CYP2C9 Genotype-guided Warfarin Prescribing Enhances the Efficacy and Safety of Anticoagulation: A Prospective Randomized Controlled Study

Transcription

1 nature publishing group CYP2C9 Genotype-guided Warfarin Prescribing Enhances the Efficacy and Safety of Anticoagulation: A Prospective Randomized Controlled Study Y Caraco 1, S Blotnick 1 and M Muszkat 1 Warfarin anticoagulation effect is characterized by marked variability, some of which has been attributed to CYP2C9 polymorphisms. This study prospectively examines whether a priori knowledge of CYP2C9 genotype may improve warfarin therapy. Patients were randomly assigned to receive warfarin by a validated algorithm ( control, 96 patients) or CYP2C9 genotype-adjusted algorithms ( study, 95 patients). The first therapeutic international normalized ratio and stable anticoagulation were reached 2.73 and 18.1 days earlier in the study group, respectively (Po0.001). The faster rate of initial anticoagulation was driven by a 28% higher daily dose in the study group (Po0.001). Study group patients spent more time within the therapeutic range (80.4 vs 63.4%, respectively, Po0.001) and experienced less minor bleeding (3.2 vs 12.5%, Po0.02, respectively). In conclusion, CYP2C9 genotype-guided warfarin therapy is more efficient and safer than the average-dose protocol. Future research should focus on construction of algorithms that incorporate other polymorphisms (VKORC1), host factors, and environmental influences. Warfarin is the most prescribed anticoagulant worldwide, and during 2003 more than 21 million prescriptions were filled in the United States alone. 1 Its popularity is attributed to its proven efficacy in the prevention of morbidity and mortality associated with thromboembolic disease. 2 4 Yet warfarin therapy entails a significant risk of bleeding, with 0.6, 3, and 9.6% annual incidence of fatal, major, and minor bleeds, respectively. 5 8 Major hemorrhages occur most frequently during the first year, but the risk is most prominent during the first month of warfarin therapy. 9 The fear of iatrogenic hemorrhagic complications often causes physicians to avoid prescribing warfarin to patients who are likely to benefit from such therapy. 10,11 The frequent occurrence of bleeding in the course of warfarin treatment reflects a combination of two unique characteristics of the drug. First, warfarin has a typical narrow therapeutic window, so the risk of recurrence of thromboembolism rises in patients with international normalized ratio (INR) values below 2, but INR values exceeding 3, and in particular above 4, greatly increase the risk of bleeding. 12,13 Second, the response to warfarin varies greatly among individuals, so that some patients may require less than 1 mg daily, whereas others may need more than 20 mg per day to achieve similar anticoagulation. 14 Warfarin is administered as a racemic mixture consisting of equal amounts of the R and S enantiomers, but about twothirds of the anticoagulation effect is attributed to the S enantiomer. 15 The metabolism of S-warfarin in the liver is predominantly mediated by the activity of CYP2C9, 16,17 which exhibits 20-fold interindividual variability, mostly ascribed to genetic polymorphisms in the gene encoding for CYP2C9. 18,19 Most of these polymorphisms encode for defective proteins, but their clinical relevance is minor as their frequency is low except for CYP2C9*2 and CYP2C9*3, which occur among Caucasians at allele frequencies of and , respectively. 20,21 Carriers of CYP2C9 variant alleles require lower maintenance and cumulative induction doses and are more likely to exhibit supratherapeutic INR (i.e., INR43) and to experience bleeding during warfarin induction Selection of the correct warfarin dose is a challenging task, particularly during the loading period. Most physicians will 1 Clinical Pharmacology Unit, Division of Medicine, Hadassah University Hospital, Jerusalem, Israel. Correspondence: Y Caraco (caraco@hadassah.org.il) Received 21 April 2007; accepted 18 June 2007; advance online publication 12 September doi: /sj.clpt VOLUME 83 NUMBER 3 MARCH

2 attempt to tailor the dosing regimen to the patient s characteristics by taking into account the impact of factors that are known to influence warfarin pharmacokinetics and/ or pharmacodynamics. Yet until recently much of the interindividual variability in the response to warfarin remained unaccounted for, leaving the physician no choice but to use a trial and error method combined with frequent INR monitoring. This study was undertaken to evaluate the hypothesis that warfarin dosing based on a priori knowledge of the CYP2C9 genotype might result in a safer and more efficacious loading of warfarin. RESULTS Recruitment was initiated in October 2001 and the last patient was enrolled in August Out of the 283 patients who signed the informed consent, 92 (45 control group and 47 study group) were later excluded from the study (Figure 1). Most patients were excluded because of the decision of the treating physician not to initiate treatment with warfarin (16 in the study group and 15 in the control group), or to stop warfarin after less than 8 days (11 in the study group and 9 in the control group), mainly because of the clinical judgment of the treating physician regarding the strength of indication for anticoagulation. Violation of monitoring protocol was the reason for exclusion in 25 additional patients (dose deviation in 12 and missing INR tests in 13). Sixteen additional patients (seven in the study group and nine in the control group) elected to withdraw from the study before the completion of the initiation phase, with the most common explanation provided by the patients being that they would prefer that warfarin dosage be determined by their primary physician. None of the patients in either study arm was excluded owing to over- or underanticoagulation. The number of patients excluded and the reasons for exclusion were similar in the study and the control groups (Figure 1). Atrial fibrillation (AFIB) was almost twice as common among the group of patients who were eventually excluded from the study (62.0 vs 34.0%, respectively, Po0.001), reflective of the decision made by the primary physician in many of these cases to discontinue warfarin or not to initiate warfarin. Pulmonary embolism and the combination of deep 283 enrolled and randomized 142 study group 141 control group 47 excluded 16 warfarin not initiated 11 warfarin discontinued after <8 days 13 protocol violations 6 missed INR 7 dose deviations 7 dropped out after <8 days 45 excluded 15 warfarin not initiated 9 warfarin discontinued after <8 days 12 protocol violations 7 missed INR 5 dose deviations 9 dropped out after <8 days 95 completed the induction 96 completed the induction 3 dropped out after induction 3 voluntary withdrawals 3 dropped out after induction 2 voluntary withdrawals 1 discontinuation of warfarin 92 followed until stabilization was reached 93 followed until stabilization was reached Figure 1 Flow of patients through the study. CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 83 NUMBER 3 MARCH

3 vein thrombosis (DVT) and pulmonary embolism (PE) were less common among the patients who were excluded from the study (PE: 13.0 vs 21.5%, respectively, P ¼ 0.10, DVT and PE: 4.3 vs 17.3%, respectively, Po0.002). Otherwise, demographic details, clinical characteristics, and CYP2C9 genotypes were similar to those of patients who completed the study. The final cohort consisted of 191 patients, 95 in the study group and 96 in the control group. Five patients, three in the study group and two in the control group, elected to withdraw from the study after completing the initiation period. Serious bleeding occurred in additional control group patient necessitating discontinuation of warfarin therapy soon after the completion of the induction period. Thus, of the 191 patients, 185 (92 in the study group and 93 in the control group) were followed from initiation to maintenance. Patients demographic details, baseline clinical characteristics, indications for warfarin therapy, and CYP2C9 genotypes are described in Table 1. Notably, a large fraction of the patients who entered the study suffered from various chronic illnesses and accordingly were treated by a median of 5 drugs (interquartile range from 2 to 8 drugs), including a median of 1 drug (interquartile range from 1 to 2 drugs) likely to alter the anticoagulant effect of warfarin. 31 The demographic details and baseline clinical characteristics were similar in the study and the control groups except for the diagnosis of diabetes mellitus and the use of calcium channel blockers (i.e., verapamil, nifedipin XL, amlodipine, diltiazem, felodipine, and lercanidipine), which were more common in the study group (P ¼ 0.07), and the higher rate of hyperlipidemia among patients in the control group (P ¼ 0.05). No significant difference was noted in the primary or secondary end points between users and non-users of calcium channel blockers (P ¼ 0.52) and patients with and without the diagnosis of diabetes mellitus (P ¼ 0.26) or hyperlipidemia (P ¼ 0.71). The fraction of patients with isolated DVT, isolated PE, and AFIB was similar (P ¼ 0.97, 0.69, 0.50, respectively), but the combination of DVT and PE was more common in the control group (P ¼ 0.03). Meaningful genotyping result was unavailable for a single patient in the control group who had undergone bone marrow transplantation 4 years before enrollment. CYP2C9 allele frequencies were in accordance with Hardy Weinberg equilibrium (P ¼ 0.998), and no significant differences were noted between the study and the control groups in either CYP2C9 allele frequencies or genotypes (P ¼ 0.15). Pharmacodynamic end points Initiation phase. Adherence to the study protocol during the initiation period was similar in the study and the control groups, so that the administered dose was concordant with the dose recommended by the appropriate algorithm in 95.0 and 95.8% of the cases in the study and the control groups, respectively (Table 2). In the study group, first therapeutic INR was reached 2.73 days (95% confidence interval (CI): days) earlier Table 1 Patient's demographic details and baseline clinical characteristics Demographics Study group N=95 Control P- group N=96 a values Age (years) Gender (M/F) 46/49 42/ Weight (kg) Body mass index (kg/m 2 ) Current smokers, no. (%) 21 (22.1) 19 (19.8) 0.70 Indication for warfarin, no. (%) Deep vein thrombosis 27 (28.4) 25 (26.0) 0.97 Pulmonary embolism 22 (23.2) 19 (19.8) 0.69 Deep vein thrombosis+pulmonary embolism 11 (11.6) 22 (22.9) 0.03 Atrial fibrillation 35 (36.8) 30 (31.3) 0.50 Clinical characteristics, no. (%) Hypertension 48 (50.5) 43 (44.8) 0.43 Diabetes mellitus 20 (21.1) 11 (11.5) 0.07 Ischemic heart disease 30 (31.6) 27 (28.1) 0.60 Congestive heart failure 24 (25.3) 21 (21.9) 0.58 Hyperlipidemia 14 (14.7) 25 (26.0) 0.05 Renal dysfunction b 10 (10.5) 16 (16.7) 0.22 Medication use, no. (%) Antibiotics 29 (30.5) 25 (26.0) 0.49 b-blockers 34 (35.8) 39 (40.6) 0.49 Calcium channel blockers 33 (34.7) 22 (22.9) 0.07 Statins 16 (16.8) 24 (25.0) 0.17 ACE inhibitors 32 (33.7) 26 (27.1) 0.32 Proton pump inhibitors 27 (28.4) 38 (39.6) 0.10 Aspirin 33 (34.7) 35 (36.5) 0.80 Antiarrhythmics 24 (25.3) 23 (24.0) 0.84 Diuretics 33 (34.7) 34 (35.4) 0.92 Total number of drug use c 6 (7) 6 (6) 0.80 CYP2C9 genotype, no. (%) CYP2C9*1/*1 60 (63.2) 51 (53.1) CYP2C9*1/*2 17 (17.9) 23 (24.0) CYP2C9*1/*3 14 (14.7) 13 (13.5) CYP2C9*2/*2 2 (2.1) 2 (2.1) 0.50 CYP2C9*2/*3 2 (2.1) 4 (4.2) CYP2C9*3/*3 0 (0.0) 2 (2.1) F, female; M, male. a Meaningful genotype was unavailable for one patient in the control group who had undergone bone marrow transplantation 4 years before enrollment. b Renal dysfunction denotes plasma creatinine 4120 mmol/l. c Data are presented as median (interquartile range). 462 VOLUME 83 NUMBER 3 MARCH

4 than in the control group (Po0.001; Table 2 and Figure 2). Kaplan Meier curves for the time required to reach the first therapeutic INR varied significantly between the study and the control groups (log-rank test, Po0.001) (Figure 3). Faster attainment of the initial therapeutic INR in the study group was confirmed in a Cox regression model (unadjusted hazard ratio (HR): 2.89, 95% CI: ). Age, gender, smoking status, CYP2C9 genotype, indication for warfarin treatment, the presence of comorbid conditions, and the use of interacting drugs were not found to alter significantly the HR in a stepwise model fitting procedure. The adjusted HR of the Cox regression model, which included average warfarin dose until first therapeutic INR was reached and weight, was 3.95 (95% CI: ). Counterintuitively, warfarin cumulative dose until first therapeutic INR was achieved was 41.7% higher in the control group, but as it was given over a longer period of time, average warfarin daily dose was 22% lower than in the study group (Table 2, Figure 4a and b). The use of higher average warfarin daily dose in the study group was not associated with higher rate of over-anticoagulation (i.e., INR43) (Table 2). Furthermore, the percent time spent within the therapeutic range was 1.85-fold higher in the study group than in the control group (Po0.001). Comparison of anticoagulation during the initiation phase between the study and control groups among carriers of two wild-type alleles or single variant allele (i.e., heterozygotes) yielded similar results (Figures 2 and 4). A similar trend was noted for the comparison of patients carrying two variant alleles, but owing to the small number of patients included in this analysis (i.e., four in the study group and eight in the control group) statistical significance was not reached. Among patients in the control group, time to first therapeutic INR, total warfarin dose, and average daily warfarin dose did not vary across the three major genotypic groups (Kruskal Wallis, P ¼ 0.21, 0.18, and 0.30, respectively). Yet among patients in the study group and as expected from the study design, warfarin cumulative dose and average daily dose up to therapeutic INR were dependent on CYP2C9 genotype (Kruskal Wallis, P ¼ 0.01 and o0.001, respectively) (Figure 4). Figure 2 Time to reach first therapeutic INR (i.e., 42) according to CYP2C9 genotype and loading protocol (study: open bars, control: closed bars). *Po0.001 for intra-genotypic comparison of study vs control. Figure 3 Kaplan Meier curve for time to reach first therapeutic INR (study: solid line, control: dashed line). Table 2 Anticoagulation details during the initiation phase Study group (N=95) Control group (N=96) P-values Time to first INR42 (days) o0.001 Cumulative loading dose (mg) o0.001 Average loading daily dose (mg) o0.001 Time spent at INR43 (days) Sum deviations of INR Time spent at INRo2 (days) o0.001 Sum deviations of INRo o0.001 Time spent in therapeutic range (%) o0.001 No. of patients with single missed INR test No. of patients with single dose deviation INR, international normalized ratio. Plus minus values are mean7sd. CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 83 NUMBER 3 MARCH

5 Stabilization phase. Pharmacodynamic steady state was reached in the study group after days (95% CI: days) and 18.1 days (95% CI: days) earlier than in the control group ( days, 95% CI: days) (Po0.001; Table 3 and Figure 5). Kaplan - Meier plots of the time required to achieve stable anticoagulation were significantly different between the study and the control groups (log-rank, Po0.001; Figure 6). The faster rate of stabilization was confirmed in a Cox regression model (HR: 4.23, 95% CI: ). No adjustment was made as none of the above-mentioned covariates was found to contribute significantly to the HR. Enhanced pace of stabilization in the study group was associated with significantly reduced number of days spent outside the therapeutic range and significantly higher percent of time spent within the range (Po0.001; Figure 7a and b). This was accounted for by 4.0- and 3.7-fold lower number of days spent below and above the recommended range, respectively (Po0.001). The extent of deviation of INR values from the desired range was more than fourfold lower in the study group than in the control group (Po0.001). Enhanced efficiency of the stabilization process in the study group could not be ascribed to more meticulous monitoring of anticoagulation. In fact, the total number of performed INR tests was more than twice as much lower in the study group than in the control group (Po0.001), but the average interval between consecutive INR tests was similar (P ¼ 0.86). Stratified comparison between patients in the study and the control groups according to CYP2C9 genotypes yielded similar results (Figures 5 and 7). The comparison of all pharmacodynamic parameters for patients carrying two Figure 4 Warfarin dosing during the induction phase according to CYP2C9 genotype and loading protocol (study: open bars, control: closed bars). (a) Cumulative warfarin dose until the first therapeutic INR was achieved. (b) Average daily warfarin dose until first therapeutic INR was reached. *Po0.001 for intra-genotypic comparison of study vs control. & Po0.005 for intra-genotypic comparison of study vs control. # Po0.04 for intra-genotypic comparison of study vs control. y P ¼ 0.01 for inter-genotypic comparison within the study group. w Po0.001 for inter-genotypic comparison within the study group. Figure 5 The length of the stabilization period according to CYP2C9 genotype and loading protocol (study: open bars, control: closed bars). *Po0.001 for intra-genotypic comparison of study vs control. Table 3 Anticoagulation characteristics from induction to stable anticoagulation phase Study group (N=92) Control group (N=93) P-values Time from induction to maintenance (days) o0.001 Maintenance dose (mg/day) Maintenance INR Time spent at INR43 (days) o0.001 Sum deviations of INR o0.001 Time spent at INRo2 (days) o0.001 Sum deviations of INRo o0.001 Time spent in therapeutic range (%) o0.001 No. of INR tests performed o0.001 Interval between INR tests (days) INR, international normalized ratio. Plus minus values are mean7sd. 464 VOLUME 83 NUMBER 3 MARCH

6 CYP2C9 genotypic groups (Kruskal Wallis, Po0.001), so that patients carrying a single or two variant allele(s) required 77 and 52% of the dose used by CYP2C9*1 homozygotes, respectively (Po0.03 and o0.001, respectively). Figure 6 Kaplan Meier curve for time to reach stable anticoagulation (study: solid line, control: dashed line). Figure 7 Anticoagulation characteristics during the stabilization period according to CYP2C9 genotype and loading protocol (study: open bars, control: closed bars). (a) Time (days) spent outside the therapeutic range (42 and o3) during the stabilization phase. (b) Percent time spent in the therapeutic range during the stabilization phase. *Po0.001 for intra-genotypic comparison of study vs control. & Po0.01 for intra-genotypic comparison of study vs control. variant alleles produced similar yet non-statistically significant results, which is most probably accounted for by the low number of enrolled patients carrying these rare genotypes. Warfarin daily dose required to maintain adequate anticoagulation exhibited a 60-fold variation ranging from 0.75 to 45 mg/day, and it varied across the three major Pharmacokinetics The pharmacokinetics of warfarin was evaluated at steady state in 60 study group patients and 54 control group patients who were able and willing to perform 24 h urine collection. The oral clearance of S-warfarin and the plasma S/R-warfarin ratio, a putative marker of CYP2C9 activity in vivo, 32 varied among patients carrying different CYP2C9 genotypes (Kruskal Wallis, Po0.001). In homozygotes for the wild-type allele, the mean oral clearance of S-warfarin was ml/min and was significantly greater than in carriers of either a single allele ( ml/min, Po0.02) or two variant alleles ( ml/min, Po0.001). Mean S/R-warfarin ratio was among CYP2C9*1 homozygotes and was significantly lower than that in heterozygotes ( , Po0.001) or carriers of two mutated alleles ( , Po0.001). The number of variant alleles was inversely correlated with S-warfarin oral clearance (Spearman s r ¼ 0.417, Po0.01) and directly correlated with S/Rwarfarin ratio (Spearman s r ¼ 0.552, Po0.01). Adverse effects The incidence of bleeding was higher in control group patients than in study group patients (12.5 vs 3.2%, respectively, Po0.02). Of the 15 bleeding episodes, 14 were minor and 5 (4 in the control group and 1 in the study group) occurred during the first 8 days of induction. Serious lower gastrointestinal bleeding occurred after 9 days in a single patient of the control group. Bleeding coincided with an INR value of 1.74, well below the therapeutic range. Warfarin was promptly discontinued and the patient received two packed cell units and vitamin K. Subsequent colonoscopy revealed multiple angiodysplasias, which were believed to be the source of the bleeding. All three study group patients who bled were homozygotes for the wild-type allele. In contrast, seven of the bleeders in the control group were carriers of a single variant allele, and the remaining five patients were CYP2C9*1/*1 homozygotes. Among study group patients, bleeding coincided with INR values that were either below or within the therapeutic range (mean7sd, , 95% CI: ). On the other hand, bleeding among control group patients coincided with INR values above the therapeutic range in 7 out of the 12 bleeders. Overanticoagulation was most prominent among control group patients who bled during the initiation phase with average INR of (95% CI: ). New thromboembolic events were not diagnosed in any of the control or study group patients. DISCUSSION The beneficial therapeutic effects of warfarin in the prevention and treatment of venous or arterial thromboembolism CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 83 NUMBER 3 MARCH

7 are indisputable. Yet its use is often associated with a high rate of bleeding complications occurring most frequently among elderly patients who are prime candidates for such treatment. 10,33 One of the major hurdles is the fact that the extent of anticoagulation exerted by warfarin varies greatly among individuals even when administered in identical doses. This is of particular importance during the initiation phase of warfarin, where common doses have been reported to induce excessive anticoagulation and bleeding. The usual approach to overcoming this difficulty is to administer average doses of warfarin, which are less likely to induce over-anticoagulation among sensitive individuals. This is why most loading protocols recommend the use of low starting warfarin doses (usually 5 mg) during the initial days of therapy. 34 This widely used approach does not eliminate the risk of bleeding 35 and unnecessarily prolongs the loading period for the majority of patients. Administration of individualized warfarin doses may constitute an attractive alternative to the average dose approach. To the best of our knowledge, the findings described in this paper are the first to illustrate this principle in a prospective randomized study. Warfarin loading during the induction guided by CYP2C9 genotype produced a faster rate of anticoagulation that was predominantly driven by the administration of higher average warfarin doses. This would be expected for carriers of two wild-type alleles but is somewhat unexpected for patients carrying a single CYP2C9*3 allele or any two variant alleles who were prescribed per algorithm lower daily doses than patients in the control group. Nevertheless, as loading was less accurate and lasted longer in the control group, mean average daily warfarin doses were still higher in the CYP2C9 genotypebased treatment group. Lengthening of the induction period among carriers of variant alleles was predominantly due to higher frequency of extreme deviations from the therapeutic range. Thus, during the induction period INR44 was noted in 5 out of 44 carriers of variant allele(s) in the control group but in none out of the 35 carriers of variant allele(s) in the study group (P ¼ 0.06). Attainment of stable anticoagulation marks the end of a process through which the patient s response to warfarin is unexposed. Fluctuations above or below the desired therapeutic range may still occur but they are less probable, as is the risk of bleeding and/or subtherapeutic anticoagulation. Time to reach stable anticoagulation was almost twofold lower in the study group than in the control group, implying that CYP2C9 genotype-guided warfarin therapy significantly shortened the vulnerable stabilization period and may potentially enhance the safety and efficacy of anticoagulation. This conclusion is further supported by the finding that study group patients spent significantly more time within the therapeutic range, a surrogate measure of the quality of anticoagulation that strongly correlates with bleeding and thromboembolic rates. 5,6 Time to reach stable anticoagulation and percent time spent within therapeutic range in the control group compare favorably with published data in similar populations and are indicative of the appropriate quality of anticoagulation monitoring. 5,30 Thus, better precision and faster rate of anticoagulation in the group treated by CYP2C9-adjusted regimen is not due to inferior results in the control group but represents an improvement from the current practice of warfarin treatment. The mode of warfarin loading (i.e., study or control algorithms) accounted for about 31% of the variability in the time required to reach INR42 (Spearman s r ¼ 0.557, Po0.01) and could explain 27.5% of the observed variability in the length of the stabilization process (Spearman s r ¼ 0.525, Po0.01). None of the patient s clinical characteristics or demographic details were correlated with the study s primary outcomes, except for weight, which was significantly correlated with the time required to reach first therapeutic INR (Spearman s r ¼ 0.190, Po0.01). Previous studies have indicated that carriers of variant alleles require a longer period of time until stable anticoagulation is reached. 20,25 The use of CYP2C9 genotype to guide dose selection in the study group completely abolished this intergenotypic difference by markedly reducing the fluctuations in INR values among carriers of variant alleles. The similar pace of stabilization among carriers and noncarriers of polymorphic alleles in the control group is most probably accounted for by the stringent monitoring protocol with frequent INR testing and the prompt response to deviations from the therapeutic range. The rationale for using CYP2C9 genotype to guide warfarin dosing is based on the assumption that it is a major determinant of CYP2C9 catalytic activity, the enzyme mediating the metabolism of the more active S enantiomer. Nevertheless, in vivo CYP2C9 activity is influenced by various non-genetic host characteristics and environmental factors. 36 These factors may have come into play in this study and potentially could have masked the effect of genotype. In reality, CYP2C9 genotype remained fairly accurate in predicting CYP2C9 activity during steady state, as indicated by the correlations noted with S-warfarin clearance and the S/R-warfarin ratio. On the basis of these findings, it may be concluded that the role played by CYP2C9 genetics overshadows the effect on non-hereditary factors on the phenotypic activity of this enzyme. This study has several potential limitations. The demographic details and baseline clinical characteristics were quite similar in the study and the control groups, but subtle differences were still present. For example, the proportion of CYP2C9*1/*1 carriers was nonsignificantly higher in the study group (63.2 vs 53.1%). As non-carriers of variant alleles are less likely to experience over-anticoagulation, this might have biased our results in favor of the study group. This possibility seems unlikely as stratified comparison between the study and the control groups according to CYP2C9 genotype status yielded similar results. In addition, when the study was initiated on October 2001, the contribution of other non-cyp2c9 genetic polymorphisms to the variability in response to warfarin was unknown. Since then, several 466 VOLUME 83 NUMBER 3 MARCH

8 retrospective studies have shown that a 1639G/A polymorphism (referred to as 3673G/A in other studies) in the gene encoding for VKORC1 may explain a significant portion (up to 30% in some papers) of the variability in warfarin maintenance dose. 37,38 On the basis of our own preliminary data, 39 this polymorphism may also explain some of the variability in the response to warfarin during the loading period that is unaccounted for by CYP2C9 genetic polymorphisms. A prospective study designed to evaluate warfarin loading algorithms based on the combined genotypes of CYP2C9 and VKORC1 is currently under way in our institute. Adoption of a modified warfarin loading protocol that takes into account CYP2C9 genotype is based on the assumption that the new scheme is not only more efficient but also confers better safety. Minor bleeding was more common in the control group, but this study was not designed to identify differences in the rate of bleeding complications, and neither was it defined as a primary study end point. Nevertheless, two surrogate markers for the risk of bleeding, namely the number of days spent at INR43 and the extent of deviation from the therapeutic range, were significantly greater in the control group. 2,5,6,40 The results in this study should be interpreted carefully. The dose required to achieve anticoagulation may vary in patients treated for different indications. 41 This study included only patients with AFIB, DVT, and PE, and therefore the applicability of the CYP2C9 genotype-based algorithms in patients with other indications should be confirmed. Furthermore, the algorithms used in this report required daily INR monitoring for the first 8 days, which is almost never possible or required under non-study conditions. A less demanding monitoring protocol requiring only four INR tests during the first days of warfarin loading is currently under investigation in our laboratory. CONCLUSION The increasing evidence regarding the major role played by CYP2C9 genetic polymorphism in determining the response to warfarin has not been translated thus far into clinical medicine. The findings in this prospective study suggest that in patients with recent-onset AFIB, DVT, and PE, the use of CYP2C9 genotype to guide warfarin administration results in faster attainment of initial anticoagulation and shorter stabilization period than the usual trial and error average dose method. Furthermore, CYP2C9 genotype-based prescribing of warfarin may be safer than the current practice of warfarin loading, as it is associated with higher percent of time spent within the therapeutic range and lower extent of deviation from this range. Interindividual variability in the response to warfarin is multifactorial, involving genetic polymorphisms in CYP2C9, VKORC1, and possibly other genes, host characteristics, and environmental influences. Major effort is currently being invested in defining the contribution of each of these factors, which may eventually be incorporated into a comprehensive loading protocol of warfarin. METHODS Study population. The study was conducted in the Division of Medicine at the Hadassah University Hospital. Warfarin-naive inpatients, diagnosed with AFIB, DVT, or PE and in whom warfarin was scheduled to be initiated, were offered the opportunity to participate in this study. Enrolled patients were randomly assigned based on the last digit of the patient s identity number to the control (uneven) or the study (even) group. The study was designed to imitate the usual clinical setting, and therefore the only exclusion criteria were age o18 years and baseline INR41.4. Patients were treated with 1 mg/kg of enoxaparin twice daily until INR42 was reached on 2 consecutive days. The protocol of enoxaparin administration was identical in the study and the control groups. The study protocol was approved by the Institutional Review Board of the Hadassah University Hospital and all participants signed a consent form before enrollment. CYP2C9 genotyping. A single blood sample was drawn for genetic analysis of CYP2C9, which was performed by two specific polymerase chain reactions, followed by digestion with restriction enzymes, as described elsewhere. 42 As the CYP2C9 genotype of patients assigned to the study arm was provided within 8 h, there was no need to delay warfarin dosing in the study group. Samples obtained from patients in the control group were not analyzed until study termination. The investigators responsible for running the study (YC and MM) were not involved in either the recruitment/randomization process or in running the genetic analysis tests. The CYP2C9 genotype of patients in the study group was disclosed to the principal investigators of this study (YC and MM) only after completion of the first 8 days of induction. Construction of warfarin loading algorithms. In the control group, the selection of warfarin dose during the first 8 days was based on a validated published algorithm. 43 According to this algorithm, warfarin dose is determined based on the result of the INR and the response to previous dose(s) of warfarin. This specific induction method has been validated and found to be as good as dose selection by expert personnel. In the study group, warfarin loading was guided by six different CYP2C9 genotype-adjusted algorithms. The first step was to construct an algorithm for patients carrying two wild-type alleles. The basic principle that guided the construction of the dosing regimen for this group was that average warfarin clearance is expected to be faster than in the control group, which also consists of individuals carrying one or even two variant CYP2C9 alleles. Therefore, it was hypothesized that higher initial doses of warfarin can be safely administered to this homogenous group without increasing the risk of exaggerated anticoagulation and bleeding rate. In keeping with this assumption, the constructed CYP2C9*1/*1 algorithm consisted of doses that were on average 25% higher than the doses recommended in the validated algorithm used for our control group. This increase corresponds to the 27% increase in S- warfarin clearance noted among CYP2C9*1/*1 carriers relative to the clearance of S-warfarin in the entire population. 23 The next step was to construct additional warfarin loading regimens for each of the remaining five CYP2C9 genotypes. As the clearance of R-warfarin is not expected to vary across different CYP2C9 genotypes, we assumed that the variability in response to warfarin among carriers of CYP2C9 variant alleles is predominantly influenced by the decrease in the clearance of S-warfarin. In patients carrying the CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3, and CYP2C9*3/*3 genotypes, S-warfarin clearance is decreased by 20, 40, 50, 60, and 85%, respectively. 23,24 These decreases were translated into proportionally lower warfarin doses relative to the recommended doses for CYP2C9*1 homozygous. No specific algorithms were constructed for potential carriers of non-cyp2c9*2 CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 83 NUMBER 3 MARCH

9 or -CYP2C9*3 variant alleles, as they are very rare among Caucasians. No specific algorithms were constructed for patients treated by drugs known to alter the response to warfarin, except for patients treated with amiodarone. The case of amiodarone coadministration is unique because amiodarone is commonly administered to patients with AFIB, and it is known to exert a strong inhibitory effect on warfarin metabolism, which might result in over-anticoagulation. 44 To avoid excessive anticoagulation, these patients were loaded by specially designed amiodarone algorithms, which were constructed by lowering the doses in all algorithms by an average of 25%. Monitoring of anticoagulation. The study period was divided into two intervals: the initiation period and the stabilization period. Initiation was defined as the first 8 days on warfarin therapy. The stabilization phase spanned from day 9 to the day when stable anticoagulation was achieved. Stable anticoagulation was declared once two consecutive INR values, 7 days apart, were within the therapeutic range, without any intervening dose alteration. Target INR was 2 3 for all patients. The response to warfarin was monitored by daily INR testing during the initiation phase and as required, but it was not less than twice weekly until stable anticoagulation has been reached. If an INR test was not available during the initial 8 days of warfarin therapy, dose was determined based on the response to previous doses and CYP2C9 genotype (study group only). Patients who missed more than a single INR test during the initiation phase were excluded. During stabilization period, additional INR tests were requested in case of bleeding, suspected new thromboembolic event, or following the introduction of a drug that might alter the response to warfarin, and when significant deviation from the therapeutic range (i.e., INRo1.8 or 43.4) was noted. 45 For all in-patients, measurement of prothrombin time was determined using HemosIL PT-Fibrinogen HS PLUS (rabbit brain thromboplastin) with an international sensitivity index of Warfarin dosing. Warfarin was routinely administered at 1800 hours and a blood sample for INR testing was drawn when required at 0800 hours. In principle, warfarin dose during the first 8 days of induction was determined by the respective algorithm. The administration of warfarin dose that was different from the recommended dose by the respective algorithm was defined as dose deviation. Single dose deviation was allowed, but patients who were administered a different dose from that recommended by the algorithm for more than 1 day were excluded. Dose adjustment during the stabilization period was determined in accordance with published guidelines and usually consisted of dose modification by an average of 5 20%, depending on the extent of deviation from the therapeutic range. 5,6,45 For patients assigned to the study group, the decision took into account his/her CYP2C9 genotype. Weekly warfarin dose was modified in advance by up to 25% if a drug known to alter warfarin dose requirement (amiodarone, antibiotics, anticonvulsants, some statins ) 31 was prescribed or discontinued, and the dose was further adjusted based upon subsequent INR measurements. In case of significant deviation (40.3) from the target therapeutic range, an additional INR test was scheduled and warfarin dose was modified accordingly. The approach to patients with excessive INR (INR45) was in accordance with published guidelines. 5,6,45 Pharmacodynamic end points. The primary end points were the time required to reach the therapeutic INR range (i.e., first INR42) and the time required to reach stable anticoagulation. Both have been used in previous pharmacogenetic warfarin studies, as they signify important clinical landmarks. 5,6,25,45 Fluctuations in INR can still occur following attainment of therapeutic INR, but the likelihood is decreased so that the frequency of INR measurements can be reduced from daily to 2 3 times weekly, and subcutaneous administration of low molecular heparin is discontinued when therapeutic INR is sustained for 2 days. 5,6 On the other hand, attainment of stable anticoagulation implies that the individual s response to warfarin has been characterized, and therefore the likelihood of fluctuations in INR and the risk of bleeding or subtherapeutic anticoagulation are significantly reduced. 5,6 The third end point was the time spent within the therapeutic range, as calculated by linear interpolation method, 46 a known surrogate measure of the quality of anticoagulation. 5,6 Secondary end points included the number of days spent outside the therapeutic range, the sum of deviations from the desired range, and the occurrence of bleeding episodes or new thromboembolic events. Bleeding complications were classified as either minor (requiring no additional testing and treatment) or major, as defined by a drop in hemoglobin requiring hospitalization and transfusion of blood. Pharmacokinetic evaluation. Once stable anticoagulation was achieved, the patients were requested to collect urine over 24 h, and a single mid-interval blood sample was drawn. Plasma S- and R-warfarin were measured by a high-performance liquid chromatography method, as published previously. 47 S- and R-warfarin oral clearance were calculated from the ratio of the enantiomer dose to the mid-interval plasma concentration of the respective enantiomer. Data analysis. Data are presented as mean7sd unless otherwise indicated. Patients demographics and baseline clinical characteristics were compared using Mann Whitney and w 2 tests for continuous and discrete variables, respectively. Deviation of CYP2C9 allele frequencies from Hardy Weinberg equilibrium was tested by the w 2 goodness-to-fit test. Time to reach therapeutic INR and stable anticoagulation was evaluated by survival analysis methodology. Time-to-event was displayed graphically by the Kaplan Meier method and comparison between the study and the control group curves was made by using the log-rank test. HR and 95% CI were computed by comparing the study and control groups, using the Cox proportional hazards model to adjust for potential confounders. Each covariate was added separately to the model and only those covariates that altered the HR by more than 5% were finally included. The proportional hazard assumption was verified by evaluation of the Schoenfeld residuals and the interaction between time and the assigned loading protocol in the Cox proportional hazards model. Safety and efficacy parameters of warfarin treatment were compared using the Mann Whitney test. Spearman s correlation test was used to evaluate the possible association between several patients variables and the pharmacodynamic end points of warfarin therapy. Intergenotypic comparison was performed using the Kruskal Wallis test. All tests were two-tailed and a P-value of less than 0.05 was considered statistically significant. Statistical analysis was performed by the SPSS software package (version 13.0; SPSS, Chicago, IL). We calculated that a sample size of at least 85 patients in each study arm will be necessary to detect a difference of 1.5 and 10 days in the time required to reach first INR42 and pharmacodynamic steady state, respectively, between the study and the control groups with a 0.90 power (a set at 0.05). This sample size also allows the detection of a 10% difference in the time spent within the therapeutic range. ACKNOWLEDGMENTS The data appearing in this paper were presented as a poster at the American Society for Clinical Pharmacology and Therapeutics 2007 annual meeting (21 24 March, Anaheim, CA). Trial registration: Clinicaltrials.gov Identifier NCT VOLUME 83 NUMBER 3 MARCH

10 The study was supported by a grant from the Bi-national US Israel Science Foundation (YC) and a grant from the Israeli Ministry of Health (YC). We greatly appreciate the administrative help of Ilanit Linzer. Author's contributions: Yoseph Caraco, declared that he participated in planning the study design, supervising the study, analysis of the data, and writing of the paper and that he has seen and approved the final version. Simha Blotnick, declared that he participated in the study design, the analysis of the samples, and writing of the paper and that he has seen and approved the final version. Mordechai Muszkat, declared that he participated in planning the study design, supervising the study, and writing of the paper and that he has seen and approved the final version. CONFLICT OF INTEREST The authors declared no conflict of interest. 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