Wild-type CYP2C9 Research Articles

Different inhibitory effects of azole-containing drugs and pesticides on CYP2C9 polymorphic forms: An in vitro study

CYP2C9 plays a major role in drug metabolism. It is highly polymorphic and among the variants, CYP2C9*2 and CYP2C9*3 have been known to encode the protein with moderately to markedly reduced catalytic activity. Azole antifungals are among the most frequently used drugs in human pharmacotherapy and represent a widely used class of pesticides to which humans are inevitably exposed. Due to the similarities in CYP organization throughout species, azoles can interact not only with the target fungal CYP51 substrate-binding site but can also modulate the catalytic activity of human cytochrome P450s, including CYP2C9, causing severe adverse effects. In the present study the potency of azole-containing drugs and pesticides to inhibit recombinant wild-type CYP2C9*1 and the allelic variants CYP2C9*2 and CYP2C9*3 was evaluated. Significant differences were found in their affinity to CYP2C9*1, CYP2C9*2, and CYP2C9*3 as well as in the catalytic activity of CYP2C9 allelic variants. Moreover, addition of cytochrome b5 resulted in a decrease of CYP2C9*3 activity to diclofenac in a concentration-dependent manner. Increasing the knowledge of how azoles influence polymorphic variants of CYP2C9 could help individualize drug treatment, leading to optimization of the selection of drugs and doses for individuals based on genetic information.

Metabolism of 7-ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants.

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6β-hydroxylation, progesterone 6β-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids.

O-16 Lean body weight based dosing achieves comparable systemic pantoprazole exposures for normal-weight and overweight/obese children

Background We previously reported increased AUCtot and decreased CL/F for the commonly prescribed pro-ton pump inhibitor (PPI) and CYP2C19 substrate, pan-toprazole, in obese vs. non-obese children. In light of increasing concerns regarding adverse events associated with high systemic exposure to PPIs in children (e.g., os-teopenia, infection, micronutrient deficiencies), we aimed to identify a pantoprazole dosing strategy appropriate for overweight/obese children, who are six times more likely to suffer from gastroesophageal reflux disease and require PPI therapy than normal weight peers. Given that most physiologic metabolic processes occur in lean body tissues, lean body weight (LBW) based doing was imple-mented in this prospective paediatric pharmacokinetic investigation. Methods 62 children (6–17 years of age; 39% Female), genotyped for CYP2C19 *2, *3, *4, *17 alleles (TaqMan), re-ceived a single oral dose of pantoprazole (1.2 mg/kg lean body weight). LBW was calculated via the Janmahasatian equation. Plasma pantoprazole and metabolite concen-trations were measured (HPLC-UV) at 10 time-points, over 8 hours, and pharmacokinetic parameters (PK) generat-ed via non-compartmental techniques (Kinetica 5.0). For children with at least one wild-type CYP2C19 allele (*1), select pantoprazole PK were compared in normal-weight (Body Mass Index (BMI) 10-84th% for age; n=29) and over-weight/obese (BMI ≥85th% for age; n=30) children, using independent student t-test (SPSS v23; α=0.05). Results No statistically significant differences were observed for pantoprazole AUCtot in normal-weight (13.9±32.2 µMolar*h) vs. overweight/obese (14.68±31.03 µMolar*h) children (p=0.9). No statistically significant dif-ferences in pantoprazole CL/F (23.9±16.6 vs. 19.8±25.8 L/h; p=0.5) or Cmax (5.66±3.8 vs. 7.76±4.4 uMolar; p=0.06) were observed between normal-weight and overweight/obese children. Conclusion LBW, rather than total body weight, based dosing is most appropriate to achieve comparable sys-temic exposures to pantoprazole for normal-weight and overweight/obese children. This dosing strategy appears to eliminate the systemic pantoprazole overexposure previously observed in obese children and will likely min-imize their risk for adverse events associated with high-dose PPI therapy. Future pharmacokinetic-pharmacody-namic studies of PPIs may be warranted for overweight and obese children.

Effects of functional CYP2C8,CYP2C9,CYP3A5,and ABCB1 genetic variants on the pharmacokinetics of insulin sensitizer pioglitazone in Chinese Han individuals.

Pioglitazone is a thiazolidinedione antihyperglycemic drug with insulin-sensitizing properties. We investigated whether the variant genotypes of cytochrome P450 2C8 (CYP2C8), CYP2C9, CYP3A5 and transporter ABCB1 influence the pharmacokinetic phenotype of the substrate pioglitazone in Chinese individuals. Single-nucleotide polymorphisms were determined by the PCR-restriction fragment length polymorphism method in 244 (CYP2C8 and CYP2C9) healthy Chinese Han individuals. After a single oral dose of 30 mg pioglitazone, the plasma concentrations of the parent drug and of two major active metabolites M-III and M-IV were measured using a validated LC-MS/MS in 21 (genotyping CYP3A5 and ABCB1) of these 244 volunteers. The results confirmed that the unique frequencies of CYP2C8*2 (0.0%), CYP2C8*3 (0.0%), and CYP2C9*2 (0.0%) alleles were significantly different from those reported in Whites and Africans, and there were only 10 variant CYP2C9*1/*3 heterozygous (CYP2C9*3 carriers) among 244 Chinese individuals. These results were similar to those reported in Asian ethnic populations, including the Chinese. Unexpectedly, the pioglitazone AUC0-48 in CYP2C9*3 carriers was lower (50.8%), whereas the AUC0-48 ratios of metabolites M-III/pioglitazone and M-IV/pioglitazone increased to 134.3 and 155.8%, respectively, compared with the wild-type CYP2C9*1/*1 homozygous. Moreover, this phenomenon was not observed in individuals with genetic variants of CYP3A5*3 and ABCB1 (C1236T). The present research suggests that the CYP2C8, CYP3A5, and ABCB1 genes play no significant role in the interindividual variation of pioglitazone pharmacokinetics, whereas CYP2C9*3 carriers are likely to accelerate the metabolism of this antidiabetic drug in the Chinese Han ethnic population.

Association of CYP2C9, CYP2A6, ACSM2A, and CPT1A gene polymorphisms with adverse effects of valproic acid in Chinese patients with epilepsy

ObjectiveTo explore the influence of CYP2C9, CYP2A6, ACSM2A, CPT1A gene polymorphisms on valproic acid (VPA) and its role in metabolism-related liver dysfunction in order to guide the clinical safety and rational use of VPA. MethodsOne hundred two patients taking sodium valproate oral solution were genotyped. To assess the genotypes of relevant genes, the CYP2C9 gene was directly sequenced; for polymorphism classification, multiple Long-PCR electrophoresis was conducted for CYP2A6; and imLDR method was used for ACSM2A and CPT1A. GC–MS-SIM was used to determine the levels of VPA and 2-propyl-4-pentenoic acid (4-ene-VPA) in human plasma simultaneously. ResultsCYP2C9 mutations had a significant impact on 4-ene-VPA concentration, in patients with wild-type CYP2C9 (CYP2C9*1), which has a greater capacity for VPA metabolism than the mutant type (CYP2C9*3), liver dysfunction was substantially higher. Patients with an ACSM2A polymorphism had higher levels of ALT and AST compared with wild-type (p<0.05), but the mutations had no effect on the VPA-related liver dysfunction (p>0.05). Among different CYP2A6 and CPT1A genotype groups, there was no significant correlation in the levels of VPA, 4-ene-VPA, ALT, AST or TB (p>0.05). The content of 4-ene-VPA had no direct correlation with the incidence of liver dysfunction. ConclusionsEarly detection of CYP2C9 gene polymorphisms may help to predict or prevent liver dysfunction caused by VPA. While the concentration of 4-ene-VPA was not suitable as an early warning index, the results provide clear theoretical guidance for the rational and safe clinical use of VPA.

Association of CYP2C19*2 with hyperlipidemia in patients with the wild-type CYP2C9

Arachidonic acids are metabolized by cytochromes P450 (CYP450) to epoxyeicosatrienoic acids (EETs). EETs have many cardiovascular protective effects such as vasodilation, anti-hypertension, anti-inflammation, lipid-lowering effects, and favorable effects on glucose homeostasis. This study aimed to investigate whether gene polymorphism of cytochrome P450 CYP2C9 and CYP2C19 are related to the risk of hypertension, hyperlipidemia, and diabetes mellitus. Four hundred and eighty patients were enrolled, and single nucleotide polymorphisms for CYP2C9 and CYP2C19 were genotyped. The percentages of patients with hypertension, hyperlipidemia, and diabetes mellitus with CYP2C9 *1/*2, *1/*3, and wild-type (CYP2C9 *1/*1) were similar, and the chi-square (χ2) analysis showed no statistically significant differences between each genotype. Further, no statistically significant differences were observed for the percentages of patients with hypertension, hyperlipidemia, and diabetes mellitus between CYP2C19 genotypes. Subgroup analysis showed that among patients with the wild-type CYP2C9 genotype (CYP2C9*1/*1, N = 297), patients with CYP2C19*1/*2 and *2/*2 genotypes have a significantly higher percentage of hyperlipidemia than those with CYP2C19 *1/*1, *1/*17, and *17/*17 genotypes. Binary logistic regression analysis also suggested that among patients with the wild-type CYP2C9 genotype, the CYP2C19*2 allele was associated with risk of hyperlipidemia (P = 0.027, odds ratio = 2.036; 95% confidence interval = 1.09-3.82). General linear model analysis showed that among our general research population, both CYP2C9 and CYP2C19 had no effect on hyperlipidemia independently, but the interaction between CYP2C9 and CYP2C19 and hyperlipidemia was statistically significant (F = 2.682, P = 0.031). Our study suggests that the association of CYP2C19*2 with hyperlipidemia was specific among patients with the wild-type CYP2C9.

Mean Stable Warfarin Doses Versus CYP2C9*2 and VKORC11639G&gt;A Genotypes in Sudanese Population

Warfarin is a potent anticoagulant with a confirmed effectiveness when anticoagulation targets are attained, an issue, that is troublesome to reach due to the fact that warfarin has a narrow therapeutic index (NTI), that means they have a narrow window between their effective doses and those at which they produce adverse toxic effects. However, oral anticoagulation throughout genetics recommended a genotype guided dosing, but is it favourable over clinical based dosing? Objectives: To analyze the mean stable warfarin doses attained clinically within CYP2C9*2 and VKORC11639G&amp;gt;A wild-type and variant genotype status in Sudanese patients. Method: Genotyping for the CYP2C9*2 and VKORC1-1639G&amp;gt;A polymorphisms were accomplished with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique. The mean stable warfarin dose per genotype was defined as the mean stable warfarin dose related to the stable INR within target range within a genotype of each of CYP2C9*2 or VKORC11639G&amp;gt; Agenes, using Analysis of Variance (ANOVA) as the statistical method. Results: Fifty-three stable patients with wild-type CYP2C9*1*1 genotype had a mean stable warfarin dose of 4.9 ± 2.1 mg, 5 patients who were heterozygous CYP2C9*1*2 genotype, had a mean stable warfarin dose of 5.0 ± 0.71mg, and 2 patients were homozygous mutant CYP2CP*2*2 genotype had a mean stable warfarin dose of 4.8 ± 0.3 mg. The results were statistically insignificant, P = 0.992.Sixteen unstable patients were of wild-type CYP2C9*1*1 genotype, 40 patients with heterozygous CYP2C9*1*2 genotype, and 4 with homozygous mutant CYP2CP*2*2 genotype, had mean stable doses of 5.32 ± 2.9, 6.5 ± 2.7 and 4.5 ± 0.71 mg respectively. The result was statistically insignificant, P = 0.508. Fifty-two stable patients were having wild-type VKORC1G/G genotype, 3 patients had heterozygous VKORC1G/A genotype and 3 patients had homozygous mutant VKORC1/AA genotype, these patients had mean stable doses of 5.41 ± 1.63, 4.8 ± 2.19 and 5.4 ± 0.99 mg respectively. The mean warfarin stable dose among homozygous mutant VKORC1A/A genotype was lower than among wild-type and heterozygous genotype profiles. This result was statistically not significant, P = 0.729.In the unstable group, 8 patients of wild-type VKORC1G/G genotype, had a mean stable warfarin dose of 7.34 ± 3.9 mg, 40 patients of heterozygous VKORC1G/A genotype had a mean stable warfarin dose of 5.21 ± 2.76 mg, and 10 patients of homozygous VKORC1A/A genotype had a mean stable warfarin dose of 4.3 ± 1.83 mg. The result was statistically insignificant, P = 0.067.Conclusion: In our study, as there were no significant differences between warfarin mean stable doses related to different CYP2C9*2 and VKORC11639G&amp;gt;A genotypes, the evidence is not satisfactory to conclude that the conventional use of genotype guided warfarin dosing will correct stable warfarin dose among Sudanese patients.

Mean Stable Warfarin Doses Versus CYP2C9*2 and VKORC11639G&gt;A Genotypes in Sudanese Population

Warfarin is a potent anticoagulant with a confirmed effectiveness when anticoagulation targets are attained, an issue, that is troublesome to reach due to the fact that warfarin has a narrow therapeutic index (NTI), that means they have a narrow window between their effective doses and those at which they produce adverse toxic effects. However, oral anticoagulation throughout genetics recommended a genotype guided dosing, but is it favourable over clinical based dosing? Objectives: To analyze the mean stable warfarin doses attained clinically within CYP2C9*2 and VKORC11639G&amp;gt;A wild-type and variant genotype status in Sudanese patients. Method: Genotyping for the CYP2C9*2 and VKORC1-1639G&amp;gt;A polymorphisms were accomplished with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique. The mean stable warfarin dose per genotype was defined as the mean stable warfarin dose related to the stable INR within target range within a genotype of each of CYP2C9*2 or VKORC11639G&amp;gt; Agenes, using Analysis of Variance (ANOVA) as the statistical method. Results: Fifty-three stable patients with wild-type CYP2C9*1*1 genotype had a mean stable warfarin dose of 4.9 ± 2.1 mg, 5 patients who were heterozygous CYP2C9*1*2 genotype, had a mean stable warfarin dose of 5.0 ± 0.71mg, and 2 patients were homozygous mutant CYP2CP*2*2 genotype had a mean stable warfarin dose of 4.8 ± 0.3 mg. The results were statistically insignificant, P = 0.992.Sixteen unstable patients were of wild-type CYP2C9*1*1 genotype, 40 patients with heterozygous CYP2C9*1*2 genotype, and 4 with homozygous mutant CYP2CP*2*2 genotype, had mean stable doses of 5.32 ± 2.9, 6.5 ± 2.7 and 4.5 ± 0.71 mg respectively. The result was statistically insignificant, P = 0.508. Fifty-two stable patients were having wild-type VKORC1G/G genotype, 3 patients had heterozygous VKORC1G/A genotype and 3 patients had homozygous mutant VKORC1/AA genotype, these patients had mean stable doses of 5.41 ± 1.63, 4.8 ± 2.19 and 5.4 ± 0.99 mg respectively. The mean warfarin stable dose among homozygous mutant VKORC1A/A genotype was lower than among wild-type and heterozygous genotype profiles. This result was statistically not significant, P = 0.729.In the unstable group, 8 patients of wild-type VKORC1G/G genotype, had a mean stable warfarin dose of 7.34 ± 3.9 mg, 40 patients of heterozygous VKORC1G/A genotype had a mean stable warfarin dose of 5.21 ± 2.76 mg, and 10 patients of homozygous VKORC1A/A genotype had a mean stable warfarin dose of 4.3 ± 1.83 mg. The result was statistically insignificant, P = 0.067.Conclusion: In our study, as there were no significant differences between warfarin mean stable doses related to different CYP2C9*2 and VKORC11639G&amp;gt;A genotypes, the evidence is not satisfactory to conclude that the conventional use of genotype guided warfarin dosing will correct stable warfarin dose among Sudanese patients.

Do Genetic Contributors to Warfarin Responsiveness or Common Thrombophilias Influence the Risk of Major Bleeding in Patients on Extended Duration Vitamin K Antagonist (VKA) for Venous Thromboembolic Disease?

Background: Recent studies have indicated that genetic factors such as polymorphisms in the CYP2C9 and the VKORC1 genes play an important role in vitamin K antagonist (VKA) response and perhaps bleeding. There are also data to suggest, especially for factor V Leiden, that thrombophilia may confer a decreased risk of bleeding (which could explain its persistence and high prevalence in the population). The influence of major genetic factors on risk of bleeding have not previously been evaluated in large prospective cohort studies of patients with VTE on extended (treatment after the first 3 to 6 months) VKA therapy.Aims: We sought to determine the effect of genetic variants that influence warfarin metabolism and thrombophilias on the rates of major bleeding during extended VKA therapy in patients with VTE disease.Methods: The Bleeding Risk study is a multicentre, multinational prospective cohort study of patients on extended VKA for unprovoked VTE, or provoked VTE with prior VTE. Patients were enrolled after at least 3 months of VKA. All major bleeding events during long term VKA were captured and adjudicated. A blood sample was taken from each patient at their first study visit and analysed in a central lab to identify the following genetic variant types: CYP2C9*2 (C/T), CYP2C9*3 (T/G), 1639G-A (G/A), Factor V Leiden (G/A: FVL), CYP4F2 (G/A), and Prothrombin gene 20210 variant (PGV). Rates of major bleeding were then evaluated for patients with each genetic variant in isolation and in combination.Results: 2290 of the 2514 patients enrolled at 12 sites have contributed over 7000 years of observation and were included in this analysis. The mean patient age was 60.2±14.7 years, 64% were male, 92% Caucasian, average BMI was 31.3, and 9% of patients were on antiplatelet agents. Patients were followed for a mean of 2.8 years (range, 0.1 to 6.8 years. 121 patients (4.8%) experienced at least one episode of major bleeding. The annual rate of bleeding was 1.7 per 100 patient-years of observation. The CYP2C9*2 variant heterozygous/homozygous versus wildtype (476 patients vs 1584) was protective against bleeding (Incidence risk ratio 0.5, p =0.01), CYP2C9*3 variant heterozygous/homozygous versus wildtype (215 patients vs 1845) was associated with major bleeding (Incidence risk ratio 2.0, p =0.01), VKORC1 and CYP4F2 had no association with major bleeding, and CYP2C9*3 variant heterozygous/homozygous in combination with wildtype CYP2C9*2 (186 patients) was associated with major bleeding (Incidence risk ratio 3.24, p =0.02). Both FVL (19% of patients) and PGV (8% of patients) had no effect on major bleeding, with p values > 0.35 for comparison of the wildtype to heterozygous/homozygous.Conclusion: This is the largest study we are aware of to determine if warfarin metabolic genotype variants and common thrombophilias influence major bleeding risk in patients followed long-term with extended duration VKA therapy for VTE. The thrombophilias do not influence bleeding risk but those with CYP2C9*3 hetero/homozygous inheritance had double the risk and CYP2C9*2 hetero/homozygous inheritance half the risk. There appears to be an interaction between the CYP2C9*2 and *3 genotypes. DisclosuresWells:BMS/Pfizer: Research Funding; Bayer Healthcare: Other: Speaker Fees and Advisory Board; Janssen Pharmaceuticals: Consultancy; Itreas: Other: Served on a Writing Committee. Kovacs:LEO Pharma: Honoraria; Bayer: Honoraria, Research Funding; Daiichi Sankyo Pharma: Research Funding; Pfizer: Honoraria, Research Funding. Anderson:Bayer Healthcare: Research Funding. Rodger:Canadian Agency for Drugs and Technologies in Health: Consultancy; Boehringer Ingelheim: Research Funding.

Quantitative Assessment of CYP2C9 Genetic Polymorphisms Effect on the Oral Clearance of S-Warfarin in Healthy Subjects.

Genetic polymorphisms in CYP2C9 account for 10-20% of the variability in warfarin dose requirement. As such CYP2C9 genetic polymorphisms are commonly included in algorithms aimed to optimize warfarin therapy as a way to account for variability in warfarin responsiveness that is due to altered pharmacokinetics. However, most of the currently available pharmacokinetic data were derived from studies among patients on chronic warfarin therapy and therefore suffer from the confounding effects of disease states and drug interactions. The purpose of the present study was to provide an accurate quantitative estimate of S-warfarin oral clearance (CLS) among healthy subjects carrying different CYP2C9 genotypes. Single dose of warfarin was administered to 150 non-smokers, age (mean±SD) 23.3±4.5years, 60% male, non-obese, healthy subjects. Blood samples were taken for up to 168h and urine was collected over the entire study period. Compared with carriers of the wild-type CYP2C9*1/*1 genotype (n=69), CLS was reduced by 25, 39 and 47% among heterozygote for CYP2C9*2 (n=41) CYP2C9*3 (n=26) and carriers of 2 variant alleles (n=14), respectively (p<0.001). The corresponding decrease in the formation clearance of 6 and 7 S-hydroxy-warfarin was 45, 65 and 75%, respectively (p<0.001). The current study provides an estimate concerning the effect of CYP2C9 polymorphisms on S-warfarin pharmacokinetics among healthy subjects. As such it is free of the confounding effects of disease states and drug interactions. Further research is needed to evaluate whether the incorporation of quantitative data obtained in the present study into pharmacogenetic warfarin algorithm may enhance its precision. Clinicaltrials.gov Identifier NCT00162474.

The Influence of CYP2C9 and VKORC1 Gene Polymorphisms on the Response to Warfarin in Egyptians

Warfarin is the most commonly used drug for chronic prevention of thromboembolic events, it also ranks high among drugs that cause serious adverse events. The variability in dose requirements has been attributed to inter-individual differences in medical, personal, and genetic factor. Cytochrome P-450 2C9 is the principle enzyme that terminates the anticoagulant effect of warfarin by catalyzing the conversion of the pharmacologically more potent S-enantiomer to its inactive metabolites. Warfarin exerts its effect by inhibition of vitamin K epoxide reductase. This protein is encoded by vitamin K epoxide reductase complex subunit 1 gene (VKORC1). The current study aimed to investigate the pharmacogenetic effect of CYP2C9 and VKORC1 gene polymorphisms on the patients response to warfarin. One hundred cases starting warfarin treatment and 20 healthy controls were enrolled. The mean daily dose of warfarin was calculated from patient's medical records. For each patient, less than 10% variability in warfarin dose and a target international normalized ratio (INR) within the therapeutic target range were required for at least 3months for one of the following indications (deep vein thrombosis, pulmonary embolism, cerebrovascular stroke and myocardial infarction) prior to inclusion in the study. Tetraprimer amplification refractory mutation system PCR was performed to determine CYP2C9*2, CYP2C9*3, and the VKORC1 1639 G>A genetic polymorphisms. Plasma warfarin determination was performed using rapid fluorometric assay. Plasma warfarin concentration ranged from 2.19 to 10.98μg/ml with a median 3.52μg/ml. Supratherpeutic INR was observed in 11% of the cases. Thromboembolic complications occurred in 7% of the cases and 8% of cases experienced major bleeding. High maintenance dose (>7mg/day) was associated with the combined non VKORC1*2 and homozygous wild type CYP2C9 (CYP2C9*1*1) alleles, while low maintenance dose was associated with the Variant (AG+AA)/Wild (*1/*1). (p value <0.001). CYP2C9 variant was a risk factor for supratherapeutic INR in the multivariate logistic regression model. Thromboembolic complication and incidence of supratherapeutic INR were observed in patients carrying combined VKORC1 Variant (AG+AA) and CYP2C9 Variant (*2/*3). Data from our study suggest that together with clinical factors, VKORC1 and CYP2C9 polymorphisms are important contributors to warfarin dosing and may help predict adverse effects in Egyptian patients.

The Effects of CYP2C19 genotype on the susceptibility for nephrosis in cardio-cerebral vascular disease treated by anticoagulation.

In recent years, the genetic factor has become one of the important predisposing factors of nephropathy susceptibility. There is a high incidence of nephropathy in CCVd. The CYP2C19 enzyme metabolizes most the drugs, including proton pump inhibitors commonly used medicines to treat CCVd, CYP2C19 genetic polymorphisms is association with multi-pathogenesis factors of nephropathy. The purpose of the study is to reveal the association between CYP2C19 genotype and the susceptibility of nephropathy in the CCVd patients. The study is composed of 623 samples from CCVd treated by anticoagulation. The patients were studied, including CCVd with hyperuricemia, coronary heart disease, diabetes, and other complication. Biochemical tests and CYP2C19 variants measurements were performed by the gene chip method. The association among CYP2C19 variants, complications, and nephropathy was analyzed in the CCVd. There is no correlation between nephropathy and complications in CCVd. In hyperuricemia, coronary heart disease and diabetes groups, the differences of renal function tests were significant between CYP2C19 mutant (P < 0.05). The nephropathy risk of wild genotype is 3.288 times higher than of mutation genotype in hyperuricemic group, 1.928 times higher than mutation genotype in coronary heart disease group, and 5.248 times higher than CYP2C19 mutation genotype in the diabetic group. There was significant correlation between the CYP2C19 wild type and the nephropathy susceptibility in CCVd patients. The CYP2C19 gene plays a potential maker to evaluate nephropathy in CCVd patients. We deduced that identification of CYP2C19 gene type may benefit for reducing and avoiding nephropathy caused by abnormal metabolism function in CCVd patients.

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24.

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4'-hydroxylation activity of CYP2C19*23 (V max 111.5±16.0pmol/min/mg, K m 158.3±88.0μM) protein relative to CYP2C19 WT (V max 101.6+12.4pmol/min/mg, K m 123.0±19.2μM) protein had no significant difference. In contrast, the K m of CYP2C19*24 (270.1±57.2μM) increased significantly as compared to CYP2C19 WT (123.0±19.2μM) and V max of CYP2C19*24 (23.6±2.6pmol/min/mg) protein was significantly lower than that of the WT protein (101.6±12.4pmol/min/mg). In vitro intrinsic clearance (CLint=V max/K m) for CYP2C19*23 protein was 85.4% of that of CYP2C19 WT protein. The corresponding CLint value for CYP2C19*24 protein reduced to 11.0% of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K m of CYP2C19*23 (1911±244.73μM) was at least 100 times higher than that of CYP2C19 WT (18.37±1.64μM) and V max of CYP2C19*23 (3.87±0.74pmol/min/mg) dropped to 13.4% of the CYP2C19 WT (28.84±0.61pmol/min/mg) level. Derived from V max/K m, the CLint value of CYP2C19 WT was 785 folds of CYP2C19*23. K m and V max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5'-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.

Exome sequencing of extreme clopidogrel response phenotypes identifies B4GALT2 as a determinant of on-treatment platelet reactivity.

Interindividual variability in platelet aggregation is common among patients treated with clopidogrel and both high on-treatment platelet reactivity (HTPR) and low on-treatment platelet reactivity (LTPR) increase risks for adverse clinical outcomes. CYP2C19 influences clopidogrel response but only accounts for ∼12% of the variability in platelet reactivity. To identify novel variants implicated in on-treatment platelet reactivity, patients with coronary artery disease (CAD) with extreme pharmacodynamic responses to clopidogrel and wild-type CYP2C19 were subjected to exome sequencing. Candidate variants that clustered in the LTPR subgroup subsequently were genotyped across the discovery cohort (n = 636). Importantly, carriers of B4GALT2 c.909C>T had lower on-treatment P2Y12 reaction units (PRUs; P = 0.0077) and residual platelet aggregation (P = 0.0008) compared with noncarriers, which remained significant after adjusting for CYP2C19 and other clinical variables in both the discovery (P = 0.0298) and replication (n = 160; PRU: P = 0.0001) cohorts. B4GALT2 is a platelet-expressed galactosyltransferase, indicating that B4GALT2 c.909C>T may influence clopidogrel sensitivity through atypical cell-surface glycoprotein processing and platelet adhesion.

Functional Characterization of Human CYP2C9 Allelic Variants in COS-7 Cells.

Variability in activity of CYP2C9, which is involved in the metabolism of approximately 15% of current therapeutic drugs, is an important contributor to interindividual differences in drug response. To evaluate the functional alternations of CYP2C9*2, CYP2C9*3, CYP2C9*8, CYP2C9*11 and CYP2C9*31, identified in our previous study in Chinese Han population, allelic variants as well as the wild-type CYP2C9 were transiently expressed in COS-7 cells. Kinetic parameters (Km, Vmax, and Clint) for S-warfarin 7-hydroxylation by these recombinant CYP2C9s were determined. Relative to CYP2C9.1, recombinant CYP2C9.3 and CYP2C9.11 exhibited significantly higher Km values, and all allelic variants showed significantly decreased Vmax and Clint values. Among all allelic variants, catalytic activity of CYP2C9.3 and CYP2C9.11 reduced the most (8.2% and 9.8% of Clint ratio, respectively; P < 0.001). These findings should be useful for predicting the phenotype profiles of CYP2C9 in Chinese Han population, comparing the functional results of these alleles accurately, and finally optimizing pharmacotherapy of drug treatment.

Cyclophosphamide treatment-induced leukopenia rates in ANCA-associated vasculitis are influenced by variant CYP450 2C9 genotypes.

Correlation of outcomes of cyclophosphamide (CP) therapy in antineutrophil cytoplasmic antibody-associated vasculitis with genotype polymorphisms in prodrug activating cytochrome P450 enzyme genes CYP2C9 and CYP2C19. One hundred and ninety six patients with antineutrophil cytoplasmic antibody-associated vasculitis treated with CP, either as intravenous pulse or as daily oral medication, were included. Genotypes of CYP2C9 and CYP2C19 were correlated with clinical outcomes (leukopenia, infection, urotoxicity and treatment response). Sixty five (33.2%) patients had variant CYP2C9 and 55 (28.1%) had variant CYP2C19 genotype. In patients bearing variant CYP2C9, leukopenia was documented significantly more frequent than in carriers of wild-type CYP2C9 (55.4 vs 37.4%; odds ratio: 2.08; 95% CI: 1.14-3.80; p = 0.017). The impact of the CYP2C9 genotype was stronger in patients treated with oral CP (69.6 vs 45.6%; odds ratio: 2.73; 95% CI: 1.27-5.89; p = 0.009), but was not present in patients treated with intravenous pulsed CP. We observed less refractory disease courses in patients with variant CYP2C9, not reaching statistical significance. Patients with variant CYP2C9 are at increased risk for cyclophosphamide-induced leukopenia but may have a better chance to respond to treatment.

Frequency of Cyp2c93 allele in patients with type 2 diabetes mellitus on sulfonylurea therapy

Background: Sulfonylureas are mainstay of pharmacotherapy for type 2 diabetes mellitus (T2DM). Individual variability exists in pharmacokinetic and pharmacodynamic responses adverse effects to sulfonylurea. Objective: To determine frequency of cytochrome P450 2C9 mutant allele CYP2C93*, in T2DM patients on sulfonylurea therapy, and to ascertain the frequency of adverse drug reactions (ADR) with respect to particular allelic distribution. Materials and Methods: A hospital-based prospective observational study was carried out in a tertiary-care teaching hospital. Study included 136 T2DM patients on sulfonylurea therapy (83 with ≥1 ADR and 53 without ADR). DNA was isolated from the blood samples taken from all 136 patients by DNA isolation. PCR-RFLP (restriction fragment length polymorphism) technique was used for detection of CYP2C93* (Ile359Leu) allele and the wild type allele CYP2C91* by digestion with restriction enzyme. Data were analyzed using Statistical Package for Social Survey (SPSS) for Windows, version 16.0 and Microsoft Excel to determine descriptive statistics. Result: Allele CYP2C93* was detected in 11 patients. All alleles negative for the nucleotide substitutions at position 42614 (*3) were presumed to be wild type CYP2C9*1. Among the patients with CYP2C93* allele 11 patients experienced hypoglycemia and one patient experienced acute visual disturbances. No CYP2C93* was detected in the subjects without ADR. Conclusion: In our study CYP2C93* was identified in 11 patients experiencing hypoglycaemia and in one patient experiencing acute visual disturbances. In view of the existence of such polymorphisms and its effects on sulfonylurea therapy further studies are required to assess the magnitude of such problems in T2DM.

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59.

CYP2C9, one of the most important drug-metabolizing enzymes, is responsible for metabolizing approximately 15% of clinically important drugs, including warfarin, diclofenac, and losartan. Similar to other CYP members, human CYP2C9 exhibits marked genetic polymorphisms among individuals of different ethnicities. In this study, a novel missense mutation (1300A>T) was identified in a warfarin-sensitive patient after a genetic screen of three candidate genes related to high variability in response to warfarin doses. This base transversion leads to an Ile-to-Phe amino acid substitution at codon 434 within the CYP2C9 protein, and this new variant has been named a novel allele, CYP2C9*59, by the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki.se/cyp2c9.htm). The exogenous expression of CYP2C9.59 in insect cell microsomes revealed that, despite a similar protein expression level as wild-type CYP2C9, variant CYP2C9.59 exhibited significantly reduced maximal velocity, Vmax, and/or increased Michaelis constant, Km, values toward three CYP2C9-specific substrates. Our data suggest that the 1300A>T mutation can greatly decrease the enzymatic activity of the CYP2C9 protein both in vitro and in vivo.

Effect of CYP2C9 Genetic Polymorphism in a Chinese Population on the Metabolism of Mestranol in vitro

Background: Mestranol is a widely used estrogen, which is converted into its active metabolite ethinyl estradiol by cytochrome P450 (CYP) 2C9. To comprehensively examine the enzymatic activity of reported CYP2C9 variants in Chinese individuals in response to mestranol, wild-type CYP2C9*1 and 35 allelic variants were highly expressed in Sf21 insect cell microsomes and used for the detection of their enzymatic values in vitro. These results showed that the majority of tested variants exhibited decreased clearance values compared to wild type, except for CYP2C9*40 and *36. Method: Insect microsomes expressing the 36 CYP2C9 variants were incubated with 0.25-8 μmol/l mestranol for 30 min at 37°C. Then, the production of the metabolite of mestranol, ethinyl estradiol, was analyzed using high-performance liquid chromatography. Results: Most CYP-catalyzed reactions were sufficiently described by classical Michaelis-Menten kinetic parameters (e.g., K_m and V_max), while 9 variants exhibited atypical or non-Michaelis-Menten kinetic values, which were largely due to the self-inhibitory effect in response to mestranol. Conclusion: This is the first report of these rare alleles for mestranol metabolism, which provides fundamental data for further clinical studies on CYP2C9 alleles for mestranol metabolism.

EFFECT OF CYTOCHROME P450 2C9 GENE POLYMORPHISMS ON INDIVIDUAL SENSITIVITY TO GLICLAZIDE IN PATIENTS WITH TYPE 2 DIABETES MELLITUS

Cytochrome P450 2C9, encoded by the gene CYP2C9 , is a key enzyme for the metabolism of most sulfonylurea (SU). It is known that the polymorphisms CYP2C9*2 and CYP2C9*3 are associated with decreased activity of cytochrome, which leads to an increase in plasma concentrations of SU and reduction in its clearance. The aim of the study was to assess the impact CYP2C9 polymorphisms on individual sensitivity to gliclazide in patients with type 2 diabetes. The study included 74 patients with newly diagnosed diabetes. For all patients gliclazide in a dose of 30 or 60 mg/day was prescribed. Dose titration was carried out for 6 months of observation. If necessary other glucose-lowering therapy (a combination of drugs or insulin) were prescribed. Following the results of study, all patients had achieved the target values of Hb A1c. In comparison with patients who had a polymorphic allele in the gene, patients with wild-type CYP2C9 had lower effective dose of gliclazide, more often required prescription of large (90-120 mg) doses of drug or other antihyperglycemic therapy. Conclusion: The presence of a polymorphic allele in the gene CYP2C9 in homo- or heterozygous state is associated with a decrease in the effective dose of gliclazide, used as monotherapy in patients with type 2 diabetes.