Wild-type Cytochrome P450 2C9 Research Articles

Identification and in vitro functional assessment of 10 CYP2C9 variants found in Chinese Han subjects.

Cytochrome P450 2C9 (CYP2C9) participates in about 15% of clinical drug metabolism, and its polymorphism is associated with individual drug metabolism differences, which may lead to the adverse drug reactions (ADRs). In this study, 1163 Chinese Han individuals were recruited to investigate their distribution pattern of CYP2C9 gene and find out the variants that may affect their drug metabolic activities. We successfully developed a multiplex PCR amplicon sequencing method and used it for the genetic screening of CYP2C9 in a large scale. Besides the wild type CYP2C9*1, totally 26 allelic variants of CYP2C9 were detected, which included 16 previously reported alleles and 10 new non-synonymous variants that had not been listed on the PharmVar website. The characteristics of these newly detected CYP2C9 variants were then evaluated after co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis revealed that except for Pro163Ser, Glu326Lys, Gly431Arg and Ile488Phe, most of newly detected variants showed comparable protein expression levels to wild type in yeast cells. Two typical CYP2C9 probe drugs, losartan and glimepiride, were then used for the evaluation of metabolic activities of variants. As a result, 3 variants Thr301Met, Glu326Lys, and Gly431Arg almost lost their catalytic activities and most of other variants exhibited significantly elevated activities for drug metabolism. Our data not only enriches the knowledge of naturally occurring CYP2C9 variants in the Chinese Han population, but also provides the fundamental evidence for its potential clinical usage for personalized medicine in the clinic.

Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient.

Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.

VKORC1 and CYP2C9 genotypes in Egyptian patients with warfarin resistance.

Warfarin is the most commonly prescribed anticoagulant drug; however, a narrow therapeutic range and a high risk of bleeding or stroke complicate its clinical use. Warfarin resistance was defined as prolonged warfarin requirements of more than 15 mg/day to achieve therapeutic anticoagulation or failure to achieve therapeutic anticoagulation with more than 20 mg/day. The resistance is associated with polymorphisms of the vitamin K epoxide reductase-oxidase complex (VKORC1) and cytochrome P450-2C9 (CYP2C9) genes, which affect warfarin pharmacodynamics and pharmacokinetics, respectively. Identification of the VKORC1 -1639 (A/G) and CYP2C9 (*1/*2/*3) allelic variants was performed using the PGX-Thrombo Strip in 41 patients with warfarin resistance compared with 30 patients with normal warfarin response out of 352 diagnosed cases of deep vein thrombosis. In warfarin-resistant patients, the VKORC1-1639 genotype frequencies were GG 0.756, GA 0.244 and AA 0.0, whereas in warfarin responder patients, they were: GG 0.333, GA 0.400 and AA 0.276 with P ≤ 0.001. The CYP2C9 genotype frequencies showed nonsignificant difference in both group of patients (P = 0.31). Our results suggest that the VKORC1-1639 GG and the wild type CYP2C9*1*1genotypes are associated with the high-dose requirement for warfarin therapy, and that VKORC1-1639 GG is responsible for warfarin resistance and failure in Egyptian patients.

Clinical application of a new warfarin-dosing regimen based on the CYP2C9 and VKORC1 genotypes in atrial fibrillation patients.

The polymorphisms of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) are important genetic factors for warfarin dose determinations. The present study aimed to investigate the contribution of the CYP2C9 and VKORC1 genotypes to warfarin dose requirement in atrial fibrillation (AF) patients, and to evaluate the clinical application of a warfarin-dosing algorithm. A total of 122 AF patients with a target international normalized ratio of 2.0 to 3.0 were included to determine the genotypes of CYP2C9 (rs1057910) and VKORC1 (rs9923231). A warfarin-dosing algorithm was developed based on age, height, and the CYP2C9 and VKORC1 genotypes of AF patients. The results indicated that the mean warfarin daily dose requirement was lower in the CYP2C9*1/*3 genotype compared with those in the homozygous wild-type CYP2C9*1/*1 patients (P<0.05), and was higher in patients with the VKORC1 AG and GG genotypes compared with those with the AA genotype (P<0.05). The multivariate regression model showed that age, height, and the CYP2C9 and VKORC1 genotypes were the best variables for estimating warfarin dose (R2=56.4%). A new warfarin-dosing algorithm was developed and its validity was confirmed in a second cohort of AF patients. During the 50-day follow-up, 63.3% (19/30) of control group patients and 86.7% (26/30) of patients in the experimental group acquired the warfarin maintenance dose. Among all the patients who acquired the warfarin maintenance dose, the mean time elapse from initiation until warfarin maintenance dose was significantly less in the experimental group (25.8±1.7 day) compared to the control group (33.1±1.9 day) (P<0.05). There was significant linear correlation between predicted warfarin maintenance dose and actual dose (r=0.822, P<0.01). In conclusion, a new warfarin-dosing algorithm was developed based on the CYP2C9 and VKORC1 genotypes, and it can shorten the time elapse from initiation until warfarin maintenance dose in AF patients with warfarin therapy.

Impact of CYP2C9 and VKORC1 genetic polymorphisms upon warfarin dose requirements in Egyptian patients with acute coronary syndrome.

Warfarin is the most widely prescribed anticoagulant drugs. Cytochrome P450-2C9 (CYP2C9) and vitamin K epoxide reductase-oxidaxe complex subunit 1 (VKORC1) contribute significantly to the variability of warfarin dose requirements among patients. We investigated the impact of CYP2C9 and VKORC1 polymorphisms on the variability of warfarin dosage requirements in Egyptian patients with acute coronary syndrome and their association with other nongenetic factors. Eighty participants with acute coronary syndrome were enrolled in this cross-sectional study. Associations between CYP2C9 and VKORC1 gene variants together with daily warfarin dose, demographic data, clinical status of patients and time to target international normalized ratio were assessed. Mean warfarin dose among patients with wild-type CYP2C91/1 genotype was significantly higher than heterozygous CYP2C91/2 and CYP2C91/3 variants (P ≤ 0.001). Patients with wild VKORC1 (G/G) genotype were treated with significantly higher daily warfarin dosages than homozygous (A/A) and heterozygous (G/A) genotypes. Patients carrying VKORC1 (G/G) genotype in combination with the CYP2C91/1 type alleles had the highest daily warfarin dosage, whereas the lowest daily warfarin dosage to achieve the required clinical effect was found among patients having CYP2C91/2 and CYP2C91/3 genotypes combined with VKORC1 (A/A) genotype (P ≤ 0.001). Regression analysis revealed that age, height, CYP2C9 and VKORC1 genotypes were significantly associated with warfarin dose. Genetic polymorphisms in VKORC1, CYP2C9 along with age and height are determinants of warfarin dose requirements in Egyptian population acute coronary syndrome. Higher warfarin loading dose is required for both wild CYP2C9 and VKORC1 gene variants which may contribute to warfarin-resistant cases.

In vitro assessment of 39 CYP2C9 variants found in the Chinese population on the metabolism of the model substrate fluoxetine and a summary of their effects on other substrates.

Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme that is responsible for clearing approximately 15% of clinically important drugs. The objective of this study was to assess the catalytic characteristics of 39 CYP2C9 isoforms found in the Chinese population and their effects on the metabolism of the model substrate fluoxetine invitro. Baculovirus-mediated expressing system was used to highly express wild-type and the 38 CYP2C9 allelic variants in insect cell microsomes. Then, the enzymatic characteristics of each variant were evaluated using fluoxetine as the substrate. Reactions were performed at 37°C with the insect microsomes and 10-200μm fluoxetine for 60min. After termination, the products were precipitated and used for signal collection by UPLC-MS/MS. Of the 39 tested CYP2C9 isoforms, only four variants (CYP2C9*3, CYP2C9*27, CYP2C9*34 and CYP2C9*37) exhibited similar relative clearance values to that of the wild-type CYP2C9*1. Moreover, five variants (CYP2C9*14, CYP2C9*36, CYP2C9*45, CYP2C9*48 and CYP2C9*55) showed a higher intrinsic clearance value than the wild-type protein, whereas the remaining 29 CYP2C9 isoforms exhibited significantly decreased clearance values (from 6·23% to 87·74%) compared to CYP2C9*1. In addition, 28 CYP2C9 isoforms including CYP2C9*3 exhibited a trend towards substrate inhibition for fluoxetine. This study provides the most comprehensive data on the enzymatic activities associated with all reported CYP2C9 variants in the Chinese population with regard to the widely used antidepressant drug, fluoxetine. Our data indicate that more attention should be paid to subjects carrying the corresponding infrequent CYP2C9 alleles when administering fluoxetine in the clinic.

Functional characterization of 32 CYP2C9 allelic variants

Genetic variations in cytochrome P450 2C9 (CYP2C9) contribute to interindividual variability in the metabolism of clinically used drugs such as warfarin and tolbutamide. We functionally characterized 32 types of allelic variant CYP2C9 proteins. Recombinant CYP2C9 proteins generated using a heterologous expression system are useful for comparing functional changes in CYP2C9 variant proteins expressed from low-frequency alleles. Wild-type CYP2C9 and its 31 variants were found to be transiently expressed in COS-7 cells, and the enzymatic activity of the CYP2C9 variants was characterized using S-warfarin as a representative substrate. Among the 32 types of CYP2C9 allelic variants tested, CYP2C9.18, CYP2C9.21, CYP2C9.24, CYP2C9.26, CYP2C9.33 and CYP2C9.35 exhibited no enzyme activity, and 12 types showed significantly decreased enzyme activity. In vitro analysis of CYP2C9 variant proteins should be useful for predicting CYP2C9 phenotypes and for application to personalized drug therapy.

The Ligands of Estrogen Receptor α Regulate Cytochrome P4502C9 (CYP2C9) Expression

Cytochrome P4502C9 (CYP2C9) is an important drug-metabolizing enzyme responsible for the metabolism of approximately 16% of all clinically relevant drugs. It was shown previously that the activity of CYP2C9 in vivo is inhibited by oral contraceptives. The mechanisms of this effect have not been elucidated. We hypothesize that this may occur because of the sex steroid-dependent activation of estrogen receptor α (ERα) with further transactivation of the CYP2C9 gene. Here, we show that the CYP2C9 promoter indeed contains a functionally relevant estrogen responsive element (ERE) half-site at position -149/-145. Its ERα binding activity was tested by the luciferase gene reporter assay. Promoter constructs bearing this site were cotransfected with ERα into Huh7 hepatoma cells and treated with various ERα ligands including 4-hydroxytamoxifen (4-OHT), raloxifene (R), 17β-estradiol (EE), and 17α-ethinylestradiol (ETE). The luciferase activity driven by the wild-type CYP2C9 promoter construct was up-regulated by 4-OHT and R and significantly or marginally suppressed by ETE and EE, respectively. An identical effect was observed in primary hepatocytes treated with these compounds. Mutations introduced into the ERE half-site abolished the observed effects in the Huh7 cells. Electrophoretic mobility-shift assay revealed sequence-specific binding of a nuclear protein to the oligonucleotide containing the ERE half-site, which was identified as ERα by antibody supershift analysis. In addition, the association of ERα with CYP2C9 promoter was strongly supported by chromatin immunoprecipitation data. Taken together, these results indicate that ERα and its ligands play an important role in the regulation of CYP2C9 expression.

Loss-of-Function CYP2C9 Variants Improve Therapeutic Response to Sulfonylureas in Type 2 Diabetes: A Go-DARTS Study

Sulfonylureas are metabolized mainly by the cytochrome p450 2C9 (CYP2C9) enzyme. Two CYP2C9 variants--*2 (Arg144Cys) and *3 (Ile359Leu)--are associated with reduced enzyme activity and impaired substrate metabolism. We identified 1,073 incident users of sulfonylureas in Tayside, Scotland, and assessed the impact of the combined CYP2C9*2 and CYP2C9*3 genotypes on early and sustained sulfonylurea response. We found that patients with two copies of a loss-of-function allele were 3.4 times (P = 0.0009) more likely to achieve a treatment hemoglobin A(1c) (HbA(1c)) level <7% than patients with two wild-type CYP2C9 alleles. This corresponds to a 0.5% (P = 0.003) greater reduction in HbA(1c) concentration. In addition, *2 and *3 allele carriers were less likely to experience treatment failure with sulfonylurea monotherapy (P = 0.04; per-allele hazard ratio 0.79; 95% confidence interval 0.63-0.99). In conclusion, CYP2C9 loss-of-function alleles are associated with greater response to sulfonylureas and decreased failure of therapy consistent with the pharmacokinetic role of CYP2C9.

Genetic Polymorphism of the Human Cytochrome P450 2C9 Gene and Its Clinical Significance

Human cytochrome P450 2C9 (CYP2C9) accounts for approximately 20% of total hepatic CYP content and metabolizes approximately 15% clinically used drugs including S-warfarin, tolbutamide, phenytoin, losartan, diclofenac, and celecoxib. To date, there are at least 33 variants of CYP2C9 (*1B through to *34) being identified. CYP2C9*2 and CYP2C9*3 differ from the wild-type CYP2C9*1 by a single point mutation: CYP2C9*2 is characterised by a 430C>T exchange in exon 3 resulting in an Arg144Cys amino acid substitution, whereas CYP2C9*3 shows an exchange of 1075A>C in exon 7 causing an Ile359Leu substitution in the catalytic site of the enzyme. CYP2C9*2 is frequent among Caucasians with approximately 1% of the population being homozygous carriers and 22% heterozygous. The corresponding figures for the CYP2C9*3 allele are 0.4% and 15%, respectively. Worldwide, a number of other variants have also to be considered. The CYP2C9 polymorphisms are relevant for the efficacy and adverse effects of numerous nonsteroidal anti-inflammatory agents, sulfonylurea antidiabetic drugs and, most critically, oral anticoagulants belonging to the class of vitamin K epoxide reductase inhibitors. Numerous clinical studies have shown that the CYP2C9 polymorphism should be considered in warfarin therapy and practical algorithms how to consider it in therapy are available. These studies have highlighted the importance of the CYP2C9*2 and *3 alleles. Warfarin has served as a practical example of how pharmacogenetics can be utilized to achieve maximum efficacy and minimum toxicity. Polymorphisms in CYP2C9 have the potential to affect the toxicity of CYP2C9 drugs with somewhat lower therapeutic indices such as warfarin, phenytoin, and certain antidiabetic drugs. CYP2C9 is one of the clinically significant drug metabolising enzymes that demonstrates genetic variants with significant phenotype and clinical outcomes. Genetic testing of CYP2C9 is expected to have a role in predicting drug clearance and implementing individualized pharmacotherapy. Prospective clinical studies with large samples are required to establish gene-dose and gene-effect relatiohsips for CYP2C9.

Use of Pharmacogenetic Factors to Predict Warfarin Dose in Children - a Retrospective Cohort Study

Background: Warfarin is the most commonly used oral anticoagulant, but its use is limited by the narrow therapeutic index. Single nucleotide polymorphisms in cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) have been shown to impact warfarin dose requirements. Use of such pharmacogenetic data to improve warfarin dosing has been widely studied in adult populations and is being integrated into clinical practice. However, there remains a paucity of data on the appropriate use of this genetic information in the pediatric population. We developed a retrospective review of pediatric patients on warfarin therapy to evaluate associations between CYP2C9 and VKORC1 polymorphisms and warfarin dose requirements in children.Methods: Patients were recruited from outpatient clinics at Texas Children's Hospital and Stollery Children's Hospital. Any patient less than 18 years of age that had taken warfarin and achieved maintenance dose was eligible. Blood sample or buccal swab was collected for genotyping and clinical data was collected from the medical record. Genotyping was performed by melting curve analysis to detect CYP2C9*2, CYP2C9*3, or VKORC1 −1639 mutations. Warfarin genotyping reagents were obtained from Idaho Technology and assays were run using the Roche LightCyler instrument.Results: This study was initiated in March 2008 and is ongoing. To date 47 children (23M: 24F) have been enrolled. Indications for warfarin therapy include: arrhythmia (n=2), artificial heart valve (n=10), pulmonary hypertension (n=10), deep vein thrombosis (n=9), pulmonary embolism (n=2), Fontan (n=10), stroke (n=2), other (n=2). The mean age is 9.3 years (range 1–18); median target International Normalized Ratio (INR) range is 2–3 (with similar target INRs across genotypes). Twenty-nine patients have fully evaluable data at the time of this report, of whom 21 (72%) are wild type CYP2C9, 6 (21%) carry at least 1 mutant allele, and 2 (7%) are homozygous or compound heterozygote. As compared with wild type patients (mean dose 0.14mg/ kg/day), patients with heterozygous (0.09mg/kg/day; p=0.04) or homozygous (0.05mg/ kg/day; p=0.001) genotype required significantly lower maintenance daily warfarin dose. For VKORC1 −1639 genotype 6 (21%) are AA, 11 (38%) are GA, and 12 (41%) are GG. As compared with GG patients (mean dose 0.16mg/kg/day), patients with 1 (0.11mg/kg/ day; p=0.12) or 2 (0.09mg/kg/day; p=0.02) A allele required lower doses.Discussion: We present the first pediatric series to evaluate the influence of CYP2C9 and VKORC1 genotype on warfarin dosing. These data demonstrate genotype frequency similar to adults, and suggests that genotype influences the maintenance warfarin dose in children. Enrollment to this study is ongoing, and will allow a more precise evaluation of the impact of genotype and additional demographic data on pediatric dosing. These data will provide for development of a pediatric dosing algorithm to predict maintenance warfarin dose a priori.

Cytochrome P450 2C9 *2 and *3 Polymorphisms and the Dose and Effect of Sulfonylurea in Type II Diabetes Mellitus

Sulfonylurea hypoglycemics are mainly metabolized by the cytochrome P450 2C9 (CYP2C9) enzyme. The CYP2C9*2 and *3 polymorphisms encode proteins with less enzymatic activity and are correlated with elevated serum levels of sulfonylurea, as demonstrated in healthy volunteers. In this study, the effect of these variants is described for patients with diabetes mellitus treated with sulfonylurea. Associations between CYP2C9 polymorphisms, prescribed doses of sulfonylurea, and change in glucose levels after the start of sulfonylurea therapy were assessed in all patients with incident diabetes mellitus starting on sulfonylurea therapy in the Rotterdam Study, a population-based cohort study of 7,983 elderly people. In CYP2C9*3 allele carriers using tolbutamide, the prescribed dose was lower compared to patients with the wild-type CYP2C9 genotype. No differences in the prescribed dose were found in tolbutamide users with the CYP2C9*1/*2 or CYP2C9*2/*2 genotype compared to wild-type patients or in patients using other sulfonylurea. In CYP2C9*3 allele carriers, the mean decrease in fasting serum glucose levels after the start of tolbutamide therapy was larger than in patients with the wild-type genotype, although not statistically significant. Patients with diabetes mellitus who are carriers of a CYP2C9*3 allele require lower doses of tolbutamide to regulate their serum glucose levels compared to patients with the wild-type genotype.

Catalytic activities of human cytochrome P450 2C9*1, 2C9*3 and 2C9*13

Cytochrome P450 2C9 (CYP2C9) is a geneticly polymorphic enzyme responsible for the metabolism of some clinically important drugs. CYP2C9*13 is an allele identified in a Chinese poor metabolizer of lornoxicam which has a Leu90Pro amino acid substitution. This paper reports on a study aimed at comparing the catalytic properties of CYP2C9*13 with those of the wild-type CYP2C9*1 and mutant CYP2C9*3 (Ile359Leu) in the COS-7 expression system using various substrates. CYP2C9*3 and *13 produced far lower luminescence than CYP2C9*1 in luciferin H metabolism. CYP2C9*13 exhibited an 11-fold increase in Km but no change in Vmax with tolbutamide as the substrate, a five-fold increase in Km and an 88.8% reduction in Vmax with diclofenac. These data indicate that CYP2C9*13 exhibits reduced metabolic activity toward all studied CYP2C9 substrates. The magnitude of the CYP2C9*13-associated decrease in intrinsic clearance (Vmax/Km) is greater than that associated with CYP2C9*3.

Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells.

Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T>C, 1146C>T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) CYP, but was undetectable in parental CHL cell. The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.