Warfarin dose and the pharmacogenomics of CYP2C9 and VKORC1 - rationale and perspectives

Warfarin is the most widely prescribed oral anticoagulant, but there is greater than 10-fold interindividual variability in the dose required to attain a therapeutic response. Information from pharmacogenomics, the study of the interaction of an individual's genotype and drug response, can help optimize drug efficacy while minimizing adverse drug reactions. Pharmacogenetic analysis of two genes, the warfarin metabolic enzyme CYP2C9 and warfarin target enzyme, vitamin K epoxide reductase complex 1 VKORC1, confirmed their influence on warfarin maintenance dose. Possession of CYP2C9*2 or CYP2C9*3 variant alleles, which result in decreased enzyme activity, is associated with a significant decrease in the mean warfarin dose. Several single nucleotide polymorphisms (SNPs) in VKORC1 are associated with warfarin dose across the normal dose range. Haplotypes based on these SNPs explain a large fraction of the interindividual variation in warfarin dose, and VKORC1 has an approximately three-fold greater effect than CYP2C9. Algorithms incorporating genetic (CYP2C9 and VKORC1), demographic, and clinical factors to estimate the warfarin dosage, could potentially minimize the risk of over dose during warfarin induction.     

老年房颤患者VKORC1和CYP2C9基因多态性对华法林代谢和药效的影响

目的：探讨维生素K环氧化物还原酶复合物1基因（VKORC1）1639G／A（rs9926231）与细胞色素P450酶2C9基因（CYP2C9）1061A／C（rs1057910）多态性对中国汉族老年房颤患者华法林代谢和药效的影响。方法：用PCR—RFLP技术检测248例≥60岁房颤患者及262例≥60岁健康对照者的VKORC1—1639G／A、CYP2C91061A／C基因多态性。用HPLC法检测190例口眼华法林14d后国际标准化比值（1NR）达标1．5～3．0患者血药浓度,比较华法林代谢的药效差异。结果：两组-1639G／A与1061A／C各基因型分布差异无统计学意义（P〉0．05）;INR达标组1061AA型比AC＋CC型华法林维持剂量高、INR值低（P〈0．05）,1639AA型比GA＋GG型华法林维持剂量低（P〈0．05）;调整体表面积和剂量后,1061AC＋CC型华法林血药浓度高于AA型（P〈0．05）;调整体表面积、剂量及血药浓度后,-1639AA型INR大干GA＋GG型（P〈0．01）。结论：对中国老年房颤患者来说,VKORC1及CYP2C9基因多态性可能影响华法林药效;VKORC1多态性可能是中国汉族人群华法林维持剂量较低的原因之。     

Impact of pharmacokinetic (CYP2C9) and pharmacodynamic (VKORC1, F7, GGCX, CALU, EPHX1) gene variants on the initiation and maintenance phases of phenprocoumon therapy

Compared to warfarin, little is known about the effect of pharmacogenomics on the inter-individual variability of phenprocoumon therapy. In a retrospective cohort study, we investigated the impact of VKORC1 c.-1639G>A; CYP2C9*2 , CYP2C9*3 ; GGCX c.214+597G>A; CALU c.*4A>G; EPHX1 c.337T>C; F7 c.-402G>A, and F7 c.-401G>T on the initiation (n=54) and maintenance phases (n=91) of phenprocoumon therapy. We assessed the following outcome parameters: time to stable international normalised ratio (INR), time to first supra-therapeutic INR, time out of INR range, probability of over-anticoagulation, number of anticoagulation clinic visits. During the initiation phase, homozygotes for the VKORC1 c.-1639 A and G alleles achieved stable INRs later (p<0.001), spent more time at supra-therapeutic INRs (p<0.001), had increased risks of over-anticoagulation (odds ratio 19.83, p=0.003 and 4.45, p=0.045, respectively), and had higher frequencies of anticoagulation clinic visits (p<0.001) compared to GA carriers. CYP2C9*2, *3 carriers reached stable INRs faster (p=0.024) with fewer anticoagulation clinic visits (p=0.001) than wild-type carriers. EPHX1 c.337 C carrier spent significantly more time above range in the initiation phase (p=0.023). GGCX , CALU , and F7 gene variants did not affect outcome parameters of the initiation phase and none of the genotypes had an impact on maintenance phase parameters. Compared to the VKORC1 genotype, early INR values were less informative in the prediction of outcome parameters such as time to stable INR and time above the INR range. Our study is limited by the retrospective study design with no standardised protocol in a usual care setting. Therefore, our findings should be validated in a larger, controlled prospective study.     

A new algorithm to predict warfarin dose from polymorphisms of CYP4F2 , CYP2C9 and VKORC1 and clinical variables: derivation in Han Chinese patients with non valvular atrial fibrillation

Few pharmacogenomic dosing regimens of warfarin have been developed for Chinese patients with non valvular atrial fibrillation (NVAF). The objective of this study was to develop a new algorithm by polymorphisms of CYP2C9, VKORC1 and CYP4F2 to predict the daily stable dose of warfarin in Chinese patients with NVAF. A total of 325 Chinese NVAF patients on stable dose of warfarin with a target international normalised ratio of 1.5 to 3.0 were recruited and divided randomly into two cohorts. CYP2C9*3, VKORC1-1639, VKORC1 1173 and CYP4F2 were detected by ligase detection reaction method. The new algorithm was developed with multivariate linear regression in cohort 1 (260 patients) and assessed with Pearson Correlation Analysis (PCA) in cohort 2 (65 patients). From 260 enrolled patients, the model (R2 = 51.7%) was developed as: Dose = 3.47 - 0.022 (AGE) + 0.017 (WT) + 0.189 (PTE) - 0.283 (β-blocker) - 0.471 (AMIO) - 0.586 (CYP2C9 *1/*3) - 0.296 (VKORC1 CT) - 0.648 (VKORC1 TT) + 0.219 (CYP4F2 TT). PCA displayed that the algorithm was good (r = 0.658). The residual plots revealed that the predicted doses by the algorithm tend to be overestimated when lower doses were administered to patients and to be underestimated in higher doses. The algorithm developed by us might predict warfarin dose used by Chinese NVAF patients.     

Changes at the CYP2C locus and disruption of CYP2C8/9 linkage disequilibrium in patients with essential tremor

To identify low-penetrance genes related to sporadic essential tremor (ET) at the CYP2Clocus, located in chromosome 10 q23.33. Leukocytary DNA from 200 ET patients and a control group of 300 unrelated healthy individuals with known CYP2C19 genotypes was studied for common CYP2C8 and CYP2C9 allelic variants by using amplification-restriction analyses. Patients with ET showed the following differences compared with healthy subjects: a 1.6-fold reduction in the frequency for CYP2C8*3 (p=0.006), a 1.35-fold reduction of CYP2C9*2 (p=0.05) and a 1.52-fold reduction in the frequency for CYP2C9*3 (p=0.07). The frequency for patients with ET carrying at least one defective allele was 1.33-fold reduced as compared with healthy subjects (p=0.002). In addition, a disruption of the CYP2C8*3/CYP2C9*2 linkage disequilibrium was observed in ET patients, with a 2.1-fold reduction in the percentage for carriers of the haplotype CYP2C8*3 plus CYP2C9*2 in ET patients (p=0.0001). These findings were independent of gender, age, age of onset, or clinical symptoms. These results suggest that alterations at the CYP2C gene locus are associated with the risk for ET.     

The risk of bleeding complications in patients with cytochrome P450 CYP2C9*2 or CYP2C9*3 alleles on acenocoumarol or phenprocoumon

The principal enzyme involved in coumarin metabolism is CYP2C9. Allelic variants of CYP2C9, CYP2C9*2 and CYP2C9*3, code for enzymes with reduced activity. Despite increasing evidence that patients with these genetic variants require lower maintenance doses of anticoagulant therapy, there is lack of agreement among studies on the risk of bleeding and CYP2C9 polymorphisms. It was, therefore, our objective to study the effect of the CYP2C9 polymorphisms on bleeding complications during initiation and maintenance phases of coumarin anticoagulant therapy. The design of the study was a population-based cohort in a sample of the Rotterdam Study, a study in 7,983 subjects. All patients who started treatment with acenocoumarol or phenprocoumon in the study period from January 1, 1991 through December 31, 1998 and for whom INR data were available were included. Patients were followed until a bleeding complication, the end of their treatment, death or end of the study period. Proportional hazards regression analysis was used to estimate the risk of a bleeding complication in relation to CYP2C9 genotype after adjustment for several potentially confounding factors such as age, gender, target INR level, INR, time between INR measurements, and aspirin use. The effect of variant genotype on bleeding risk was separately examined during the initiation phase of 90 days after starting therapy with coumarins. The 996 patients with analysable data had a mean follow-up time of 481 days (1.3 years); 311 (31.2%) had at least 1 variant CYP2C9 allele and 685 (68.8%) had the wild type genotype. For patients with the wild type genotype, the rate of minor bleeding, major bleeding and fatal bleeding was 15.9, 3.4 and 0.2 per 100 treatment-years, respectively. For patients with a variant genotype, the rate of minor, major and fatal bleeding was 14.6, 5.4 and 0.5 per 100 treatment-years. Patients with a variant genotype on acenocoumarol had a significantly increased risk for a major bleeding event (HR 1.83, 95% CI: 1.01-3.32). During the initiation phase of therapy we found no effect of variant genotype on bleeding risk. In this study among outpatients of an anticoagulation clinic using acenocoumarol or phenprocoumon, having a variant allele of CYP2C9 was associated with an increased risk of major bleeding events in patients on acenocoumarol, but not in patients on phenprocoumon. Although one might consider the assessment of the CYP2C9 genotype of a patient for dose adjustment before starting treatment with acenocoumarol, a prospective randomised trial should demonstrate whether this reduces the increased risk of major bleeding events.     

VKORC1 haplotypes and their impact on the inter-individual and inter-ethnical variability of oral anticoagulation

In order to elucidate the role of VCORC1 sequence variants in warfarin sensitivity, we established a complete SNP map of the VKORC1 gene locus in 200 blood donors from Western Germany. Nearly all of the genetic variability of the VKORC1 gene in Europeans is reflected by three main haplotypes. Recently described polymorphisms associated with low warfarin dose requirement (dbSNP:rs9934438; dbSNP:rs17878363) were found in complete linkage disequilibrium with the VKORC1*2 haplotype. In two patient cohorts of European origin with either increased coumarin sensitivity (n= 14) or partial coumarin resistance (n=36) the VKORC1*2 frequency varied highly significant between the two groups and also when compared to 200 blood donor controls (coumarin sensitive 96%, coumarin resistant 7%, controls 42%) thus demonstrating a strong association between these two phenotypes and the VKORC1 haplotype (p = 1.6 x 10(-8) for coumarin sensitive and p = 1.9 x 10(-8) for coumarin resistant). Analysis of database derived VKORC1 genotypes of African Americans and Chinese revealed that haplotype frequencies in these populations differ significantly from the European sample (for VKORC1*2: Europeans 42%, Chinese 95%, African Americans 14%). These observations suggest VKORC1 as principal genetic modulator of the ethnic differences in warfarin response. Since hereditary pharmacodynamic (VKORC1) and pharmacokinetic (CYP2C9) factors account for up to 50% of the inter-individual variability of the warfarin response, these genetic markers may serve as clinically relevant predictors of warfarin dosing in future studies.     

A regression model to predict warfarin dose from clinical variables and polymorphisms in CYP2C9, CYP4F2, and VKORC1: Derivation in a sample with predominantly a history of venous thromboembolism

Background

Pharmacogenomic warfarin dosing has been suggested to produce more accurate dosing and an improved patient safety profile; however, very few models have been derived in patients with venous thromboembolism. We sought to develop a new algorithm to predict maintenance dose in a cohort of patients, using clinical variables and genetic polymorphism in CYP2C9, VKORC1, and CYP4F2.

Methods

Patients on a stable maintenance dose of warfarin, with observed dose ranging from 0.6 to 12 mg were recruited from a specialized anticoagulation clinic (Ottawa Hospital Thrombosis Clinic) with genotyping and standardized patient interviews being conducted to collect clinical and genomic variables known to impact warfarin dose. Multivariate linear regression was used to develop the model using a stepwise backwards elimination approach.

Results

From 249 enrolled patients with a mean clinical maintenance dose of 5.58 mg/day, a model with an R² of 58% was developed as: Dose = 1.85-0.048(Age) + 0.041(BMI) + 0.05(Height in cm) - 0.73(Less Exercise) - 1.13(2C9*2 Hetero) - 2.09(2C9*2 Homo) - 1.51(2C9*3 Hetero) -1.43(VKORC1 GA) - 2.86(VKORC1 AA) - 1.33(4F2 CC) -1.24(4F2 CT) - 1.46(Angiotensin II Receptor Antagonist) - 0.84(β-Blockers). Analysis of residual plots revealed that prediction errors were a function of observed maintenance dose with the model tending to predict higher doses than observed in those with low dose requirements and lower doses than observed in those with higher dose requirement.

Conclusion

Our study confirms the importance of the CYP4F2 polymorphism. Our model may prove useful in clinical practice but further validation studies are required before implementation into clinical practice.     

The influence of cytochrome oxidase CYP2A6, CYP2B6, and CYP2C9 polymorphisms on the plasma concentrations of valproic acid in epileptic patients

Objectives

To investigate influences of the functional polymorphisms of Cytochrome P450 isozymes 2A6 (CYP2A6), 2B6 (CYP2B6), and 2C9 (CYP2C9) on pharmacokinetics of VPA in vivo.

Patients and methods

In the study, we analyzed the genotypes of CYP2A6, CYP2B6, and CYP2C9 and their contribution to the steady-state standardized plasma VPA concentrations in 179 subjects with epilepsy of a Northern Han Chinese population. The genotypes were detected by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Results

The subjects with one or two variant CYP2A6*4 alleles showed higher mean plasma VPA concentrations compared with non-*4 alleles [(3.4 ± 0.4) μg kg ml⁻¹ mg⁻¹ vs. (3.6 ± 0.4) μg kg ml⁻¹ mg⁻¹, p = 0.0055]. A significant difference [one-way ANOVA (p = 0.0203)] was also found between mean plasma VPA concentrations and the CYP2B6 genotypes. In addition, subjects with the heterozygous genotype CYP2C9*3 had higher mean plasma VPA concentrations than did those subjects with the wild-type genotype [(3.9 ± 0.4) μg kg ml⁻¹ mg⁻¹ vs. (3.4 ± 0.4) μg kg ml⁻¹ mg⁻¹, p = 0.0001].

Conclusion

The presently evaluated variant alleles in the CYP2A6, CYP2B6, and CYP2C9 genes may explain part of the substantial variability in VPA pharmacokinetics between different subjects.     

回、汉族癫痫患者CYP2C9和CYP2C19基因多态性与苯巴比妥血药浓度的相关性研究

目的研究CYP2C9*3和CYP2C19*2的单核苷酸多态性在回、汉族癫痫人群中的分布特点;探讨两种基因型与苯巴比妥血药浓度的关系。方法对宁夏地区回、汉族癫痫患者185例采用聚合酶链反应-限制性片段长度多态性技术(PCR-RFLP)分析CYP2C9*3和CYP2C19*2基因型,并进行回、汉族间基因型及等位基因频率的比较;应用反相高效液相色谱法(RP-HPLC)测定其中113例单用苯巴比妥患者的血药浓度,再将其标准化后,分析两种基因型与苯巴比妥血药浓度的关系。结果 (1)回、汉族癫痫患者中CYP2C9*3和CYP2C19*2基因型及等位基因频率均无统计学差异(P>0.05)。(2)根据所携带的CYP2C9和CYP2C19突变等位基因的数量,将113例单用苯巴比妥的患者分为强代谢(EM)组、中间代谢(IM)组和弱代谢(PM)组,IM组和PM组苯巴比妥血药浓度明显高于EM组,且突变基因携带数量与血药浓度呈正相关。结论苯巴比妥血药浓度在CYP2C9和CYP2C19变异基因携带者中增高,根据患者CYP2C9和CYP2C19基因型可以预测患者药物浓度,指导临床选择合适的苯巴比妥初始剂量。     

Relationship between haloperidol plasma concentration, debrisoquine metabolic ratio, CYP2D6 and CYP2C9 genotypes in psychiatric patients

BACKGROUND: Around seven percent of Caucasians are poor metabolizers of cytochrome P450, CYP2D6 due to genetically impaired activity of the enzyme. Haloperidol in vitro and in vivo inhibits the activity of CYP2D6 and also the involvement of the enzyme in haloperidol metabolism has been reported. The present study was aimed to evaluate the possible inhibition of CYP2D6 during haloperidol treatment, and to determine the effect of CYP2D6 and CYP2C9 genotypes on the plasma concentration of haloperidol. METHODS: Thirty Caucasian psychiatric patients under haloperidol monotherapy were studied. CYP2D6 activity was evaluated by the debrisoquine metabolic ratio (MR), subjects with MR > 12.6 were named as poor metabolizers. Haloperidol plasma concentration was determined by high performance liquid chromatography. RESULTS: The number of patients with debrisoquine MR > 12.6 was higher than the expected comparing to healthy volunteers (13 % vs. 6.6 %, respectively). Debrisoquine MR was correlated with the dose of haloperidol (r = 0.40, p < 0.05), and also with the plasma concentration (r = 0.58, p < 0.001). Additionally, three patients comedicated with inhibitors of CYP2D6 were studied, all of them had a debrisoquine MR > 12.6, however only one was genetically poor metabolizer of CYP2D6. CYP2D6 and CYP2C9 genotypes were not related to the dose or plasma concentration of haloperidol. CONCLUSIONS: The present data support the dose-dependent inhibitory effect of haloperidol on CYP2D6, and the influence of this enzyme activity on haloperidol plasma concentration under steady-state conditions. The inhibitory effect of haloperidol on CYP2D6 enzyme activity may result in drug interactions and unexpected high plasma concentrations when drugs metabolized by the same enzyme are given concomitantly with haloperidol.     

CYP2C19*2 and other genetic variants affecting platelet response to clopidogrel in patients undergoing percutaneous coronary intervention

Introduction

Information regarding any possible additional effect of genetic variants other than CYP2C19*2 on platelet reactivity in patients undergoing percutaneous coronary intervention (PCI), while on dual antiplatelet therapy, is sparse.

Materials and Methods

Genotyping for CYP2C19*2, CYP2C19*17, CYP2C9*3, CYP2B6*5, ABCB1 and P2RY12 (c.-217 + 2739 T > C) variants was performed in 146 consecutive PCI patients receiving clopidogrel. Platelet reactivity was assessed by the Verify Now P2Y12 point-of-care assay and high on-treatment platelet reactivity (HTPR) was defined as a Platelet Reactivity Unit (PRU) ≥ 235.

Results

We identified 65(44.5%) patients with HTPR and 38(26%) carriers of at least one CYP2C19*2 allele, which had higher platelet reactivity compared to non-carriers [least square (LS) mean difference 44.5, 95%CI 15.8-77.3, p = 0.003]. In the entire study population, the presence of at least one CYP2C19*2 or P2RY12 allelic variant was independently associated with HTPR (OR = 3.02, 95%CI 1.16-7.86, p = 0.023 and OR = 3.11, 95%CI 1.03-9.39, p = 0.05 respectively). In CYP2C19*2 non-carriers, carriers of at least one CYP2B6*5 allelic variant had higher platelet reactivity compared to the remainders (LS mean difference 35.6, 95%CI 3.7-67.6, p = 0.03) and the presence of at least one CYP2B6*5 or P2RY12 allelic variant was independently associated with HTPR (OR = 3.26, 95%CI 1.08-9.86, p = 0.04 and OR = 4.27, 95%CI 1.11-16.4, p = 0.04 respectively).

Conclusions

Apart from the CYP2C19*2, other genetic variants involved in clopidogrel metabolism and action like CYP2B6*5 and P2RY12 seem to have an important association with HTPR.     

A candidate gene study of antiepileptic drug tolerability and efficacy identifies an association of CYP2C9 variants with phenytoin toxicity

Background and purpose: It is widely acknowledged that individual response to antiepileptic drugs (AEDs) is influenced by genetic factors. However, most of the underlying genes and genetic variants remain unidentified to date. The purpose of this study is to examine the role of common variants in a number of candidate genes in the response to commonly prescribed AEDs. Methods: We recruited 495 patients with epilepsy. Patients were classified according to their response to several AEDs. We genotyped 104 polymorphisms in 17 candidate genes for AED response. We looked for statistically significant associations between these polymorphisms and well‐defined AED response phenotypes. Results: We identified significant associations of CYP2C9 variant alleles with presence of phenytoin (PHT) adverse drug reactions (ADRs) and of GSTM1 copy number variation with the presence of carbamazepine ADRs. The latter association could not be confirmed in a replication study. Conclusions: Our study is the first comprehensive candidate gene association study in epilepsy pharmacogenetics. Our results confirm the role of CYP2C9 variants in PHT toxicity. No other definite associations were identified. Large‐scale efforts are needed to unravel the genetic determinants of AED response.