Introduction
In 2007, the Food and Drug Administration (FDA) announced that warfarin’s label will carry new information describing the role of genetics in drug dosing. The label states that a lower initial warfarin dose “should be considered for patients with certain genetic variations”. Genotyping of CYP2C9, and vitamin K epoxide reductase complex C1 (VKORC1) is recommended for dosing algorithm (1). This is the first FDA recommendation to consider genetic testing when initiating a commonly prescribed medication and may set a precedent for the future use of genetic technologies in clinical practice. Many efforts are driving this technology into practice with companies offering tests, academic institutions trying to be on the cutting edge of clinical medicine, and patients interested in the potential of personalized medicine.
Why warfarin? There are several reasons that make this drug attractive target of personalized medicine. Warfarin is the most widely used anticoagulant, prescribed to more than 2 million new warfarin patients per year. Warfarin is commonly used as life-long therapy in the prevention of systemic embolism in patients with atrial fibrillation, valvular heart disease, and in the primary and secondary prevention of venous thromboembolism. It is also used for the prevention of thromboembolic events in patients with acute myocardial infarction and with angina pectoris, in patients with biological heart valves, and after some types of orthopedic surgery. Clinical management is difficult because of a narrow therapeutic range and considerable interpatient variability. A combination of genetic and non-genetic factors is responsible for up to 20-fold variation in the warfarin dose required to achieve the usual therapeutic level of anticoagulation as measured by the prothrombin international normalized ratio (INR). The optimal INR range is 2 to 3, with ratios < 2 increasing thrombotic events and those > 4 increasing hemorrhagic events (2,3). Several common genetic variants affect warfarin metabolism and activity (4,5). In the absence of genetic testing data or clinical information to predict the warfarin dose required in each individual patient, the initial doses prescribed may be too low, increasing the risk of thrombosis, or too high, which may be associated with the risk of overanticoagulation and severe bleeding. Up to 800 reportable adverse drug events associated with warfarin usage per year occur in the United States (6).
The warfarin risk of serious side effects, narrow therapeutic range, and wide interindividual variation in warfarin dose have imposed the need of algorithms to better predict the dose in the initial stage(s) of treatment.
The role of CYP2C9 in warfarin metabolism
CYP2C9 is predominantly expressed in the liver, representing about 20% of the hepatic CYP content. It metabolizes more than 20% of all therapeutic drugs as well as a number of endogenous compounds. Drug-substrates of large clinical importance include angiotensin-2 antagonist, nonsteroid antiinflammatory drugs (NSAIDs), oral antidiabetics, antimicrobials, antiepileptics, and oral anticoagulants (Table 1) (7,8). Warfarin is a racemic mixture and CYP2C9 metabolizes S-warfarin, which is the most active enantiomer (Fig. 1). Genetic polymorphisms of CYP2C9 have been reported; more than 34 alleles have been found (seehttp://www.cypalleles.ki.se/). The CYP2C9*2, *3, *4,*5, and *30 alleles encode for protein variants with amino acid replacement and have been reported to reduce catalytic activities in vitro and/or in vivo (9,10). There is a large interindividual variation in CYP2C9 activity in the population with resulting interindividual variations in drug response and also in adverse effects. The poor metabolizer phenotype has a frequency of 3%-5% in Caucasians (11,12).The most frequent variants are CYP2C9*2 (rs4917639, p.R114C) and CYP2C9*3 (rs1057910, p.I359L) alleles. Of special interest are drug substrates with a narrow therapeutic range, such as S-warfarin, tolbutamide and phenytoin, where impaired CYP2C9 metabolic activity might cause difficulties in dose adjustment as well as toxicity (13,14). CYP2C9 polymorphisms can also be associated with an increased rate of NSAID-induced adverse events (15).
Table 1. Other important drugs that are CYP2C9 substrates and can interact with the oral anticoagulant warfarin
Figure 1. Warfarin pharmacokinetic and pharmacodynamic pathway. Warfarin is administered as a racemic admixture of R- and S-enantiomers. The more potent S-enantiomer is metabolized principally by cytochrome P450 (CYP) 2C9. The pharmacological effect of warfarin is mediated by the inhibition of vitamin K epoxide reductase complex 1 (VKORC1). This results in decreased concentrations of activated clotting factors (II, VII, IX and X) producing therapeutic anticoagulation (according to: www.medscape.com; 2008 Pharmacotherapy Publications).
Besides genetic polymorphisms, interindividual variability in CYP2C9 activity can also be caused by environmental factors (16). A variety of xenobiotics such as phenobarbital, rifampicin, and hyperforin have been shown to induce transcriptional expression ofCYP2C9 in primary human hepatocytes and to increase the metabolism of CYP2C substrates in vivo in man (17,18). This induction can result in drug-drug interactions, drug tolerance, and therapeutic failure. Several drug-activated nuclear receptors including constitutive androgen receptor (CAR) and pregnane X-receptor (PXR) recognize drug responsive elements within the 5’ flanking promoter region of CYP2C genes to mediate the transcriptional up-regulation of these genes in response to xenobiotics and steroids (19). Inhibition of CYP2C9 is also feasible, as was shown in vitro for synthetic preservative substances such as benzethonium chloride (20). Clinically significant inhibition may occur with coadministration of amiodarone, fluconazole, phenylbutazone, certain sulfonamides and oral contraceptives (17,21).
The effects of genetic polymorphisms on the rates of drug metabolism are well known, but how these polymorphisms influence the susceptibility to drug-drug interactions is less clear, in particular considering warfarin-drug interactions, whether reduced function proteins (e.g., CYP2C9*3) are inhibited to the same extent in vivo as wild-type proteins (e.g., CYP2C9*1). Pharmacogenetic variations of metabolic enzymes and drug transporters can have a major role in drug interactions with oral anticoagulants. This is of special importance for drug substrates such as NSAIDs, fluvastatin, tolbutamide, and phenytoin. Genotype should be considered when attempting to predict the potential CYP2C9 drug-drug interactions because of the possible genotype-dependent inhibition (22,23). Studies confirmed that genetic CYP2C9 polymorphism contributed to the variability in warfarin dosage requirements in the presence of drug-disease and drug-drug interactions (24).
VKORC1 – a target of oral anticoagulants
The target of oral anticoagulants is the protein vitamin K reductase complex subunit 1 (VKORC1) encoded by the homonymous gene VKORC1. Vitamin K epoxide reductase (VKOR) reduces vitamin K 2,3-epoxide to the biologically active vitamin K hydroquinone, which catalyzes the production of carboxylated blood-clotting proteins II, VII, IX and X (Figure 1). Anticoagulants of the coumarin type act by inhibiting VKOR activity. VKORC1 was identified in 2004 (25,26). Rost et al. identified the VKORC1 gene from four independent families whose members were resistant to warfarin. One patient needed more than 17 mg/day of warfarin to achieve adequate anticoagulation, two others about 40 mg/day, and the fourth did not respond to any dose of warfarin. Analysis confirmed they carried rare mutations of VKORC1, g.85G>T (p.V29L), g.134T>C (p.V45A), g.172A>G (p.A58G) and g.3487T>G(p.L128A) (25). Other groups described the common VKORC1 SNPs to be strongly associated with oral anticoagulant sensitivity (4,27-30). VKORC1 genetic polymorphism has a similar effect on oral anticoagulants (4,31). In clinical practice, two SNPs, -1639G>A and 1173C>T, are usually genotyped to identify VKORC1 haplotypes. Evidence suggest that -1639 G>A promoter polymorphism reduces hepatic VKORC1expression and therefore decreases drug dose requirements (29,30). The -1639G allele is present in the VKORC1*1, *3, and *4 haplotypes and is typically associated with a ‘normal’ warfarin dose (32,33). In contrast, the -1639A allele, which is in strong linkage disequilibrium with 6484C>T, 6853G>C, 7566C>T and 1173C>T, is present in theVKORC1*2 haplotype and predisposes to warfarin sensitivity and lower drug doses (5,27,34,35). In a sample of 200 volunteers, a German group identified 28 SNPs within the VKORC1 genomic sequence and found six of these SNPs in complete linkage disequilibrium with each other, forming three main haplotypes *2, *3, *4 (32,36). The estimated distribution in Caucasians is as follows: VKORC1*2, 42%; VKORC1*3, 38%; and VKORC1*4, 20%. There are large interethnic differences in the allelic frequency of VKORC1. In Asians, the frequency of the VKORC1 -1639 AA genotype (č83%) is significantly different from that recorded in Caucasians (14%) (4,37). The VKORC1 *2 haplotype was found to be rather low among subjects of African origin (0.15), high in Caucasians (0.42), and extremely frequent among Asians (0.95) (29,32,38,39).
According to data from pharmacogenetic association studies of CYP2C9 and VKORC1, several major clinical consequences on the pharmacokinetics-pharmacodynamics of oral anticoagulants have been observed.
VKORC1 and CYP2C9 polymorphisms and daily dose requirements
Carriers of alleles coding for reduced CYP2C9 and/or VKORC1 enzyme activity require lower warfarin doses and have been observed to be more difficult to titrate to a stable maintenance dose than those needing higher doses. The relative contribution of each polymorphism to the variation in warfarin dosage requirement must be considered in relation to the frequencies of the polymorphisms. Consequently, VKORC1 is likely to contribute more than CYP2C9 due to the markedly higher frequency of some tested variants (1173C<T, -1639G>A) as compared to the CYP2C9*2 and CYP2C9*3 variants. However, in individual patients, the CYP2C9*2 and especially CYP2C9*3 variant will be expected to markedly influence the warfarin dosage requirement (40,41). Compared with homozygous carriers of CYP2C9*1, patients homozygous for CYP2C9*3 were estimated to need 3.3-times lower mean doses of warfarin to achieve the same INR, with *2 carriers and heterozygous patients in-between (42). Two studies analyzing the first few weeks of warfarin treatment showed the proportion of patients with INR values of more than 3 to be higher among carriers of the CYP2C9*2 or *3 allele than in patients that were not carriers of these alleles (43,44). In a study of patients starting warfarin therapy, a major finding was that genetic variation in VKORC1 but not in CYP2C9 modulated the early response to warfarin (45). However, both the VKORC1 haplotype and the CYP2C9 genotype had a significant effect on the warfarin dose after the first 2 weeks. VKORC1 genotype was found to determine up to 40% of individual coumarin dose requirement (4,5,33,46,47). In Caucasians, the 1173 C>T polymorphism was associated with lower warfarin dosage requirements (27): the mean warfarin dose was higher (6.2 mg/day) in patients with the VKORC1 1173 CC genotype than in those with the CT genotype (4.8 mg/day; P= 0.002) or TT genotype (3.5 mg/day; p < 0.001). Study results confirmed VKORC1*2 to be the most important haplotype for warfarin dosage (33). Patients with VKORC1*2 haplotype had more frequent visits to clinic than patients with VKORC1*3 or *4 haplotypes, higher coefficient of variation of prothrombin time-INR and higher percentage of INR values outside the therapeutic interval than patients with VKORC1*3 or *4 haplotypes. Also, there was a statistically significant difference in warfarin dose (p < 0.001) and R-warfarin plasma concentrations (p < 0.01) between VKORC1*2 and VKORC1*3 or *4haplotypes. Patients with VKORC1*2 haplotype required much lower warfarin doses than other patients. The combination of genotype and clinical factors explains approximately 50% to 60% of the variance in warfarin dose requirements in Caucasians and Asians, but only 25% to 40% in African Americans. Racial differences in the association between genotype and patient response to warfarin treatment may be caused by racial differences in the frequencies of the variant CYP2C9 and VKORC1 alleles and/or by the influence of non-genetic factors.
Warfarin resistance
Warfarin resistance is a relatively uncommon phenomenon in humans and has been related to several missense mutations in theVKORC1 gene that have been discovered so far. Results obtained by Rost group corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance (25,48). Other researchers confirmed these findings (26,37,49,50). The missense mutations p.D36Y, p.V29L, p.A41S, p.V45A, p.R58G and p.V66M, responsible for warfarin resistance, are located in the conserved luminal loop (L1) VKORC1 region (51). It was found for a coding VKORC1 Asp36Tyr polymorphism that carriers of Asp36Tyr required significantly higher warfarin doses of 80.9 ± 10.1 mg/wk as compared with 42.7 ± 7.5 mg/wk in non-carriers (F = 9.79; P = 0.002) (52). Another group documented an Irish case of true warfarin resistance as the result of a mutation in VKORC1(383 T>G transition in exon 2) (53). However, the mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo remains to be elucidated.
CYP4F2 is a vitamin K(1) (VK1) oxidase and carriers of the CYP4F2 V433M allele (rs2108622) have a reduced capacity to metabolize VK1, secondary to an rs2108622-dependent decrease in steady-state hepatic concentrations of the enzyme. Therefore, patients with the rs2108622 polymorphism are likely to have elevated hepatic levels of VK1, necessitating a higher warfarin dose to elicit the same anticoagulant response (54). Other researchers have also confirmed the relevant role of CYP4F2 V433M polymorphism in the pharmacogenetics of coumarin anticoagulants (55,56).
Effect of CYP2C9 and VKORC1 genotype on hemorrhage risk
In the United States, the annual major bleeding risk in patients on oral anticoagulants is estimated to 1%-5% (57,58). A combination of the coumarin-sensitive alleles of both the VKORC1 and CYP2C9 genes may strongly indicate a high risk of severe overanticoagulation (43). Patients with the CYP2C9*2 and *3 allele experienced the first bleeding event sooner and showed a higher bleeding rate than patients with the wild-type genotype (59-61). In a study conducted among African and European Americans (38), the variant CYP2C9 genotype conferred an increased risk of major but not minor hemorrhage. The risk of major hemorrhage was 5.3-fold before therapy stabilization, 2.2-fold after therapy stabilization, and 2.4-fold during all periods when anticoagulation was not stable. The variant VKORC1 1173C/T genotype did not confer a significant increase in the risk of major or minor hemorrhage. The risk of gastrointestinal bleeding during acenocoumarol therapy in carriers of any of the CYP2C9 orVKORC1 variants was severely increased with exposure to weekly doses of acenocoumarol higher than 15 mg or the use of amiodarone or aspirin (62).
Although the incorporation of genotype information improves the accuracy of dose prediction, no convincing improvement has been demonstrated in the reduction of hemorrhagic complications.
Genome-wide association study
A genome-wide association study (GWAS) of common genetic variants with major effect on warfarin maintenance dose, performed by Cooper et al. from approximately 550,000 polymorphisms tested, found the most significant independent effect to be associated with VKORC1 polymorphisms (P = 6.2 x 10-13) (63). CYP2C9 (CYP2C9*3 and CYP2C9*2) was associated with the dose at moderate significance levels (P = 10-4). The authors conclude that common SNPs with large effects on warfarin dose are unlikely to be discovered outside the CYP2C9 and VKORC1 genes. Takeuchi et al. (64) conducted the first GWAS sufficiently powered to detect DNA variants with a modest influence on the warfarin dose needed to achieve therapeutic anticoagulation. On univariate analysis of GWAS SNPs they also identified extremely strong association signals (p = 10-78 to 10-13) at SNPs in and nearVKORC1 and CYP2C9. By applying multivariate regression adjusting for known genetic and nongenetic dose predictors, they also detected genome-wide significance of p < 8.3 x 10-10 at CYP4F2 (rs2108622) that accounted for approximately 1.5% of the dose variance. CYP4F2 association with dose variance has also been reported by other authors (65).
Genetic polymorphism in further enzymes and structures involved in the effect of anticoagulants such as gamma-glutamylcarboxylase, glutathione S-transferase A1, microsomal epoxide hydrolase and apolipoprotein E appear to be of negligible importance.
Pharmacogenetics-based algorithms
Algorithms based on pharmacogenetic data have been proposed for oral anticoagulant dosing. The majority of models accounted for age, sex, body surface area, concomitant medications, comorbidities and clinical indications. One of the most frequently cited equations that estimate warfarin dose (mg/day) was the dosing algorithm based on a regression model:
[0.628 - 0.0135 x age (years) - 0.24 x CY2C9*2 – 0.37 x CYP2C9*3 – 0. 241 x VKORC1 + 0.062 x height (cm)]2,
where CYP2C9 genotype is 0, 1, or 2 for the number of *1, *2 and *3 alleles within the patient’s genotype and VKORC1 genotype is 1 for GG, 2 for GA, and 3 for AA.
The authors found a R2 of 54% (the equation explained 54% of the variability in the warfarin dose in the derivation cohort) (5). Several published warfarin-dosing algorithms (Washington University, UCSF, Louisville, and Newcastle) were compared for accuracy in predicting warfarin dose in a retrospective analysis of a local patient population on long-term, stable warfarin therapy. Validation of pharmacogenetic testing for warfarin responses included demonstration of analytical validity of testing platforms and of the clinical validity of testing (66). Gage et al. developed and validated a pharmacogenetic algorithm in the derivation cohort of 1015 participants (67). The independent predictors of therapeutic dose were: VKORC1 polymorphism -1639/3673 G>A (-28% per allele), body surface area (BSA) (+11% per 0.25 m2, CYP2C9*3 (-33% per allele), CYP2C9*2 (-19% per allele), age (-7% perdecade), target INR (+11% per 0.5 unit increase), amiodarone use (-22%), smoker status (+10%), race (-9%), and current thrombosis (+7%). The pharmacogenetic equation proposed explained 53%-54% of the variability in the warfarin dose in the derivation and validation (N = 292) cohorts. For comparison, a clinical equation explained only 17%-22% of the dose variability (P < 0.001). To facilitate the use of these pharmacogenetic and clinical algorithms, a nonprofit website has been developed (http://www.WarfarinDosing.org). VKORC1 and/or CYP2C9 polymorphisms have been introduced in several other clinical dosing algorithms and prospective clinical studies (68-72). In a study conducted by the International Warfarin Pharmacogenetics Consortium (73), data from 4.043 patients were used to create a dose algorithm that was based on clinical variables only and an algorithm in which genetic information was added to the clinical variables. Most recent data are published. The main conclusions were as follows: the use of a pharmacogenetic algorithm to estimate the appropriate initial dose of warfarin produces recommendations that are significantly closer to the required stable therapeutic dose than those derived from a clinical algorithm or a fixed-dose approach. The greatest benefits were observed in 46.2% of the population that required 21 mg or less of warfarin per week or 49 mg or more per week for therapeutic anticoagulation.
Cost-effectiveness
Although the American College of Medical Genetics Working Group on Pharmacogenetic Testing of CYP2C9 and VKORC1 alleles for warfarin use have not endorsed the routine use of warfarin genotyping at this time, they pointed that in certain situations,CYP2C9 and VKORC1 testing may be useful, and warranted, on determining the cause of unusual therapeutic responses to warfarin therapy (74). Eckman et al. (75) conclude that warfarin-related genotyping is unlikely to be cost-effective for typical patients with non-valvular atrial fibrillation, but may be cost-effective in patients at a high risk of hemorrhage that are starting warfarin therapy. Lippi et al. (76) point out that limitations of genetic testing include still largely unknown optimal composition of test panels, information concerning interindividual variability, lack of analytical and quality specifications, lack of comprehensive outcome analyses to enable assessment of cost-effectiveness, lack of universal agreement related to reliable dosing algorithms, and other ethical and social issues. Results obtained by Wadelius group, which genotyped 183 polymorphisms in 29 candidate genes in 1.496 Swedish patients starting warfarin treatment, and tested for association with response, strongly support that warfarin initiation be guided by pharmacogenetics (77). Whether genotype-guided dosing is clinically beneficial remains unclear, but studies are currently underway that will help elucidate this issue (78).
Conclusions
The effect of oral anticoagulants is influenced by genetic and environmental factors. Common mutations in the genes coding for the cytochrome P450 (CYP2C9) and vitamin K epoxide reductase with one or more combinations of its polymorphisms are responsible for the reduced warfarin requirements or warfarin resistance. Racial differences in the association between genotype and patient response to warfarin treatment have been observed. Genotypes influence the time required to attain therapeutic anticoagulation and the risk of overanticoagulation and hemorrhage. Although the incorporation of genotype information improves the accuracy of dose prediction, an improvement in anticoagulation control or a reduction in hemorrhagic complications has yet to be demonstrated in terms of efficacy and cost-benefit (79). While individuals at the extremes of dose requirements are likely to benefit, the overall clinical merits of a genotype-adapted anticoagulant treatment regimen in the entire patient population remain to be determined in future prospective clinical studies.
