Pharmacokinetic and Pharmacodynamic Variation Associated with VKORC1 and CYP2C9 Polymorphisms in Thai Patients Taking Warfarin

Drug Metab. Pharmacokinet. 25 (6): 531–538 (2010).

Regular Article Pharmacokinetic and Pharmacodynamic Variation Associated with VKORC1 and CYP2C9 Polymorphisms in Thai Patients Taking Warfarin Alisara SANGVIROON1,2, Duangchit PANOMVANA1,*, Wichittra TASSANEEYAKUL3 and Jule NAMCHAISIRI4 1 Department

of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand 2 Department of Pharmacy, Police General Hospital, Bangkok, Thailand 3Department of Pharmacology, Faculty of Medicine, Khon kaen University, Khon kaen, Thailand 4Cardiothoracic Unit, Department of Surgery, Faculty of Medicine Chulalongkorn University, Bangkok, Thailand Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: We investigated the influence of genetic polymorphisms of CYP2C9 and VKORC1 genotypes on the pharmacokinetics and pharmacodynamics of warfarin and established an equation for predicting the maintenance dose of warfarin in the Thai population using genetic and non-genetic factors. The CYP2C9*2, CYP2C9*3, VKORC1 C1173T and VKORC1 G1639A genotypes were detected by realtime PCR using fluorogenic hybridization probes. The associations between genetic and demographic factors with respect to warfarin dosage were analyzed. CYP2C9 polymorphisms affect warfarin metabolism as shown by a significant difference in warfarin clearance, whereas VKORC1 genotypes cause a significant difference in warfarin sensitivity index (INR:Cp). The mean weekly warfarin dose was significantly different among different VKORC1 and CYP2C9 genotypes. Patients with the VKORC1 BB haplotype and CYP2C9*1/*1 required about twice the warfarin dose compared to those with the VKORC1 AA haplotype and CYP2C9*1/*1. Using stepwise multiple linear regression, clinical factors (age and weight) and genetic factors (CYP2C9 and VKORC1) could explain about 53.8z of the variance of the warfarin maintenance dose. CYP2C9 and VKORC1 genotypes played an important role in the inter-individual variation in warfarin maintenance dose in a Thai population. Keywords: CYP2C9; VKORC1; Warfarin; Thai; Pharmacokinetics; Pharmacodynamics

and pharmacodynamic parameters of warfarin can be influenced by many factors and this fact makes it difficult to adjust or predict the appropriate dose for individual patients; the correct dose can depend on intra- and interindividual differences and ethnicity. Recently, at least 30 alleles of CYP2C9 have been identified (http://www.imm.ki.se/CYPalleles). CYP2C9*2 and CYP2C9*3 have reduced enzymatic activity by about 30% and 80%, respectively.5,6) Single nucleotide polymorphisms in CYP2C9 are associated with a lower dose requirement of warfarin during the induction phase and maintenance phase.7–12) Lindh, et al.13) have shown a systematic review and meta-analysis of the influence of

Introduction Warfarin, an oral anticoagulant, is metabolized to an inactive form by cytochrome P450 (CYP) enzymes in the liver. While S-isomer warfarin is oxidized by CYP2C9, CYP2C19 and CYP2C18, the R-isomer is oxidized by CYP1A1, CYP1A2 and CYP3A4.1) Warfarin exerts its anticoagulant effect by inhibiting vitamin K epoxide reductase (VKOR), encoded by vitamin K epoxide reductase complex subunit 1 (VKORC1), thus interrupting the conversion of vitamin K epoxide to vitamin KH2, a cofactor in the gamma-carboxylation process of activation of the vitamin K-dependent proteins.2–4) The pharmacokinetic

Received; June 29, 2010, Accepted; July 28, 2010, J-STAGE Advance Published Date; October 1, 2010 *To whom correspondence should be addressed: Duangchit PANOMVANA, Ph.D. Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. Tel. ＋662-2188405, Fax. ＋662-2188403, E-mail: duangchit.p＠chula.ac.th This study was supported by Chulalongkorn University, Bangkok, Thailand. The VKORC1 reagents were supported by Roche Diagnostic, Thailand.

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CYP2C9 genotype on warfarin dose requirement and found that patients with CYP2C9*1/*2, *1/*3, *2/*2, *2/*3, and *3/*3 genotypes required warfarin doses about 19.6, 33.7, 36.0, 56.7, and 78.1% lower than those with the CYP2C9*1/*1 genotype (wild type), respectively. It has been reported that VKORC1 is associated with an interindividual variability in the dose of warfarin.14) As Rieder, et al.15) have identified, VKORC1 haplotype group A is consisted of H1 and H2 haplotypes which is associated with a low warfarin dose requirement and is commonly found in the Asian-American population. Haplotype group B comprises H7, H8 and H9 haplotypes and is associated with a high warfarin dose requirement; this group is common in African-Americans. In Asians, Yuan et al.16) have shown that the G allele in the VKORC1 promoter (G-1639A) had 44% higher activity than the A allele. Moreover, the frequencies of GG, GA and AA were significantly different from those in Caucasians (pº0.0001). Several studies have investigated the association between warfarin doses and CYP2C9 and VKORC1 genotypes in different populations. The contribution of clinical factors and genetic factors to variability in the warfarin requirement was reported to be about 30–60%.17–22) However, a best-fit equation to predict warfarin dose requirement for the Thai population has not yet been proposed. In addition, the frequencies of CYP2C9 variants are different among various ethnic groups. CYP2C9*2 is found in about 8–20% of Caucasians but is not found in Asians (Japanese, Chinese, Taiwanese, Korean, Malaysian and Singaporean); also, the frequencies of CYP2C9*3 are higher in Caucasians (5–16%) than in Asians (about 1–5%).23) Therefore, the purpose of this study was to investigate the frequencies of CYP2C9 and VKORC1 genotypes in a Thai population, to evaluate the effect of CYP2C9 and VKORC1 genotypes on the pharmacokinetic and pharmacodynamic parameters of warfarin, and to establish an equation for prediction of the maintenance dose of warfarin by using genetic and nongenetic factors in the Thai population.

Methods Patients: The subjects enrolled in this study were outpatients at the cardiovascular thoracic unit, The King Chulalongkorn Memorial Hospital, Bangkok, Thailand who had taken warfarin for at least 2 months and had taken a stable dose for at least the last 2 visits. Patients who had congestive heart failure, cancer, hepatic or thyroid disorders were excluded. The study protocol was approved by The Ethics Committee of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. All patients gave informed consent to participate in the study. The patient data, including gender, age, weight, under-

lying diseases, dose of warfarin, co-medications, INR level, time of taking the last dose of warfarin and vitamin Kcontaining food consumption (within the week before INR testing), were collected. Determination of warfarin concentrations: The total concentrations of warfarin in plasma were determined by high-performance liquid chromatography (HPLC), as previously described with some modifications.24) In brief, 1 ml of plasma sample was added to a tube and 50 ml of internal standard (naproxen 100 mg/ml), 0.7 ml of 1 M sulfuric acid and 5 ml of diethyl ether were then added. The mixture was briefly mixed by vortex for 5 seconds and rotary mixed at 300 rpm for 30 minutes. The tubes were then centrifuged at 600 rpm for 10 minutes and kept in a freezer at －209C for 3 hours. The diethyl ether layer was decanted from a frozen aqueous layer to a clean tube and then evaporated to dryness in a heat box at 459C. The residue was dissolved in 250 mL of acetonitrile in water (25:75 v/v). A volume of 100 mL of the solution was injected into the chromatograph, a HyperClone C18-BDS 5 mm, column 150×4.6 mm (Phenomenex). The mobile phase consisted of 15 mM of phosphate buffer (pH 3.0, adjusted by 1 M HCl), methanol, acetonitrile (52:32:16 v/v). The flow rate was 1.2 ml/min. Validation of the HPLC method including linearity, specificity, selectivity, precision, accuracy, and stability were performed (data not shown). gDNA preparation: Nine milliliters of whole blood from a peripheral vein was drawn into an EDTA tube. The genomic DNA was extracted by the standard phenolchloroform extraction protocol. CYP2C9 and VKORC1 genotyping: The genotypes of CYP2C9 and VKORC1 were determined by realtime polymerase chain reaction using fluorogenic hybridization probes (TIB MOLBIOL, Berlin, Germany), as previously described.25–27) Data and statistical analysis: Since warfarin is a long half-life drug taken once daily, the total warfarin concentration in the plasma sample was assumed to be the average steady state concentration (Cssav); the clearance (Cl) was then calculated using the following formula: Cl＝Dose/Cssav* dosing interval Descriptive statistics and inferential statistics were performed using the SPSS program version 14. The warfarin dose and prevalence of CYP2C9 and VKORC1 genotypes are shown as means and 95% confidence intervals. The warfarin dose, clearance, warfarin sensitivity index between different CYP2C9 and VKORC1 genotypes were compared by independent t-test, ANOVA, MannWhitney U test or Kruskal-Wallis test. The association of all factors to doses of warfarin were analyzed by forward stepwise multiple linear regressions. The characteristic factors analyzed included age, gender, weight, and co-

533

VKORC1-CYP2C9 Variation on Warfarin Kinetic & Dynamic

Table 1. Demographic data Characteristics

Frequency, (mean±SD)

Total number (N)

89

Female

48

Table 2. CYP2C9 and VKORC1 genotype frequencies in a Thai population %, (range) Alleles 53.9

Age (years)

(49.25±13.09)

(21–78)

Weight (kg)

(57.10±10.62)

(30–96)

(22.04±4.02)

(13.67–36.21)

2

BMI (kg/m )

CYP2C9

VKORC1

43

48.3

Aortic Valve Replacement

21

23.6

Double Valve Replacement

21

23.6

4

4.5

Lasix

34

47.9

HCTZ

13

18.3

Aspirin

9

12.7

Aldactone

6

8.4

Statins

4

5.6

Allopurinol

2

2.8

Omeprazole

2

2.8

Celebrex

1

1.4

INR

(2.2±0.5)

(1.5–3.5)

Warfarin dose (mg/day)

(3.7±1.5)

(0.64–10)

(0.466±0.209)

(0.062–1.591)

Others Co-medications

Warfarin dose (mg/kg/wk)

[95%CI]

*1

173

*2

0

0

*3

5

2.8

[1.2–6.4]

C

40

22.5

[17.0–29.2]

97.2

[93.6–98.8] —

1173CÀT

Indications Mitral Valve Replacement

(89 patients×2 alleles) N＝178 %

VKORC1 －1639GÀA

T

138

77.5

[70.9–83.0]

G

40

22.5

[17.0–29.2]

A

138

77.5

[70.9–83.0]

r＝1.0*** *** Significant at pº0.0001.

HCTZ, hydrochlorothiazide.

medication. A p-value of less than 0.05 was considered statistically significant for all analyses.

Results Eighty nine Thai patients with an established stable warfarin maintenance dose who met the criteria were enrolled into this study. The major indication for taking warfarin (95.5%) was prosthetic heart valve replacement. Other indications were Bental's operation, pulmonary embolism, PA thrombectomy, and commissurotomy. Patient characteristics are summarizes in Table 1. The CYP2C9 and VKORC1 genotypes of these patients are shown in Table 2. The CYP2C9*2 genotype was not found in the studied population (0%). The allele frequency of CYP2C9*3 observed in these patients was 0.028 [95%CI 0.012–0.064]. The most common genotype in Thais was the wild type, CYP2C9*1/*1 (95.5%). One patient had the CYP2C9*3/*3 genotype (1.1%) and 3 patients had CYP2C9*1/*3 (3.4%). The frequencies of VKORC1 1173T and －1639A were both 0.775 [95%CI 0.709–0.830], indicating that VKORC1 1173CÀT was completely correlated with VKORC1 －1639GÀA (Pearson's correlation＝1.0, pº0.0001). Among these

Fig. 1. Comparison of warfarin clearances among different CYP2C9 (***pº0.0001) (a) and VKORC1 genotypes (no significant difference, p＝0.909) (b) (N＝89)

patients, 55 (61.8%) carried VKORC1 AA haplotypes (``low dose group'') whereas 28 patients (31.5%) had the AB genotype and 6 patients (6.7%) had the VKORC1 BB haplotype. All allele frequencies were in Hardy-Weinberg equilibrium. The mean total warfarin concentration in plasma of all

534

Alisara SANGVIROON, et al.

patients was 948.7±451.8 ng/ml (range, 238.8 to 2,488.1 ng/ml). Concomitant drugs were reported as not interfering with the determination of warfarin concentration. Clearance (Cl) was 0.052±0.021 ml/min/kg (range 0.009–0.114 ml/min/kg). Figure 1 shows that warfarin clearance was significantly lower in the variant group of CYP2C9 polymorphisms (CYP2C9*1/*3 and *3/*3) as compared to wild type (CYP2C9*1/*1) (pº0.0001). However, clearances were not significantly different among VKORC1 haplotypes (p＝0.909). The ratio of INR:warfarin plasma concentration (INR:Cp), which is defined as the warfarin sensitivity index (WSI), was analyzed as a pharmacodynamic parameter. In this study, the total plasma warfarin concentration (a mixture of S- and R-warfarin) was used instead of plasma S-warfarin concentration in the calculation of the INR:Cp ratio. Patients with a high INR:Cp ratio need a lower dose of warfarin to control the INR in range as compared to those with a low INR:Cp ratio. Therefore, patients with a high WSI are more sensitive to warfarin than those with low WSI. The effect of VKORC1 genotypes on WSI were compared among 85 patients with the same CYP2C9 genotype (CYP2C9*1/*1) to eliminate the effect of CYP2C9 polymorphism on WSI. The WSI in the VKORC1 AA group was significantly greater than those in the VKORC1 BB & AB group, 3.02±1.08 and 2.05±0.95, respectively (pº0.0001). However, WSI was not significantly different between CYP2C9*1/*1 and the CYP2C9*1/*3 & CYP2C9*3/*3 group among the 55 patients with the same VKORC1 AA haplotype, p＝0.213 (Fig. 2). The mean warfarin doses among different CYP2C9 genotypes were significantly different. The warfarin dose in the CYP2C9*3 group (0.229±0.195 mg/kg/wk) was significantly lower than that in the wild type group (0.477±0.204 mg/kg/wk), p＝0.020 [95%CI 0.041– 0.455] (Table 3). The mean warfarin dose in the VKORC1 high-dose group (BB) was 0.842±0.403 mg/kg/wk, in the AB group it was 0.550±0.148 mg/kg/wk, while in the low-dose group (AA) it was 0.381 ±0.135 mg/kg/wk (Table 4). There were significant differences between warfarin maintenance doses among different VKORC1 haplotype groups, pº0.0001. Patients with the VKORC1 BB haplotype and CYP2C9*1/*1 required the highest warfarin maintenance dose, while the patient with the VKORC1 AA haplotype and CYP2C9*3/*3 required the lowest warfarin maintenance dose. Most of this Thai population were VKORC1 AA haplotype, CYP2C9*1/*1 and required only about 50% of the warfarin dose required by the highest dose group (VKORC1 BB haplotype, CYP2C9*1/*1) (Table 4). A warfarin dose of 21 mg/wk (about 0.4 mg/kg/wk), which was the most common optimum dosage used, was set as a reference line. The weekly warfarin dose of each different genotype group was compared with this refer-

Fig. 2. Warfarin sensitivity index for different CYP2C9 genotypes of the same VKORC1 AA genotype (N＝55, p＝0.213) (a) and different VKORC1 genotypes of the same CYP2C9 *1 /*1 genotype (N＝85, ***pº0.0001) (b).

Table 3. Comparison of weekly warfarin doses between CYP2C9 genotypes CYP2C9 genotypes

N (89)

Warfarin dose (mg/kg/wk)

INR

Wild type group

*1 /* 1

85 (95.5%)

0.477±0.204

2.1±0.5

Variant group

*1 /*3

3 (3.4%)

0.285±0.196

2.4±0.3

*3 /*3

1 (1.1%)

0.062

3.5

*1/*3 and *3/*3

4 (4.5%)

0.229±0.195

2.7±0.6

Sig.

p＝0.020*

p＝0.026*

* Significant at pº0.05 between variant and wild type groups.

ence group and is shown (Fig. 3) as the percentage difference in warfarin dose. The warfarin doses for VKORC1 AA/CYP2C9*1/*3 and VKORC1 AA/CYP2C9*3/*3 were about 50% and 80% less than the reference dose, respectively, but for VKORC1 BB/CYP2C9*1/*1, VKORC1 AB/CYP2C9*1/*1 and VKORC1 AB/CYP2C9*1/*3, the doses of warfarin were about 120%, 40% and 30% higher

535

VKORC1-CYP2C9 Variation on Warfarin Kinetic & Dynamic

Table 4. Comparisons of weekly warfarin doses among VKORC1 genotypes VKORC1

CYP2C9

N (89)

Weekly warfarin dose (mg/kg/wk)

INR

Approximate % of dose compare to ref.

6

0.842±0.403

2.3±0.3

220%

(1173CC, －1639GG)

* 1/*1 * 1/*3

0

—

—

* 3/*3

0

—

—

Total

6 (6.7%)

0.842±0.403

2.3±0.3

* 1/*1 * 1/*3

27

0.552±0.151

2.1±0.5

140%

1

0.509

2.3

130%

* 3/*3

0

—

—

Total

28 (31.5%)

0.550±0.148

2.1±0.5

AA

* 1/*1

52

* 1/*3

2

0.395±0.124 0.173±0.041

2.2±0.5 2.5±0.4

100% (Ref.)

(1173TT, －1639AA)

* 3/*3

1

0.062

3.5

20%

55 (61.8%)

0.381±0.135ab

2.2±0.5

Sig.

pº0.0001***

p＝0.402

BB

AB

(1173CT, －1639GA)

Total

50%

*** Significant at pº0.0001. pº0.0001 between AB and AA groups. b pº0.0001 between BB and AA groups. a

Table 5. Multivariate analysis: coefficients of factors in the best fit equation for prediction of warfarin dose requirement Weekly warfarin dose (mg/wk)＝exp [1.846＋(0.412×VKORC1 AB)＋(0.559 ×VKORC1 BB)＋(1.512×CYP2C9*1/*1)＋(1.136×CYP2C9*1/*3)－(0.007× age)] Factors

Coefficient

SE

p value

1.846

0.314

º0.0001

0.412

0.068

º0.0001

VKORC1 BB CYP2C9*1/*1

0.559

0.125

º0.0001

1.512

0.292

º0.0001

CYP2C9*1/*3

1.136 －0.007

0.334

0.001

0.002

0.007

nd

0.055

Constant VKORC1 AB

Age Body weight (Kg)

0.149

nd＝no data.

Fig. 3. Mean weekly warfarin dose requirement associated with different genotypes as compared to standard dose (warfarin 21 mg/week (0.4 mg/kg/wk) was use as the standard dose). nd＝no data.

than the reference dose, respectively. Patients older than 65 years required a lower dose of warfarin to achieve the optimum INR as compared to those patients who were younger. Using stepwise multiple linear regressions (Table 5), age and genetic factors (CYP2C9 and VKORC1) could explain approximately 53.8% of the variance in warfarin maintenance dose based on the following equation:

Weekly warfarin dose (mg/wk)＝exp [1.846＋(0.412 ×VKORC1 AB)＋(0.559×VKORC1 BB)＋(1.512× CYP2C9*1/*1)＋(1.136×CYP2C9*1/*3)－(0.007×age)]. Input the age in years and 1 or 0 if present or absent in terms of CYP2C9*1/*1, CYP2C9*1/*3, VKORC1 AB or VKORC1 BB. There was a highly significant correlation between the observed warfarin dose and the predicted dose using the aforementioned equation (r＝0.734, pº0.0001) (Fig. 4). In addition, this study compared the percentage of patients whose predicted weekly warfarin doses were within 15% of the observed dose for 3 different prediction methods. The first method set the predicted dose to be a

536

Alisara SANGVIROON, et al.

Fig. 4. Scatter plot of observed warfarin dose versus predicted warfarin dose from equation (log-transformed data)

Fig. 5. Bar charts show percentage of patients whose predicted weekly warfarin doses were within 15% of the observed doses Fixed dose: 21 mg/week Simple method: based on data from Figure 3 Equation method: based on equation from Table 5

fixed dose of warfarin equal to 21 mg/week. The second method, called the simple method, predicted the dose from the percentage difference in dosage requirement associated with different genotypes as shown in Figure 3. The third method, called the equation method, predicted the dose from the equation generated above. The equation method and the simple method could predict warfarin dose better than the fixed-dose method (Fig. 5).

Discussions In this study, we found that CYP2C9*3 and VKORC1 AA groups were associated with a lower required dose of warfarin. CYP2C9*3 was associated with lower clearance

of warfarin and, therefore, required a lower dose of warfarin than the wild type group. In addition, the warfarin sensitivity index (INR:Cp) in the VKORC1 AA group was higher than those in VKORC1 BB and AB groups; thus, VKORC1 AA group required a lower dose of warfarin. It was demonstrated that warfarin clearance was not significantly different among VKORC1 AA, AB and BB haplotypes; this finding suggested that VKORC1 haplotypes had no association with the pharmacokinetics of warfarin, but they are likely to have some effect on the pharmacodynamics of warfarin. VKORC1 AA patients were more sensitive to the same concentration of warfarin as compared to VKORC1 AB or BB patients. In Asians, variations in VKORC1 haplotypes were found more frequently than variations in CYP2C9 polymorphisms.21,28) The allele frequencies of CYP2C9 and VKORC1 genotypes found in this study were not different from those reported for a Northern Thai population.28) It has been reported previously that variation in VKORC1 is one of the factors causing variation in warfarin doses among Asians.21,22,29,30) VKORC1 haplotype frequencies in Thais were different from other Asians such as Chinese, Japanese, Malaysian and Indian.21,28–31) Mushiroda, et al.30) reported that VKORC1 BB, AB and AA groups in 828 Japanese patients were 0.8%, 15.9% and 83.3%, respectively. The present study found that the frequency of VKORC1 BB, AB and AA groups in Thai population was 6.7%, 31.5% and 61.8%, respectively. It was significantly different from those of the Japanese (chi square＝35.34, pº0.0001). Moreover, these were also significantly different from the Chinese population, in which the reported frequencies of VKORC1 BB, AB and AA groups were 1.5%, 21.1% and 77.4%, respectively32) (chi square ＝11.77, p＝0.003). Genetic factors played an important role in the interindividual variation in warfarin maintenance dose in a Thai population. The warfarin dose depended about 31.4% on VKORC1 and about 22.5% on CYP2C9. VKORC1 was associated with pharmacodynamic parameters such as the warfarin sensitivity index (INR:Cp). S-warfarin plasma concentration should be used in the calculation of warfarin sensitivity index, not the racemic mixture of S- and R-warfarin, to reflect genuine warfarin sensitivity; however, due to the limitation of the analytical instruments of this study, the total warfarin concentration was determined. It should be noted that the same brand of warfarin was used throughout the study to ensure uniformity in the ratio of S- to Rwarfarin that each patient received. Therefore, the use of WSI calculated from the total warfarin concentration might be justified for comparison of the differences between genotypes. CYP2C9 polymorphism was apparently associated with the clearance of warfarin. The warfarin sensitivity index appeared to be higher in the CYP2C9 variant group, but

VKORC1-CYP2C9 Variation on Warfarin Kinetic & Dynamic

did not reach statistical significance because only a few patients with variant CYP2C9 (n＝3) were enrolled in this study comparison (Fig. 2a). A larger number of patients with CYP2C9*1/*3 or *3/*3 is needed if a significant difference in warfarin sensitivity index is to be detected. This study also gives a stepwise multiple linear regression model for estimation of the warfarin maintenance dose for Thai patients. Using simplified factors including age, CYP2C9*3 and VKORC1 genotypes could explain about 53.8% of the variance of warfarin maintenance dose. A rapid genotyping method giving results within an hour may increase the possibility of incorporating these factors in setting the individual warfarin dose in clinical practice.33) Recently, a prospective study has shown that a pharmacogenetics-based dosing method could reach the stable dose in a shorter time than the traditional method (p＝0.001).32) However, future research with prospective randomized controlled clinical studies are required to determine the cost-benefit outcome from a genetic-based dosing regimen.

Acknowledgments: We are grateful to all patients who took part in this study. Our thankfulness is extended to Chulalongkorn University, Bangkok, Thailand for financial support. The VKORC1 reagents were supported by Roche Diagnostic, Thailand.

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