Expert Opinion on Drug Metabolism & Toxicology

ISSN: 1742-5255 (Print) 1744-7607 (Online) Journal homepage: http://www.tandfonline.com/loi/iemt20

Genetically based impairment in CYP2C8-

and CYP2C9-dependent NSAID metabolism
as a risk factor for gastrointestinal bleeding:
Is a combination of pharmacogenomics and
metabolomics required to improve personalized
medicine?

Jos AG Agndez, Elena Garca-Martn & Carmen Martnez

To cite this article: Jos AG Agndez, Elena Garca-Martn & Carmen Martnez (2009) Genetically
based impairment in CYP2C8- and CYP2C9-dependent NSAID metabolism as a risk factor for
gastrointestinal bleeding: Is a combination of pharmacogenomics and metabolomics required to
improve personalized medicine?, Expert Opinion on Drug Metabolism & Toxicology, 5:6, 607-620,
DOI: 10.1517/17425250902970998

To link to this article: http://dx.doi.org/10.1517/17425250902970998

Published online: 08 May 2009.

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1. Introduction
2. Role of CYP2C8 and CYP2C9
enzymes in the metabolism of
NSAID
3. CYP2C8 and CYP2C9 gene
polymorphisms
4. Effect of CYP2C8 and CYP2C9
polymorphisms on the
pharmacokinetics of NSAIDs
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5. Effect of CYP2C8 and CYP2C9
polymorphisms on the risk of
developing acute gastrointestinal
bleeding
6. Expert opinion
10.1517/17425250902970998 2009 Informa UK Ltd ISSN 1742-5255 607
All rights reserved: reproduction in whole or in part not permitted
Review
Genetically based impairment in
CYP2C8- and CYP2C9-dependent
NSAID metabolism as a risk
factor for gastrointestinal
bleeding: Is a combination
of pharmacogenomics and
metabolomics required to
improve personalized medicine?
Jos AG Agndez, Elena Garca-Martn & Carmen Martnez
University
of Extremadura, Medical School, Department of Pharmacology, Avda. de Elvas s/n,
E-06071, Badajoz, Spain
Polymorphisms in CYP2C8 and CYP2C9 are common in all the human
populations and many CYP2C8 and CYP2C9 gene variations cause decreased
enzyme activity towards the NSAIDs aceclofenac, celecoxib, diclofenac,
ibuprofen, indomethazine, lornoxicam, meloxicam, naproxen, piroxicam,
tenoxicam and valdecoxib. This impairment in drug biodisposition alters
drug pharmacokinetics, with carriers of detrimental mutations displaying
increased values of AUC and decreased drug clearance. Individuals carrying
the gene variants CYP2C8*3 (rs11572080; rs10509681), CYP2C9*2 (rs1799853)
or CYP2C9*3 (rs1057910) show increased risk of developing acute gastro
intestinal bleeding during the use of NSAID that are CYP2C8 or CYP2C9
substrates. However, it is not known whether parent drugs or products of
alternative metabolic pathways are responsible for bleeding. We present an
overview of the current knowledge of relevant polymorphisms of CYP2C8 and
CYP2C9 genes, their association with NSAID metabolism and pharmacokinetics
and a meta-analysis that confirms the clinical significance of these gene
variations with regard to gastrointestinal bleeding.
Keywords: CYP, CYP2C8, CYP2C9, NSAID, pharmacogenomics, polymorphisms
Expert Opin. Drug Metab. Toxicol. (2009) 5(6):607-620
1. Introduction
Over 30 million people receive NSAIDs daily[1], and 25% of the population
has experienced NSAID-related side effects that require medical care[2]. The clinical
relevance of NSAID-induced adverse effects is obvious: acute gastrointestinal
bleeding associated with the use of NSAIDs may cause > 30,000 hospitalizations
per year and accounts for one-third of gastrointestinal bleeding events in the US
population[3]. The economic implications are also of high relevance. The cost
derived from the adverse effects of NSAID is higher than the cost of the NSAID
therapy itself. In this regard it has been reported that NSAID-related side effects
cause a mean increase in medical costs of $0.9 1.2/day and per patient receiving
these drugs[4,5]. It is not surprising, therefore, that there is growing interest in the
identification of at-risk individuals among patients receiving NSAID.
 Genetically based impairment in CYP2C8- and CYP2C9-dependent NSAID metabolism as a risk factor for
gastrointestinal bleeding
In recent years, considerable advances in the knowledge
of the enzymes involved in NSAID metabolism and in the
identification of NSAID metabolites have been achieved. In
addition, pharmacogenomic techniques have been developed
to identify individuals with genetically determined impair-
ment in the metabolism of NSAID. Individuals carrying
enzyme-inactivating mutations display higher drug plasma
concentrations, and lower clearance rates occur in carriers of
inactivating mutations when treated at standard doses[6,7].
Therefore, an increased prevalence and severity of adverse
drug reactions is expected among subjects carrying enzyme-
inactivating mutations, when receiving drugs that are substrates
of the defective enzyme.
Much of the interindividual variability in adverse reactions
related to altered metabolic drug inactivation is attributable
to the parent drug but, in some cases, toxicity is caused by
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metabolites rather than parent drugs. This is the case, for
instance, of toxic hepatitis caused by inhalational anesthetics[8].
A substantial amount of information with regard to the phar-
macogenomics of NSAIDs on the main metabolic pathways
and on major metabolites is available. In contrast, information
with regard to secondary metabolic pathways and minor metab-
olites is surprisingly scarce and requires further studies. These
secondary pathways may be of special relevance in individuals
with genetically based impairment in the main NSAID
metabolizing enzymes, and we cannot rule out that some of the
adverse effects caused by NSAID may be related to metabo-
lites. Surprisingly, no studies analyzing the role of NSAID
metabolites in adverse drug effects invivo have been carried out.
With the earlier-mentioned limitations regarding lack of
information on secondary metabolic pathways and minor
metabolites, this invited review summarizes the current
knowledge of the polymorphisms of CYP2C8 and CYP2C9,
which are the most relevant enzymes involved in the biodis-
position of NSAID, and the potential role of these drug-
metabolizing enzymes and variant genotypes in the
pharmacokinetics and gastrointestinal bleeding risk during
the use of such drugs.
2. Role
of CYP2C8 and CYP2C9 enzymes in the
metabolism of NSAID
NSAIDs constitute a chemically heterogeneous group of
drugs that differ in their therapeutic efficacy and toxicity.
CYP2C8 and CYP2C9 enzymes are the chief enzymes
involved in the first steps of their metabolism. However, the
relative role of CYP2C enzymes in primary metabolism differs
among different NSAIDs.
Table 1 summarizes the relevance of CYP2C8, CYP2C9
and other enzymes in the primary metabolism of NSAID. The
data summarized in the table refer to primary metabolism,
and not to the subsequent metabolic steps that are detailed in
the following paragraphs.
Four NSAID, namely celecoxib, ibuprofen, lornoxicam and
piroxicam, are extensively metabolized by CYP2C enzymes,
608 Expert Opin. Drug Metab. Toxicol. (2009) 5(6)
these enzymes being responsible for > 90% of the primary
metabolism of the drug.
Celecoxib is extensively metabolized in liver by methyl
hydroxylation catalyzed by the CYP2C9 enzyme to hydroxycele-
coxib. The involvement of other CYP enzymes in celecoxib
hydroxylation, such as CYP3A4, CYP2C8 and CYP2C19, has
been suggested, and of these CYP3A4 seems to be the most
relevant[9]. Hydroxycelecoxib undergoes further oxidation to
a carboxycelecoxib, which is the main metabolite in man.
Carboxycelecoxib is metabolized by glucuronic acid conjugation
and excreted in bile[10,11].
Ibuprofen is a chiral drug that is extensively hydroxylated.
CYP2C9 is the main enzyme involved in the hydroxylation of
S-(+)-ibuprofen, and both CYP2C8 and CYP2C9 are involved
in the hydroxylation of R-(-)-ibuprofen[12,13]. Part of the
drug and metabolites are excreted conjugated and the involve-
ment of several forms of UDP-glucuronosyltransferase enzymes
has been demonstrated in ibuprofen conjugation[14].
Lornoxicam is almost completely metabolized in man.
The major lornoxicam metabolite is 5-hydroxylornoxicam, a
CYP2C9 product[15-17]. Other polar metabolites have been
identified in urine, but these account for a minor fraction of
the drug[18].
Piroxicam metabolism occurs primarily by hydroxylation
to 5-hydroxypiroxicam, which is further conjugated with
glucuronic acid. Urinary excretion of unchanged piroxicam
is negligible[19]. CYP2C9 plays a predominant role in the
piroxicam 5-hydroxylation pathway[20], although a contribution
of other CYP2C enzymes cannot be ruled out[21].
A second group of NSAIDs is constituted by drugs
predominantly, but not exclusively, metabolized by CYP2C
enzymes, with a contribution of CYP2C ranging from 50 to
90% of the metabolism of the drug. These include ace-
clofenac, diclofenac, flurbiprofen, indomethacin, meloxicam
and tenoxicam.
Aceclofenac is metabolized to 4-hydroxyaceclofenac by
the CYP2C9 enzyme[22], and to diclofenac through plasma
esterases. 4-Hydroxyaceclofenac is finally converted to
4-hydroxydiclofenac through plasma esterases. Diclofenac
metabolism is carried out by diverse hydroxylation pathways
and by acyl glucuronidation. About 50% of diclofenac is
eliminated as 4-hydroxydiclofenac, a CYP2C9 product[23].
The other metabolite in humans, 5-hydroxydiclofenac, seems
to be the product of several enzymes, including CYP2C8,
CYP2C18, CYP2C19 and CYP2B6[24,25]. A role for CYP3A4
in diclofenac metabolism has also been claimed[24,25], although
this issue has not been investigated in sufficient detail. The rest
of the drug is metabolized by uridine 5-diphosphoglucuronosyl
transferases UGT2B4 and UGT2B7[14,26]. Diclofenac
glucuronide is further metabolized by CYP2C8[25,27].
Flurbiprofen is metabolized mainly by CYP2C9 enzyme to
4-hydroxiflurbiprofen, which represents the main metabolite
in humans. Nearly 20% of the drug is excreted unchanged or
conjugated. Other hydroxylated metabolites are 3-hydroxy-
4-methoxiflurbiprofen and 3,4-dihydroxyflurbiprofen[28,29].
 Agndez, Garca-Martn & Martnez

Table1. Chief enzymes involved in the metabolism of NSAID.

Drug Relevance of CYP2C enzymes Main enzyme Secondary enzyme

Aceclofenac Partial CYP2C9 Plasma esterases

Aspirin Secondary UGT1A6 CYP2C9
Celecoxib Predominant CYP2C9
Diclofenac Partial CYP2C9 UGT2B7 and diverse CYPs
Dipyrone Secondary CYP2C19, CYP2C8 CYPs
Etoricoxib Secondary CYP3A4
Flurbiprofen Partial CYP2C9
Ibuprofen Predominant CYP2C8, CYP2C9
Indomethacin Partial CYP2C9 Carboxyl esterases
Ketoprofen Secondary UGTs CYP
Lornoxicam Predominant CYP2C9
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Meloxicam Partial CYP2C9 CYP3A4

Naproxen Secondary UGT2B7 CYP2C9, CYP1A2
Parecoxib Secondary Hydrolisis to valdecoxib CYP3A4, CYP2C9
Piroxicam Predominant CYP2C9
Rofecoxib Secondary UGT2B7, UGT2B15 CYP2C9, CYP3A4
Sulindac Secondary UGTs CYP2C9
Tenoxicam Partial CYP2C9
Valdecoxib Secondary CYP3A4 CYP2C9

Relevance of CYP2C enzymes in primary NSAID metabolism (see the text for details).
Predominant: > 90% of the drug is metabolized by CYP2C8 or CYP2C9 enzymes; partial: 50 90% of the drug is metabolized by CYP2C8 or CYP2C9 enzymes;
secondary: < 50% of the drug is metabolized by CYP2C8 or CYP2C9 enzymes.

A small part of the parent drug and all metabolites undergo
further conjugation.
Indomethacin is metabolized in the liver mainly through
O-demethylation. The formation of the inactive metabolite
O-desmethylindomethacin, which represents 50% of the
total drug eliminated in urine, is carried out by CYP2C9.
Another metabolic pathway for indomethacin is carried out by
carboxyl esterases although this pathway is minoritary[30,31].
Meloxicam is metabolized through two sequential steps to
hydroxymeloxicam and to a carboxylated metabolite after
oxidation of the methyl group of hydroxymeloxicam. The
enzymes CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2E1
and CYP3A4 can hydroxylate meloxicam. CYP2C9 displays
the highest activity, and the contribution of the rest of the
enzymes is of slight importance[9,32,33].
Tenoxicam is metabolized by CYP2C9 to 5-hydroxy-
tenoxicam, which is the chief urinary metabolite in humans.
In addition, tenoxicam and 5-hydroxytenoxicam undergo
glucuronidation. The amount of tenoxicam excreted as
unchanged drug is < 0.5%[21,34-36].
The third group of NSAID include drugs that are
metabolized only partially by CYP2C enzymes.
Aspirin is deacetylated to salicylic acid, which is further
metabolized by glucuronidation by UGT1A6, glycine conjugation
and hydroxylation. The role of CYP enzymes in aspirin
metabolism is of minor relevance, because gentisic acid, a
CYP2C9 oxidation product of salicylic acid, is a minor
metabolite[37-39]. Etoricoxib is metabolized by CYP3A4 and
no major implication of CYP2C enzymes in its metabolism
has been demonstrated[9,40]. Naproxen is metabolized by
acyl glucuronidation to form naproxen acyl glucuronide and
by O-demethylation to form 6-O-desmethylnaproxen. The
demethylated metabolite undergoes subsequent conjugation
steps[41]. UGT2B7 is the main enzyme in naproxen metab-
olism[42]. The demethylation pathway is carried out by
CYP2C9 and CYP1A2[43].
Ketoprofen, rofecoxib and sulindac are glucuronidated by
diverse UGT enzymes[14,44,45]. In contrast, the role of CYP2C
enzymes seems to be of minor relevance in the metabolism of
these drugs[9]. Parecoxib is an inactive prodrug of valdecoxib.
After administration, it is converted to valdecoxib[46]. Valde-
coxib is hydroxylated, although N-glucuronidation of valdecoxib
also occurs. Hydroxyvaldecoxib is an active metabolite that is
also glucuronidated to inactive metabolites[47]. CYP3A4, and
to a lesser extent CYP2C9, participate in the formation of
hydroxyvaldecoxib[9].
Dipyrone is partially related to CYP2C enzyme metabolism.
The drug is hydrolyzed in the gastrointestinal tract to

Expert Opin. Drug Metab. Toxicol. (2009) 5(6) 609

 Genetically based impairment in CYP2C8- and CYP2C9-dependent NSAID metabolism as a risk factor for
gastrointestinal bleeding

Table2. Nonsynonymous variants for the CYP2C8 gene.

Allele designation Amino acid change Effect on enzyme activity SNP designation

CYP2C8*2 I269F Increased Km rs11572103

CYP2C8*3 R139K; K399R Decreased rs11572080; rs10509681
CYP2C8*4 I264M Inconclusive rs1058930
CYP2C8*5 159 Frame shift Absent No designation
CYP2C8*7 R186X Absent No designation
CYP2C8*8 R186G Decreased No designation
CYP2C8*9 K247R Unknown No designation
CYP2C8*10 K383N Unknown No designation
CYP2C8*12 V461deletion Unknown No designation
CYP2C8*13 I223M Unknown No designation
CYP2C8*14 A238P Unknown No designation
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No designation A82S Unknown rs17851796

No designation I244V Unknown rs11572102
No designation L361F Unknown rs45438799

The rs numbers for single nucleotide polymorphisms are shown when officially assigned. Further information with regard to the mutations without an rs number can
be accessed on the website www.imm.ki.se.CYPalleles.
SNP: Single nucleotide polymorphism.

4-methylaminoantipyrine, which is transformed by N-demethy-
lation to 4-aminoantipyrine and formylation to 4-formylam-
inoantipyrine. The main metabolite 4-aminoantipyrine is
further acetylated to 4-acetylaminoantipyrine[48]. All meta-
bolic pathways seem to be carried out by CYP enzymes,
with the exception of the acetylation of 4-aminoantipyrine,
which is carried out by arylamine N-acetyltransferase 2[49,50].
Among the CYP enzymes involved in 4-methylaminoantipyrine
demethylation, CYP2C19 and CYP2C8, as well as CYP17,
seem to play a role[51], although the relative contribution
of each enzyme to the metabolism in vivo remains to
be explained.
3. CYP2C8 and CYP2C9 gene polymorphisms
Several CYP2C8 polymorphisms have been reported as
occurring with a relevant allele frequency. These are summarized
in Table 2 (for an updated database, see[52]). The variant
alleles CYP2C8*2, *3, *5, *7 and *8 bring about altered
enzyme activity. The commonest of these are CYP2C8*2 in
African subjects, CYP2C8*3 and CYP2C8*4 in Caucasian
subjects and CYP2C8*5 in Oriental subjects[53]. The rest of
the variant alleles shown in Table2, including three nonsyn-
onymous polymorphisms that have not yet received a formal
designation, seem to occur at extremely low frequencies[51,53,54].
It is to be noted that both interethnic and intraethnic variability
for common CYP2C8 polymorphisms occur[53].
With regard to CYP2C9, Table 3 summarizes the relevant
polymorphisms. Many CYP2C9 gene variations lead to
decreased enzyme activity. However, most of the CYP2C9
gene variants shown in Table3 are extremely rare[55-60]. Many
of these gene variants have been identified only once and it
cannot be ruled out that some of these may correspond to
artifacts. The most common CYP2C9 gene variants unam-
biguously associated to decreased enzyme activity are
CYP2C9*2, *3, *5, *6 and *11. Common CYP2C9 variant
alleles greatly vary in their frequencies across the human pop-
ulations. Among Caucasian individuals the most common are
CYP2C9*2 and CYP2C9*3. In Oriental subjects CYP2C9*2
is absent whereas CYP2C9*3 occurs with low frequency. In
African subjects many CYP2C9 gene variants, including
CYP2C9*2, *3, *5, *6 and *11, occur with frequencies
between 1 and 3%[53]. Intraethnic variability exists in the
frequencies for common CYP2C9 single nucleotide polymor-
phisms (SNPs). For instance, the variant allele CYP2C9*3 shows
a frequency of 11% in South-European Caucasians[61], and
50% of this frequency in North-European Caucasians[53].
In addition to alterations in drug metabolism, the CYP2C9
polymorphism has been associated with colorectal cancer
risk[62], and it has been proposed that the mechanism under-
lying this association may be related to altered metabolism of
NSAIDs[63].
It has been demonstrated in several human populations that
there is linkage between CYP2C8 and CYP2C9 polymorphisms.
Figure 1 shows the linkage disequilibrium for the gene areas
that contain nonsynonymous SNPs. Of particular interest is
the linkage between CYP2C8*3 and CYP2C9*2, which has been
described in several human populations. Individuals carrying
simultaneously both variant alleles show the lowest functional
activity in the metabolism of CYP2C8 and CYP2C9

610 Expert Opin. Drug Metab. Toxicol. (2009) 5(6)

 Agndez, Garca-Martn & Martnez

Table3. Nonsynonymous variants for the CYP2C9 gene.

Allele designation Amino acid change Effect on enzyme activity SNP designation

CYP2C9*2 R144C Decreased rs1799853

CYP2C9*3 I359L Decreased rs1057910
CYP2C9*4 I359T Unknown rs56165452
CYP2C9*5 D360E Decreased rs28371686
CYP2C9*6 273 Frame shift Absent rs9332131
CYP2C9*7 L19I Unknown No designation
CYP2C9*8 R150H Inconclusive rs7900194
CYP2C9*9 H251R Unknown rs2256871
CYP2C9*10 E272G Unknown rs9332130
CYP2C9*11 R335W Decreased rs28371685
CYP2C9*12 P489S Decreased rs9332239
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CYP2C9*13 L90P Decreased No designation

CYP2C9*14 R125H Decreased No designation
CYP2C9*15 S162X Absent No designation
CYP2C9*16 T299A Decreased No designation
CYP2C9*17 P382S Unknown No designation
CYP2C9*18 I359L; D397A Decreased rs1057910; no designation
CYP2C9*19 Q454H Unknown No designation
CYP2C9*20 G70R Unknown No designation
CYP2C9*21 P30L Unknown No designation
CYP2C9*22 N41D Unknown No designation
CYP2C9*23 V76M Unknown No designation
CYP2C9*24 E354K Unknown No designation
CYP2C9*25 118 Frame shift Absent No designation
CYP2C9*26 T130R Decreased No designation
CYP2C9*27 R150L Unknown No designation
CYP2C9*28 Q214L Decreased No designation
CYP2C9*29 P279T Unknown No designation
CYP2C9*30 A477T Decreased No designation
CYP2C9*31 I327T Unknown rs57505750
CYP2C9*32 V490F Unknown No designation
CYP2C9*33 R132Q Decreased No designation
CYP2C9*34 R335Q Unknown No designation
No designation I112L Unknown rs5030781
No designation R124Q Unknown rs12414460
No designation R150C Unknown rs17847037
No designation P337L Unknown rs58368927
No designation Y358C Unknown rs1057909
No designation L413P Unknown rs28371687
No designation L447F Unknown rs59485260

The rs numbers for single nucleotide polymorphisms are shown when officially assigned. Further information with regard to the mutations without an rs number can
be accessed on the website www.imm.ki.se.CYPalleles.
SNP: Single nucleotide polymorphism.

Expert Opin. Drug Metab. Toxicol. (2009) 5(6) 611

 Genetically based impairment in CYP2C8- and CYP2C9-dependent NSAID metabolism as a risk factor for
gastrointestinal bleeding
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Linkage between SNPs rs10509681 (CYP2C8*3)

and rs1799853 (CYP2C9*2). LOD: 4.97; D = 0.565

Figure1. The SNP single marker association of CYP2C8 and CYP2C9. LD structure in chromosome 10q23.33. Shown in each box
are the estimated statistics of linkage disequilibrium according to the standard color scheme. The gene areas shown correspond to those
that contain the nonsynonymous SNPs in CYP2C8 and CYP2C9 shown in Tables 1 and 2 (positions 96687810 9673883 for CYP2C9
and 96788739 96818150 for CYP2C8). The figure has been obtained with Haploview (v4.1) with release 21 of HapMap data from
Caucasian individuals of European origin [101].
LD: Linkage disequilibrium; SNP: Single nucleotide polymorphism.

substrates[12]. The linkage between CYP2C8*3 and CYP2C9*2
is disrupted in some pathologies[64], but it is consistently
observed in healthy subjects[65-67] and among individuals
with hypertension[68]. In addition to this linkage, Figure 1
shows many CYP2C8 and CYP2C9 SNPs that are strongly
linked (dark squares in the figure). As allele frequencies and
functional effects of many SNPs both in CYP2C8 and
CYP2C9 have not been investigated in detail, it is likely that
in the near future the analysis of new SNP associations will
bring about relevant information useful for clinical practice.
4. Effect of CYP2C8 and CYP2C9 polymorphisms
on the pharmacokinetics of NSAIDs
The presence of CYP2C8 gene variants has relevant consequences
in the biodisposition of CYP2C8 typical substrates, including
paclitaxel, amodiaquine and ibuprofen[12,65,69-72]. Typical
CYP2C9 substrates are warfarin, NSAIDs, COX-2 inhibitors,
phenytoin or glipizide and many other drugs[73]. Regarding
adverse effects, it has been demonstrated that the presence
of CYP2C8 variant alleles is related to diclofenac-induced
hepatotoxicity[74] and with acute gastrointestinal bleeding
after the use of NSAIDs[75]. The presence of CYP2C9 variant
alleles is related to increased risk of bleeding in patients
treated with warfarin at standard doses[76,77] and with acute
gastrointestinal bleeding after the use of NSAIDs[78,79].
Figure 2 shows a summary of the effect of common
CYP2C9 gene variants in the clearance of NSAID. Overall
findings indicate that CYP2C9*3 has a high impact on the
clearance of most drugs, as compared to that of CYP2C9*2.
This is not surprising as the CYP2C9*2 gene variant does
not contain mutations that may affect the substrate binding

612 Expert Opin. Drug Metab. Toxicol. (2009) 5(6)

 Agndez, Garca-Martn & Martnez

CYP2C9*2 CYP2C9*3

100 100
Celecoxib
50 50

0 0

100 100
Diclofenac
50 50

0 0

100 100
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Flurbiprofen 50 50

0 0

100 100

Ibuprofen 50 50

0 0

100 100

Lornoxicam 50 50

0 0

100 100

Piroxicam 50 50

0 0

100 100

Tenoxicam 50 50

0 0
s

us
us

s
ted

ted
ou

ou
go
go
uta

uta
yg

yg
y
zy
oz

oz

oz
nm

nm
mo
ter

ter

m
No

No
Ho

Ho
He

He

Figure2. Effect of common CYP2C8 and CYP2C9 gene variants in the pharmacokinetics of NSAIDs. Data are compiled from
available literature (see the text for details). Clearance (y-axis) refers to the clearance of individuals lacking variant alleles (100%). The effect
of CYP2C8 variant alleles has not been included in the figure because available information with regard to the effect of CYP2C8 variant
alleles on drug clearance is limited to ibuprofen only. The effect of CYP2C9*2 alleles on ibuprofen clearance corresponds to individuals
lacking CYP2C8*3 variant alleles, whereas for the rest of the drugs CYP2C8 genotypes of carriers of CYP2C9*2 are unknown.

Expert Opin. Drug Metab. Toxicol. (2009) 5(6) 613

 Genetically based impairment in CYP2C8- and CYP2C9-dependent NSAID metabolism as a risk factor for
gastrointestinal bleeding
capacity, and because experiments carried out invitro do not
support a major effect of the CYP2C9*2 allele on the intrin-
sic clearance for NSAIDs[80]. In addition, the effect of
CYP2C9*2 on the clearance of most drugs shown in Figure2
has been tested in individuals with unknown CYP2C8 geno-
types, with the exception of those receiving ibuprofen[12].
Because of the linkage disequibrium between CYP2C8*3 and
CYP2C9*2 (Figure1), it cannot be ruled out that the marginal
effect of CYP2C9*2 observed in the clearance of flurbiprofen,
piroxicam and tenoxicam may be related to the presence of
CYP2C8*3 variant alleles in these individuals.
4.1 Celecoxib
Invitro studies indicate that the intrinsic clearance for celecoxib
is slightly reduced in the case of the CYP2C9*2 gene product,
whereas the CYP2C9*3 gene product has a marked reduction
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to clearance values of 20% as compared to the wild-type
gene product[11,81,82]. However, Figure 2 shows that in vivo
the CYP2C9*2 variant allele does not significantly reduce
celecoxib clearance[81,83,84]. Moreover, liver samples from
subjects carrying the CYP2C9*2 allele did not differ signifi-
cantly from CYP2C9*1 in any of the systems studied[11]. In
contrast, celecoxib clearance is decreased among carriers of
the variant CYP2C9*3 allele, either in heterozygosis (mean
clearance 68% as compared with non-carriers of the allele)
or in homozygosis (mean clearance 23% as compared with
non-carriers of this allele)[73]. Recently, Kusama et al.[85]
predicted the effects of CYP2C9 polymorphisms in the
pharmacokinetics of certain CYP2C9 substrates from invitro
data and their findings regarding celecoxib pharmacokinetics
indicate that the effect of CYP2C9*2 is lower than that of
CYP2C9*3. No information with regard to the effect of
CYP2C8 polymorphisms is available.
4.2 Diclofenac
Current evidence indicates that the polymorphism on the
CYP2C9 gene does not bring about major changes in
diclofenac pharmacokinetics. The CYP2C9*2 variant allele
causes no impairment in diclofenac clearance[83,86,87], and
the influence of the CYP2C9*3 allele is very limited, carriers
displaying a mean clearance of 95% and 85% for heterozygous
and homozygous individuals, respectively[73].
4.3 Flurbiprofen
Significant differences in flurbiprofen pharmacokinetics
among individuals with different CYP2C9 genotypes have
been reported. Individuals carrying CYP2C9*2 alleles in
heterozygosis had oral clearances of 75%, and individuals
carrying CYP2C9*3 alleles in heterozygosis had oral clearances
of 60%, as compared to non-carriers[88]. Although no
studies involving homozygous carriers of CYP2C9 gene vari-
ants have been performed, from findings obtained invitro it
has been estimated that individuals homozygous for CYP2C9*2
and for CYP2C9*3 would show an average oral clearance
of 66% and 41%, respectively[85].
614 Expert Opin. Drug Metab. Toxicol. (2009) 5(6)
4.4 Ibuprofen
The pharmacokinetics of ibuprofen is strongly related to
CYP2C8 and CYP2C9 genotypes. The effect of CYP2C8*3 in
ibuprofen clearance is prominent (not shown in Figure 2);
individuals carrying this variant allele display clearances of 62
and 10% as compared with individuals lacking any mutations
in CYP2C8 and CYP2C9 genes[12,65]. The role of CYP2C9*2
in ibuprofen clearance is controversial. Although initial findings
indicated association of CYP2C9*2 genotypes with ibuprofen
clearance, it has been shown that CYP2C9*2 alone, when it is
not linked to CYP2C8*3, does not bring about a major impair-
ment in ibuprofen clearance[12]. Clearance values for subjects
who are heterozygous and homozygous for CYP2C9*2 and
who do not carry any other mutations are 96 and 84%,
respectively, as compared to individuals lacking any mutations
in CYP2C8 and CYP2C9 genes[12]. Individuals carrying
CYP2C9*3 variant alleles display a mean reduction of clearance
to values of 65 and 17% for heterozygous and homozygous
individuals, respectively[12,73,89]. It is of note that the relative
importance of CYP2C8 and CYP2C9 enzymes varies in ibu-
profen enantiomers. The main genetic factor for reduced
clearance of R-(-)-ibuprofen is the CYP2C8*3 allele, whereas
the clearance for S-(+)-ibuprofen is influenced by CYP2C8*3
and CYP2C9*3 alleles to a similar extent[12].
4.5 Lornoxicam
The clearance of this drug is impaired among individuals carrying
the CYP2C9*3 allele in heterozygosis, with clearance values < 40%
as compared to non-carriers[16,17]. No data with regard to the
effect of CYP2C9*3 in homozygosis are available. No studies
have addressed the putative role of CYP2C9*2 or CYP2C8
polymorphisms in the pharmacokinetics of lornoxicam.
4.6 Piroxicam
Piroxicam pharmacokinetics show a strong association with
CYP2C9 genotype. Both, CYP2C9*2 and CYP2C9*3 alleles
in heterozygosis are associated with reduced clearance values
representing 65% of that of individuals lacking mutations[20].
Unfortunately in the study cited the CYP2C8 genotypes
have not been analyzed, and it cannot be ruled out that the
decreased clearance associated with the CYP2C9*2 allele
may be actually related to CYP2C8*3.
4.7 Tenoxicam
The in vitro clearance of tenoxicam for the CYP2C9 variant
allele represents < 10% of that of the wild-type allele[90]. These
findings are partially supported by invivo studies that have indi-
cated that oral clearance among carriers of CYP2C9*2 and
CYP2C9*3 in heterozygosis represents 70 and 55%, respectively,
as compared with non-carriers of CYP2C9*3, whereas the effect
of the CYP2C9*2 and CYP2C8*3 alleles is less relevant[36,91].
4.8 Other NSAID
It is surprising that no studies have addressed the role of
genetic variation in the CYP2C8 or CYP2C9 genes and

pharmacokinetic parameters or adverse effects during the use
of drugs such as aceclofenac, indomethacin or meloxicam in
spite of the relevant role that CYP2C9 plays in its metabolism.
Regarding aceclofenac, as a substantial part of this drug is
converted to diclofenac, it is likely that the weak association
of the CYP2C9*3 variant allele and the pharmacokinetic
variability observed for diclofenac (Figure2) may also be valid
for patients receiving aceclofenac. Nevertheless, studies in
patients receiving aceclofenac should be carried out to confirm
this hypothesis. No studies have analyzed whether either
CYP2C8 or CYP2C9 gene variants influence indomethacin
pharmacokinetics or effects. Interindividual differences in
the pharmacokinetics of meloxicam have been described
among Chinese individuals[92]; unfortunately, no detailed
studies with regard to the role of CYP2C8 or CYP2C9 poly-
morphisms either with regard to meloxicam pharmacokinetics
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or adverse effects have been published so far.
No studies with regard to the effect of CYP2C8 or
CYP2C9 polymorphisms in the pharmacokinetics of NSAID,
whose metabolism is carried out marginally for these enzymes,
including aspirin, dipyrone, etoricoxib, ketoprofen, naproxen,
parecoxib, rofecoxib, sulindac or valdecoxib, have been pub-
lished. Nevertheless, owing to the secondary role of CYP2C8
and CYP2C9 in the metabolism of such drugs (Table1), it is
unlikely that such studies would give clinically relevant infor-
mation on the pharmacokinetics or on the risk of developing
adverse effects with the use of these drugs.
5. Effect
of CYP2C8 and CYP2C9
polymorphisms on the risk of developing
acute gastrointestinal bleeding
Acute gastrointestinal bleeding is an unwanted side effect with
a high rate of hospitalization and mortality in developed
countries. It is a common adverse drug reaction to all chemical
types of NSAID, and occurs either with oral or parenteral
administration of the drugs, thus indicating that a local irritation
mechanism is not sufficient to explain gastrointestinal bleeding
after NSAIDs use.
As many NSAID are extensively metabolized by CYP2C8 and
CYP2C9 enzymes (Table1), it is conceivable that individuals
with impaired ability to metabolize NSAID could be more
prone to develop adverse effects with these drugs, including
acute gastrointestinal bleeding. In 2003 a case was reported of
a 71-year-old patient under long-term therapy with aceno-
coumarol who presented gastrointestinal bleeding after treat-
ment with indomethacin[93]. This patient was homozygous
for the CYP2C9*3 allele and the authors speculated that the
interaction of genetically determined impaired metabolism
of indomethacin and acenocoumarol, as well as a putative
interaction in the metabolism of both CYP2C9 substrates, was
the cause of gastrointestinal bleeding. In 2004, Martinez etal.
reported the first study that analyzed the putative role of
CYP2C9 polymorphisms in gastrointestinal bleeding after
the use of NSAIDs[79]. The study concluded that carriers of
Expert Opin. Drug Metab. Toxicol. (2009) 5(6) 615
Agndez, Garca-Martn & Martnez
CYP2C9 variant alleles, in particular CYP2C9*2, were more
prone to develop acute gastrointestinal bleeding when they
received NSAIDs that were CYP2C9 substrates. It is intriguing
why in the study by Martinez etal. the variant allele confer-
ring the highest risk was CYP2C9*2 rather than CYP2C9*3,
which plays a more relevant role in the pharmacokinetics of
NSAIDs (Figure 2). In 2006, Vonkeman et al. reported
another study that concluded that the CYP2C9 genotype
was not significant as a risk factor for NSAID-related ulcers[94].
This study has been carried out with a reduced sample size
and the findings were striking as the CYP2C9*3 variant
allele was under-represented among patients, in contrast to
what would have been expected. In 2007, Pilotto et al.[95]
found a high frequency of carriers of CYP2C9*3 alleles and,
to a least extent, of carriers of CYP2C9*2 alleles among
patients who developed gastrointestinal bleeding with NSAIDs.
Although some issues in the study by Pilotto et al. were
controversial[78,96], this has been the first one that con-
firmed a relevant role of the CYP2C9*3 allele in the risk of
developing acute gastrointestinal bleeding with NSAIDs. In
2008, Blanco et al. published another study that confirmed
a role for CYP2C9*3 in NSAID-related gastrointestinal
bleeding[75] and that also demonstrated a prominent role
for CYP2C8*3 alleles in this risk. Ma et al. performed a
study on an Oriental population that was not conclusive[97]
because of the low frequency for variant CYP2C9 alleles
among Oriental subjects[53].
Three studies[75,94,95] were combined for a meta-analysis.
The study by Martinez et al.[79] has been excluded because
these patients also participated in the study by Blanco etal.[75].
The study by Ma et al.[97] has been excluded because only
1 of the 109 participants carried a variant CYP2C9 allele.
Figure 3 presents the results of the meta-analysis and shows
a significant role of CYP2C9*2 as a risk factor, with an odds
ratio = 1.58 (95% confidence interval, CI, 1.08 2.32) for
patients receiving any NSAID and 1.96 (95% CI 1.18 3.24)
for patients receiving NSAID that are CYP2C8 or CYP2C9
substrates. The role of CYP2C9*3 seems to be less signifi-
cant, with odds ratio = 1.60 (95% CI 1.00 2.56) for
patients receiving any NSAID and 1.74 (95% CI 0.98 3.10)
for patients receiving NSAID that are CYP2C8 or CYP2C9
substrates. If the two most common CYP2C9 alleles are
taken together, the statistical significance increases, with
odds ratio = 1.78 (95% CI 1.24 2.54) for patients receiving
any NSAID and 2.33 (95% CI 1.45 3.75) for patients
receiving NSAID that are CYP2C8 or CYP2C9 substrates.
Unfortunately, only one study analyzed the risk related to
CYP2C8*3[75], and it has reported an odds ratio = 1.91
(95% CI 1.16 3.15) for patients receiving any NSAID,
and 3.40 (95% CI 1.53 7.56) for patients receiving
NSAID that are CYP2C8 or CYP2C9 substrates. Although
more studies are necessary and in particular the relevance of
the risk related to CYP2C8*3 allele requires confirmation,
overall findings indicate that for patients receiving NSAIDs
that are CYP2C8 or CYP2C9 substrates, three variant alleles,
 Genetically based impairment in CYP2C8- and CYP2C9-dependent NSAID metabolism as a risk factor for
gastrointestinal bleeding

ALL NSAID CYP2C8/9 substrates

CYP2C9*2 CYP2C9*2
OR = 1.58 (1.08 2.32) OR = 1.96 (1.18 3.24)

0.10 1 10.00 0.10 1 10.00

CYP2C9*3 CYP2C9*2
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OR = 1.60 (1.00 2.56) OR = 1.74 (0.98 3.10)

0.10 1 10.00 0.10 1 10.00

Any variant allele Any variant allele

OR = 1.78 (1.24 2.54) OR = 2.33 (1.45 3.75)

0.10 1 10.00 0.10 1 10.00

Odds ratio Odds ratio

Figure3. Meta-analysis of the effect of CYP2C9*2 and CYP2C9*3 variant alleles in acute gastrointestinal bleeding after NSAID use.
Data correspond to the following studies: , Vonkeman etal., , Pilotto etal., , Blanco etal. [75,94,95].

namely, CYP2C8*3, CYP2C9*2 and CYP2C9*3, are associated
with the risk of developing acute gastrointestinal bleeding.
6. Expert opinion
After five decades of research in pharmacogenetics and
genomics[6], the initiation of genotype-guided clinical trials
and the FDAs approval of genetic tests to determine metabolizer
status, the routine use of pharmacogenomic tests in clinical
practice seems to be close, but not as close as we initially
expected. The conversion of genomic data into clinically use-
ful information on how individuals vary in their response to
drugs still needs further development. Thiopurine methyl-
transferase (TPMT) is a good example of this: whereas there
is a strong phenotype/genotype association for TPTM[98],
most of the azathioprine/mercaptopurine-induced adverse
reactions and the efficacy of therapy are not explained by TPMT
gene polymorphisms[99]. It seems that pharmacogenomics
alone is not sufficient to explain interindividual variability
in drug response. The question is of how we can improve
the use of pharmacogenomic information in clinical practice.
Perhaps we are missing two main issues: the presence of
alternative metabolic pathways, and the potential effect of
metabolites in adverse drug reactions.
Evidence summarized in this review indicates that
genetically determined impaired drug biotransformation
may play a prominent role in the development of acute
gastrointestinal bleeding with NSAIDs. However, much
further research is required to determine the putative role
of polymorphisms in other relevant enzymes that partici-
pate in the metabolism of NSAIDs (Table 1). It should be
borne in mind that drugs initially thought to be specific
substrates for an enzyme are actually metabolized by several
enzymes. Rapidly increasing evidence indicates that the
term enzyme-specific to define a drug should be used cau-
tiously. As our understanding of drug metabolism increases
with the help of highly sensitive detection methods such as
NMR+LC/MS or other similar techniques, perhaps we
should be ready to admit that a fully enzyme-specific drug
may actually not exist, and that minor metabolic pathways,

616 Expert Opin. Drug Metab. Toxicol. (2009) 5(6)

 Agndez, Garca-Martn & Martnez

usually overlooked in clinical studies, may play a relevant
role in adverse drug reactions.
In this regard, another issue that requires further investigation
in NSAID-related gastrointestinal bleeding is whether adverse
effects are related to parent drugs or to metabolites. The
simplest hypothesis is that impairment in drug metabolism
would bring about an increase in NSAID plasma concentra-
tions and hence would increase the risk of developing adverse
effects. This mechanism would be relevant in long-term
therapy because the drug would accumulate on a multiple
dose schedule but, in many cases, patients with acute gastro-
intestinal bleeding receive the NSAID only once, and the
effect of impaired metabolism in single-dose pharmacokinetics
is less relevant as can be expected in multiple-dose pharma-
cokinetics. An alternative mechanism for adverse drug effects
that should be explored is whether impaired function of
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metabolites may participate in the adverse effects. Probably
a definitive answer to these questions would come from
further development of metabolomics (or metabonomics)[100],
a rapidly emerging field that combined with other omics
such as pharmacogenomics and proteomics will be of great
value in understanding the interindividual variability in drug
response and adverse effects.
Acknowledgments
We thank James McCue for assistance in language editing.
Declaration of interest
The work in the authors laboratory is financed by Grants
FIS 05/1056, 06/1252 and RETICS RD07/0064/0016 from

main enzymes could drive the metabolism of the NSAIDs Fondo de Investigacin Sanitaria, Instituto de Salud Carlos III,
to alternative metabolic pathways and whether alternative Spain and FUNDESALUD, Mrida, Spain.

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Affiliation
Jos AG Agndez1 MD PhD,
Elena Garca-Martn2 & Carmen Martnez1
Author for correspondence
1University of Extremadura,
Medical School,
Department of Pharmacology,
Avda. de Elvas s/n, E-06071,
Badajoz, Spain
Tel: +34924289458; Fax: +34924289676;
E-mail: jagundez@unex.es
2University of Extremadura,
School of Biological Sciences,
Department Biochemistry,
Avda. de Elvas s/n, E-06071,
Badajoz, Spain