Warfarin is an anticoagulant drug with narrow therapeutic index and high interindividual variability in dose requirement. S-warfarin is metabolized mainly by polymorphic cytochrome P450 (CYP) 2C9. We systematically quantified the influence of CYP2C9 genotype, demographic factors and concomitant drug treatment on warfarin metabolism and maintenance dose. The mean warfarin doses were lower in carriers of one (2.71 mg/day, 59 patients) and two polymorphic alleles (1.64 mg/day, 11 patients) than in carriers of two wild-type alleles (4.88 mg/day, 118 patients). Multiple regression analysis demonstrated that CYP2C9 genotype, age, concomitant treatment with warfarin metabolism inducers and lean body weight contributed significantly to interindividual variability in warfarin dose requirement (adjusted R 2 ¼ 0.37). The same factors, except for age, significantly influenced S-warfarin clearance (adjusted R 2 ¼ 0.42). These results can serve as a starting point for designing prospective studies in patients in the initiation phase of genotype-based warfarin therapy.

The degree of interpatient variability in the warfarin dose required to achieve the desired anticoagulant response can only partly be explained by polymorphisms in the CYP2C9 gene, suggesting that additional genetic factors such as polymorphisms in genes involved in blood coagulation may influence warfarin dose requirement. In total, 165 Caucasian outpatients on stable maintenance warfarin treatment previously genotyped for CYP2C9 were analysed for common polymorphisms in FVII, GGCX and VKORC1 genes. The -402G > A polymorphism and a variable number of repeats in intron 7 of FVII gene did not significantly influence warfarin dose. The mean warfarin doses increased with the number of (CAA) repeats in the GGCX gene, but the differences were significant only in the CYP2C9*1/*1 subgroup of patients (p = 0.032). Common polymorphism (6484C > T) in intron 1 of the VKORC1 gene led to lower warfarin dose requirement; the means were 5.70 (95% C.I. 4.95-6.45), 3.49 (3.07-3.90) and 2.11 (1.80-2.42) mg/day for 6484 CC, CT and TT genotypes, respectively (p < 0.001). In contrast, 9041G > A polymorphism in 3'UTR of theVKORC1 gene led to higher warfarin dose requirement; the means were 3.09 (2.58- 3.60), 4.26 (3.69-4.82) and 5.86 (4.53-7.19) mg/day for 9041 GG, GA and AA genotypes, respectively (p < 0.001). With a regression model we explained 60.0% of variability in warfarin dose, which was due to gene polymorphisms (CYP2C9, VKORC1), age and body-surface-area. When aiming for individualised warfarin therapy, at least VKORC1 polymorphisms should be included in predictive genotyping besides CYP2C9.

We confirmed important interaction between carbamazepine and warfarin metabolism which can be of major clinical importance. If treatment with carbamazepine cannot be avoided, patients taking warfarin should be frequently monitored, especially when initiating or stopping carbamazepine therapy.

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ABSTRACT:CYP2C9 is one of the major drug-metabolizing enzymes, and it is involved in the oxidative metabolism of approximately 10% of clinically important drugs, among which some, such as the anticoagulant warfarin, have a narrow therapeutic index. The human CYP2C9 gene is highly polymorphic. We found a new sequence variation in exon 7 of the CYP2C9 gene (1060G>A) resulting in a substitution of acidic amino acid glutamate to basic lysine (E354K) when translated. The allele, designated CYP2C9*24, was present in heterozygous state in one warfarin-treated patient. To characterize the CYP2C9*24 allele, we expressed the wild-type and CYP2C9.24 protein in a recombinant yeast expression system and a human embryonic kidney (HEK)-293 cell system. Carbon monoxide difference spectra were recorded on dithionite-reduced microsomes, and protein was determined by Western blotting. Transfection with CYP2C9.1 cDNA resulted in detectable CYP2C9 protein in yeast or HEK-293 cells, whereas only small amounts of the protein were detected in yeast transfected with CYP2C9.24 cDNA. A strong differential absorption peak at 450 nm was observed with microsomes of yeast transfected with CYP2C9.1 cDNA, whereas no peak was detected with microsomes of yeast transfected with CYP2C9.24 cDNA or empty pYeDP60 plasmid. These results suggest that CYP2C9.24 may be improperly folded, both in yeast and mammalian cells, resulting in improper heme incorporation and rapid intracellular degradation. The data obtained in the expression systems are consistent with our findings in vivo. In conclusion, we have identified a novel defective CYP2C9 variant allele of potential importance for drug metabolism in vivo.CYP2C9 is involved in the metabolism of more than 100 currently used drugs, corresponding to about 10 to 20% of commonly prescribed drugs (Kirchheiner and Brockmoller, 2005). Some of them (e.g., anticoagulant warfarin) have a narrow therapeutic index (Miners and Birkett, 1998). Clinically available warfarin is a racemic mixture, and CYP2C9 is responsible for the metabolism of more potent Senantiomer (Kaminsky and Zhang, 1997). The human CYP2C9 gene is highly polymorphic (http://www.imm.ki.se/CYPalleles/). The two most common variant alleles in Caucasian populations are CYP2C9*2, with a point mutation in exon 3 (430CϾT), and CYP2C9*3, with a point mutation in exon 7 (1075AϾC). Both are associated with a decrease in the catabolic activity of the enzyme (Rettie et al., 1994;Sullivan-Klose et al., 1996). It was shown in many studies that the patients with polymorphic alleles require significantly lower doses of warfarin and are more susceptible to bleeding complications than the carriers of two wild-type alleles (Kirchheiner and Brockmoller, 2005).We have previously reported a novel 1060GϾA sequence variant in exon 7 of CYP2C9 gene, leading to substitution of acidic amino acid glutamate to basic lysine (E354K). The new allele, designated CYP2C9*24, was present in the heterozygous state in one warfarintreated patient, who was also a heterozygous carrier of t...

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