Pharmacogenetics of acenocoumarol: CYP2C9, CYP2C19, CYP1A2, CYP3A4, CYP3A5 and ABCB1 gene polymorphisms and dose requirements

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Background and objective: Acenocoumarol (AC) is a coumarin derivative, vitamin K antagonist anticoagulant drug. It has a narrow therapeutic index and shows large pharmacokinetic and pharmacodynamic interindividual variability. Our objective was to
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  PHARMACOGENETICS Pharmacogenetics of acenocoumarol:  CYP2C9, CYP2C19,CYP1A2, CYP3A4, CYP3A5  and  ABCB1  genepolymorphisms and dose requirements R. B. Saraeva*  MS , I. D. Paskaleva   MD PhD , E. Doncheva   MD , C. B. Eap   PhD ,V.S. Ganev*§  MD DSc * Department of Chemistry and Biochemistry, Medical University of Sofia, Sofia, Bulgaria,   National Heart Hospital, Sofia, Bulgaria,   Unit of Pharmacogenetics and Clinical Psychopharmacology, Center forPsychiatric Neurosciences, Department of Psychiatry – University Hospital Center, University of Lausanne,Prilly-Lausanne, Switzerland and  §  Institute of International Health, Michigan State University, EastLasting, MI, USA SUMMARY Background and objective:   Acenocoumarol (AC) isa coumarin derivative, vitamin K antagonistanticoagulant drug. It has a narrow therapeuticindex and shows large pharmacokinetic andpharmacodynamic interindividual variability.Our objective was to investigate the associationbetween AC dose requirements to achieve a targetlevel of anticoagulation and genetic polymor-phisms of genes possibly associated with itsmetabolism ( CYP1A2, CYP2C9, CYP2C19,CYP3A4, CYP3A5 ) and transport (  ABCB1 ).  Methods:   Ninety-six Bulgarian patients treatedorally with AC for at least 3 months wereincluded. They were separated into three groupsaccording to their AC dose requirement, i.e. low,medium and high. Results and discussion: CYP2C9 * 1   ⁄   *3 (associatedwith an intermediate CYP2C9 activity), CYP2C9*2   ⁄   *2, and  CYP2C9 * 2   ⁄   *3 genotypes (asso-ciated with a low CYP2C9 activity) were moreprevalent in the group with low dose requirementof AC compared with the other two groups( P   = 0 Æ 003). The frequency of  CYP2C9 * 1   ⁄   *1 geno-type, which is associated with an extensiveCYP2C9 activity, was higher in the group ofpatients with high dose requirements (79   ),compared with the groups of the medium andlow dose requirements (67   and 21   respec-tively). In addition, the  ABCB1 2677GG   ⁄   3435CChaplotype was associated with use of lower ACdose, whereas the  2677TT    ⁄   3435TT and 2677GT    ⁄   3435TT haplotypes were associated withuse of higher AC dose ( P   = 0 Æ 03). The distributionof polymorphisms of other genes did not showsignificant differences between the three groups. Conclusion: In vivo  , cytochromes P450 isoformsother than CYP2C9, and the permeability glyco-protein transporter, which is encoded by the  ABCB1  gene, were not significantly associatedwith dose requirement of AC. In our Bulgarianpatients, the presence of  CYP2C9*2  or    ⁄   and CYP2C9*3  alleles, as well as the  ABCB12677GG   ⁄   3435CC haplotype were associated withlow dose requirement of AC. Keywords :  ABCB1  , acenocoumarol,  CYP2C9  ,cytochromes P450s, dose requirement  ,  haplotype  , pharmacogenetics INTRODUCTION Oral anticoagulant drugs (AD) are commonly usedto treat patients with thromboembolic diseases. TheADs have a narrow therapeutic index and showlarge pharmacokinetic and pharmacodynamicinterindividual variability. Their variability is due Received 7 July 2007, Accepted 3 September 2007Correspondence: Radoslava B. Saraeva, 2, Zdrave Str Depart-ment of Chemistry and Biochemistry, Medical University of Sofia, 1431 Sofia, Bulgaria. Tel.: +359 2 9172 268; fax: +359 2 9172469; e-mail: r_saraeva@yahoo.comGrant Support: This work was supported in part by the ‘AgenceUniversitaire de la Francophonie’ (International Bursary forResearch: University year 2004–2005), and by internal fundsfrom the Unit of Pharmacogenetics and Clinical Psychopharma-cology.  Journal of Clinical Pharmacy and Therapeutics  (2007)  32 , 641–649   2007 The Authors. Journal compilation    2007 Blackwell Publishing Ltd 641  to several factors including age, sex, diet and ethnicfactors (1–3). Recent pharmacogenetic studies pro-vide evidence that genetic variations leading topharmacokinetic (i.e. cytochrome P450 2C9 orCYP2C9) and pharmacodynamic (i.e. vitamin Kepoxide reductase complex subunit I or VKORC1)variations could contribute to interindividual vari-ability in response to AD (4). Acenocoumarol (AC)isacoumarin,derivativevitaminKantagonist,usedas anticoagulant therapy mainly in Central Europeand Latin America. Chemically AC is a 4 ¢ -nitroanalogue of warfarin, the most widely prescribedoral AD, principally in North America and GreatBritain. Like warfarin, AC is available as a racemicmixture of two enantiomers [(R)- and (S)-AC] but,unlike warfarin, the  in vitro  pharmacological activi-ties of the two enantiomers of AC are comparable.However, as a result of a faster elimination of the(S)-enantiomer, (R)-AC is largely responsible forthe overall anticoagulant response (5, 6).  In vitro , CYP2C9 has been found to be the mainenzyme implicated in the metabolism of AC (6-,7-, and   ⁄   or 8-hydroxylation of both enantiomers)whereas CYP1A2 (6-hydroxylation) and CYP2C19(6-, 7-, 8-hydroxylation) are also involved in themetabolism of the (R)-enantiomer (6). To ourknowledge, no  in vitro  studies have yet assessedthe possible implication of CYP3A5 in ACmetabolism. In humans, the genetic polymor-phism of   CYP2C9  is well described with over 30different alleles (7). The most common allelicvariants in Caucasians are  CYP2C9*2  ( 430C>T, Arg144Cys) and  CYP2C9*3  ( 1075A>C,  Ile359Leu),which code for enzymes with reduced activitycompared with the wild-type allele (8–10). Thepresence of   CYP2C9  variant alleles has beenassociated with an increased risk of major bleed-ings in patients on AC treatment (11). Doseadjustment has been found to be more difficult incarriers of the  CYP2C9*3  allele than in individualshomozygous for the wild type allele or in patientscarrying the  CYP2C9*2  allele (12). The  CYP2C9*3 allele has also been associated with a lower ACdose-requirement to reach the same level of anti-coagulation in comparison with the wild typeallele. Carriers of the  CYP2C9 *3  allele required a1 Æ 5-fold lower AC dose when compared withcarriers of the wild type (13). In contrast, the effectof the  CYP2C9*2  allele on AC sensitivity appearedto be absent or less clinically relevant (13–15), afinding which was confirmed recently, with car-riers of   CYP2C9*2  allele requiring lower dailydoses of AC in comparison with wild-type sub- jects, with the lowest doses given to carriers of the CYP2C9*3  allele (16–18). All of those studies sug-gested that  CYP2C9  genotyping may be useful forpredicting the dose requirement of AC and sen-sitivity to AC treatment, thereby preventingpotentially life-threatening complications becauseof AC overdose. So far, no  in vivo  study has yettested whether genetic polymorphism of CYPisoforms other than  CYP2C9  (with the exceptionof CYP2C19 15) influences AC pharmacologicalactivity.P-glycoprotein (PGP), a transporter encoded bythe  ABCB1  gene, plays a major role in drug dis-position. PGP is a 170 kDa membrane glyco-protein that mediates ATP-dependent substrateefflux of a wide variety of natural compoundsand lipophilic xenobiotics (19). PGP is expressedin the intestinal mucosa, in the liver canalicularmembrane, in the kidney proximal tubules, in the blood–brain barrier and in the placenta (20).There is some evidence that PGP contributes towarfarin sensitivity (21), but no study has yetexamined the influence of PGP on AC distributionand pharmacological action. Such studies arewarranted because of the similarity in the chem-ical structure of the two drugs. The aim of thepresent study was to investigate the association between the AC dose requirements to achieve atarget level of anticoagulation and allelic variantsof   CYP2C9 ,  CYP1A2 ,  CYP2C19 ,  CYP3A4 ,  CYP3A5 and  ABCB1 . MATERIALS AND METHODS Subjects This study included 96 Bulgarian patients: 40women and 56 men, treated at the National HeartHospital in Sofia, who had been receiving a stableAC dose for at least 3 months to achieve an Inter-national Normalized Ratio (INR) for anticoagula-tion between 2 Æ 0 and 3 Æ 0. The mean (±SD) age of thepatients was 60 ± 12 years (range: 17–86 years) andthe mean weight (±SD) was 80 ± 15 kg (range:45–120 kg). They were classified into three groupsaccording to the prescribed AC dose: low-doserequirement [ £ 7 mg   ⁄   week; 14 patients; mean dose   2007 The Authors. Journal compilation    2007 Blackwell Publishing Ltd,  Journal of Clinical Pharmacy and Therapeutics ,  32 , 641–649 642  R. B. Saraeva  et al.  (±SD): 6 ± 1 mg   ⁄   week, range: 3 Æ 5–7 mg   ⁄   week];medium-dose requirement [>7 mg and <28 mg   ⁄   week; 63 patients; mean dose (±SD): 16 Æ 7 ± 5 Æ 6,range: 7 Æ 5–27 mg   ⁄   week]; high-dose requirement[ ‡ 28 mg   ⁄   week; 19 patients; mean dose (±SD):37 Æ 6 ± 7 Æ 7, range: 28–64]. The indications for treat-ment were: heart valve prosthesis – 40 patients,atrial fibrillation – 27 patients, pulmonary throm- boembolism – 11 patients, arterial embolism – 3patients, cerebral embolism – 4 persons, deepvenous thrombosis – 6 persons and myocardialinfarction – 5 patients. The patients were notco-medicated. The medical ethics committee at theMedical University of Sofia, Bulgaria, approvedour study. All subjects participating in this studygave their written informed consent. Genotype determination Genomic DNA was extracted from whole blood bya salting out protocol (22). All the SNPs weredetected by real-time polymerase chain reactionwith the use of 5 ¢ -nuclease allelic discriminationassays (ABI PRISM 7000 Sequence Detection Sys-tem; Applied Biosystems, Rotkreuz, Switzerland)with primers and probes obtained from AppliedBiosystems. The study included the analyses of  CYP1A2*1F  ( -163C>A ) ; CYP2C9*2  ( 430C>T  ) and  *3 ( 1075A>C );  CYP2C19*2  ( 681G>A ) and  *3  ( 636G>A ); CYP3A4*1B  (- 392A>G );  CYP3A5*3  ( 6986A>G ) ; ABCB1 61A>G, 2677G>T,  and  3435C>T   polymor-phisms.  CYP1A2*1F ;  CYP2C9*2  and  *3 ;  CYP2C19*2 and  *3 ;  CYP3A4*1B  and  CYP3A5*3  were analysedas previously described (23–25). The  ABCB161A>G ,  2677G>T   and  3435C>T   SNPs were analysedas previously reported (26). Statistical analysis Values for age and weight were expressed asmean ± SD. One-way analysis of variance wasused to compare age and weight between groups.Differences in the distribution of alleles andgenotypes between the groups of patients wereassessed by  v 2-test using SPSS software version13 Æ 0 (SPSS, Chicago, IL, USA). Combination of genotypes (haplotypes) of   ABCB1  was calculatedalso using SPSS software version 13 Æ 0. A  P -valueinferior to 0 Æ 05 was considered to indicate statis-tical significance. RESULTS The mean age in the low-, medium- and high-dosegroups was significantly different (71 ± 9, 59 ± 13and 53 ± 7 years respectively,  P  < 0 Æ 0001), as wasthe mean weight (73 ± 11, 77 ± 13 and 92 ± 15 kgrespectively,  P  < 0 Æ 0001). The gender distributiondid not differ between the three groups ( P  > 0 Æ 05).Age and weight were important and independentfactors modulating the sensitivity to AC. CYP and ABCB1 genotypes The observed allelic frequencies of   CYP2C9*2  and CYP2C9*3  were 16 % (95 % CI = 11–22 % ) and 6 % (95 %  CI = 3–11 % ), respectively, similar to previ-ously reported values in Caucasian populations(11–18 % and 5–11 % respectively) (21, 27–30). Theallelic frequency of   CYP2C19*2  was 14 %  (95 % CI = 6–20 % ), again similar to those of other Cau-casian populations (11–15 % ) (31–34). The observedallelic frequencies of   CYP3A4*1B  and  CYP3A5*3 were 2 % (95 % CI=1–5 % ) and 94 % (95 % CI=87–98),respectively, similar to those previously describedin Caucasian populations (2–10 %  and 91–95 % respectively) (35–39). The observed allelic fre-quencies of   CYP1A2*1F  was 72 % (95 % CI = 65–78),also similar to those of previous reports for Cau-casian populations (68–74 % ) (40). The observedallelic frequencies were 6 % (95 % CI = 3–11 % ) for  ABCB1 61G , 36 % (95 % CI = 29–43) for  2677T   and43 % (95 % CI = 36–50) for  3435T  , again similar tothose previously described for Caucasian popula-tions (39–46 %  and 46–48 %  for  ABCB1 2677T   and 3435T  ) (41–44). The frequencies of the differentalleles and genotypes are presented in Tables 1–3.Comparison of the distribution of the differentgenotypesindicatedastronglinkagedisequilibrium between the three SNPs in  ABCB1  ( P  = 0 Æ 294 for 61A>G   ⁄   3435T>C,  P  = 0 Æ 074 for  61A>G   ⁄   2677G>Tand  P  = 0 Æ 03 for  3435C>T    ⁄   2677G>T), in agreementwith published data (45). CYP2C9 genotypes and AC dose CYP2C9  genotypes and allele frequencies showedstatistically different distributions among the dif-ferent dose groups ( P  = 0 Æ 003 and  P  < 0 Æ 0001respectively) (Table 4). The frequency of the wildtype genotype ( CYP2C9*1   ⁄   *1) was highest in the   2007 The Authors. Journal compilation    2007 Blackwell Publishing Ltd,  Journal of Clinical Pharmacy and Therapeutics ,  32 , 641–649  AC dose requirement: impact of CYP2C9 and ABCB1 polymorphisms  643  group of patients on high dose (79 % ), followed bythe group on medium dose (67 % ), and the groupon low dose (21 % ). Accordingly, the carriers of  CYP2C9*1   ⁄   *3,  CYP2C9*2   ⁄   *2 and  CYP2C9*2   ⁄   *3genotypes were more common in the group onlow-dose requirement of AC compared with theother two groups. Also, while the carriers of  CYP2C9*1   ⁄   *2 genotype were distributed equally between the low- and medium-dose requirementgroups (29 % and 25 % respectively), its frequencywas lower in the high-dose group (11 % ). Finally,the slow-metabolizer genotypes ( CYP2C9*2   ⁄   *2 and CYP2C9*2   ⁄   *3) were not detected in the high-dosegroup, whereas their frequencies were 2 %  in themedium-dose group and 14 %  in the low-doserequirement group.  ABCB1 genotypes and AC dose Proximity to statistical significance was observedfor the distribution of   ABCB1 61A>G  and  2677G>T  genotypes ( P -values of 0 Æ 061 and 0 Æ 080 respec-tively) among the AC-dose groups (data notshown). Approach to statistical significance wasalso observed for differences in the distribution of the haplotypes combining  ABCB1 61A>G  and 2677G>T   SNPs among the AC-dose groups Table 1.  Frequencies of   CYP2C9  and  CYP2C19  allelesand genotypes n  Frequency ( % ) 95 % CI CYP2C9  allele*1 150 78 71–84*2 31 16 11–22*3 11 6 3–11 CYP2C9  genotype*1   ⁄   *1 60 63 52–72*1   ⁄   *2 22 23 15–33*1   ⁄   *3 8 8 4–16*2   ⁄   *2 3 3 1–9*2   ⁄   *3 3 3 1–9 CYP2C19  allele*1 166 86 80–90*2 26 14 6–20 CYP2C19  genotype*1   ⁄   *1 70 73 63–81*1   ⁄   *2 26 27 19–37 CI, confidence interval. Table 2.  Frequencies of   CYP3A4*1B, CYP3A5*3  and CYP1A2*1F  alleles and genotypes n  Frequency ( % ) 95 % CI CYP3A4  allele *1  188 98 95–99 *1B  4 2 1–5 CYP3A4*1B  genotype *1   ⁄   *1 92 96 89–99 *1   ⁄   *1B 4 4 1–11 CYP3A5*3  allele *1  11 6 2–13 *3  181 94 87–98 CYP3A5*3  genotype *1   ⁄   *3 11 11 6–19 *3   ⁄   *3 85 89 81–94 CYP1A2*1F  allele *1  54 28 22–35 *1F  138 72 65–78 CYP1A2*1F  genotype *1   ⁄   *1 8 8 4–16 *1   ⁄   *1F 38 40 30–51 *1F   ⁄   *1F 50 52 42–62 CI, confidence interval. Table 3.  Frequencies of   ABCB1  alleles and genotypes  ABCB1  genotype  n  Frequency ( % ) 95 % CI 61A>G  allele 61A  181 94 89–97 61G  11 6 3–11 61A>G  genotype  AA  86 90 82–95  AG  9 9 4–17 GG  1 1 0–6 2677G>T   allele 2677G  123 64 57–71 2677T   69 36 29–43 2677G>T   genotypeGG 36 38 28–49 GT   51 53 43–63 TT   9 9 4–17 3435C>T   allele 3435C  109 57 50–64 3435T   83 43 36–50 3435C>T   genotypeCC 29 30 21–40 CT   51 53 43–63 TT   16 17 10–26 CI, confidence interval.   2007 The Authors. Journal compilation    2007 Blackwell Publishing Ltd,  Journal of Clinical Pharmacy and Therapeutics ,  32 , 641–649 644  R. B. Saraeva  et al.  ( P  = 0 Æ 074, data not shown). Although the distri- bution of   ABCB1 61A>G   ⁄   3435C>T   haplotypes didnot show any statistically significant difference between the different dose-requirement groups( P  = 0 Æ 294), that of   ABCB1 2677G>T    ⁄   3435C>Thaplotypes did ( P  = 0 Æ 03, Table 5). For the twomost frequent haplotypes ( 2677GG   ⁄   3435CC and 2677GT    ⁄   3435CT), the  2677GG   ⁄   3435CC haplotypewas predominant in the low- and medium-dosegroups (29 % and 32 % respectively), and lower inthe high-dose groups (11 % ). The 2677GT   ⁄   3435CThaplotype was distributed equally among the threedose-groups (50 % , 41 %  and 47 %  respectively).The  2677TT    ⁄   3435TT haplotype was not found inthe low-dose group, but was present with increas-ing frequency in the medium- and high-dosegroups (0 % , 8 %  and 11 %  respectively). The 2677GT    ⁄   3435TT haplotype was found principallyin the high-dose group (21 % ) and with lowerfrequency in the medium- and low-dose groups(3 %  and 7 %  respectively). Taken as a group,  ABCB1  haplotypes containing  3435TT   genotype, Table 4.  Frequencies of   CYP2C9  genotypes and allelic variants in patients with different AC dose requirementsVariantDoseLow ( n  = 14) Medium ( n  = 63) High ( n  = 19) n  Frequency ( % ) 95 % CI  n  Frequency ( % ) 95 % CI  n  Frequency ( % ) 95 % CICYP2C9*1   ⁄   *1 3 21 5–51 42 67 54–78 15 79 54–93CYP2C9*1   ⁄   *2 4 29 10–58 16 25 15–38 2 11 2–35CYP2C9*1   ⁄   *3 3 21 5–51 3 5 0–14 2 11 2–35CYP2C9*2   ⁄   *2 2 14 2–44 1 2 0–10 0 0 0CYP2C9*2   ⁄   *3 2 14 2–44 1 2 0–10 0 0 0 P -value 0 Æ 003CYP2C9*1 13 46 28–65 103 82 74–85 34 90 75–97CYP2C9*2 10 36 20–56 19 15 9–23 2 5 1–19CYP2C9*3 5 18 7–38 4 3 1–8 2 5 1–19 P -value <0 Æ 0001 Table 5.  Frequencies of   ABCB1 2677G>T    ⁄   3435C>T haplotypes distribution in patients with different AC doserequirements [haplotype nomenclature was previously described by Johne  et al.  (49)]Haplotype  ABCB1 DoseLow ( n  = 14) Medium ( n  = 63) High ( n  = 19) n  Frequency ( % ) 95 % CI  n  Frequency ( % ) 95 % CI  n  Frequency ( % ) 95 % CI(2677GG   ⁄   3435CC) 4 29 10–58 20 32 21–45 2 11 2–35(2677TT   ⁄   3435CC) 1 7 0–36 0 0 0 0 0 0(2677GT   ⁄   3435CC) 0 0 0 0 0 0 2 11 2–35(2677GG   ⁄   3435TT) 0 0 0 2 3 0–12 0 0 0(2677TT   ⁄   3435TT) 0 0 0 5 8 3–18 2 11 2–35(2677GT   ⁄   3435TT) 1 7 0–36 2 3 0–12 4 21 7–46(2677GG   ⁄   3435CT) 1 7 0–36 7 11 5–22 0 0 0(2677TT   ⁄   3435CT) 0 0 0 1 2 0–10 0 0 0(2677GT   ⁄   3435CT) 7 50 24–76 26 41 29–54 9 47 25–70 P -value 0 Æ 03   2007 The Authors. Journal compilation    2007 Blackwell Publishing Ltd,  Journal of Clinical Pharmacy and Therapeutics ,  32 , 641–649  AC dose requirement: impact of CYP2C9 and ABCB1 polymorphisms  645
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