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Journal of Digestive Diseases 2008; 9; 7983 doi: 10.1111/j.1751-2980.2008.00326.x

BlackwellPublishingAsiaMelbourne,AustraliaCDDChineseJournal ofDigestiveDiseases1443-96111443-95732008TheAuthorsJournalcompilation 2008Chinese MedicalAssociationShanghai Branch,Chinese Societyof Gastroenterologyand BlackwellPublishingAsia PtyLtd.XXXOriginalArticlesCYP2C9genotype inNSAID gastropathyJMa etal .

CYP2C9 polymorphism in non-steroidal anti-inflammatorydrugs-induced gastropathy

Juan MA,* Xiu Yan YANG, Liang QIAO, Liu Qin LIANG & Min Hu CHEN*

*Division of Gastroenterology and Hepatology, Division of Rheumatology and Immunology, First AffiliatedHospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China, and Division of Gastroenterology

and Hepatology, Department of Medicine, Queen Mary Hospital, University of Hong Kong, Hong Kong

OBJECTIVE: Non-steroidal anti-inflammatory drugs(NSAID) induce gastroduodenal mucosal injury andare metabolized by cytochrome P450 2C9 (CYP2C9).It is postulated that CYP2C9 genotype is associated

with NSAID-induced gastropathy. This study aimsto determine whether individuals with a CYP2C9allele mutation are susceptible to NSAID-inducedgastropathy.

METHODS: A total of 109 patients diagnosed as hav-ing rheumatic diseases and taking NSAID wereappraised as having gastropathy by endoscopy, stooloccult blood test and questionnaire two weeks afterentering the study. Their peripheral blood was ana-lyzed by polymerase chain reaction-restriction frag-ment length polymorphism (PCR-RFLP).

RESULTS: A total of 47.7% gastropathy (33% erosions,14.7% ulcers, 2.75% ulcer bleeding) and 56% dyspepticsymptoms were presented. Only one CYP2C9*2 heter-ozygote (*1/*2) was found in the group with gastro-pathy and two variant alleles (CYP2C9*2 and CYP2C9*3) could not be found in the group without gastro-pathy. There was no significant difference in bothCYP2C9 genotype (0.96% vs 0%) and CYP2C9 variantallele frequency (1.92% vs 0%) between patients withand without gastropathy.

CONCLUSION: These results confirm the high prev-alence of NSAID-induced gastropathy but do not sup-port the postulation that CYP2C9*2 and CYP2C9*3contribute to the development of NSAID-inducedgastropathy. This may be due to the low frequency ofthe two alleles in the population studied.

KEY WORDS: CYP2C9 genotype, CYP2C9*2, CYP2C9*3, NSAID-induced gastropathy, Non-steroidal anti-inflammatory drugs.

INTRODUCTION

Non-steroidal anti-inflammatory drugs (NSAID) arewidely used in rheumatoid arthritis (RA) and osteo-arthritis.1 The notorious gastrointestinal toxicity of

NSAID limits their clinical use, as various symptoms

and lesions including dyspepsia, mucosal erosion, ulcersand even life-threatening perforation and severehemorrhage can occur.2,3 It is now believed that thegastrointestinal toxicities of NSAID are the results ofthe non-specific inhibition of cyclooxygenase 1 (COX-

1);4

however, the underlying mechanism has not yetbeen fully understood.

CYP2C9, one of the cytochrome P450 subfamilies,is responsible for the metabolism of many drugs suchas warfarin, phenytoin, tolbutamide, losartan and NSAID,including specific COX-2 inhibitors.5 The gene encod-ing CYP2C9 is highly polymorphic and more than 50single nucleotide polymorphisms have been found.6,7

Correspondence to: Min Hu CHEN, Division of Gastroenterologyand Hepatology, First Affiliated Hospital of Sun Yat-sen University,58 Zhongshan Road II, Guangzhou, 510080, Guangdong Province,China. Email: [email protected] 2008 The AuthorsJournal compilation 2008 Chinese Medical AssociationShanghai Branch, Chinese Society of Gastroenterology andBlackwell Publishing Asia Pty Ltd.

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Journal compilation 2008 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology and Blackwell Publishing Asia Pty Ltd.

Two common alleles are CYP2C9*2 and CYP2C9*3, which are closely related to the attenuation of theenzymatic activity of CYP2C9 and may be responsiblefor enhancing the toxicity of its substrates.69 A singlebase substitution of the CYP2C9*2 allele (T430C) andCYP2C9*3 allele (C1075A) lead to amino acid changesof Cys144Arg and Leu359Ile, respectively.10

Although the clinical relevance of CYP2C9 poly-morphism to some substrates such as warfarin hasbeen confirmed, the relationship between the CYP2C9genotype and NSAID-induced gastropathy remainsunclear. Limited evidence indicates that there is noassociation between the frequency of CYP2C9 allelic

variants and the risk of NSAID-induced gastric ulcers.11

The potential association between CYP2C9 poly-morphism in the Chinese population and NSAID-induced gastropathy has not yet been determined.

We hypothesized that Chinese individuals carryingCYP2C9*2 or CYP2C9*3 alleles are susceptible to

NSAID-induced gastroduodenal lesions. In this study,we aimed to examine the incidence of NSAID-inducedgastropathy and elucidate the relationship between theCYP2C9 genotype and NSAID-induced gastropathy inthe Chinese population.

MATERIALS AND METHODS

Study population

Enrolled in the study were 118 consecutive Chinesepatients admitted to the Division of Rheumatologyand Immunology of the First Affiliated Hospital of Sun

Yat-sen University (Guangzhou, China). These patientshad different rheumatic disease and needed to receiveregular NSAID treatment for at least two weeks. Excludedfrom this study were patients taking drugs affectingthe function of CYP2C9 enzyme, such as warfarin andlosartan, those with other independent diseases affectingthe gastrointestinal tract besides rheumatic disorders andthose who had poor compliance and were pregnant.

Two weeks after entering the study all the participantsunderwent endoscopy and occult blood test. Theirdetails, including their dyspeptic symptoms (epigastricpain or discomfort, nausea, vomiting, bloating, sensa-tion of fullness, regurgitation and dysphagia), historyof peptic ulcer, smoking, alcohol abuse, concomitantdrugs, category and the duration of administration ofNSAID, were recorded. All the participants understoodthe significance of the study and gave their written,informed consent before participation. The studyprotocol had received the approval of the local ethicscommittee.

Sampling and genotyping

Peripheral blood samples were collected, immediatelyfrozen and kept at 20C till analysis. Genomic DNA

was extracted from the blood samples by conventionalphenol/chloroform/isoamyl alcohol extraction andanalyzed by polymerase chain reaction-restriction

fragment length polymorphism (PCR-RFLP) describedby Wang et al.10 Primers used to amplify exon3 andexon7 were designed by Primer 5.0 software (PremierCompany, Canada). The sequences of the primers usedin PCR-RFLP were 5-GTATTTTGGCCTGAAACCCATA-3 (sense) and 5-GGCCTTGGTTTTTCTCAACTC-3(antisense) for CYP2C9*2, and 5-TGCACGAGGTC-CAGAGGTAC-3 (sense) and 5-ACAAACTTACCTT-GGGAATGAGA-3 (antisense) for CYP2C9*3.

The amplified products, 454 bp and 105 bp, weredigested by two restriction enzymes,AvaII and KpnI,respectively, at 37C for 1.5 h. The digested PCR

products were separated by 12% polyacrylamide gelelectrophoresis and stained by ethylene dibromide.Compared with CYP2C9*1 (wild type), CYP2C9*2lost one AvaII site whereas CYP2C9*3 generated aKpnI site.

Statistics

The inter-group comparisons were conducted with2-test or Fishers exact test. A P-value of

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caused by ulcers. Only 28 (25.7%) patients withdyspeptic symptoms had endoscopic gastroduodenallesions. There was no significant difference in NSAIDgastropathy between the patients with dyspepsia andthose without (45.9% vs 50.0%) (Table 2).

CYP2C9 genotyping

Of the 109 patients, only one CYP2C9*2 allele wasdetected and no CYP2C9*3 allele was found (Figs 1and 2). The frequencies of the CYP2C9*2 allele andCYP2C9*1/*2 genotype in the study population were0.46 and 0.92%, respectively (Tables 3 and 4).

There were no significant differences in the genotypeand allele frequency of CYP2C9*2 between the groups

with and without NSAID gastropathy (1.92% vs 0%and 0.96% vs 0%, P> 0.05).

When risk factors such as gender, history of smoking oralcohol drinking, a gastroduodenal ulcer or concom-

itant drugs and the category and duration of NSAIDswere considered, no statistical differences in the geno-type and allele frequency of CYP2C9*2 were observedbetween both groups.

DISCUSSION

Our data revealed a whole rough estimate of pre- valence of NSAID-induced gastropathy among the

population with rheumatic diseases. In this study, 56%of patients with dyspepsia symptoms and 47.71% ofpatients with NSAID gastropathy, including 14.7%

with ulcers and 2.75% with upper gastrointestinal tractbleeding, were reported. As was seen in previous studies,1530% of high-risk NSAID users developed ulcers12

whereas NSAID-associated dyspepsia and ulcers werefound in 79% and 16% of primary care patients,respectively with an average risk.13 Although the parti-cipants, the endoscopic lesions and the endpoint ofthese studies were different, a high incidence of NSAID-associated gastroduodenal toxicity was consistentlyfound.

Table 1. General characteristics of the study population

Variable

Gender (male/female, n) 42/67Age (mean SE, range, years) 48 17, 1176Smokers (n, %) 12 (11)

Alcohol drinkers (n, %) 4 (3.7)History of peptic ulcer (n, %) 3 (2.8)

Rheumatic diseases (n, %)Rheumatoid arthritis (n, %) 93 (85.3)Systematic lupus erythematosus (n, %) 9 (8.3)

Angiitis (n, %) 5 (4.6)Myositis (n, %) 2 (1.84)

NSAID n mg/day daysMeloxicam 79 7.515 1913Lornoxicam 13 816 276Nimesulide 18 50300 136Nabumetone 14 40 2180Loxoprofen 12 60180 2380

Acemetacin 8 90180 117Diclofenac 8 75 7330Ibuprofen 4 300600 412Indomethacin 5 75 1365Rofecoxib 17 2550 2730Celecoxib 9 200400 390

Table 2. NSAID-induced gastropathy of the studypopulation

(n, %)

NSAID-induced gastropathy 52/109 (47.7)

Lesions

Mucosal erosion 36 (33.0)Ulcer 16 (14.7)Upper gastrointestinal tract bleeding 3 (2.8)

Area

Stomach only 29 (26.6)Duodenum only 11 (10.1)Both stomach and duodenum 12 (11.0)

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At least 16 different NSAID are metabolized by CYP2C9.4-hydroxylation of diclofenac and flurbiprofen,5-hydroxylation of piroxicam and tenoxicam, methyl-hydroxylation of celecoxib are mainly or partly catalyzed

by the enzyme.6,7

Previous studies have shown that there was no signi-ficant association between the CYP2C9 genotype andrisk of NSAID-induced gastric ulceration.11 A recentstudy showed that individuals with CYP2C9*2 andCYP2C9*3 who had NSAID were more likely to haveacute gastrointestinal bleeding.14 However, these find-ings were from a Caucasian population. The clinical

influence of CYP2C9 polymorphism in the Chinesepopulation is poorly addressed.

We hypothesize that NSAID users carrying the CYP2C9*2 or CYP2C9*3 allele are at a higher risk for developmentof endoscopic mucosal lesions. Unfortunately, we failed

to confirm our hypothesis in the present study. This is probably due to ethnic difference and thegeographical distribution of the CYP2C9 polymor-phism. In a Caucasian population, higher frequenciesof CYP2C9*2 and CYP2C9*3 alleles were observed(19.0 and 16.2%, respectively), whereas in African-

Americans the frequencies were much lower (1.0 and0.5%, respectively).7 Lee et al. noted that the allele fre-quencies of CYP2C9*2 and CYP2C9*3 in a Chinesepopulation were no more than 0.04 and 3.30% whencompared with those of the Caucasian population.6

Similarly, a recent study from a large population reportedthat the frequencies of CYP2C9*2 and CYP2C9*3among Chinese were as low as 0.10 and 3.60%, respec-tively.15 We found that the CYP2C9*2 allele frequency

was only 0.46% in 109 patients. Since the allele fre-quencies of CYP2C9*2 and CYP2C9*3 are so lowamong Chinese people, the clinical value of these twoalleles may require further investigation. More samplesare becoming available to discover more individualscarrying the two variant alleles.

This study cannot yet exclude the relationship betweenthe CYP2C9 genotype and NSAID-induced gastropathy.

The CYP2C9 gene is highly polymorphic and many

new allelic variants in addition to CYP2C9*2 andCYP2C9*3 have been found. CYP2C9*13, initiallyfound in 2% of the Chinese population, is a novel

variant CYP2C9 allele involving a T269C transversionin exon2 that leads to a Leu90Pro substitution in theencoded protein.16 The CYP2C9*13 allele couldreduce 5-hydroxylation of lornoxicam.17 In addition,Leunget al. reported genetic polymorphism in exon4of CYP2C9, including 608TTG>GTG (Leu208Val),

Figure 1. CYP2C9*2 genotyping by polyacrylamide gelanalysis of exon3 PCR products digested withAvaII.

Table 3. Results of CYP2C9 genotyping in patients with and without gastropathy

Table 4. CYP2C9 variant allele frequencies in patients with and without gastropathy

Genotype Total (n, %) Patients with gastropathy (n, %) Patients without gastropathy P-value

CYP2C9*1/*1 108 (99.08) 51 (98.08) 57CYP2C9*1/*2 1 (0.92) 1 (1.92) 0 0.477

Allele frequency Total (n, %) Patients with gastropathy (n, %) Patients without gastropathy P-value

CYP2C9*1 217 (99.54) 103 (99.04) 114CYP2C9*2 1 (0.46) 1 (0.96) 0 0.476

Figure 2. CYP2C9*3 genotyping by polyacrylamide gelanalysis of exon7 PCR products digested with Kpn I.

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561CAG>CCG (Gln192Pro), 537CAT>CCT (His184Pro)and 527AAT>CTT (Ilu181Leu) at frequencies of 75, 20,10 and 9%, respectively, in 89 Chinese patients receiving

warfarin after a prosthetic heart valve replacement.18

They also found that the patients with heterozygousLeu208Val or homozygous Val208 seemed to require alower dose of warfarin than those with homozygous

Leu208. In contrast, patients who are heterozygousIle181Leu require a higher dose of warfarin than thehomozygous Ile181. Hence, the relationship betweenother CYP2C9 allelic variants and NSAID gastro-intestinal toxicity may warrant further study.

The role of CYP2C9s substrate-specificity in the rela-tionship of CYP2C9 polymorphism with correspond-ing NSAID should be considered. One study reportedthat the oral clearance rate of S-ibuprofen was relativeto the CYP2C9 genotype,19 whereas in other studies theCYP2C9 genetic polymorphism in pharmacokineticsrevealed no correlation or only a weak one with the

hepatic toxicity of diclofenac.20,21

It would be useful inthe future to examine the association of the CYP2C9genotype with different NSAID-induced gastropathies.

In conclusion, individuals carrying a CYP2C9*2 orCYP2C9*3 allele may not necessarily be at a higherrisk for gastroduodenal mucosal lesions followingNSAID administration. Compared to the wild type, thefrequency of CYP2C9*2 and CYP2C9*3 alleles in theChinese population are very low. Our study could helpelucidate further the relationship between the CYP2C9genotype and NSAID-induced gastrointestinal toxicity.

ACKNOWLEDGMENTS

We thank Dr Zhong Ping ZHAN (Division of Rheumatologyand Immunology, First Affiliated Hospital of Sun Yat-senUniversity, Guangzhou, China) and Dr Yin Xue XI (Divisionof Medicine, People Hospital of Buji, Shenzhen, China)for collecting blood samples.

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