenz, CYP2B6 genotype also affects plasma
levels of the antiretroviral drug nevirapine (Penzak et al., 2007;
Mahungu et al., 2009). The impact of the CYP2B6 516G>T poly-
morphism on nevirapine exposure was confirmed and quantified
in a pharmacometric analysis of nevirapine plasma concentrations
from 271 patients genotyped for 198 SNPs in 45 ADME (absorp-
tion, distribution, metabolism, and excretion) genes and covari-
ates (Lehr et al., 2011). Moreover, nevirapine-related cutaneous
adverse events, which are most likely major histocompatibil-
ity complex (MHC) class I-mediated, were significantly influ-
enced by CYP2B6 polymorphism while hepatic side effects,
most likely MHC class II-mediated, were unaffected by CYP2B6
(Yuan et al., 2011).
CYP2B6 allele variants were also investigated in the context
of the synthetic μ-opioid receptor agonist, methadone, which
is metabolized by CYPs 3A4/5, 2B6, and 2D6, and used as a
maintenance treatment for opioid addiction. In *6/*6 carriers
(S)-methadone plasma levels were increased leading to potentially
higher risk of severe cardiac arrhythmias and methadone asso-
ciated deaths (Crettol et al., 2005;Eap et al., 2007;Bunten et al.,
2011). Methadone dose requirement for effective treatment of
opioid addiction was shown to be significantly reduced in carriers
of this genotype (Levran et al., 2011).
CONCLUSION
The polymorphism of the CYP2B6 gene has initially been studied
by reverse genetics approach, i.e., starting from the identification
of genetic variants in DNA and liver samples, followed by in vitro
characterization of genotyped livers and expressed variant pro-
teins. Clinical studies have then contributed to identify the variants
that are important in vivo, and in vitro studies are again needed to
identify and mechanistically explain causal variants. Nevertheless,
CYP2B6 pharmacogenetics has yet to be fully explored, especially
with respect to combined effects of the involved variants on both
expression and catalytic properties, the latter of which additionally
depend on the substrate. While the relevance for HIV-1 therapy
with efavirenz is well established and translational approaches have
already been clinically tested, an increasing number of studies
suggest clinical relevance for additional drug substrates.
ACKNOWLEDGMENTS
Work in the authors’ laboratory was supported by the Ger-
man Federal Ministry of Education and Research (Virtual Liver
Network grant 0315755), the 7FP EU Initial Training Network
program‚ FightingDrugFailure’ (GA-2009–238132), and by the
Robert-Bosch Foundation, Stuttgart, Germany.
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Conflict of Interest Statement: Ulrich
M. Zanger is a named coinventor of
a patent on the detection of specific
CYP2B6 polymorphisms for diagnos-
tic purposes. Kathrin Klein declares no
conflict of interest.
Received: 19 October 2012; accepted:
14 February 2013; published online: 05
March 2013.
Citation: Zanger UM and Klein K (2013)
Pharmacogenetics of cytochrome P450
2B6 (CYP2B6): advances on polymor-
phisms, mechanisms, and clinical rele-
vance. Front. Genet. 4:24. doi: 10.3389/
fgene.2013.00024
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