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Advanced Drug Delivery Reviews 54 (2002) 1271–1294
www.elsevier.com / locate / drugdeliv
Genetic contribution to variable human CYP3A-mediated
metabolism
Jatinder K. Lamba a , Yvonne S. Lin b , Erin G. Schuetz a , Kenneth E. Thummel b , *
b
a
St. Jude Children’ s Research Hospital, Memphis, TN 38105, USA
Department of Pharmaceutics, Box 357610, University of Washington, Seattle, WA 98195, USA
Received 29 April 2002; accepted 20 May 2002
Abstract
The human CYP3A subfamily plays a dominant role in the metabolic elimination of more drugs than any other
biotransformation enzyme. CYP3A enzyme is localized in the liver and small intestine and thus contributes to first-pass and
systemic metabolism. CYP3A expression varies as much as 40-fold in liver and small intestine donor tissues. CYP3Adependent in vivo drug clearance appears to be unimodally distributed which suggests multi-genic or complex gene–
environment causes of variability. Interindividual differences in enzyme expression may be due to several factors including:
variable homeostatic control mechanisms, disease states that alter homeostasis, up- or down-regulation by environmental
stimuli (such as smoking, drug intake, or diet), and genetic mutations. This review summarizes the current understanding and
implications of genetic variation in the CYP3A enzymes. Unlike other human P450s (CYP2D6, CYP2C19) there is no
evidence of a ‘null’ allele for CYP3A4. More than 30 SNPs (single nucleotide polymorphisms) have been identified in the
CYP3 A4 gene. Generally, variants in the coding regions of CYP3 A4 occur at allele frequencies , 5% and appear as
heterozygous with the wild-type allele. These coding variants may contribute to but are not likely to be the major cause of
inter-individual differences in CYP3A-dependent clearance, because of the low allele frequencies and limited alterations in
enzyme expression or catalytic function. The most common variant, CYP3 A4*1 B, is an A-392G transition in the 59-flanking
region with an allele frequency ranging from 0% (Chinese and Japanese) to 45% (African-Americans). Studies have not
linked CYP3 A4*1 B with alterations in CYP3A substrate metabolism. In contrast, there are several reports about its
association with various disease states including prostate cancer, secondary leukemias, and early puberty. Linkage
disequilibrium between CYP3 A4*1 B and another CYP3 A allele (CYP3 A5*1 ) may be the true cause of the clinical phenotype.
CYP3A5 is polymorphically expressed in adults with readily detectable expression in about 10–20% in Caucasians, 33% in
Japanese and 55% in African-Americans. The primary causal mutation for its polymorphic expression (CYP3 A5*3 ) confers
low CYP3A5 protein expression as a result of improper mRNA splicing and reduced translation of a functional protein. The
CYP3 A5*3 allele frequency varies from approximately 50% in African-Americans to 90% in Caucasians. Functionally,
microsomes from a CYP3 A5*3 /*3 liver contain very low CYP3A5 protein and display on average reduced catalytic activity
towards midazolam. Additional intronic or exonic mutations (CYP3 A5*5, *6, and *7 ) may alter splicing and result in
premature stop codons or exon deletion. Several CYP3 A5 coding variants have been described, but occur at relatively low
allelic frequencies and their functional significance has not been established. As CYP3A5 is the primary extrahepatic CYP3A
isoform, its polymorphic expression may be implicated in disease risk and the metabolism of endogenous steroids or
Abbreviations: CV, coefficient of variation; CYP, cytochrome P450
*Corresponding author. Tel.: 1 1-206-543-0819; fax: 1 1-206-543-3204.
E-mail address: [email protected] (K.E. Thummel).
0169-409X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0169-409X( 02 )00066-2
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xenobiotics in these tissues (e.g., lung, kidney, prostate, breast, leukocytes). CYP3A7 is considered to be the major fetal liver
CYP3A enzyme. Although hepatic CYP3A7 expression appears to be significantly down-regulated after birth, protein and
mRNA have been detected in adults. Recently, increased CYP3 A7 mRNA expression has been associated with the
replacement of a 60-bp segment of the CYP3 A7 promoter with a homologous segment in the CYP3 A4 promoter
(CYP3 A7*1 C allele). This mutational swap confers increased gene transcription due to an enhanced interaction between
activated PXR:RXRa complex and its cognate response element (ER-6). The genetic basis for polymorphic expression of
CYP3A5 and CYP3A7 has now been established. Moreover, the substrate specificity and product regioselectivity of these
isoforms can differ from that of CYP3A4, such that the impact of CYP3A5 and CYP3A7 polymorphic expression on drug
disposition will be drug dependent. In addition to genetic variation, other factors that may also affect CYP3A expression
include: tissue-specific splicing (as reported for prostate CYP3A5), variable control of gene transcription by endogenous
molecules (circulating hormones) and exogenous molecules (diet or environment), and genetic variations in proteins that may
regulate constitutive and inducible CYP3A expression (nuclear hormone receptors). Thus, the complex regulatory pathways,
environmentally susceptible milieu of the CYP3A enzymes, and as yet undetermined genetic haplotypes, may confound
evaluation of the effect of individual CYP3 A genetic variations on drug disposition, efficacy and safety.
 2002 Elsevier Science B.V. All rights reserved.
Keywords: Cytochrome P450; Pharmacogenetics; Genetic polymorphism; Drug metabolism; Biotransformation; Toxicogenetics
Contents
1. Overview................................................................................................................................................................................
1.1. Multiplicity of CYP3A enzymes .......................................................................................................................................
1.2. Hepatic and intestinal CYP3A expression ..........................................................................................................................
1.3. Interindividual differences in CYP3A probe clearance ........................................................................................................
2. CYP3A4 genetic variation........................................................................................................................................................
2.1. CYP3 A4 59-flanking domain .............................................................................................................................................
2.1.1. CYP3 A4 59-flanking mutations................................................................................................................................
2.1.2. Phenotype of CYP3 A4 59-flanking mutations ...........................................................................................................
2.2. CYP3 A4 coding domains ..................................................................................................................................................
2.2.1. CYP3 A4 coding mutations ......................................................................................................................................
2.2.2. Phenotype of CYP3 A4 coding mutations..................................................................................................................
2.3. CYP3 A4 intronic domains ................................................................................................................................................
2.3.1. CYP3 A4 intronic mutations ....................................................................................................................................
2.3.2. Phenotype of CYP3 A4 intronic mutations ................................................................................................................
3. CYP3A5 genetic variation........................................................................................................................................................
3.1. CYP3 A5 59-flanking domain .............................................................................................................................................
3.2. CYP3 A5 coding domains ..................................................................................................................................................
3.3. Intronic domains ..............................................................................................................................................................
3.3.1. CYP3 A5 intronic mutations ....................................................................................................................................
3.3.2. Phenotype of CYP3 A5 intronic mutations ................................................................................................................
3.4. Implications for polymorphic extrahepatic CYP3A5 and disease risk ...................................................................................
3.5. Extrahepatic CYP3 A5 splicing variation ............................................................................................................................
4. CYP3A7 genetic variation........................................................................................................................................................
4.1. Mutations in CYP3 A7 59-flanking, coding and intronic domains ..........................................................................................
4.2. Relationship of CYP3 A7*1 C to phenotypic expression of CYP3A7 in adult liver..................................................................
5. CYP3A43 splicing variation .....................................................................................................................................................
6. Summary ................................................................................................................................................................................
Acknowledgements ......................................................................................................................................................................
References ..................................................................................................................................................................................
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1. Overview
1.1. Multiplicity of CYP3 A enzymes
The human CYP3A subfamily, CYP3A4,
CYP3A5, CYP3A7 and CYP3A43, is one of the
most versatile of the biotransformation systems that
facilitate the elimination of drugs, other xenobiotic
compounds, and endogenous molecules from the
body. Although there has been no systematic analysis of the extent of its contribution, it is generally
accepted that CYP3A enzymes play a dominant role
in the metabolic elimination of more drugs than any
other biotransformation enzyme. CYP3A metabolic
versatility may be due to a large active site that
permits the binding of structurally diverse molecules
[1]. Moreover, CYP3A enzymes are known to
accommodate multiple ligands (different molecules
or two or more of the same molecule) in the active
site. This phenomenon can result in enhanced product formation (activation), or reduced product formation (non-competitive inhibition), depending on the
concentration of substrate and nature of the second
or third ligand [2].
There is a rich body of literature regarding
CYP3A-mediated drug clearance. It has developed,
in part, because of the important contribution of
CYP3A to first-pass metabolism and reduced systemic bioavailability of orally administered drugs
[3], and also as a consequence of the susceptibility of
CYP3A to the effects of numerous drugs that either
induce or inhibit its catalytic activity [4,5]. For many
drugs, CYP3A activity accounts for the majority of
total body clearance. Accordingly, inter-individual
variability in enzyme content or function can have a
profound effect on systemic drug exposure and,
potentially, drug efficacy and safety. Among adults,
CYP3A4 is the dominant CYP3A enzyme in the
liver and small intestine. CYP3A5 is also found in
the adult liver and small intestine (and other organs)
but its expression is clearly polymorphic, with
individuals exhibiting a relatively high or low level
of protein [6,7]. CYP3A5 is also polymorphically
expressed in fetal liver [8]. CYP3A7 is the major
fetal liver CYP3A enzyme, whereas CYP3A4 is
absent [9]. Although hepatic CYP3A7 expression
appears to be significantly down-regulated after birth
[9], protein has been detected in some adults [10],
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and it may contribute to drug / xenobiotic clearance.
Because of differences in substrate specificity and
product regioselectivity for these three major human
CYP3A enzymes, and variability in their hepatic and
intestinal expression, one can expect significant
inter-individual differences in the disposition of
drugs cleared exclusively by the CYP3A subfamily.
The identification of a new member of the human
CYP3 A gene locus, CYP3 A43, has recently been
reported [11,12]. Gene transcription was detected in
liver, kidney, prostate and pancreas, but the level of
mRNA in liver was much less than that for CYP3 A4.
Heterologous expression of CYP3A43 and subsequent characterization has revealed a much lower
catalytic activity towards the classical CYP3A4
substrate testosterone. Thus, CYP3A43 is unlikely to
contribute much to the systemic clearance of drugs
or other xenobiotics, but if it possesses a unique
substrate profile, it may still play an important role in
the intra-cellular disposition of xenobiotics in extrahepatic organs.
1.2. Hepatic and intestinal CYP3 A expression
There have been several published studies involving the characterization of CYP3A4 content in
‘banks’ of liver and small intestinal mucosa. The
range of hepatic protein expression (specific content:
mole enzyme / mass tissue or cell fraction) varies
considerably, as much as 40-fold among liver (Fig.
1) and small intestinal tissues obtained surgically or
by organ donation [7,13,14]. Large inter-individual
differences in CYP3A4-dependent in vitro intrinsic
clearances (Vmax /Kmax ) are also evident. Such extreme variability may be partly artificial as a consequence of changes in constitutive gene regulation
that may occur prior to tissue procurement (e.g.,
down-regulation of CYP genes secondary to systemic cytokine release and diminished pituitary
function and hormone secretion in brain-dead
donors), or CYP3A4 induction by concomitant medications. However, much of this variability may also
be attributed to differences in CYP3A4 regulation,
with a significant component controlled by genetic
factors [15].
Polymorphic expression of CYP3A5 in human
liver and small intestine has been recognized for
several years [6,7]. Although CYP3A5 protein can
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Fig. 1. Variable CYP3A4 and CYP3A5 specific content in Caucasian livers. Microsomal CYP3A4 and CYP3A5 contents were measured by
Western blot analysis. Closed, partially filled and open symbols correspond to livers with a CYP3 A5*1 /*1, CYP3 A5*1 /*3 and
CYP3 A5*3 /*3 genotype, respectively. Reproduced with the permission of authors and journal [19].
be detected in most livers [7,16], the presence of
readily detectable CYP3A5 bands by Western blot
analysis occurs in only 10–40% of Caucasians (Fig.
1) [6,7,10,17,18], 33% of Japanese [10] and 55% of
African-Americans [17]. The maximum microsomal
CYP3A5 protein content reported for livers in which
CYP3A5 protein has been detected is variable: 60
pmol / mg (n 5 20 Caucasians [6]); 69 pmol / mg (n 5
15 Caucasians [10]; 25 pmol / mg (n 5 15 Japanese;
[10]); 185 pmol / mg (n 5 20 African-Americans
[17]); and 351 pmol / mg (n 5 60 Caucasians [19]).
More importantly, in these studies, CYP3A5 accounted for approximately 6–68% [6], 20–48% [10],
41–99% [17], and 46–85% [19] of the total CYP3A
content in the livers in which it was readily detected.
CYP3A5 is also polymorphically expressed in
human small intestinal mucosa, where it can contrib-
ute significantly to the total CYP3A pool [7,19].
Thus, one might anticipate that genetic factors
controlling polymorphic CYP3A5 expression contribute to inter-individual differences in the clearance
of some CYP3A substrates.
Because CYP3A5 and CYP3A4 have overlapping
substrate specificity, it is difficult to segregate the
relative contributions of the two enzymes to CYP3Amediated metabolism. There are some substrates that
are metabolized equally well by CYP3A4 and
CYP3A5: e.g., midazolam [20], lidocaine [21] and
fentanyl [22]. However, results from other experiments indicate lower intrinsic activity for CYP3A5
(metabolite formation rate or intrinsic clearance) than
that of CYP3A4 for many substrates [6,23–25].
There is also published data to suggest that purified
CYP3A5 and CYP3A4 are sensitive to reconstitution
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
conditions and that, when optimized, the metabolic
activity of CYP3A5 towards many drugs is similar to
that of CYP3A4 for many of the molecules found
previously to be poor CYP3A5 substrates [26].
The catalytic activity of both CYP3A4 and
CYP3A5 is affected significantly by the presence of
cytochrome b 5 [27–29]. Moreover, the extent of
enhancement of catalytic activity by cytochrome b 5
may be dependent on whether cytochrome b 5 is
co-expressed with CYP3A and P450 reductase or is
added to the incubation mixture with CYP enzyme
prior to activity measurements. Hirota et al. reported
a 25-fold higher intrinsic clearance for alprazolam
hydroxylation catalyzed by CYP3A4 expressed in a
baculovirus system when enzyme was co-expressed
with cytochrome b 5 , compared to the activity resulting after addition of purified cytochrome b 5 to
the CYP3A4 membrane preparation [30]. Given this
result, it may not be appropriate to compare the
activity of commercial CYP3A enzymes expressed
under different conditions, with variable enzyme /
coenzyme-specific contents. Future in vitro and in
vivo studies are needed to resolve the relative
importance of CYP3A5, compared to CYP3A4, in
drug clearance.
The expression of CYP3A7 in adult livers (based
on mRNA content) is generally thought to be
negligible, in comparison to CYP3A4 and CYP3A5
content [31]. Direct assessment of hepatic CYP3A7
content has been hampered by the fact that the
protein is difficult to resolve from CYP3A4 under
one-dimensional Western blot analysis conditions,
and most CYP3A-antibodies cross-react with other
members of the CYP3A subfamily. However, data
from a recent publication where investigators employed a CYP3A7-specific antibody suggests that, in
Japanese, CYP3A7 contribution can be quite significant [10]. Among the 15 Japanese livers examined,
CYP3A7 accounted for 5–40% (mean 5 20%) of the
total CYP3A protein. The same authors reported that
the average hepatic CYP3A7 content in Caucasians
was much lower than that of Japanese and it did not
contribute significantly to the total liver CYP3A
pool. Little is known about the substrate specificity
of CYP3A7; however, it does catalyze the metabolism of some drugs, including midazolam [32],
warfarin [33], and promutagens [34]. Thus, genetic
factors affecting CYP3A7 expression or function
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may also contribute to inter-individual differences in
CYP3A-dependent drug clearance and cancer risk in
the adult.
During pregnancy, fetal CYP3A7 is an important
catalyst of 16a-hydroxy dehydroepiandrosterone sulfate formation from DHEA-sulfate [35]. This metabolic product represents the major source of estriol
for the maternal–fetal unit [36]. CYP3A7 is also a
very efficient catalyst of retinoic acid metabolism, an
endogenous ligand for RXRa [37]. Again, there is
only limited data available on interindividual variability of fetal hepatic CYP3A7 expression and
activity towards the metabolic clearance of endogenous and exogenous substrates. Nonetheless, genetic
factors controlling CYP3A7 expression in the fetus
may be important determinants of metabolic capacity
towards endogenous and exogenous (drugs) substrates.
1.3. Interindividual differences in CYP3 A probe
clearance
Of more clinical relevance than the characterization of catalytic activity from tissue banks is the
extent of inter-individual variability observed in vivo
for CYP3A-mediated drug metabolism pathways.
The most useful information comes from studies
utilizing putative CYP3A-selective probes. These are
drugs for which elimination following intravenous or
oral administration can be attributed predominantly
to CYP3A-catalyzed metabolism. However, it is
important to recognize that there may be limitations
to the generalizable conclusions that can be drawn
from such data given what we know about drugspecific allosteric phenomena. For example,
midazolam and erythromycin are two drugs that have
been studied extensively and used to characterize
CYP3A–drug interactions, and constitutive CYP3A
regulation [5,38]. Although the metabolism of both
substrates will reflect the expected change in enzyme
function in response to an exogenous or endogenous
perturbation, there is only a weak or negligible
correlation between the two substrates under a basal /
constitutive state [39,40]. Although confounding
factors such as cell efflux or uptake processes may
contribute to the lack of correlation [41], it is
interesting to note that midazolam and erythromycin
appear to bind to different domains within the
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CYP3A active site [42]. Thus, these drugs may be
affected differentially by exogenous or endogenous
ligands of CYP3A that modify (activate or inhibit)
enzyme function.
Despite the caveats cited above, it is still mechanistically useful to consider the cause of interindividual variability for any single CYP3A probe
substrate. In this regard, there is no strong evidence
for pronounced bimodal or trimodal distribution in a
CYP3A-dependent clearance process. For example,
in a retrospective study of midazolam disposition
following intravenous and oral administration, Lin et
al. found that weight normalized systemic clearance
varied 11-fold among a population of 137 healthy
volunteers of predominantly Caucasian ethnicity
[43]. Inspection of the data revealed a unimodal
log-normal clearance distribution (Fig. 2). Approximately 85% of subjects exhibited a clearance between 2 and 8 ml / min per kg, but there were
apparent outliers with an unusually high clearance of
16–18 ml / min per kg. A similar population distribution was observed for oral midazolam clearance,
which reflects variability in both hepatic and intesti-
nal CYP3A4 / 5 activity. The range of oral clearances
in 148 subjects was 48-fold, but the majority (84%)
of subjects had a weight-normalized clearance between 10 and 40 ml / min per kg.
Population data available for nifedipine, one of the
first selective CYP3A substrates identified [44], is
similar to that described for midazolam. Schellens et
al. [45] reported a lack of bimodality in nifedipine
oral AUC in 130 healthy subjects, with only a
10-fold range in metabolic activity observed. The
majority of subjects fell within a 4-fold AUC range.
Interestingly, there was evidence for a skewed
distribution towards higher nifedipine AUCs (lower
clearance), in contrast to midazolam data. However,
it should be noted that the oral nifedipine and
midazolam data was complicated by the possibility
of sequential intestinal and hepatic first-pass metabolism. Since hepatic and intestinal CYP3A expression
do not appear to be concordant [7,46], the impact of
genetic factors may be masked to a significant degree
by multiple (tissue-specific) regulatory factors.
There are many other drugs for which CYP3A
makes an important, if not dominant, contribution to
Fig. 2. Population distribution of i.v. midazolam clearance. Clearance values were generated from plasma concentrations measured after
administration of a 1-mg intravenous midazolam dose to healthy adult volunteers. Reproduced with the permission of authors and journal
[43].
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
oral or systemic clearance. A high degree of interindividual variability in AUC values following a
fixed dosing regimen is often reported, particularly
for those drugs with low oral bioavailability [47].
For example, the coefficient of variation (CV) for
oral terfenadine AUC was reported to be 85% in a
study of 130 healthy, predominantly Caucasian volunteers [48]. This is similar to CV values of 80 and
49% for midazolam and nifedipine oral AUC, respectively [43,45]. The CV for lovastatin [49] and
buspirone [50] oral AUC, two very low bioavailability CYP3A substrates, was 49–118 and 112%,
respectively. In contrast, CV values for the oral AUC
of two well absorbed, high bioavailability CYP3A
substrates, alprazolam [51] and zolpidem [52], were
11 and 10–28%, respectively. Taken together, the
available clinical data suggests that functional in
vivo CYP3A activity varies significantly between
people with similar demographic and health characteristics. However, the majority of people will exhibit metabolic clearances within a 4- to 6-fold
range, but outliers with unusually high or low
metabolic activity can be expected.
It has been suggested that genetic variability will
account for approximately 90% of the inter-individual differences in hepatic CYP3A activity, based
on an analysis of inter- and intra-subject variability
in the elimination of CYP3A-selective probes [15].
In addition, there is a predicted 60% genetic contribution to the variability in composite metabolic
activity for a sampling of known CYP3A substrates
that are not necessarily selective probes. Although
there are clear limitations to the statistical method
used for assigning genetic contribution by these
investigators (i.e., assessment of intra-subject variability), the analysis is the most comprehensive
performed to date. Surprisingly, there have been no
reports of CYP3A activity in monozygotic and
dizygotic twins, studies that could be extremely
valuable in guiding current and future research on
CYP3A inter-subject variability. However, based on
the unimodal population distribution of CYP3A
activity, one might predict that no single genetic or
environmental factor determines the metabolic clearance of CYP3A substrates.
Interindividual variability in constitutive CYP3A
expression and intrinsic clearance could arise from
multiple causes [53]. These include variable control
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of gene transcription by endogenous molecules such
as circulating hormones and exogenous molecules
from the diet or environment, and genetic polymorphisms. As will be discussed later, the 59-flanking region of all three major human CYP3A enzymes
contains putative transcription factor binding elements [54]. The function of some of these (e.g.,
PXRE) may be controlled by variability in systemic
or local concentrations of endogenous molecules
[54–56]. Within the context of endogenous factors
that affect gene transcription, one should also consider pathophysiological states that arise from acute
and chronic disease and result in CYP modifying
levels of various cytokines [57]. Finally, at the core
of interindividual differences in CYP3A activity is
the possibility of variation within the CYP3 A genes
themselves (flanking, intronic and coding mutations)
or in the genes that code for proteins that may
regulate constitutive and inducible CYP3 A expression (e.g., PXR [56], VDR [58] and growth hormone
[59]). Thus, the likelihood that an individual’s
CYP3A activity will be determined by multiple
genetic and non-genetic factors will make it quite
challenging to establish genotype–phenotype relationships.
For the remainder of this article, we will review
current understanding of the multiplicity and functional consequence of CYP3 A genetic variation. The
information presented has been compiled from current literature and the contents of the P450 website
(www.imm.ki.se / CYPalleles.htm). Clearly, the field
is evolving rapidly and one can expect an explosion
of new information in the near future that may force
a revision of our interpretations. Of high probability
is the identification of numerous ‘rare’ SNPs from
large study populations that may or may not contribute importantly to inter-individual difference in
CYP3A activity.
2. CYP3A4 genetic variation
Unlike other human P450s (e.g., CYP2 D6,
CYP2 C19 ), there is no evidence of a ‘null’ allele for
CYP3 A4. Genetic variation found in the flanking,
intronic and exonic regions of the gene may influence the level or function of CYP3A4 protein, but
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
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full length mRNA has been detected in all adults
studied to date.
2.1. CYP3 A4 59 -flanking domain
2.1.1. CYP3 A4 59 -flanking mutations
Five different SNPs in the 59-flanking region of
CYP3 A4 [60] have been identified (Table 1). By far
the most common is the A-392G transition
(CYP3 A4*1 B) in the putative nifedipine response
element (NFSE) [61]. CYP3 A4*1 B allele frequency
varies among different ethnic groups: 0% in Taiwanese [62], Chinese [63], Chinese Americans [64] and
Japanese Americans [64]; 2–9.6% in Caucasians
[17,62,64,65]; 9.3–11% in Hispanic Americans
[64,66]; and 35–67% in African Americans
[17,62,64,67,68]. The other allelic variants in the
59-flanking region appear to be of much lower
Table 1
CYP3A4 Alleles
Allele
Location Nucleotide
Amino Acid
Substitution
CYP3A4*1
CYP3A4*1B 59 UTR A-392G
CYP3A4*1C
CYP3A4*1D
CYP3A4*1E
CYP3A4*1F
CYP3A4*2
59 UTR
59 UTR
59 UTR
59 UTR
Exon 7
CYP3A4*3
CYP3A4*4
CYP3A4*5
CYP3A4*6
CYP3A4*7
Exon
Exon
Exon
Exon
Exon
CYP3A4*8
CYP3A4*9
Exon 5 G389A
Exon 6 G508A
in vitro
Effect
in vivo
Effect
Caucasian African Other
American
no effect (testosterone),
increased luciferase expression
no effect (midazolam,
ERMBT, nifepidine,
cyclosporine)
2–9.6% 35–67% Japanese 0%,
Chinese 0%,
Hispanic
9.3–11%
1%
0%
1%
0%
, 1%
20%
Iranian 18%
2.7%
0%
Chinese 0%
T-444G
C-62A
T-369A
C-747G
12 T1334G
5 A352G
7 C653G
9 831 insA
3 G167A
CYP3A4*10 Exon 6 G520C
CYP3A4*11 Exon 11 C1088T
CYP3A4*12 Exon 11 C1117T
S222P
lower Clint (nifedipine),
no effect (testosterone)
no effect (testosterone)
M445T
0.47–4%
I118V
decreased activity (cortisol)
P218R
decreased activity (cortisol)
stop codon at 285
decreased activity (cortisol)
G56D
no effect (progesterone,
1.41
testosterone, 7BFC)
R130Q
no expression
0.33
V170I
no effect (progesterone,
0.24
testosterone, 7BFC)
D174H
no effect (progesterone,
0.24–2% 2%
testosterone, 7BFC)
T363M
low expression, no effect
0.34%
(progesterone, testosterone, 7BFC)
L373F
low expression, altered activity
0.34%
(testosterone, midazolam),
no effect (progesterone)
P416L
no expression
0.34%
L15P
R162Q
0%
2–4.2%
R162Q
, 1%
CYP3A4*13 Exon 11 C1247T
CYP3A4*14 Exon 1 T44C
CYP3A4*15A Exon 6 G485A
CYP3A4*15B
A-392G, G485A,
2 845 ins ATGGAGTGA
CYP3A4*16 Exon 7 C554G
T185S
CYP3A4*17 Exon 7 T566C
F189S
CYP3A4*18 Exon 10 T878C
L293P
CYP3A4*19 Exon 12 C1399T;VS10 1 12 G . A P467S
no effect
lower activity (testosterone)
higher activity (testosterone)
no effect (testosterone)
2.1%
0%
0%
0%
0%
0%
Chinese
Chinese
Chinese
Chinese
Refs.
1.5%
1.5%
0.98%
0.5%
[60]
[17,61,62,64,
66,67,72–78]
[67]
[67]
[69]
[69]
[67]
[67,71,82,83]
[63]
[63]
[63]
[71]
[71]
[71]
Mexican 5%
[68]
[71]
[71]
Asian 0%
Asian 0%
[71]
[68]
[68,82]
[69]
Mexican 5%,
[68]
Japanese 5%
0%
[82]
Chinese 10%
[82]
Indo-Pakistani 12% [82]
Accession number for alleles CYP3A4*1B-CYP3A4*1F: AF280107 (gene); Accession number for alleles CYP3A4*2-CYP3A4*19:
M18907 (cDNA). Designated base changes are numbered relative to the A in the ATG initiation codon.
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
frequency among Caucasians: CYP3 A4*1 C (1%),
CYP3 A4*1 D (1%), CYP3 A4*1 E ( , 1%), and
CYP3 A4*15 B ( , 1%) [17,69], with the exception of
CYP3 A4*1 F (20%) [69]. CYP3 A4*1 C and
CYP3 A4*1 D were not detected in African Americans [17]. Of particular note, investigators [69]
searched for but did not find SNPs in the upstream
distal enhancer element of the CYP3 A4 gene required for maximal CYP3A4 induction by exogenous
PXR ligands [70]. Moreover, no SNPs have been
identified in the proximal PXR enhancer motif
[17,71] that is essential for transcriptional activation
by exogenous PXR ligands [56].
2.1.2. Phenotype of CYP3 A4 59 -flanking mutations
Of the five promoter SNPs identified, only one
(CYP3 A4*1 B) has been studied to ascertain the
effect of the mutation on transcriptional activity and
in vivo catalytic activity. Amirimani et al. [72]
examined the effect of the A-392G mutation on
luciferase reporter transcription in HepG2 hepatoma
and MCF7 breast cancer cell lines. The investigators
demonstrated that luciferase expression from the
mutant G-392 promoter construct occurred at a rate
that was 1.4- to 1.9-fold higher than that observed
for the wild-type A-392 construct. This finding is
supported by an observation of 1.6- and 2.1-fold
higher level of nifedipine oxidation and CYP3A4
protein in human livers carrying at least one variant
G-392 allele, although the difference was not statistically significant [73]. However, results from studies
with a larger sample size demonstrate no clear
association between the A-392G mutation and
CYP3A4-specific content or catalytic activity among
tissue banks from predominantly Caucasian donors
[68,74].
The functional significance of the CYP3 A4*1 B
mutation has also been evaluated in vivo, but with
populations of relatively limited size. Ball et al. [64]
studied the metabolic fate of CYP3A probe substrates erythromycin and nifedipine in healthy African American volunteers. The investigators found
no difference in the mean erythromycin demethylation rate or mean oral nifedipine clearance measured
in individuals with a homozygous A-392 (n 5 8) or
homozygous G-392 (n 5 23) CYP3 A4 genotype.
Wandel et al. [75] examined the disposition of i.v.
and oral midazolam to assess the effect of the
1279
CYP3 A4*1 B allele on hepatic and intestinal CYP3A
activity in African Americans and Caucasian Americans (n 5 15 subjects in each ethnic group). All of
the Caucasian Americans were homozygous A-392
and all but one of the African Americans carried at
least one G-392 variant allele. Although the investigators reported no relationship between genotype
and oral midazolam clearance, there was a 30%
lower systemic midazolam clearance for subjects
homozygous for the variant G-392 allele, compared
to those homozygous for the wild-type A-392 allele.
However, this difference could be spurious since the
distribution of CYP3 A4*1 B genotypes was not balanced for each ethnic group. That is, there may be
alternative causes (genetic or non-genetic) for a
lower midazolam clearance in African Americans
compared to Caucasians, as suggested by the 14%
lower clearance for the entire African American
group [75].
In a study of cyclosporine disposition in stable
Caucasian renal transplant patients, von Ahnsen et al.
[76] reported no significant difference in the mean
trough cyclosporine blood level / oral dose ratio for
patients with a heterozygous G-392 /A-392 genotype
and those homozygous for the A-392 allele. Although cyclosporine is a substrate for both CYP3A
and P-glycoprotein, the same conclusion was reached
when the comparison of CYP3 A4*1 B genotypes was
controlled for the common P-glycoprotein mutation
in exon 26 (C3435T) that has been associated with
altered transporter activity.
Finally, the CYP3 A4*1 B allele genotype–phenotype relationship was examined recently by GarciaMartin et al. in a population of Caucasian Spanish
volunteers [65]. In their study of dextromethorphan
N-demethylation activity, as reflected by the urinary
dextromethorphan / 3-methoxymorphinan ratio, the
investigators reported no significant difference between individuals that were heterozygous or homozygous for the G-392 allele and those homozygous
for the wild-type A-392 allele.
The absence of any major pharmacokinetic consequence for the CYP3 A4*1 B mutation is somewhat at
odds with recent literature linking the polymorphism
to altered clinical outcomes. In their original report,
Rebbeck et al. noted that African American prostate
cancer patients were more likely to be homozygous
for the mutant CYP3 A4*1 B allele than matched
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J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
healthy controls [61]. This finding was confirmed by
Paris et al. [66] in their study of African American
men with prostate cancer, and more recently by
Tayeb et al. [77], in a study of Caucasian men with
benign prostatic hyperlasia who went on to develop
prostate cancer. Rebbeck and co-workers [78] also
reported that individuals with a homozygous wildtype CYP3 A4*1 genotype were at greater risk of
encountering secondary leukemia after treatment of
the primary cancer with epipodophyllotoxin therapy
than individuals with a mutant CYP3 A4*1 B genotype. The odds ratio for the association of the
CYP3 A4*1 B genotype among treatment related cases
was significantly less than one (0.07; 95% CI, 0.01–
0.08), even after adjustment for race, age and gender.
The investigators went on to speculate that inheritance of the variant allele would confer reduced
CYP3A4 activity and decreased production of toxic
epipodophyllotoxin metabolites. However, this hypothesis is at odds with in vitro data indicating
increased transcriptional activity associated with the
variant allele [72].
Since these initial reports, abstracts have appeared
at national meetings describing significant associations between the CYP3 A4*1 B allele and clinical
response to cyclophosphamide therapy [79] and early
onset puberty [80]. In the latter study, the investigators found that girls with a homozygous G-392
genotype were far more likely to enter puberty
prematurely than girls with a wild-type genotype. In
this case, the authors argued that a higher level of
CYP3A4 enzyme, associated with increased gene
transcription for the variant G-392 allele, would
enhance steroidogenesis and breast maturation. However, this finding has been challenged by results of a
comprehensive study of early menarche and polymorphisms in CYP3 A4, CYP1 B1, CYP17 and
CYP1 A2 genes, in multiple ethnic groups [81].
Comparing subjects homozygous for the mutant G392 allele and those heterozygous or homozygous for
the wild-type A-392 allele, the authors reported no
difference in the mean age of menarche after controlling for ethnic group and year of birth. For the
unadjusted data, a statistically significant and slightly
earlier mean age of menarche was seen in the
homozygous G-392 group.
Although the clinical data indicate convincingly
that there is an association between the CYP3 A4*1 B
polymorphism and disease risk / treatment toxicity,
the lack of compelling in vivo evidence for altered
CYP3A probe substrate disposition for the
CYP3 A4*1 B genotype suggests the possibility of
linkage disequilibrium between CYP3 A4*1 B and
another genetic mutation that is the true cause of the
clinical phenotype. A possible candidate gene for
this role is CYP3 A5, as presented later in this review.
2.2. CYP3 A4 coding domains
2.2.1. CYP3 A4 coding mutations
A total of 18 unique mutations have been identified within the coding regions of the CYP3 A4 gene
(Table 1). Coding SNPs are present in nine of the 13
CYP3A4 exons, and seem particularly concentrated
in exons 5–7 and 11–12 (Fig. 3). All of the coding
SNP allele frequencies are relatively low ( , 5%) in
most of the populations studied and no homozygotes
for these mutations have been reported
[65,67,68,71,82,83]. Although the number of studies
reported is small, in some cases the frequency of
coding variants is lower for Caucasians compared to
other ethnic groups. Several of the coding variants
reported for Caucasians (CYP3 A4*8, *9, *11, *12,
*13, and *15 ) appeared at an allele frequency of less
than 1%. The most common coding variants reported
for Caucasians are CYP3 A4*2 (2.7%) [67],
CYP3 A4*10 (0.24–2%) [68,71], and CYP3 A4*17
(2%) [82], whereas they are CYP3 A4*15 A (2–4%)
[68,82] for African Americans, CYP3 A4*16 and
Fig. 3. Distribution of mutations in the CYP3 A4 gene. Single point mutations and identifying nomenclature abstracted from the P450
website (www.imm.ki.se / CYPalleles.htm).
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
CYP3 A4*10 for Mexicans (5%)
CYP3 A4*16 for Japanese (5%) [68].
[68],
and
2.2.2. Phenotype of CYP3 A4 coding mutations
Many of the identified coding CYP3 A4 SNPs have
been studied in vitro to determine the effect of the
SNP on enzyme production / degradation rates and
catalytic function. For simplicity, these SNPs are
identified by the predicted amino acid change. Their
location within the cDNA is also reported in Table 1.
Direct evaluations have been constrained largely to
an assessment of catalytic activity of cDNA-expressed enzyme, purified and reconstituted with lipid,
cytochrome P450 reductase and cytochrome b 5 . As
seen in Table 1, the majority of the amino acid
substitutions appear to have no pronounced effect on
drug metabolism kinetics. Six of 19 variants,
CYP3 A4*3
(M445T),
CYP3 A4*7
(G56D),
CYP3 A4*9
(V170I),
CYP3 A4*10
(D174H),
CYP3 A4*11 (T363M), and CYP3 A4*19 (P467S),
metabolized the probe substrates testosterone, progesterone
or
7-benzyloxy-4-(trifluoromethyl)
coumarin at rates comparable to that observed for
wild-type enzyme [67,71,82]. However, four of 19
structural variants were associated with altered catalytic function, an effect that was sometimes drugspecific. For example, in the first publication on
CYP3 A4 SNPs, Sata et al. [67] reported that, compared to wild-type enzyme, CYP3 A4*2 (S222P)
exhibited a 6-fold increase in Km and corresponding
decreased intrinsic clearance for nifedipine oxidation
but no change in testosterone 6b-hydroxylation
activity. Another structural variant, CYP3 A4*12
(L373F), showed a comparable rate of testosterone
6b-hydroxylation as wild-type enzyme but was far
more efficient than CYP3 A4*1 at hydroxylations in
the 15b, 2b-positions [71]. The L373F variant
enzyme also displayed a change in product regioselectivity with a reduction in the intrinsic clearance for midazolam 19-hydroxylation coupled to an
increased 4-hydroxylation clearance. Modestly enhanced catalytic activity towards testosterone (6bhydroxylation) and chlorpyrofos (desulfuration) was
also reported for CYP3 A4*18 (L293P). In contrast, a
reduction in testosterone 6b-hydroxylation and
chlorpyrofos desulfuration was observed for
CYP3 A4*17 (F189S) [82].
Two of the rare CYP3 A4 coding variants,
CYP3 A4*8 (R130Q) and CYP3 A4*13 (P416L), were
1281
reported to be associated with low or no CYP3A4specific protein content, respectively, in a heterologous cell expression system [71]. Although there
are alternative explanations for this finding, the
authors argued convincingly that the Q130 and L416
substitutions would affect steady-state enzyme levels
by altering heme binding and / or protein stability.
The functional significance of coding SNPs in the
CYP3 A4 gene has also been assessed by comparison
of genotype and observed phenotype in a bank of 74
livers from Caucasian and African American donors.
Livers heterozygous for CYP3 A4*2, *10, *14, *15
and *16 were identified among the African American group, whereas CYP3 A4*10 was the only structural variant seen in the Caucasian cohort. There was
no significant association between midazolam hydroxylation activity and any of the observed genetic
variants, although the absolute number of variant
livers studied was low [68].
Most of the coding variants reported by other
research groups were not found in the study of
Lamba et al. [68] and vice versa. This is a consequence of the low variant CYP3 A4 coding allele
frequencies. Indeed, it was estimated that only 14%
of Caucasians, 10% of Japanese and 15% of Mexicans carry a CYP3 A4 allele with at least one coding
change. With such low allele frequencies, it will be
exceedingly difficult to link genotype to in vitro
phenotype, except by the heterologous expression
studies described above. A more tenable approach
will assess the association between genotype and in
vivo phenotype in a large population, through the use
of a CYP3A4-selective probe. For example, in their
description of novel CYP3 A4 mutations among
Chinese, Hsieh et al. [63] reported that subjects
carrying a CYP3 A4*4 (I118V), CYP3 A4*5 (P218R)
or CYP3 A4*6 (exon 9 insertion) allele exhibited
below average 6b-hydroxycortisol / cortisol ratios,
implying reduced catalytic activity for the corresponding variant proteins.
Of the structural variants of CYP3A4 identified to
date, none involve a mutation of amino acids comprising the putative substrate binding domain. However, many of the functionally significant changes
might result from perturbations of the tertiary protein
structure that directly or indirectly alter substrate
access to the active site, substrate binding affinity or
protein–protein electron transfer. Indeed, most of the
changes in catalytic activity observed for CYP3 A4
1282
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
gene variants are relatively modest, in comparison to
the functional consequences of coding mutations in
the CYP2 C9 gene associated with a poor metabolizer
phenotype (CYP2 C9*3 and CYP2 C9*5 ) [84]. These
catalytic findings and consideration of the low allele
frequencies for the known structural CYP3 A4 variants, implies that they are not the major cause of
inter-individual differences in CYP3A-dependent
drug clearance observed for the general population.
sion phenotype [68]. However, as discussed earlier,
physiological changes that occur in an organ donor
may mask any true association. Although results
from in vivo studies might be helpful in addressing
the question, the extent of linkage disequilibrium
between multiple alleles on both CYP3 A4 and
CYP3 A5 genes may preclude any definitive conclusions without additional supporting in vitro data.
2.3. CYP3 A4 intronic domains
3. CYP3A5 genetic variation
2.3.1. CYP3 A4 intronic mutations
A number of intronic SNPs have been identified in
the CYP3 A4 gene. They are identified in the text
below by their position relative to the translation
start site (A in ATG, 1 1). None have been definitively linked to altered CYP3A4 expression and
function, and most are rare ( , 1% allelic frequency). However, Lamba et al. [68] reported a
G20338A transition within intron 10 and a T15871C
transition within intron 7 at an allele frequency of 48
and 50%, respectively, among African Americans,
and at 6.5% for both alleles among Caucasians.
Other investigators have also reported an A20338
allele frequency of 9.5% for Caucasians [71]. Finally, three different SNPs in the 39-UTR of CYP3A4
have also been identified [68].
The CYP3 A5 cDNA sequence was first described
independently by Aoyama et al. [23] and Schuetz et
al. [85]. The allele corresponding to this cDNA and
the respective expressed protein was designated
wild-type, CYP3 A5*1 A. Subsequent work described
59-flanking sequence of two distinctly different genomic clones from a human liver containing CYP3A5
protein [86]. The clones contained identical sequences for exon 1 but differed slightly in the 59flanking sequence. The flanking sequence corresponding to CYP3 A5*1 A was notable for minor
differences compared to the CYP3 A4 gene, in particular an A-44G substitution (relative to the transcription start site) in the Basic Transcription Element (BTE). More recent work described below
revealed that the putative CYP3 A5*1 A promoter
¨ et al. actually corresequence described by Jounaıdi
sponds to that of a closely related CYP3 A pseudogene, CYP3 AP1 [87]. Thus, literature published
between 1994 and 2001 describing the CYP3 A5
promoter should be interpreted to reflect regulation
of CYP3 AP1.
2.3.2. Phenotype of CYP3 A4 intronic mutations
The most prevalent intronic mutations within the
CYP3 A4 gene occur with a frequency sufficiently
high enough to engender further interest. Intriguingly, the G20338 (intron 10) allele was observed in
combination with the T15871 (intron 7) allele and
the A-392 allele in 97 out of 106 Caucasian alleles
examined. The close association between the three
alleles was also present for African Americans, with
concordance among 40 of 52 alleles examined.
Moreover, it was previously noted that the variant
G-392 allele of CYP3 A4 was in linkage disequilibrium with the A6986 allele (intron 3) of CYP3 A5*1 A
that confers significant expression of CYP3A5 protein [17].
An analysis of CYP3A4 protein content and
catalytic activity in Caucasian and African American
liver banks has not revealed any striking association
between the CYP3 A4 intronic mutations and expres-
3.1. CYP3 A5 59 -flanking domain
In 2000, Paulussen et al. [88] described two linked
mutations in the proximal 59-flanking region of the
CYP3 A5 gene that were in complete concordance
with polymorphic hepatic CYP3A5 protein content.
One of the mutations, identified as G-45A (also
described as G-44A by Kuehl et al. [17]), occurred
in the BTE element of CYP3 A5. It was proposed that
mutation at this site might perturb Sp1 binding,
reducing gene transcription, and result in low
CYP3A5 protein expression in individuals homozygous for the variant A-45 allele. However, recent
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
publication of the sequence for the entire CYP3 A
gene locus has revealed the presence of two CYP3 A
pseudogenes, CYP3 AP1 and CYP3 AP2 [87]. Importantly, the 59-flanking and exon-1 sequence for
¨
CYP3 AP1 is identical to that described by Jounaıdi
et al. [86] as the putative CYP3 A5 promoter sequence. The true 59-flanking sequence for
CYP3 A5*1 A (AC005020), is homologous to that of
CYP3 AP1 (89%), but contains important differences.
In particular, it was shown that allelic variation in
the BTE of CYP3 A5 is not present, and is found only
on CYP3 AP1 [17]. However, the observation of an
extraordinarily close association between the
CYP3 AP1 -44 SNP and CYP3A5 protein expression
led to the identification by Kuehl et al. of the now
recognized causal mutation within intron-3 of the
CYP3 A5 gene [17]. There are three mutations within
the true 59-flanking region of CYP3 A5 (CYP3 A5*1 B
and CYP3 A5*1 C) (Table 2), but none are associated
with polymorphic CYP3A5 protein expression
[17,18].
3.2. CYP3 A5 coding domains
The first report of a potentially important CYP3 A5
¨ et al. [16] in their
coding SNP came from Jounaıdi
1283
study of livers obtained from Caucasian donors.
These investigators described a C1280A transversion
in exon 11 (CYP3 A5*2 ) that co-segregated with the
absence of CYP3A5 protein in two of five individuals. The frequency of the CYP3 A5*2 allele in
Caucasians was reported as 1.9% [18], but was not
seen in another study [17]. In their communication,
¨ et al. [16] suggested that the resulting
Jounaıdi
amino acid change, T398A, might affect the stability
of the CYP3A5 protein and account for reduced
hepatic content.
New CYP3 A5 coding mutations (Q200R, H30Y,
S100Y) have been reported recently (Table 2), but
all occur at a relatively low allelic frequency [18,89].
The functional significance of these mutations is
unknown.
3.3. Intronic domains
3.3.1. CYP3 A5 intronic mutations
The most frequent and functionally important SNP
in the CYP3 A5 gene consists of an A6986G transition within intron 3 (CYP3 A5*3 ) (Fig. 4) [17]. This
mutation creates an alternative splice site in the
pre-mRNA and production of aberrant mRNA (SV1mRNA) that contains 131 bp of intron 3 sequence
Table 2
CYP3A5 Alleles
Allele
Location
Nucleotide changes
CYP3A5*1A
CYP3A5*1B
CYP3A5*1C
CYP3A5*1D
CYP3A5*2
CYP3A5*3A
59 UTR
59 UTR
39 UTR
Exon 11
Intron 3
G-86A
C-74T
C31611T
C27289A
A6986G,
C31611T
CYP3A5*3B
Intron 3
CYP3A5*3C
Intron 3
C3705T, 3709 ins G,
A6986G, C31611T
A6986G
CYP3A5*4
CYP3A5*5
CYP3A5*6
CYP3A5*7
Exon 7
Intron 5
Exon 7
Exon 11
A14665G
T12952C
G14690A
27131 ins T
Amino Acid Substitution
Expression
T398N
Caucasian
African American
3%
3%
0%
7%
[23]
[17]
[17,18]
1.9 – 5%
0%
[16,18]
splicing defect
None
70%
27–50%
H30Y, splicing defect
None
95%
27%
splicing defect
None
Q200R
splicing defect
splicing defect
stop codon at 348
Alternatively spliced mRNA
None (skip Exon 7)
None
0%
0%
13%
10%
Other
Japanese 71–85%,
Chinese 65–73%,
Koreans 70%,
Mexicans 75%
Refs.
[17,18]
[18]
Japanese 71%,
Chinese 73%,
Koreans 70%
Chinese 0.9%
Chinese 0.9%
0%
0%
Accession number: AC005020. Designated base changes are numbered relative to the A in the ATG initiation codon.
[18]
[89]
[89]
[17,18]
[18]
1284
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
Fig. 4. Distribution of mutations in the CYP3 A5 gene. Single point mutations and identifying nomenclature abstracted from the P450
website (www.imm.ki.se / CYPalleles.htm).
(exon 3B) inserted between exon 3 and exon 4 (Fig.
5). The exon-3B insertion results in a frameshift and
predicted truncation of the translated protein at
amino acid 102. Among Caucasians, a CYP3 A5*3 /
*3 genotype was perfectly concordant with very low
or undetectable hepatic CYP3A5 protein content
(Fig. 1), a finding that has been confirmed by other
investigators [18]. In addition, homozygosity for the
CYP3 A5*3 allele was also strongly associated with
low CYP3A5 protein content in African American
livers and Caucasian intestinal mucosa [17].
As seen in Table 2, the variant CYP3 A5*3
(G6986) allele is common to all ethnic groups
studied, although it is present with different allele
frequencies. In this case, the variant allele is actually
more prevalent than the wild-type allele for most
populations. Focusing on the functional wild-type
allele (A6986), the frequency is reported as 5–15%
for Caucasians and 45–73% for African Americans
[17,18]. Hustert et al. also reported an A6986 allele
frequency of 27% in Chinese and 30% in Koreans
[18]. Sequence information from more limited data
sets suggested a A6986 allele frequency of 35% in
Chinese, 15% in Japanese, 25% in Mexicans and
60% in Southwestern American Indians [17]. Overall, the available data suggests significant ethnic
genetic diversity for the CYP3 A5*1 allele that may
have important clinical consequences, as discussed
below.
There are mutations within intron 4 and intron 5 of
the CYP3 A5 gene which also create cryptic splice
sites that can affect the production of functional
mRNA (SV2 and SV3) [17]. However, these rarer
mutations have, to date, always been seen in combination with the intron 3 SNP and, thus, represent
CYP3 A5*3 haplotypes (*3 A, *3 B and *3 C). Additional rare mutations that also give rise to splicing
defects have been reported. A G14690A transition in
exon 7 seen initially in African American livers,
results in the splicing deletion of exon 7
(CYP3 A5*6 ) [17]. CYP3 A5*5, first described in
Chinese [89], represents a T12952C transition (intron 5 donor splice site) that results in multiple
alternatively spliced mRNA and presumably decreased CYP3A5 protein accumulation. CYP3 A5*7
represents a base insertion in exon 11 (27131–32
insT), resulting in a frameshift and a predicted
premature stop codon and truncated protein [18,89].
Fig. 5. Alternative mRNA splicing that controls polymorphic CYP3A5 expression. A single point mutation within intron 3 (A6986G)
creates a cryptic acceptor splice site that leads to the insertion of exon 3B (131 bp) into a variant SV1 -CYP3 A5 mRNA. Properly spiced
wild-type mRNA is produced by CYP3 A5*1 A and to a small degree by CYP3 A5*3.
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
Both CYP3 A5*6 and CYP3 A5*7 variants occur at a
relatively low allelic frequency in Caucasians and
Chinese, but are relatively common among African
Americans (10–13%). Several other intronic and
flanking CYP3 A5 SNPs have also been described
[18], although the haplotype for these mutations has
not yet been determined.
3.3.2. Phenotype of CYP3 A5 intronic mutations
In their original publication Kuehl et al. [17]
reported on the close association between high
hepatic CYP3A5-specific content and the presence of
the CYP3 A5*1 A allele (Fig. 1). Moreover, they also
demonstrated that Caucasian and African American
livers with at least one copy of the CYP3 A5*1 A
allele exhibited a mean midazolam hydroxylation
activity that was approximately 2-fold higher than
that observed for the corresponding CYP3 A5*3 /*3
livers. While there was considerable phenotypic
variability within different CYP3 A5 genotype
groups, presumably as a result of variable CYP3A4
expression, livers carrying a functional CYP3 A5
allele were more likely to exhibit high catalytic
activity than those that did not. Hustert et al. [18],
reported a similar concordance between hepatic
CYP3A5 protein content and the CYP3 A5*1 A genotype, however, the presence of CYP3A5 protein was
less prevalent than that seen in other Caucasian liver
banks (10 vs. 20–25%) [6,19].
The impact of CYP3 A5 splice variants on the
production of mRNA is quite interesting. Although
livers with a homozygous CYP3 A5*1 genotype
produce only properly spliced, wild-type (wt)
mRNA, those homozygous for the CYP3 A5*3 variant allele produce both wt and variant (SV1) mRNA
[17]. Utilizing a quantitative Taqman assay, Lin et
al. [19] found that the mean wt-CYP3 A5 mRNA
level was 4-fold higher in CYP3 A5*1 /*3 livers
compared to CYP3 A5*3 /*3 livers. SV1 -CYP3 A5
mRNA was absent from CYP3 A5*1 /*1 tissue, and
present in heterozygous livers at levels that were
approximately 10-fold lower than wt-CYP3 A5
mRNA. Moreover, inter-individual differences in wtCYP3 A5 mRNA content could explain 53% of the
variability in CYP3A5 protein content. A similar
correlation was seen between hepatic CYP3 A4
mRNA and protein content, demonstrating that variable transcriptional control is the dominant source of
1285
inter-individual differences in hepatic CYP3A levels
[19]. Interestingly, the relative amount of wt- and
SV1 -CYP3 A5 mRNA were highly correlated with
each other, for both CYP3 A5*1 /*3 and CYP3 A5*3 /
*3 sub-populations, suggesting that the probability
of proper and improper splicing at the exon-3 / exon4 boundary is pre-determined by the interaction of
the pre-mRNA with the spliceosome.
Because the substrate specificity and product
regioselectivity of CYP3A5 can differ from that of
CYP3A4, one expects that the impact of CYP3 A5
genetic polymorphism on drug disposition will be
drug dependent. There is currently no CYP3A5selective substrate probe. As mentioned earlier in
this review, good substrates for CYP3A5 include
midazolam [20], lidocaine [21], and fentanyl [22].
Unpublished data from our lab and that of colleagues
at University of Washington School of Pharmacy
(John T. Slattery, personal communication) indicate
that CYP3A4 and CYP3A5 exhibit comparable
catalytic activity towards flunitrazepam (3-hydroxylation) and cyclophosphamide (4-hydroxylation).
There are also substrates for which CYP3A5 catalytic activity is appreciably lower than that of
CYP3A4. These include cyclosporine [23] and
etoposide [90]. Again, strict interpretation of these
data is difficult since CYP3A5 function appears
highly sensitive to reconstitution environment [26].
For example, Gillam et al. [26] reported that
nifedipine, testosterone and erythromycin are excellent CYP3A5 substrates, whereas results from other
investigators indicate reduced or non-detectable activity [6,24,25].
Even when CYP3A5 is shown to be as efficient a
catalyst of drug metabolism as CYP3A4, one can
still identify significant differences in product regioselectivity. For example, at a nominal concentration of 8 mM, midazolam 19-hydroxylation is
favored over 4-hydroxylation by a ratio of 16:1 for
CYP3A5, compared to 5:1 for CYP3A4 [17]. Similar
product regioselectivity is reported for alprazolam
[30]. The intrinsic clearance for alprazolam a-hydroxylation is 3-fold higher for CYP3A5, compared
to CYP3A4, whereas CYP3A4 is 2-fold better at the
4-hydroxylation pathway. Finally, CYP3A5 will
catalyze the formation of the M1 metabolite of
cyclosporine, but it does not make AM9 and AM4N
[23]. CYP3A4 produces all three metabolites. These
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J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
similarities and differences in substrate selectivity
and product regioselectivity extend into the area of
CYP3A cooperativity. Both CYP3A5 and CYP3A4
undergo homotropic activation in vitro, since the
19- / 4-hydroxymidazolam product ratio produced
from a single enzyme system changes as a function
of substrate concentration [17,20]. Moreover, there is
also evidence of heterotropic cooperativity for
CYP3A5, as a-naphthoflavone has also been shown
to activate CYP3A5-catalyzed aflatoxin B 1 8,9-epoxide formation [91]. Whether or not putative CYP3 A5
coding polymorphisms will affect this phenomenon
is unknown.
There is little information in the literature pertaining to the in vivo significance of newly identified
CYP3 A5 polymorphisms with respect to metabolic
drug clearance. Undoubtedly, many pharmacogenetic
studies are currently underway and results can be
expected to appear in the literature in the near future.
Based on in vitro data with midazolam, one can
anticipate a modest (2- to 3-fold) enhancement of
activity in any given ethnic population.
3.4. Implications for polymorphic extrahepatic
CYP3 A5 and disease risk
CYP3A5 is the primary, if not exclusive, CYP3A
family member expressed in cells outside of the liver
and intestine (e.g., in kidney [92,93], lung [94],
prostate [95], breast [96] and polymorphonuclear
leukocytes [97]). This feature suggests that CYP3A5
may have an important physiological function in
these tissues. For example, CYP3A5 can mediate the
metabolism of cortisol to 6b-hydroxycortisol, a
physiological regulator of Na 1 transport in renal
epithelia [98]. Variable and polymorphic renal expression of CYP3A5 [92,93] could contribute to
individual differences in the localized generation of
6b-hydroxycortisol within the nephron and could
play an etiological role, for example, in salt-sensitive
hypertension by increasing renal retention of Na 1 . It
is interesting to note that salt-dependent renal hypertension is more prevalent in African Americans
than in Caucasian Americans, as is the CYP3 A5*1
allele. Thus, CYP3A5 is not just a catalyst of drug
detoxification, but in organs such as the kidney,
CYP3A5 may serve an important function in regulat-
ing the pool of endogenous paracrine or endocrine
factors that influence disease risk.
Polymorphic expression of CYP3A5 could also
contribute to variable metabolism of steroids in the
prostate and breast and to differences in the concentrations of circulating steroids, and hence, risk of
disease in these tissues. For example, CYP3A4 and
CYP3A5 metabolize estrone [99] and 17b-estradiol
[100] to the respective catechol estrogens (2-hydroxy
and 4-hydroxy), and to 16a-hydroxylated estrogens
[101], all of which have been implicated in estrogenmediated carcinogenicity. Moreover, CYP3A5 exhibits a different product regiospecificity than
CYP3A4, with a greater preference for formation of
the 4-hydroxy estrogens. The 4-hydroxycatechol
estrogens are of particular importance because they
appear to have greater carcinogenic effects than 2hydroxycatechol estrogens [102,103]. Thus, it is
possible that regioselective metabolism by polymorphically expressed CYP3A5 in breast and prostate
contribute to variable metabolism of steroids in
prostate and breast and thus to disease risk in these
tissues.
As discussed above, using CYP3 A4*1 B allele as a
marker, results from several studies have implicated
CYP3A4 as a candidate gene in several disorders
including prostate cancer and epipodophyllotoxininduced secondary acute myelogenous leukemia
[61,66,78]. Although the CYP3 A4*1 B allele was
originally proposed to be associated with altered
CYP3A4 hepatic activity [72,73], that relationship is
controversial [64,74]. Kuehl et al. hypothesized that
the CYP3 A4*1 B and CYP3 A5*1 alleles can be
present in the same person and that CYP3 A5*1
might influence local or systemic functional CYP3A
activity towards endogenous and exogenous substrates [17]. Indeed, African Americans frequently
carry both the CYP3 A5*1 and CYP3 A4*1 B alleles.
Using data from study subjects homozygotic for
CYP3 A4 or CYP3 A5 the investigators concluded
there was linkage disequilibrium between the two
alleles [17]. Although the same trend was seen for
co-occurrence of these two alleles in Caucasians, the
low frequency of these alleles in this population
necessitates analysis of larger populations to achieve
statistical significance.
The simultaneous occurrence of CYP3 A4*1 B and
CYP3 A5*1 alleles in the same African Americans
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
suggests the need to reevaluate this association and
to determine whether the CYP3 A5*1 allele, and thus
CYP3A5 expression, may be more closely associated
with these disease risks. For example, a reinterpretation of the finding that no persons with the
CYP3 A4*1 B alleles (i.e., no persons who expressed
CYP3A5 protein) developed secondary AML after
epipodophyllotoxin treatment is that expression of
CYP3A5 enhances clearance of this leukemogenic
agent. Also, the association between the CYP3 A4*1 B
allele and increased risk of prostate cancer in African
Americans could be due to the formation of mutagenic molecules catalyzed by the CYP3 A5*1 allele
gene product. Indeed, Chang et al. reported in
abstract form of a higher frequency of CYP3 AP1
T-369G heterozygotes among patients with hereditary prostate cancer (HPC), compared to unaffected
controls [104]. Since the implicated CYP3 AP1
G-369 allele was strongly associated with increased
CYP3A5 activity [88], and is likely to be in linkage
disequilibrium with the wild-type CYP3 A5*1 allele,
the authors’ suggestion that ‘‘sequence variants in
the CYP3 A5 gene may increase prostate cancer risk,
especially in HPC’’ has a plausible mechanistic
basis. In contrast, Liu et al. found no difference
between the CYP3 A5*1 allele frequency of control
subjects and patients with acute or chronic myeloid
leukemia [105].
3.5. Extrahepatic CYP3 A5 splicing variation
Recently, a cDNA for CYP3 A5 was isolated out of
human prostate [95] and found to have identical
sequence homology with cDNAs from human liver
between the ATG start codon through the 39-UTR
region. However, the prostate cDNA sequence differed substantially from that of the liver cDNA
within the 59-UTR between the transcription and
translation start sites. As expected, protein expressed
from the prostate cDNA was functionally active
1287
towards known CYP3A5 substrates. The authors
argued that their prostate cDNA was the result of
tissue-specific mRNA splicing between the CYP3 A5
gene and an alternate gene, and that this may confer
epigenetic control of enzyme synthesis. Presumably,
the CYP3 A5*3 allele (and other inactivating
CYP3A5 mutations) will still dictate whether a
functional transcript is produced in any tissue. However, the alternative splicing of 59-flanking noncoding sequence and the potential for SNPs in that
regulatory region, may confer tissue-specific expression, and add yet another level of complexity to a
CYP3 A5 genotype–phenotype relationship.
4. CYP3A7 genetic variation
4.1. Mutations in CYP3 A7 59 -flanking, coding and
intronic domains
The identification of SNPs in the CYP3 A7 gene
has lagged somewhat behind research on CYP3 A4
and CYP3 A5. Compared to the wild-type sequence
(CYP3 A7*1 A [106]), Kuehl et al. [17] identified four
unique mutations in the CYP3 A7 59-flanking region.
Three of the mutations represent SNPs (CYP3 A7*1 B,
*1 D and *1 E) and occurred in regions outside of
those associated with the regulation of CYP3 A
transcription. The fourth mutation (CYP3 A7*1 C)
consists of the replacement of 60 bp from the
CYP3 A4 gene with the corresponding sequence from
the CYP3 A7 gene (Fig. 6). Embedded within this
swapped sequence are 7 bases changes, compared to
the wild-type CYP3 A7 gene. Moreover, the swapped
sequence encompasses the CYP3 A4 ER-6 response
element that is known to be activated by PXR and
VDR [56,58], and HNF-5 and octamer binding
motifs. The CYP3 A7*1 C allele was reported at a
frequency of 3% in a Caucasian population, and 6%
in African Americans (Table 3) [17]. A similar
Fig. 6. CYP3 A7 promoter polymorphisms. Triangles indicate SNP variants at the specified nucleotide. The white box indicates the region of
CYP3 A4 -promoter swapped into the CYP3 A7*1 C variant allele.
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J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
Table 3
CYP3A7 Alleles
Allele
CYP3A7*1A
CYP3A7*1B
CYP3A7*1C
CYP3A7*1D
CYP3A7*1E
Nucleotide
C-314T
G-291T, T-284A, T-282C, A-281T,
T-270G, T-262A, A-232C
G-91A
G-49A
Effect
increased
expression
Caucasian
African-American
Refs.
1%
3%
0%
6%
[106]
[17]
[17,107]
0%
0%
8%
8%
[17]
[17]
Accession number: AF280107. Designated base changes are numbered relative to the A in the ATG initiation codon.
frequency of 3.5% was described by Burk et al.
[107] in their study of 127 Caucasians of German–
Austrian origin. The allele frequency for each of the
CYP3 A7*1 B and CYP3 A7*1 D variants was found to
be | 1% for Caucasians [17,107] and was not
detected in African Americans [17].
4.2. Relationship of CYP3 A7*1 C to phenotypic
expression of CYP3 A7 in adult liver
CYP3A7 is the dominant CYP3A isoform found
in fetal liver [9,108]. Gene transcription and protein
expression appears to be reduced after birth [9] and
mRNA is often very low or undetectable in
Caucasian adults [31]. Because the CYP3 A7*1 C
variant has an altered promoter sequence compared
to the wild-type allele, it was suggested that these
base changes, particularly those found in the ER-6
motif, would result in increased protein expression in
the adult liver [17]. Indeed, an analysis of CYP3 A7
mRNA from five livers with a CYP3 A7*1 C genotype
and four livers homozygous for the wild-type allele,
revealed that the two livers with the highest level of
CYP3 A7 mRNA carried the CYP3 A7*1 C allele [17].
Moreover, four of five livers with a CYP3 A7*1 C
allele had detectable message, whereas those homozygous for CYP3 A7*1 A had either undetectable or
low CYP3 A7 mRNA. It was concluded that the
CYP3 A7*1 C variant was associated with significant
CYP3A7 expression in adult liver, but that this
variant may not be the sole determinant of expression.
The association between the CYP3 A7*1 C mutation and increased gene transcription was confirmed
by Burk et al. [107]. These investigators found that
CYP3 A7 mRNA levels varied widely among the
different Caucasian livers, but that 11% of the
population represented a clear subgroup with the
highest level of transcript expression. Subsequent
genetic analysis revealed that eight of 14 high
mRNA expression livers carried at least one copy of
the CYP3 A7*1 C allele. However, one heterozygote
CYP3 A7*1 C liver exhibited a lower mRNA level.
The presence of a CYP3 A7*1 B allele was also found
in two individuals in the high mRNA cohort and no
others in the study population. The investigators also
looked at the genotype–phenotype relationship in 23
human small intestines and found two specimens
with a much higher level of mRNA than all others
( . 30-fold)
and
both
were
CYP3 A7*1 C
heterozygotes. Thus, the authors concluded that the
CYP3 A7*1 C and CYP3 A7*1 B alleles accounted for
two-thirds of cases of increased hepatic CYP3A7
mRNA expression, a ratio similar to that first reported by Kuehl et al. [17], and all of the cases of
increased intestinal mRNA expression. Moreover,
Burk et al. [107] also showed that activated PXR–
RXR heterodimer bound more strongly to the
CYP3 A7*1 C promoter than to the wild-type
CYP3 A7*1 A promoter, and that the CYP3 A7*1 C
promoter was much more effective at activating gene
transcription in a reporter assay.
Although an analysis of CYP3A7 promoter mutations in Japanese DNA has not been reported to date,
it is interesting to speculate that the CYP3 A7*1 C or
CYP3 A7*1 B allele frequency will be higher in the
Japanese population, since significant levels of
CYP3A7 protein were more frequently encountered
in Japanese compared to Caucasian livers [10]. No
data are available regarding the functional significance of the CYP3 A7*1 C variant with regard to drug
metabolism in vivo, but again one might anticipate
increased metabolic clearance of its drug substrates
(e.g., midazolam).
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
As mentioned before, fetal liver CYP3A7 plays a
major role in the production of estrogens for the
maternal–fetal unit during pregnancy [36]. It is not
clear what effect the CYP3 A7*1 C variant would
have on gene transcription in the fetus, but one could
anticipate no change (control by other fetal-specific
transcription factors) or an enhanced enzyme expression and functional activity in liver and in other sites
of expression, such as the endometrium and placenta
[109].
5. CYP3A43 splicing variation
One of the most interesting aspects of the
CYP3 A43 research to emerge is the apparent propensity for alternative splicing events. Sequence analysis
of different cDNA isolated from human liver indicated exon skipping and complete or partial intron
exclusion [11]. Subsequent work from the same
research group demonstrated the formation of
CYP3 A43 –CYP3 A4 mRNA hybrids [110]. Some of
these CYP3 A43 hybrids (exon 1 of CYP3 A43 joined
to exons 2–13 of CYP3 A4 ) retained catalytic activity, although the corresponding mRNA occurred at a
relatively low abundance compared to CYP3 A43
message. Because the CYP3 A43 and CYP3 A4 genes
are oriented head-to-head [110], production of the
hybrid is presumed to occur through alternative
splicing of pre-mRNA generated from the two genes.
While this represents yet another level of complexity
to our understanding of CYP3 A genetics, the functional significance of the phenomenon is unknown.
6. Summary
Numerous different mutations in the CYP3 A genes
have been identified. The most significant of these
mutations are found in CYP3 A5 and CYP3 A7. For
CYP3 A5, a SNP within intron-3 (CYP3 A5*3 ) results
in aberrant mRNA splicing and a pronounced reduction in protein synthesis. Depending on ethnicity, the
wild-type CYP3 A5*1 allele frequency varies between 5 and 45%. Livers and small intestines that
have at least one CYP3 A5*1 allele exhibit, on
average, increased metabolic clearance of
midazolam, compared to those tissues with a
1289
CYP3 A5*3 /*3 genotype. For CYP3 A7, replacement
of a 60-bp segment homologous to the CYP3A4
promoter into the CYP3 A7 promoter confers increased constitutive transcription of the CYP3 A7
gene, and presumably increased protein expression.
The substituted region encompasses an ER-6 motif
that binds PXR–RXR heterodimer, and potentially
other nuclear hormone receptors that may regulate
gene expression. An additional single point mutation
in the CYP3 A7 promoter (CYP3 A7*1 B) is also
associated with high levels of gene transcription, but
the mechanistic basis for this observation is not
apparent. The CYP3 A7*1 C and CYP3 A7*1 B allele
frequency also varies depending on ethnicity. It is
not known at this time whether these mutations
affect protein expression and catalytic activity towards CYP3A substrates, but a positive association
seems likely based on observed polymorphic enzyme
expression.
Coding mutations in the CYP3 A4 gene can affect
enzyme activity, but these allelic variants appear to
be relatively rare and exert, in most cases, only a
limited effect on the intrinsic clearance of CYP3A
substrates. Nonetheless, they may contribute to the
overall spectrum of variable drug clearance observed
in vivo, along with genetic variation in CYP3 A5 and
CYP3 A7 and other CYP3A regulatory factors. One
obvious area of interest is the identification of
mutations in genes coding for the nuclear hormone
receptors. However, an extensive discussion of this
literature is beyond the scope of this review. The
interested reader is referred to the following articles
for more information on PXR [56,111] and VDR
[112–114] genetic variability. Moreover, non-genetic
factors (e.g., variability in circulating hormone concentrations and cell signaling pathways) will likely
contribute importantly to interindividual differences
in CYP3A-catalyzed drug clearance. Only more
extensive and carefully controlled research will
determine the ultimate impact of CYP3 A genetic
variation on human drug disposition, efficacy and
safety.
Acknowledgements
This work was supported in part by grants from
the National Institutes of Health (GM07750,
1290
J.K. Lamba et al. / Advanced Drug Delivery Reviews 54 (2002) 1271–1294
GM63666,
ES07033,
GM32165,
GM60346,
GM61393, ES08658, P30 CA21765 and CA51001);
and by the American Lebanese Syrian Associated
Charities.
[13]
[14]
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