Chang-Woo Yeo

The effect of SLCO1B1*15 on the disposition of pravastatin and pitavastatin is substrate dependent: the contribution of transporting activity changes by SLCO1B1*15.

Objective This study was addressed to understand the underlying mechanism of the substrate-dependent effect of genetic variation in SLCO1B1, which encodes OATP1B1 (organic anion transporting polypeptide) transporter, on the disposition of two OATP1B1 substrates, pravastatin and pitavastatin, in relation to their transport activities. Methods The uptake of pravastatin, pitavastatin, and fluvastatin was measured in oocytes overexpressing SLCO1B1*1a and SLCO1B1*15 to compare the alterations of in-vitro transporting activity. After 40-mg pravastatin or 4-mg pitavastatin was administered to 11 healthy volunteers with homozygous genotypes of SLCO1B1*1a/*1a and SLCO1B1*15/*15, the pharmacokinetic parameters of pravastatin and pitavastatin were compared among participants with SLCO1B1*1a/*1a and SLCO1B1*15/*15 genotypes. Results The uptake of pravastatin and pitavastatin in SLCO1B1*15 overexpressing oocytes was decreased compared with that in SLCO1B1*15, but no change occurred with fluvastatin. The fold change of in-vitro intrinsic clearance (Clint) for pitavastatin in SLCO1B1*15 compared with SLCO1B1*1a was larger than that of pravastatin (P<0.0001). The clearance (Cl/F) of pitavastatin was decreased to a greater degree in participant with SLCO1B1*15/*15 compared with that of pravastatin in vivo (P<0.01), consistent with in-vitro study. As a result, Cmax and area under the plasma concentration–time curve of these nonmetabolized substrates were increased by SLCO1B1*15 variant. The greater decrease in the transport activity for pitavastatin in SLCO1B1*15 variant compared with SLCO1B1*1a was, however, associated with the greater effect on the pharmacokinetics of pitavastatin compared with pravastatin in relation to the SLCO1B1 genetic polymorphism. Conclusion This study suggests that substrate dependency in the consequences of the SLCO1B1*15 variant could modulate the effect of SLCO1B1 polymorphism on the disposition of pitavastatin and pravastatin.

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.

Determination of acetylsalicylic acid and its major metabolite, salicylic acid, in human plasma using liquid chromatography–tandem mass spectrometry: application to pharmacokinetic study of Astrix® in Korean healthy volunteers

The first liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of acetylsalicylic acid (aspirin, ASA) and one of its major metabolites, salicylic acid (SA), in human plasma using simvastatin as an internal standard has been developed and validated. For ASA analysis, a plasma sample containing potassium fluoride was extracted using a mixture of ethyl acetate and diethyl ether in the presence of 0.5% formic acid. SA, a major metabolite of ASA, was extracted from plasma using protein precipitation with acetonitrile. The compounds were separated on a reversed-phase column with an isocratic mobile phase consisting of acetonitrile and water containing 0.1% formic acid (8:2, v/v). The ion transitions recorded in multiple reaction monitoring mode were m/z 179 --> 137, 137 --> 93 and 435 --> 319 for ASA, SA and IS, respectively. The coefficient of variation of the assay precision was less than 9.3%, and the accuracy exceeded 86.5%. The lower limits of quantification for ASA and SA were 5 and 50 ng/mL, respectively. The developed assay method was successfully applied for the evaluation of pharmacokinetics of ASA and SA after single oral administration of Astrix (entero-coated pellet, 100 mg of aspirin) to 10 Korean healthy male volunteers.

Effect of CYP2C19 Genetic Polymorphism on Pharmacokinetics and Pharmacodynamics and Pharmacodynamics of a New Proton Pump Inhibitor, Ilaprazole

It is well known that the CYP2C19 genetic polymorphism influences the pharmacokinetics and pharmacodynamics of proton pump inhibitors (PPIs), but no report has addressed the effects on ilaprazole, a newly developed PPI. To investigate the effects of the CYP2C19 genetic polymorphism on the disposition and pharmacodynamics of ilaprazole, multiple doses of once‐daily 10 mg ilaprazole were repeatedly administered for 7 days to 27 healthy Korean participants, comprising 9 homozygous CYP2C19 extensive metabolizers (homo EMs), 10 heterozygous EMs (hetero EMs), and 8 homozygous poor metabolizers (PMs). The plasma concentration and pharmacodynamic response were measured in the last dose interval. Each genotype group was matched for gender and thus was composed of 4 male and 4 female participants when the analysis was conducted. The pharmacokinetic parameters estimated from the plasma concentrations of ilaprazole and its metabolite ilaprazole sulfone, the serum gastrin level, and the 24‐hour intragastric pH were compared among the CYP2C19 genotype groups. No statistically significant differences in the maximum plasma concentration at steady state(Css, max) and the area under the concentration‐time curve from zero to 24 hours (AUCτ) of ilaprazole and ilaprazole sulfone were observed among the homo EM, hetero EM, and PM CYP2C19 genotypes. In addition, the mean 24‐hour intragastric pH, the percentage of time at pH>4, and the AUCτ of serum gastrin showed no significant differences among the CYP2C19 genotype groups. The data suggests that the pharmacokinetics and pharmacodynamics of ilaprazole are not significantly influenced by the CYP2C19 genetic polymorphism in healthy participants.

Association between Genetic Polymorphisms of CYP2D6 and Outcomes in Breast Cancer Patients with Tamoxifen Treatment

The aim of the study was to evaluate the association between genetic polymorphisms of CYP2D6 and outcomes in breast cancer patients with tamoxifen treatment. We evaluated the CYP2D6 genetic polymorphisms in 766 breast cancer patients. Among them, 110 patients whose samples were prospectively collected before surgery and treated with tamoxifen were included to evaluate the association between CYP2D6 and outcomes. The genotypes of CYP2D6 were categorized as extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) according to the activity score. The clinicopathologic features of 110 patients were not significantly different among the three groups except for the T-stage and nodal status. The high T-stage and axillary metastasis were more frequent in the PM group. While recurrence-free and overall survival in the PM group was poorer than the other groups, there was no significant difference between the EM and the IM group. The difference between the PM and the other groups on univariate analysis disappeared on multivariate analysis. These conflicting results suggest that the clinical value of CYP2D6 polymorphisms is still unclear and more large-sized and comprehensively designed trials are necessary.

Genetic polymorphism of hepatocyte nuclear factor-4α influences human cytochrome P450 2D6 activity†

Hepatocyte nuclear factor‐4 alpha (HNF4A) is an essential transcriptional regulator for many genes that are expressed preferentially in the liver. Among the important functions of the liver is drug metabolism in response to xenobiotic exposure. Recent studies have suggested that HNF4A regulates the expression of cytochrome P450 (CYP), including CYP2D6 and CYP3A4, which show large individual variations in their activities. To understand the genetic factors that influence individual CYP activities, a genetic variant of HNF4A and the effects of genetic variants of HNF4A on CYP activity were investigated. Here, we report the identification of a novel coding variant of HNF4A that influences CYP2D6 activity in humans. After direct sequencing, a polymorphism search revealed the HNF4A G60D variant in Koreans. This variant was unable to bind to the recognition site in the CYP2D6 promoter and therefore lacked the regulatory function for this gene. Human liver specimens with the heterozygous HNF4A G60D genotype showed a tendency toward lower levels of CYP2D6 activity than the wild‐type genotype in the same genetic background of CYP2D6. Furthermore, human subjects with the HNF4A G60D genotype tended to have lower CYP2D6 activity than those with the wild‐type HNF4A. The HNF4A G60D variant was detected at low frequency in Asian populations, including Koreans, Chinese, and Vietnamese, and was not found in Africans or Caucasians. Conclusion: This is the first report to show that the genetic polymorphism of liver‐enriched nuclear receptor HNF4A influences downstream CYP2D6 function in human subjects. (HEPATOLOGY 2008;48:635–645.)

Robust CYP2D6 genotype assay including copy number variation using multiplex single-base extension for Asian populations

BACKGROUND We developed a CYP2D6 genotyping method that includes copy number variation (CNV) and recently known functional haplotypes using multiplex single-base extension (SBE). METHODS Twelve CYP2D6 alleles (*1, *2, *5, *10, *14, *18, *21, *41, *49, *52, *60, and a duplication of CYP2D6) were genotyped using 2 PCR reactions followed by multiplex SBE with 10 primers and singleplex SBE with 1 primer. The result from 758 Korean samples was validated by comparison with the results of direct sequencing or other genotyping methods. We also genotyped 89 Chinese and 122 Vietnamese subjects to determine the presence of recently identified functional alleles. RESULTS All 12 CYP2D6 alleles, including gene deletion and duplication, were obviously discriminated. The concordance rate was 100% between our method and other methods. Our method also covered over 98% of the CYP2D6 genotypes in Japanese and Chinese subjects based on reported data. In addition to published genotypes, *14, *21, *41, *49, and *52 were found in about 5% in Chinese and Vietnamese. CONCLUSIONS The CYP2D6 genotyping method may be clinically applicable for Asian populations. The method can be improved easily to cover other ethnic groups by utilizing additional haplotype tagging SNPs.

Discovery of a novel allelic variant of CYP2C8, CYP2C8*11, in Asian populations and its clinical effect on the rosiglitazone disposition in vivo

The objectives of this study were to identify the genetic variants of CYP2C8, analyze CYP2C8 single nucleotide polymorphisms (SNPs), and characterize their functional consequences in the CYP2C8 substrate drug rosiglitazone in humans. The direct full sequencing of CYP2C8 genomic DNA was performed in a Korean population (n = 50). A total of 17 CYP2C8 variants including a novel coding variant (E274Stop) were identified. The novel CYP2C8 E274Stop variant was assigned as CYP2C8*11 by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee. Seventeen SNPs were used to characterize linkage disequilibrium, haplotype structures, and haplotype tagging SNPs. Genotyping for CYP2C8*11 in an extended set of Koreans (n = 400), whites (n = 100), Han Chinese (n = 348), Vietnamese (n = 100), and African Americans (n = 93) was performed by a newly developed pyrosequencing method. The frequency of CYP2C8*11 was 0.3% in Koreans, 1% in Vietnamese, and 0.14% in Chinese. However, none of the whites or African Americans contained the CYP2C8*11 allele. Subjects with CYP2C8*1/*11 exhibited higher plasma concentration-time profiles of rosiglitazone than those of nine control subjects carrying CYP2C8*1/*1. The area under the concentration-time curve and peak plasma concentration of rosiglitazone in individuals carrying CYP2C8*1/*11 (n = 5) were 54 and 34% higher than the mean values observed in the control subjects carrying CYP2C8*1/*1 (P = 0.015 and P = 0.025, respectively). In summary, this is the first report to characterize the allele frequency and haplotype distribution of CYP2C8 in a Korean population, and it provides functional analysis of a new variant CYP2C8*11. Our findings suggest that individuals carrying CYP2C8*11, a null allele found in Asians only, may have lower activity for metabolizing CYP2C8 substrate drugs.

Pharmacogenetic Study of Deferasirox, an Iron Chelating Agent

Transfusion-associated iron overload induces systemic toxicity. Deferasirox, a convenient long acting oral agent, has recently been introduced in clinical practice with a promising efficacy. But there are some patients who experience drug-related toxicities and cannot tolerate it. To investigate effect of genetic variations on the toxicities and find optimal target population, we analyzed the genetic polymorphisms of UDP-glucuronosyltransferase 1A (UGT1A) subfamily, multi-drug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). A total of 20 functional genetic polymorphisms were analyzed in 98 patients who received deferasirox to reduce transfusion-induced iron overload. We retrospectively reviewed the medical records to find out the drug-related toxicities. Fifteen (15.3%) patients developed hepatotoxicity. Patients without wild-type allele carrying two MRP2 haplotypes containing −1774 del and/or −24T were at increased risk of developing hepatotoxicity compared to patients with the wild-type allele on multivariate analysis (OR = 7.17, 95% CI = 1.79–28.67, P = 0.005). Creatinine elevation was observed in 9 patients (9.2%). Body weight ≥40 kg and homozygosity for UGT1A1*6 were risk factors of creatinine elevation (OR = 8.48, 95% CI = 1.7–43.57, P = 0.010 and OR = 14.17, 95% CI = 1.34–150.35, P = 0.028). Our results indicate that functional genetic variants of enzymes to metabolize and transport deferasirox are associated with drug-related toxicities. Further studies are warranted to confirm the results as the pharmacogenetic biomarkers of deferasirox.

Itraconazole and rifampin alter significantly the disposition and antihistamine effect of ebastine and its metabolites in healthy participants.

The present study was performed to elucidate the effects of itraconazole and rifampin on the pharmacokinetics and pharmacodynamics of ebastine, a nonsedative H1 receptor antagonist. In a 3‐way crossover sequential design with 2‐week washouts, 10 healthy participants were pretreated with itraconazole for 6 days, rifampin for 10 days, or neither. After oral administration of 20 mg ebastine, blood and urine samples were collected for 72 and 24 hours, respectively, and histamine‐induced wheal and flare reactions were measured to assess the antihistamine response for 12 hours. Itraconazole pretreatment decreased the oral clearance of ebastine to 10% (P < .001) and increased the AUC∞ of the active metabolite, carebastine, by 3‐fold (P < .001). On the other hand, rifampin pretreatment decreased the AUC∞ of carebastine to 15% (P < .001), with an enormous reduction in the oral bioavailability of ebastine and significantly reduced histamine‐induced skin reactions. From these results, the disposition of ebastine and carebastine seems to be significantly altered by coadministration of itraconazole or rifampin. The antihistamine response after ebastine dosing would be decreased following rifampin pretreatments.