Identification and characterization of novel alternative splice variants of human constitutive androstane receptor in liver samples of Koreans and Caucasians.

Human constitutive androstane receptor (hCAR, NR1I3) is a member of the orphan nuclear receptor family and regulates the transcription of many drug-metabolizing enzymes and drug transporters. Previous studies have shown that the hCAR gene produces a number of different kinds of mRNA splicing variants (SVs) in non-Asian ethnicities. In the present study, we identified 18 hCAR SVs (SV1-SV18), including four novel SVs in Korean human livers. Among the four novel SVs, SV2 showed enhanced transactivation activity when cotransfected with CYP2B6 reporter gene, whereas other SVs were nonfunctional. When profiles of major hCAR SVs were compared among 30 livers from Korean patients and 20 livers from Caucasian patients, the relative composition of each SV showed interethnic variation as well as interindividual variation. The most predominant form of hCAR SV was not wild type, but either SV4 or SV7. The summed relative amounts of SV4 and SV7 ranged from 34.5 to 57.6% in the 30 Korean livers and from 47.2 to 82.6% in the 20 Caucasian livers, suggesting large interindividual variation. The mean relative amount of nonfunctional SV9 was significantly higher in Koreans (29.8%) than in Caucasians (12.8%). The mean relative amount of novel SV2 was 9.7% in Korean livers and 3.5% in Caucasian livers. Expression profiling of hCAR proteins in human livers also supported large interindividual variation in the expressional ratio of wild-type and SVs. Our results describe for the first time the direct comparison of hCAR SV profiles between Koreans and Caucasians. The functional relevance of these interindividual and interethnic variations of hCAR mRNA expression needs to be further characterized.

Capsaicin induces CYP3A4 expression via pregnane X receptor and CCAAT/enhancer-binding protein ß activation.

SCOPE: Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the principal pungent ingredient in hot red and chili peppers. Many studies have focused on the anticarcinogenic or chemopreventive activities of capsaicin. However, the influence of capsaicin on CYP3A4, its involvement in drug metabolism, and the underlying mechanisms remain unclear. METHODS AND RESULTS: Here, we examined the effect of capsaicin on CYP3A4 expression and the metabolism of CYP3A1 substrate, nifedipine in male Sprague-Dawley rats. Capsaicin induced the enzymatic activity and expression of CYP3A4 in HepG2 cells. Treatment with a human pregnane X receptor (hPXR) inhibitor reduced the inductive effects of capsaicin on CYP3A4 expression. Capsaicin also induced the activation of CCAAT/enhancer-binding protein ß (C/EBPß). Moreover, capsaicin increased the activation of the transient receptor potential vanilloid type-1 receptor downstream signaling components Ca²âº/calmodulin-dependent protein kinase and Akt. Capsaicin elevated the level of CYP3A1 in rat liver and significantly increased the biotransformation of nifedipine to dehydronifedipine. CONCLUSION: From these data, we conclude that capsaicin induces CYP3A4 expression in vitro and in vivo. This induction was achieved by the activation of hPXR and C/EBPß. Our results suggest that capsaicin might induce CYP3A4 expression; thus, exposure to capsaicin may increase the metabolism of CYP3A4 substrate and potentially cause food-drug interactions.

A haplotype of CYP2C9 associated with warfarin sensitivity in mechanical heart valve replacement patients.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * CYP2C9 single nucleotide polymorphisms (SNPs) are important in safe and effective oral anticoagulation with warfarin use. * Although CYP2C9*2 and *3 are important genetic factors for the warfarin dose, one of the CYP2C9 SNPs, IVS-65G>C, has been suggested to be associated with warfarin sensitivity. However, as of yet, there has been no explanation about the possible mechanism and linkage analysis. WHAT THIS PAPER ADDS * New information on CYP2C9 SNPs and their occurrences in common haplotype structures in healthy unrelated Koreans and in individuals who require low warfarin dose after mechanical heart valve replacements (MHVRs) were studied. * Additional evidence showed that an Asian dominant haplotype consisting of -1565C>T, -1188T>C, IVS3+197G>A, IVS3-334C>T, IVS3-65G>C, IVS4-115A>G and IVS5-73A>G could be associated with a low warfarin maintenance dose in mechanical heart valve replacement (MHVR) patients. AIMS The objectives of this study were to determine the distribution of CYP2C9 variants in Koreans and investigate their association with warfarin dose requirements in patients who received MHVRs. METHODS All nine exons, intron-exon junction, and promoter region of CYP2C9 were amplified and directly sequenced in 50 healthy normal Koreans. Additional direct DNA sequencing of the CYP2C9 gene was conducted in 36 of the 267 MHVR patients who required low maintenance warfarin doses without carrying CYP2C9*3 and VKORC1 1173T mutations. The effects of CYP2C9 genetics on warfarin maintenance dose were assessed in 267 MHVR patients. RESULTS Thirty-nine single nucleotide polymorphisms (SNPs) including seven previously unidentified SNPs were identified in 50 Koreans by direct DNA sequencing. One of the CYP2C9 haplotypes exhibited an association with warfarin low dose requirement. The adjusted odds ratio for the haplotype between the low dose group and the normal subjects was 2.5 (95% confidence interval 1.05, 6.16). This haplotype consisting of -1565C>T, -1188T>C, IVS3+197G>A, IVS3-334C>T, IVS3-65G>C, IVS4-115A>G, and IVS5-73A>G was found in 15% of 36 MHVR patients who required low warfarin doses, while 4% of 50 normal healthy subjects exhibited this haplotype. One of the SNPs comprising this haplotype, -1565C>T, apparently changed a protein binding pattern as observed in electrophoretic mobility shift assay. CONCLUSION The haplotype including -1565C>T, -1188T>C, IVS3+197G>A, IVS3-334C>T, IVS3-65G>C, IVS4-115A>G, and IVS5-73A>G seems to be associated with low warfarin dose requirement and this haplotype could be considered in the development of a warfarin dose prediction model for Asian populations.

Effect of CYP2C9 and VKORC1 genotypes on early-phase and steady-state warfarin dosing in Korean patients with mechanical heart valve replacement.

OBJECTIVES: The effect of CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKORC1) genotypes was evaluated for the early-phase and steady-state warfarin dosing in Korean patients with mechanical heart valve replacement. METHODS: The genotypes of CYP2C9 variants including CYP2C9*3, CYP2C9*13, and CYP2C9*14, and VKORC1 1173C>T were assessed for the association with warfarin dosing in 265 patients whose data were collected for warfarin dose; international normalized ratio (INR), comedication, comorbidity, and other clinical characteristics. RESULTS: In the early phase of warfarin therapy, the combined genotypes of CYP2C9 and VKORC1 caused statistically significant difference in warfarin dose from day 7 of warfarin dosing and the subsequent time course of dose increase showed significant difference among the three different genotypes (P<0.001). Compared with patients with CYP2C9 wild type, the patients with heterozygous CYP2C9 variants have delayed time to reach stable dose [adjusted hazard ratio (HRadj): 0.48; 95% confidence interval (CI): 0.27-0.85] and tended to have high risk for the first INR greater than 3.5 (HRadj: 1.64; 95% CI: 0.98-2.75). The patients with the VKORC1 CT genotype showed no significant difference in the time to reach stable dose but statistically significant low HR for time to first INR greater than 3.5 compared with those with VKORC1 TT genotype (HRadj: 0.25; 95% CI: 0.13-0.51). The observed warfarin maintenance dose was best explained by a model including covariates of age, weight, concurrent congestive heart failure/cardiomyopathy, INR-increasing drugs, aspirin, dietary supplements, and CYP2C9 and VKORC1 genotypes (R=0.56). CONCLUSION: The heterozygous CYP2C9 and VKORC1 genotypes influence warfarin dosing in an early phase as well as steady state of warfarin therapy in Korean patients with mechanical heart valve replacement.

Duplex pyrosequencing of the TPMT*3C and TPMT*6 alleles in Korean and Vietnamese populations.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) genetic polymorphisms have been studied intensively in relation to thiopurine toxicity. In the present study, an improved pyrosequencing method was developed for TPMT genotyping and used to investigate the genotype frequency in Korean and Vietnamese populations. METHODS: Four-hundred Korean and 159 Vietnamese subjects were genotyped by pyrosequencing for TPMT 238G>C, 460G>A, 539A>T, and 719A>G variants, in order to determine the TPMT*2, *3A, *3B, *3C, and *6 alleles. RESULTS: TPMT*3C was found to be the most frequent allele in both the Korean (0.88%) and Vietnamese (2.83%) populations. TPMT*2, *3A, and *3B were not found in either of the study populations. Two Korean subjects were genotyped as TPMT*1/*6 (0.25%), while none of the Vietnamese had this genotype. The direct comparison of the frequencies of functional TPMT1/3C genotype showed significant difference between Korean and Vietnamese populations (chi2 test, p=0.0124). Duplex pyrosequencing methods for the detection of TPMT3C and TPMT6 were developed and validated to show 100% concordance with the results obtained by direct sequencing. CONCLUSIONS: The functional TPMT alleles in Korean and Vietnamese populations are TPMT*3C and TPMT*6. Duplex pyrosequencing for these alleles appears to be an accurate, rapid, and cost-effective method for genotyping TPMT in these Asian populations.

CYP2S1 gene polymorphisms in a Korean population.

Cytochrome P450 2S1 (CYP2S1) is a drug-metabolizing enzyme that shows interindividual variations in response to treatments for psoriasis and is regarded as a putative prognostic marker for colorectal cancer treatment. To gain insight into the genetic basis for the interindividual variations, CYP2S1 gene polymorphisms were analyzed in Korean subjects. Using direct sequencing of the CYP2S1 gene, 12 genetic variations, which included the two novel nonsynonymous mutations CYP2S1 S61N (0.3%) and CYP2S1 L230R (0.8%), were identified in 50 Korean subjects. Homology modeling predicted the location of the L230R change to be near two conserved alpha-helices in the hood of the substrate-binding site. Linkage disequilibrium (LD) analysis with seven common CYP2S1 variations showed two discrete LD blocks in CYP2S1 gene and consequently a limited number of haplotypes. Although the importance of novel CYP2S1 variants and haplotypes remains to be discovered, CYP2S1 polymorphisms would provide useful information on interindividual variations with respect to CYP2S1 function, which facilitates drug response predictions and disease prognosis.