Determination of unbound warfarin enantiomers in human plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase liquid chromatography with ultraviolet or fluorescence and on-line circular dichroism detection

Takahashi, Harumi; Kashima, Toshitaka; Kimura, Sosuke; Muramoto, Nagisa; Nakahata, Hiromi; Kubo, Suzuko; Shimoyama, Yutaka; Kajiwara, Masahiro; Echizen, Hirotoshi

doi:10.1016/s0378-4347(97)00346-0

Cited by 70 publications

(51 citation statements)

References 21 publications

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“…Rats were given 15 mg/kg warfarin orally, and blood was taken from the tail at 0 and 60 min. Warfarin in plasma was extracted according to the method of Takahashi et al (1997). We added 30 l of 2 N HCl, 150 l of diethyl ether, and 19.5 l of 5 M dicoumarol into 15 l of plasma in the tube as the internal standard (500 nM, final concentration).…”

Section: Methodsmentioning

confidence: 99%

Elevated Warfarin Metabolism in Warfarin-Resistant Roof Rats (Rattus rattus) in Tokyo

Ishizuka

Okajima²,

Tamada³

et al. 2006

Drug Metab Dispos

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ABSTRACT:Wild roof rats (Rattus rattus) live in proximity to human habitats, and they may carry numerous pathogens of infectious diseases. Pest control is important for public health, and warfarin is a commonly used rodenticide worldwide. However, continual use of warfarin may cause drug resistance in rodents and lead to failure of their control, especially in urbanized areas. In warfarin-resistant rats, the warfarin level in plasma was significantly lower after oral administration than that in the control warfarin-sensitive rats. Warfarin is metabolized by cytochrome P450 (P450), and hydroxylation of warfarin by P450 isoforms was significantly higher in warfarinresistant rats (2-fold). Western blot analysis indicated that the level of CYP3A2 expression in warfarin-resistant rats was significantly larger than in warfarin-sensitive rats. The NADPH-P450 reductase activities in resistant rats were 8-fold higher than those in sensitive rats. In vivo, the administration of the P450 potent inhibitor proadifen (SKF-525A) increased the mortality of warfarin in the warfarin-resistant roof rats. We concluded that the mechanism of warfarin resistance in Tokyo roof rats is caused by increased clearance of warfarin.

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Section: Methodsmentioning

confidence: 99%

Elevated Warfarin Metabolism in Warfarin-Resistant Roof Rats (Rattus rattus) in Tokyo

Ishizuka

Okajima²,

Tamada³

et al. 2006

Drug Metab Dispos

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“…These samples were analyzed for the separate R-and S-enantiomers of warfarin, along with the extent of their plasma binding, resulting in estimates of the plasma unbound concentration (Cu) and unbound clearance (CLpo,u) of S-warfarin. [7][8][9] Variants in the 5Ј-flanking region of CYP2C9 up to Ϫ2137 bp were analyzed according to the method of Shintani et al 6 using DNA previously collected from the patients. 2 Coding region SNPs (CYP2C9*2, CYP2C9*3, CYP2C9*4, CYP2C9*5, and CYP2C9*6) were analyzed as previously described.…”

Section: Methodsmentioning

confidence: 99%

5′-Flanking region polymorphisms of CYP2C9 and their relationship to S-warfarin metabolism in white and Japanese patients

Takahashi

Ieiri

Wilkinson

et al. 2004

Blood

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White and Japanese patients require different warfarin dosages to achieve therapeutic anticoagulation, but this can be only partly explained by genetic variability in the coding region of CYP2C9-a critical enzyme in the drug's metabolism. Accordingly, analysis of the ؊2.1-kb 5-flanking region of CYP2C9 was undertaken in 22 white and 38 Japanese patients whose unbound oral clearance of S-warfarin had been previously determined. Thirteen single nucleotide polymorphisms (SNPs) were identified, some of which were in linkage disequilibrium with functionally defective coding region variants. Those 5-flanking patterns linked with at least one CYP2C9*3 allele or CYP2C9*2/*3 were associated with reduced CYP2C9 activity and warfarin dose. Japanese patients possessing the wildtype promoter and coding sequences had significantly (P < .01) greater CYP2C9 activity than white patients with the corresponding genotype. In conclusion, either unidentified polymorphisms further upstream in the promoter region or environmental factor(s) account for the differences in the warfarin doses between whites and Japanese. IntroductionPopulation differences in the drug-metabolizing enzymes are important in bridging therapeutic doses and safety profiles of drugs from one population to another. Warfarin is a widely used oral anticoagulant and its effect is largely attributable to the pharmacologically more active S-enantiomer. 1 Recently, we observed that Japanese patients receiving warfarin therapy had a significantly greater body weight-normalized plasma unbound clearance (CLpo,u) of S-warfarin than white patients, 2 which is predominantly reflective of CYP2C9-mediated hepatic metabolism. 3 At present, 11 coding region variant alleles of the gene have been reported. 4 Because whites have greater allelic frequencies than Southeast Asians 5 of the 2 most common functionally defective variants (CYP2C9*2 and CYP2C9*3), it is possible that the population differences in S-warfarin metabolism may be attributed to this distribution difference. However, in our previous study a difference in S-warfarin metabolism was noted even when the 2 populations were matched with respect to the homozygous wildtype CYP2C9 genotype (CYP2C9*1/*1). 2 This indicates that such a coding region polymorphism cannot fully account for the population differences in the CYP2C9 activity. Recently, Shintani et al 6 reported 7 single nucleotide polymorphisms (SNPs) in the 5Ј-flanking region of CYP2C9 in Japanese subjects, some of which were associated with an altered level of gene transcription. Accordingly, we investigated SNPs within the Ϫ2.1-kb promoter region of CYP2C9 in white and Japanese patients receiving warfarin and assessed their contribution to the variability of in vivo CYP2C9 activity within and between the 2 populations. Study designThe promoter region analysis was performed in the participants of an earlier investigation, 2 22 white (9 men and 13 women) and 38 Japanese patients (25 men and 13 women), whose DNA samples were available. Details of the study have be...

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“…20 Internal standard [diclofenac, 100 L of a 10 g/mL solution in a 10/90 (vol/vol) mixture of isopropanol-hexan solution], 1 mL of HCL (2 N), and 5 mL of ether were added to a 0.5-mL plasma sample. The mixture was shaken for 10 minutes and then centrifuged at 1500g for 10 minutes.…”

Section: Assay Of (S)-and (R)-warfarin Plasma Concentrationsmentioning

confidence: 99%

Association of pharmacokinetic (CYP2C9) and pharmacodynamic (factors II, VII, IX, and X; proteins S and C; and γ-glutamyl carboxylase) gene variants with warfarin sensitivity

Shikata

Ieiri

Ishiguro

et al. 2004

Blood

146

116

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We analyzed mutations of 7 vitamin K-dependent protein and cytochrome P450 2C9 genes in 45 patients and investigated whether any contribute to the large interpatient variability in the warfarin dose-effect relationship. Total clearance and daily dose, INR and INR/Cp, were used as pharmacokinetic and pharmacodynamic indexes, respectively. Patients were grouped by genotype based on a single polymorphism and combinations of polymorphisms. Among the 30 sequence variants identified, CYP2C9*3, 165Thr 3 Met of the factor II gene, ؊402G 3 A, (37-bp repeat) n , and ؊746T 3 C of the factor VII gene, and (CAA repeat) n of the ␥-glutamyl carboxylase gene were selected as candidate polymorphisms. As the analysis of single polymorphisms implied, the highest INR/Cp mean values and the lowest warfarin maintenance doses were observed in patients homozygous for the 165Met, ؊402G, (37-bp repeat) 6 and ؊746T alleles. Multiple regression analysis revealed that warfarin sensitivity was independently associated with ؊402G 3 A, (CAA repeat) n , CYP2C9*3, and 165Thr 3 Met, which accounted for 50% of variance. These results suggest that part of the considerable interpatient variation is attributable to genetic variation, and the combined genotyping of CYP2C9 and certain vitamin K-dependent protein genes is useful for predicting anticoagulant responses.

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Determination of unbound warfarin enantiomers in human plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase liquid chromatography with ultraviolet or fluorescence and on-line circular dichroism detection

Cited by 70 publications

References 21 publications

Elevated Warfarin Metabolism in Warfarin-Resistant Roof Rats (Rattus rattus) in Tokyo

Elevated Warfarin Metabolism in Warfarin-Resistant Roof Rats (Rattus rattus) in Tokyo

5′-Flanking region polymorphisms of CYP2C9 and their relationship to S-warfarin metabolism in white and Japanese patients

Association of pharmacokinetic (CYP2C9) and pharmacodynamic (factors II, VII, IX, and X; proteins S and C; and γ-glutamyl carboxylase) gene variants with warfarin sensitivity

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