Su-Jun Lee

Discovery of Novel Functional Variants and Extensive Evaluation of CYP2D6 Genetic Polymorphisms in Koreans

Our objectives were to identify CYP2D6 genetic polymorphisms in a Korean population, to compare the allele frequencies with those of other ethnic groups, and to evaluate variant-induced functional variations in dextromethorphan (DM) metabolism in vitro and in vivo. Thirty-eight single nucleotide polymorphisms of CYP2D6 were identified by direct DNA sequencing in 51 Koreans. An extended set of 707 subjects were screened for the identified variants. A group of 202 healthy subjects was subjected to phenotypic analysis on DM metabolism. CYP2D6*10 was found to be the most frequent allele (45.6%), followed by CYP2D6*1 (32.3%), *2 (9.9%), *5 (5.6%), *41 (2.2%), *49 (1.4%), and some other rare alleles (<1%). The newly identified E418K and S183Stop were assigned as CYP2D6*52 and CYP2D6*60, respectively, by the Human P450 (CYP) Allele Nomenclature Committee. Individuals having the CYP2D6*10/*49 genotype (n = 5) exhibited a significant decrease in CYP2D6 metabolic activity compared with those with the CYP2D6*1/*1 genotype (n = 31) (P < 0.019). Variations in CYP2D6 protein levels in liver tissues (n = 49) were observed with CYP2D6 genotypes, and correlation between the CYP2D6 protein content and the activity was significant (r2 = 0.7). Given the importance of CYP2D6 in drug metabolism, subjects with the CYP2D6*10/*49 genotype may benefit from genotype analysis to achieve optimal drug therapy.

Identification and functional assessment of BCRP polymorphisms in a Korean population.

The breast cancer resistance protein (BCRP) is a member of the ATP-binding cassette transporters. The aim of the present study was to identify genetic variants of BCRP in Koreans and to assess the functional consequences of BCRP polymorphisms. Twenty single nucleotide polymorphisms (SNP), including four nonsynonymous SNP, were identified by DNA sequencing of the BCRP gene in 92 Korean subjects. BCRP V12M, Q141K, P269S, and Q126Stop were detected at frequencies of 23, 28, 0.2, and 1.9%, respectively. These four coding variants were also screened in Chinese and Vietnamese subjects; the allelic frequencies among the three populations were compared; and predictions were made as to the potential frequency of each variant. In vitro functional analyses of the P269S protein and the promoter SNP –19031C>T (mutated in the hypoxia-inducible factor-1α binding site) were performed and compared with those of the wild type. P269S exhibited a 35 to 40% decrease in vesicular uptake of [3H]estrone-3-sulfate and [3H]methotrexate compared with the wild type. The promoter SNP –19031C>T did not affect BCRP promoter activity in either the presence or absence of chemical-induced hypoxic stress. Our results suggest that the P269S variant could be a functionally altered variant. Genotyping of this variant in clinical studies is needed to address its phenotypic role. Genetic polymorphisms of BCRP were found to be very common in Koreans, as well as in other ethnic groups. Comparative analyses among three Asian populations revealed different frequencies for the four functional BCRP variants.

Identification of New CYP2C19 Variants Exhibiting Decreased Enzyme Activity in the Metabolism of S-Mephenytoin and Omeprazole

Although many cases of interindividual variation in the metabolism of CYP2C19 drugs are explained by the CYP2C19*2, *3, and *17, a wide range of metabolic variation still occurs in people who do not carry these genetic variants. The objectives of this study were to identify new genetic variants and to characterize functional consequences of these variants in metabolism of CYP2C19 substrates. In total, 21 single-nucleotide polymorphisms including three new coding variants, V394M, E405K, and D256N, were identified by direct DNA sequencing in 50 randomly selected subjects and in individuals who exhibited an outlier phenotype response in the omeprazole study. Recombinant proteins produced from the coding variants V394M, E405K, and D256N were prepared by using an Escherichia coli expression system and purified. Metabolism of S-mephenytoin and omeprazole by V394M was comparable with that of the wild-type protein. E405K showed a moderate decrease in metabolism of the substrates. However, D256N exhibited a significantly decreased activity in S-mephenytoin metabolism, resulting in 50 and 76% decreases in Vmax and intrinsic clearance, respectively, compared with the wild type. This variant also exhibited a significant decrease in omeprazole metabolism in vivo. CYP2C19 D256N and E405K were assigned as CYP2C19*26 and *2D, respectively, by the Cytochrome P450 Nomenclature Committee. In summary, this report characterizes the allele frequency and haplotype distribution of CYP2C19 in a Korean population and provides functional analysis of new coding variants of the CYP2C19 gene. Our findings suggest that individuals carrying CYP2C19*26 would have lower activity for metabolizing CYP2C19 substrate drugs.

Inhibition of human cytochrome P450 isoforms and NADPH‐CYP reductase in vitro by 15 herbal medicines, including Epimedii herba

Objective:  We evaluated the potential of 15 herbal medicines (HMs), commonly used in Korea, to inhibit the catalytic activities of several cytochrome P450 (CYP) isoforms and microsomal NADPH‐CYP reductase.

Comparisons of CYP2C19 genetic polymorphisms between Korean and Vietnamese populations.

It is well known that CYP2C19 is an enzyme showing genetic polymorphism that may cause marked interindividual and interethnic variation in the metabolism and disposition of its substrates. This study compared the frequency distribution of CYP2C19*1, *2, and *3 alleles in Korean and Vietnamese populations, representing Far Eastern and Southwestern Asian populations, respectively. The presence of the CYP2C19 variant alleles was analyzed in 377 Korean and 165 Vietnamese healthy subjects using a new pyrosequencing method. The respective allele frequencies of CYP2C19*1, *2, and *3 were 64%, 28%, and 8% in Koreans and 69%, 24%, and 5% in Vietnamese. The frequency of poor metabolizer genotype (*2/*2, *2/*3, *3/*3) in Korean (12.5%, 95% confidence interval 11.4-13.6) was not significantly different from that of Vietnamese population (7.2%, 95% confidence interval 6.2-8.2) (P = 0.074). These results obtained from a large number of subjects can be used in comparative studies with other ethnic groups in future clinical research.

The CYP3A4*18 allele, the most frequent coding variant in asian populations, does not significantly affect the midazolam disposition in heterozygous individuals.

The objective of this study was to identify CYP3A4 variants in Koreans and to characterize their functional consequences in vitro and in vivo. Four single nucleotide polymorphisms were identified in 50 Koreans by direct DNA sequencing. In an additional genotyping using 248 subjects, CYP3A4*18 was confirmed as the most frequent coding variant in Koreans at 1.7%, and its frequency was similar to that of Asians, suggesting that CYP3A4*18 would be the highest coding variant in Asians. The recombinant CYP3A4.18 protein prepared in baculovirus expression system showed 67.4% lower Vmax and 1.8-fold higher Km for midazolam 1′-hydroxylation compared with the wild type. The mean values of Cmax and area under the concentration curve (AUC) in the CYP3A4*1/*18 and CYP3A5*1/*3 subjects (n = 8) were 63% and 32% higher than in CYP3A4*1/*1 and CYP3A5*1/*3 carriers (n = 8), respectively. Although the in vitro assay exhibited a significant reduction of the enzyme activity for midazolam, the in vivo differences associated with the CYP3A4*1/*18 tend to be low (P < 0.07 in Cmax and P < 0.09 in AUC). In summary, the heterozygous CYP3A4*1/*18 does not appear to cause a significant change of midazolam disposition in vivo; however, the clinical relevance of CYP3A4*18/*18 remains to be evaluated.

CYP2J2 and CYP2C19 Are the Major Enzymes Responsible for Metabolism of Albendazole and Fenbendazole in Human Liver Microsomes and Recombinant P450 Assay Systems

ABSTRACT Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

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.

Potential of pranlukast and zafirlukast in the inhibition of human liver cytochrome P450 enzymes.

1. The potential of zafirlukast to inhibit several human cytochrome P450 enzymes is well known. However, pranlukast, a structural analogue of zafirlukast, has not been studied. Accordingly, the inhibitory potential of pranlukast was evaluated and compared with that of zafirlukast, a known CYP2C9 inhibitor, in in vitro microsomal incubation studies. 2. Both pranlukast and zafirlukast showed moderate inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, competitively inhibiting tolbutamide 4-methylhydroxylation with estimated mean Ki values of 3.82 ± 0.50 and 5.86 ± 0.08 μM, respectively. 3. Pranlukast had no effect on CYP2C19-catalysed S-mephenytoin 4′-hydroxylation or CYP3A4-catalysed midazolam 1-hydroxylation. However, zafirlukast showed minor inhibition of these reactions. Neither pranlukast nor zafirlukast inhibited CYP1A2-catalysed phenacetin O-deethylation, CYP2D6-catalysed dextromethorphan O-demethylation or CYP2E1-catalysed chlorzoxazone 6-hydroxylation. 4. The results suggest that like zafirlukast, pranlukast also has the potential moderately to inhibit CYP2C9-catalysed tolbutamide 4-methylhydroxylation. Therefore, the inhibitory potential of pranlukast should be considered when it is co-administered with CYP2C9 substrates with narrow therapeutic ranges (e.g. S-warfarin, phenytoin).

Identification of cytochrome P450s involved in the metabolism of arachidonic acid in human platelets

Although cytochrome P450s (CYPs) have been identified in most human cells, identification of CYPs in human platelets remains poorly explored. CYP expressions in human platelets were screened by using reverse transcriptase-polymerase chain reaction and western blot analysis followed by functional assays using arachidonic acid (ARA). CYP1A1, 2U1, 2J2, 4A11, 4F2, and 5A1 were expressed as both proteins and mRNAs in platelets. Ethoxyresorufin-O-deethylase activity was observed in platelets and this activity was significantly decreased after treatment with the general P450 inhibitor SKF-525A and the CYP1A inhibitor, α-naphthoflavone (40-45%, P<0.001). Seventeen ARA metabolites were detected in ARA-treated platelets. Among these, the levels of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids were significantly decreased with the treatment of the P450 ω-hydroxylase inhibitor 17-octadecynoic acid (P<0.05-0.001). In summary, multiple ARA-metabolizing P450s were identified in human platelets. These findings may provide an important resource for understanding physiological function of platelet.