Search

Background: Clinical pharmacogenetics is a rapidly growing field that focuses on the study of genetic variations and their impact on drug metabolism, efficacy, and safety. Angiotensin II receptor blockers (ARBs) are commonly used to treat hypertension in Iraq but not all patients respond equally to these drugs. Aim: This article aims to review the current evidence on the clinical pharmacogenetics of ARBs in Iraq and its implications for personalized medicine. Materials and Methods: We conducted a literature review of studies on the genetic variations that affect the response to ARBs in Iraq. We also reviewed the prevalence of these genetic variants in the Iraqi population and discussed the potential clinical implications for personalized medicine. Results: The most studied genetic variations associated with ARB response in Iraq are the angiotensin-converting enzyme gene insertion/deletion polymorphism and the angiotensin II type 1 receptor gene A1166C polymorphism. The angiotensin-converting enzyme gene insertion/deletion polymorphism is associated with variability in response to ARBs, while the angiotensin II type 1 receptor A1166C polymorphism is associated with an increased risk of cardiovascular events in patients treated with ARBs. The prevalence of these genetic variants in the Iraqi population varies widely depending on the region and ethnic group. Conclusion: The clinical pharmacogenetics of ARBs in Iraq suggests that pharmacogenetic testing could improve the selection and dosing of ARBs in Iraqi patients, leading to better patient outcomes and cost-effective healthcare.

Background: Clinical pharmacogenetics is a rapidly growing field that focuses on the study of genetic variations and their impact on drug metabolism, efficacy, and safety. Angiotensin II receptor blockers (ARBs) are commonly used to treat hypertension in Iraq but not all patients respond equally to these drugs. Aim: This article aims to review the current evidence on the clinical pharmacogenetics of ARBs in Iraq and its implications for personalized medicine. Materials and Methods: We conducted a literature review of studies on the genetic variations that affect the response to ARBs in Iraq. We also reviewed the prevalence of these genetic variants in the Iraqi population and discussed the potential clinical implications for personalized medicine. Results: The most studied genetic variations associated with ARB response in Iraq are the angiotensin-converting enzyme gene insertion/deletion polymorphism and the angiotensin II type 1 receptor gene A1166C polymorphism. The angiotensin-converting enzyme gene insertion/deletion polymorphism is associated with variability in response to ARBs, while the angiotensin II type 1 receptor A1166C polymorphism is associated with an increased risk of cardiovascular events in patients treated with ARBs. The prevalence of these genetic variants in the Iraqi population varies widely depending on the region and ethnic group. Conclusion: The clinical pharmacogenetics of ARBs in Iraq suggests that pharmacogenetic testing could improve the selection and dosing of ARBs in Iraqi patients, leading to better patient outcomes and cost-effective healthcare. [ABSTRACT FROM AUTHOR]

The human cytochrome P450 (CYP) 1, 2 and 3 families of enzymes are responsible for the biotransformation of a majority of the currently available pharmaceutical drugs. The highly polymorphic CYP2C9 predominantly metabolizes many drugs including anticoagulant S-warfarin, anti-hypertensive losartan, anti-diabetic tolbutamide, analgesic ibuprofen, etc. There are >80 single nucleotide changes identified in CYP2C9, many of which significantly alter the clearance of important drugs. Here we report the structural and biophysical analysis of two polymorphic variants, CYP2C9*14 (Arg125His) and CYP2C9*27 (Arg150Leu) complexed with losartan. The X-ray crystal structures of the CYP2C9*14 and *27 illustrate the binding of two losartan molecules, one in the active site near heme and another on the periphery. Both losartan molecules are bound in an identical conformation to that observed in the previously solved CYP2C9 wild-type complex, however, the number of losartan differs from the wild-type structure, which showed binding of three molecules. Additionally, isothermal titration calorimetry experiments reveal a lower binding affinity of losartan with *14 and *27 variants when compared to the wild-type. Overall, the results provide new insights into the effects of these genetic polymorphisms and suggests a possible mechanism contributing to reduced metabolic activity in patients carrying these alleles. Structural overlay of human cytochrome P450 (CYP) 2C9*14 (Yellow) and *27 (Green) in complex with losartan. One losartan is bound in the active site and another on the surface shown in stick representation. The Arg125His variation in CYP2C9*14, and Arg150Leu in CYP2C9*27 are represented as sticks and marked with a circle. [Display omitted] • The X-ray structures of cytochrome P450 (CYP) 2C9*14 and *27 variants were solved in complex with the drug losartan. • Isothermal titration calorimetry showed reduced affinity of losartan than the wild-type enzyme. • The results yield insight into the effect of genetic variation on losartan binding. [ABSTRACT FROM AUTHOR]

Purpose: S-warfarin is used to phenotype cytochrome P450 (CYP) 2C9 activity. This study evaluated S-warfarin limited sampling strategy with a population pharmacokinetic (PK) approach to estimate CYP2C9 activity in healthy adults. Methods: In 6 previously published studies, a single oral dose of warfarin 10 mg was administered alone or with a CYP2C9 inducer to 100 healthy adults. S-warfarin concentrations were obtained from adults during conditions when subjects were not on any prescribed medications. A population PK model was developed using non-linear mixed effects modeling. Limited sampling models (LSMs) using single- or 2-timepoint concentrations were compared with full PK profiles from intense sampling using empiric Bayesian post hoc estimations of S-warfarin AUC derived from the population PK model. Preset criterion for LSM selection and validation were a correlation coefficient (R2) >0.9, relative percent mean prediction error (%MPE) >−5 to <5%, relative percent mean absolute error (%MAE) ≤ 10%, and relative percent root mean squared error (%RMSE) ≤ 15%. Results: S-warfarin concentrations (n=2540) were well described with a two-compartment model. Mean apparent oral clearance was 0.56 L/hr and volume of distribution was 35.5 L. Clearance decreased 33% with the CYP2C9 *3 allele and increased 42% with lopinavir/ritonavir co-administration. During CYP2C9 constitutive conditions, LSMs at 48 hr and at 72 hr as well as 2-timepoint LSMs were within acceptable limits for R2, %MPE, %MAE, and %RMSE. During CYP2C9 induction, S-warfarin LSMs had unacceptable %MPE, %MAE, and %RMSE. Conclusions: Phenotyping studies with S-warfarin in healthy subjects can utilize a single- and/or a 2-timepoint LSM with a population PK approach to estimate constitutive CYP2C9 activity. [ABSTRACT FROM AUTHOR]

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.

In Middle East, There is a little knowledge about the cytochrome P450s genotyping and there has not found any investigation about the CYP2C9 allelic diversity in Iraqi population nowadays .CYP2C9 genotypes were established in a total of 400 subjects of healthy Iraqis volunteers from different areas in Iraq (Arab,Kurd and Turkmen). After extraction of DNA from blood cells and polymerase chain reaction, SNP genotyping for CYP2C9 variants (2,3,4 and 5) was performed by using the primer extension KASP® genotyping assay. The agreement with “Hardy-Weinberg equilibrium” for detected the distribution of CYP2C9 genotype was proved by (X2 – test) .The CYP2C9*2, CYP2C9*3 and CYP2C9*5 alleles variant were identified in Iraqi population, whereas the CYP2C9*4 was absent, The results revealed that there was non- significant association observed in all CYP2C9 genotypes among Iraqi ethnic groups .then The CYP2C9 genotypes results in our study were compared to other populations and The results exhibited that the frequency of Iraqi population CYP2C9 variants were comparable with other countries and ethnic groups. This is the first report of CYP2C9 variants from Iraq; the results are assist as a database on CYP2C9 gene polymorphisms and clinical data baseline for dosing and avoiding adverse reactions of drugs metabolized by CYP2C9 in Iraqis patients. [ABSTRACT FROM AUTHOR]

The influence of vitamins with antioxidant properties (vitamins A, E, C), B vitamins (B1, B2, B6) and vitamin-like substances (coenzyme Q10, taurine and L-carnitine) on the enzymes of the first phase of xenobiotic metabolism - cytochromes P450 3A4 and P450 2C9 has been studied. The experiments with informed volunteers have shown that B vitamins can shorten the duration of nonsteroidal anti-inflammatory drug diclofenac therapy and reduce the daily need for it. The positive effect of B vitamins in reducing the pain syndrome, shortening the duration of therapy and reducing the need for daily intake of diclofenac. Pharmacodynamic and pharmacokinetic data have been confirmed by electrochemical tests of electrocatalytic activity of cytochrome P450 3A4 (CYP3A4). Electrochemical approach to the study of catalytic activity of cytochrome P450 and the impact of vitamins and natural compounds on electrocatalysis is an accurate and effective touch-sensitive method allowing to use low concentrations of protein at an electrode (10-15 mol/electrode), to conduct the analysis without using protein pairs (cytochrome B5, NADPH-dependent reductase) and to identify the interaction of drugs in preclinical studies. When comparing the influence of B vitamins (B1, B2, B6) in the same concentration (300 μM) according to the electrochemical analysis, riboflavin (vitamin B2) is most effectively inhibits the interaction of diclofenac with cytochrome P450 3A4. Vitamin-like substance taurine with antioxidant properties and antioxidant vitamins stimulated electrochemical reduction of cytochromes P450 3A4 and P450 2C9. The obtained data confirm that it is possible that the influence of vitamins on cytochromes P450 3A4 (CYP3A4) и P450 2C9 (CYP2C9) allows to regulate pharmacokinetic parameters and the pharmacodynamic effect intensity.

The majority of Mexican patients with diabetes mellitus type 2 (DMT2) (67.9-85.0%) are prescribed sulphonylureas (SUs), which are metabolized by cytochrome P450 2C9 (abbreviated as CYP2C9). SUs are a type of oral anti-diabetic compound which inhibit ATP-sensitive potassium channels, thus inducing glucose-independent insulin release by the β-pancreatic cells. The wide variability reported in SU responses has been attributed to the polymorphisms of CYP2C9. The present study aimed to describe CYP2C9 polymorphisms (*2, *3 and IVS8-109T) within a sample of Mexican patients with DMT2, while suggesting the potential clinical implications in terms of glibenclamide response variability. From a sample of 248 patients with DMT2 who initially consented to be studied, those ultimately included in the study were treated with glibenclamide (n=11), glibenclamide combined with metformin (n=112) or metformin (n=76), and were subsequently genotyped using a reverse transcription-quantitative polymerase chain reaction (PCR), end-point allelic discrimination and PCR amplifying enzymatic restriction fragment long polymorphism. Clinical data were gathered through medical record revision. The frequencies revealed were as follows: CYP2C9*1/*1, 87.5%; *1/*2, 6.5%; *1/*3, 5.2%; and CYP2C9, IVS8-109A>T, 16.1%. Glibenclamide significantly reduced the level of pre-prandial glucose (P<0.01) and the percentage of glycated hemoglobin (%HbA1c; P<0.01) for IVS8-109A>T compared with combined glibenclamide and metformin treatment. Concerning the various treatments with respect to the different genotypes, the percentages obtained were as follows: Glibenclamide A/A, HbA1c<6.5=33.3%; glibenclamide + metformin A/A, HbA1c<6.5=24.6%; glibenclamide A/T, HbA1c<6.5=33.3%; glibenclamide + metformin A/T, HbA1c<6.5=25%; glibenclamide T/T, HbA1c<6.5=100%; and glibenclamide + metformin T/T, HbA1c<6.5=12.5%. Altogether, these results revealed that, although genetically customized prescriptions remain a desirable goal to increase the chances of therapeutic success, within the studied population neither allelic variants nor dosages demonstrated a clear association with biomarker levels. A key limitation of the present study was the lack of ability to quantify either the plasma concentrations of SU or their metabolites; therefore, further, precise experimental and observational studies are required. [ABSTRACT FROM AUTHOR]

Aim - to assess the influence of genetic polymorphisms of CYP2C9 and VKORC1 on warfarin dosing in patients with permanent form of atrial fibrillation. Methods. The study included examination of 100 patients with coronary heart disease and permanent form of atrial fibrillation; mean age 60.5±5.8 years. Conclusion. In patients with permanent form of atrial fibrillation, genotypes CYP2C9*1/*3, TT and GG genotypes of VKORC1 are associated with low warfarin dose, compared to other genotypes. Genotype CYP2C9*1/*3 increases the risk of bleeding complication during warfarin therapy.

Background/Aims: Data from previous reports, addressing the significance of genotype-guided dosing of warfarin in Egyptian patients, are infrequent and controversial. This study is aimed at demonstrating the validity of genetic dosing algorithms in Egyptian patients on warfarin therapy. Methods: A total of 100 Egyptian patients on a stable maintenance daily dose of warfarin were enrolled. The predicted warfarin dose for each patient was calculated using the warfarin dosing table, the Gage and the International Warfarin Pharmacogenetics Consortium (IWPC) clinical algorithms and the Gage and the IWPC genetic algorithms and compared to the actual dose. The accuracy of warfarin dosing algorithms was assessed by using the linear regression analysis. Results: The most accurate model in predicting the ideal dose was the Gage genetic algorithm by R2 of 50.4% and the IWPC genetic algorithm by R2 of 42.3%, followed by the warfarin dosing table by R2 of 19.1%, and the Gage clinical algorithm by R2 of 18.9% and the least accurate was the IWPC clinical algorithm by R2 of 9.4%. Conclusions: The Gage ­genetic warfarin dosing algorithm is the best model that could be implemented in Egyptian patients starting warfarin therapy. [ABSTRACT FROM AUTHOR]

Abstract: Many dietary supplements are promoted to patients with osteoarthritis (OA) including the three naturally derived compounds, glucosamine, chondroitin and diacerein. Despite their wide spread use, research on interaction of these antiarthritic compounds with human hepatic cytochrome P450 (CYP) enzymes is limited. This study aimed to examine the modulatory effects of these compounds on CYP2C9, a major CYP isoform, using in vitro biochemical assay and in silico models. Utilizing valsartan hydroxylase assay as probe, all forms of glucosamine and chondroitin exhibited IC50 values beyond 1000 μM, indicating very weak potential in inhibiting CYP2C9. In silico docking postulated no interaction with CYP2C9 for chondroitin and weak bonding for glucosamine. On the other hand, diacerein exhibited mixed‐type inhibition with IC50 value of 32.23 μM and Ki value of 30.80 μM, indicating moderately weak inhibition. Diacerein's main metabolite, rhein, demonstrated the same mode of inhibition as diacerein but stronger potency, with IC50 of 6.08 μM and Ki of 1.16 μM. The docking of both compounds acquired lower CDOCKER interaction energy values, with interactions dominated by hydrogen and hydrophobic bondings. The ranking with respect to inhibition potency for the investigated compounds was generally the same in both in vitro enzyme assay and in silico modeling with order of potency being diacerein/rhein > various glucosamine/chondroitin forms. In vitro‐in vivo extrapolation of inhibition kinetics (using 1 + [I]/Ki ratio) demonstrated negligible potential of diacerein to cause interaction in vivo, whereas rhein was predicted to cause in vivo interaction, suggesting potential interaction risk with the CYP2C9 drug substrates. [ABSTRACT FROM AUTHOR]

Background:CYP2C9gene polymorphisms modulate inter-individual variations in the human body’s responses to various endogenous and exogenous drug substrates. To date, little is known about theCYP2C9gene polymorphisms among the aboriginal populations of the world, including those in Malaysia. Aim: To characterise and compare theCYP2C9polymorphisms (CYP2C9*2, CYP2C9*3, CYP2C9*4andCYP2C9*5) between one of Malaysia’s aboriginal populations, Jahai, with the national major ethnic, Malay. To also compare the allele frequencies from these two populations with available data of other aboriginal populations around the world. Subjects and methods: The extracted DNA of 155 Jahais and 183 Malays was genotyped forCYP2C9polymorphisms using a nested multiplex allele-specific polymerase chain reaction technique. The results were confirmed by DNA direct sequencing. Results: Genotyping results revealed thatCYP2C9*2, CYP2C9*4andCYP2C9*5were absent in Jahais, while only the latter two were absent in Malays. TheCYP2C9*3allelic frequency in Jahais was 36.2%, making them the most frequent carriers of the allele thus far reported in any ethnic group from Southeast Asia. Conclusions: The high frequency ofCYP2C9*3and the absence ofCYP2C9*2in Jahais suggest that genetic drift may be occurring in this ethnic group. This is the first study to determine theCYP2C9polymorphisms in an aboriginal population in Malaysia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective To develop and evaluate the warfarin-dosing algorithm based on cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) genotypes, age and height of patients with atrial fibrillation (AF). Methods A total of 122 AF patients were included and their genotypes of CYP2C9 (rs1057910) and VKORC1 (rs9923231) were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP). Multiple linear regression was performed to develop a warfarin dosing algorithm which was based on age, height, CYP2C9 and VKORC1 genotypes of AF patients. Another 60 AF patients were enrolled and randomly assigned into the control group (n = 30) and experimental group (n = 30). The experimental group received warfarin of " predicted warfarin dose" based on warfarin dosing algorithm, and the control group received warfarin of 2. 5 mg / d. Both groups received warfarin for 7 days and then were adjusted to INR until reaching the stable warfarin maintenance dose. The time to reach a stable warfarin maintenance dose was compared between the control group and experimental group. Results The warfarin-dosing = 0. 438- 0. 014 3 × age + 0. 014 9 × height-0. 324 × CYP2C9 × 3 + 0. 209 × VKORC1, R2= 0.564. During the 50 day follow-up, there were 26 patients in the control group and 19 patients in the experimental group who acquired warfarin maintenance dose, with significant difference (P<0.05). The mean time from initiation to warfarin maintenance dose was significantly shorted in the experimental group (25. 8 ± 1. 7 d) than that in the control group (33. 1 ± 1. 9 d), P<0.05. Conclusion A new warfarin-dosing algorithm is developed based on CYP2C9 and VKORC1 genotypes, age and height and it can shorten the time from initiation to warfarin maintenance dose in AF patients with warfarin therapy. [ABSTRACT FROM AUTHOR]

CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, and 57 cytochrome P450 2C9 alleles have been previously reported. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for 4′-hydroxyflurbiprofen were determined using recombinant human P450s CYP2C9 microsomes from insect cells Sf21 carrying wild-type CYP2C9*1 and other variants. The results showed that the enzyme activity of most of the variants decreased comparing with the wild type as the previous studies reported, while the enzyme activity of some of them increased, which were not in accordance with the previous researches. Of the 36 tested CYP2C9 allelic isoforms, two variants (CYP2C9*53 and CYP2C9*56) showed a higher intrinsic clearance value than the wild-type protein, especially for CYP2C9*56, exhibited much higher intrinsic clearance (197.3%) relative to wild-type CYP2C9*1, while the remaining 33 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.6 to 83.8%) compared to CYP2C9*1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used non-steroidal anti-inflammatory drug, flurbiprofen (FP). The results indicated that most of the tested rare alleles decreased the catalytic activity of CYP2C9 variants toward FP hydroxylation in vitro. This is the first report of all these rare alleles for FP metabolism providing fundamental data for further clinical studies on CYP2C9 alleles for FP metabolism in vivo. [ABSTRACT FROM AUTHOR]

Objectives The aim of this study is to establish the inhibitory effects of 14 commonly used complementary and alternative medicines ( CAM) on the metabolism of cytochrome P450 2C9 ( CYP2C9) substrates 7-methoxy-4-trifluoromethyl coumarine ( MFC) and tolbutamide. CYP2C9 is important for the metabolism of numerous drugs and inhibition of this enzyme by CAM could result in elevated plasma levels of drugs that are CYP2C9 substrates. Especially for anticancer drugs, which have a narrow therapeutic window, small changes in their plasma levels could easily result in clinically relevant toxicities. Methods The effects of CAM on CYP2C9-mediated metabolism of MFC were assessed in Supersomes, using the fluorometric CYP2C9 inhibition assay. In human liver microsomes ( HLM) the inhibition of CYP2C9-mediated metabolism of tolbutamide was determined, using liquid chromatography coupled to tandem mass spectrometry ( LC-MS/MS). Key findings The results indicated milk thistle as the most potent CYP2C9 inhibitor. For milk thistle, silybin (main constituent of milk thistle) was mainly responsible for the inhibition of CY2C9. Conclusions Milk thistle and green tea were confirmed as potent inhibitors of CYP2C9-mediated metabolism of multiple substrates in vitro. Clinical studies with milk thistle are recommended to establish the clinical relevance of the demonstrated CYP2C9 inhibition. [ABSTRACT FROM AUTHOR]

Abstract: Background: The study aimed at evaluating the contribution of genetic variations in the drug metabolizing enzyme, CYP2C9, and the influence of co-medication with the antiepileptic drug, phenytoin, to variability in acenocoumarol response, in patients with cerebral venous thrombosis (CVT). Methods: 476 acenocoumarol-treated CVT patients (153 males and 323 females) were genotyped for CYP2C9*2 and CYP2C9*3 polymorphisms by PCR-RFLP method. Mean acenocoumarol dose required for achieving and maintaining a stable international normalized ratio (INR) was calculated for different genotypes. The effect of co-administration with phenytoin was determined. Results: Genotype distributions of CYP2C9 were as follows: 83%CYP2C9*1/*1, 8.6%CYP2C9*1/*3, 5.9%CYP2C9*1/*2, 1.9%CYP2C9*3/*3, 0.4%CYP2C9*2/*3 and 0.2%CYP2C9*2/*2. During the initiation phase of anticoagulation the CYP2C9*2 allele was independently associated with low acenocoumarol dose requirement (Adjusted OR 5.38; 95%CI 1.65-17.49; p =0.005). Similarly, the adjusted odds ratio for requiring a low dose during the induction phase in patients bearing the CYP2C9*3 allele was 12.79 (95%CI 4.74-34.57; p <0.0001). During the maintenance phase, CYP2C9*2 and CYP2C9*3 alleles were associated with 19-fold (Adjusted OR 19.67; 95%CI 2.46-157.19; p =0.005) and 11.9-fold odds (Adjusted OR 11.98; 95%CI 2.61-55.08; p =0.001) of requiring a low dose. Clinical covariates such as age, alcohol consumption, postpartum state and oral contraceptive intake also influenced acenocoumarol dosage. Co-medication with phenytoin was associated with lower dose requirement across genotypes during the initiation phase. However, during the maintenance phase, phenytoin-treated patients of all genotypes required higher doses of acenocoumarol. Conclusion: This study emphasizes the fact that polymorphisms in CYP2C9 gene and co-medication with phenytoin alter the anticoagulant effect of acenocoumarol. [Copyright &y& Elsevier]

Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly(methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: (1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and (2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process. [ABSTRACT FROM AUTHOR]

Abstract: Electrochemical behavior of human cytochrome P450 2C9 (CYP2C9) incorporated in polyacrylamide (PAM) hydrogel films cast on glassy carbon electrodes (GCE) was investigated. CYP2C9–PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks. The electron-transfer rate constant was about 6.96s−1 in pH 7.0 buffers, and the formal potential (E 0′) was −0.427V (vs. SCE). The dependence of E 0′ on solution pH indicated that one-proton transfer was coupled to each electron transfer in the direct electron-transfer reaction. UV–vis absorption spectroscopy demonstrated that CYP2C9 retained a near native conformation in PAM films at medium pH. The CYP2C9 in the PAM film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of its substrate bisphenol A (BPA) to the air-saturated solution, the reduction peak current of dissolved oxygen increased, which indicates the catalytic behavior of CYP2C9 to BPA. By amperometry a calibration linear range for BPA was obtained to be 1.25–10.0μM with a sensitivity of 18.21μAmM−1. And the apparent Michaelis–Menten constant for the electrocatalytic activity of CYP2C9 was estimated to be 3.90μM for BPA. [Copyright &y& Elsevier]

Abstract: The anti-inflammatory drug ibuprofen (Ibu) is metabolized in the human liver to a number of metabolites including 1-hydroxyibuprofen (1-OH-Ibu), 2-OH-Ibu, and 3-OH-Ibu, respectively. The only human CYP known to produce relevant amounts of 3-OH-Ibu is CYP2C9 and as genetic polymorphisms of CYP2C9 influence the metabolization of numerous drugs, the availability of reference standards for CYP2C9-specific metabolites is of considerable interest. The aim of this study was to develop a biological production process for 3-OH-Ibu and to affirm its NMR characteristics. The recombinant fission yeast strain CAD68 coexpressing human CYP2C9 and CPR was used for the whole-cell biotransformation of Ibu to 3-OH-Ibu in 1L batch-scale for 75h. The average space-time yield for the bioproduction of 3-OH-Ibu (125±34μmol/Ld) considerably exceeded that of 2-OH-Ibu (44±10μmol/Ld). Accordingly, average biotransformation activities normalized to dry biomass weight were 5.0±0.8μmol/gd (3-OH-Ibu) and 1.9±0.7μmol/gd (2-OH-Ibu). The metabolite was prepurified on preparative TLC-plates, isolated by HPLC fractionation, and characterized by LC–MS and NMR. As expected, differential fragmentation patterns of 2-OH-Ibu and 3-OH-Ibu were detected in ESI-LC–MS analysis. 44mg of 3-OH-Ibu was efficiently purified from four 1L batch cultures and its structure was clearly confirmed by one- and two-dimensional NMR. [Copyright &y& Elsevier]

Abstract: While Hispanics are the largest and most rapidly growing minority population in the United States, they are underrepresented in pharmacogenomic studies with warfarin. We sought to determine the combination of clinical and genetic influences of warfarin dose requirements in Hispanics. In addition, we tested the performance of published warfarin dosing algorithms derived from largely non-Hispanic cohorts in an inner-city U.S. Hispanic population. Genetic samples and clinical data were obtained from 50 Hispanics on a stable dose of warfarin. The contribution of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex-1 (VKORC1) genotypes and clinical factors to warfarin dose requirements was determined. The correlation between the predicted dose using published algorithms and therapeutic dose was also assessed. Compared to the VKORC1-1639 GG genotype, warfarin dose requirements were 30% and 62% lower with the GA and AA genotypes, respectively (p =0.001). The combination of the VKORC1-1639G>A and CYP2C9 genotypes and clinical factors explained 56% of the inter-patient variability in warfarin dose. Warfarin dose predicted using algorithms derived from mostly non-Hispanic cohorts was significantly correlated with the therapeutic dose in our Hispanic cohort (r 2 =0.43 to 0.49; p <0.001); the predicted dose was within 1.0 mg/day of the therapeutic dose for 40% to 50% of patients. Our data suggest that factors influencing warfarin dose requirements in Hispanic Caucasians are similar to those previously described in European Caucasians and that dosing algorithms derived from non-Hispanic Caucasian cohorts are applicable to Hispanics living in the U.S. [Copyright &y& Elsevier]

Several reports suggest garlic supplements may inhibit the metabolism of cytochrome P450 (CYP) 2C9 and CYP3A4 substrates, such as warfarin and saquinavir. To characterize the effects of garlic extract on CYP2C9 and CYP3A4 enzyme activity immortalized human hepatocytes (Fa2N-4 cells) were exposed to garlic extract (0-200 μg/mL). CYP2C9 and CYP3A4 enzyme activities were evaluated in parallel with enzymatic activities, expression of respective RNA transcripts was also assessed. Exposure to increasing concentrations of garlic extract led to progressive reduction in Fa2N-4 CYP2C9 activity as detected by diclofenac hydroxylation. CYP2C9 mRNA expression also revealed a concentration-dependent reduction. Greater than 90% reduction in CYP2C9 activity was observed following four days of exposure to 50 μg/mL garlic extract. In contrast, exposure to garlic extract had no effect on the CYP3A4 enzymatic activity or RNA transcript concentration in Fa2N-4. Therefore, suppression of CYP2C9 expression and activity is a heretofore unrecognized mechanism by which garlic extract may modulate CYP activity. Exposure of hepatocytes to garlic extract may reduce the expression and activity of CYP2C9 with no detectible effects on CYP3A4. [ABSTRACT FROM AUTHOR]

Cytochrome P450 2C9 (CYP2C9) enzymes metabolize warfarin and arachidonic acid. We hypothesized that the CYP2C9*2 (rs.1799853) and CYP2C9*3 (rs.1057910) polymorphisms with decreased enzyme activity affect risk of subclinical atherosclerosis (reduced ankle brachial index and increased C-reactive protein), ischemic vascular diseases (ischemic heart disease, myocardial infarction, ischemic cerebrovascular disease and ischemic stroke) and death after an ischemic heart disease diagnosis. We genotyped the Copenhagen City Heart Study, a prospective study including 10 398 participants with 30–32 years of follow-up; the Copenhagen General Population Study, a cross-sectional study including 21 629 participants; and the Copenhagen Ischemic Heart Disease Study, a case–control study including 5082 cases and 14 904 controls. CYP2C9 carriers versus noncarriers did not associate with subclinical atherosclerosis. Furthermore, the odds/hazard ratios for ischemic vascular disease did not differ from 1.0 for CYP2C9 carriers versus noncarriers. Finally, we found no altered risk of early death after a diagnosis of ischemic heart disease. For all end points, we could exclude even minor changes in risk of disease with 90% power. In conclusion, in three independent studies totaling more than 52 000 individuals, we found no association between CYP2C9*2 and CYP2C9*3 polymorphisms and risk of subclinical atherosclerosis, ischemic vascular disease or death after ischemic heart disease. [ABSTRACT FROM AUTHOR]

Losartan is used for anti-proteinuric as well as blood pressure effects in chronic kidney disease (CKD). It is metabolized by cytochrome P450 (CYP) 2C9 to active E-3174. Single nucleotide polymorphisms in CYP2C9 that reduce catalytic activity could reduce clinical benefits. The study aims were to determine whether CYP2C9 variant alleles ( *2 and *3) altered urinary protein excretion, glomerular filtration rate, and blood pressure in Caucasian patients prescribed losartan. Differences between baseline and 6-month follow-up outcomes were compared by CYP2C9 genotypes in 59 patients using unpaired t test or Mann–Whitney U test. Primary renal disease patients had a trend toward less favorable antiproteinuric response (−31.7 ± 156 vs. −125 ± 323%; p = 0.123) when carrying variant alleles. Patients with secondary renal diseases had less favorable diastolic blood pressure (9.8 ± 16.0 vs. −3.2 ± 10.6 mmHg; p = 0.043) and systolic blood pressure (16.2 ± 27.1 vs. −5.5 ± 17.5 mmHg; p = 0.044) with CYP2C9 variants. These preliminary results suggest a possible influence of CYP2C9 genotype on proteinuria and blood pressure in Caucasian CKD patients treated with losartan. [ABSTRACT FROM AUTHOR]

The electrochemical properties of cytochrome P450 2C9 (CYP2C9) and polymorphic modifications P450 2C9*2 (CYP2C9*2) and P450 2C9*3 (CYP2C9*3) were studied. To analyze the comparative electrochemical and electrocatalytic activity, the enzymes were immobilized on electrodes modified with a membrane-like synthetic surfactant (didodecyldimethylammonium bromide (DDAB)). An adequate choice of the type of modified electrode was confirmed by cyclic voltammetry of cytochromes P450 under anaerobic conditions, demonstrating well-defined peaks of reduction and oxidation of the heme iron. The midpoint potential, Emid, of cytochrome P450 2C9 is −0.318 ± 0.01 V, and Emid = −0.324 ± 0.01 V, and Emid = −0.318 ± 0.03 V for allelic variant 2C9*2 and allelic variant 2C9*3, respectively. In the presence of substrate diclofenac under aerobic conditions, cytochrome P450 2C9 and its polymorphic modifications P450 2C9*2 and P450 2C9*3 exhibit catalytic properties. Stimulation of the metabolism of diclofenac by cytochrome P450 2C9 in the presence of antioxidant medications mexidol and taurine was shown. [ABSTRACT FROM AUTHOR]

C-reactive protein (CRP) is a nonspecific marker of inflammation linked to cardiovascular disease and possibly colon cancer. Polymorphisms in CRP have been associated with differential CRP concentrations among healthy adults, with some evidence for functional effects on CRP expression.A linkage disequilibrium-based tag single nucleotide polymorphism (SNP)-selection algorithm identified six tagSNPs for Europeans (-821A>G, -390C>T/A, 90A>T, 838G>C, 2043G>A, and 4363C>A), defining six haplotypes with more than 1% frequency. In a case-control study of adenomatous (n=491) or hyperplastic (n=184) polyps versus polyp-free controls (n=583) we investigated these SNPs in relation to colorectal polyp risk.Individuals with 838 GC or CC genotypes had a modestly, although not statistically significantly, increased risk of adenomas (odds ratio: 1.4 95% confidence interval: 0.9-2.1) and a nearly 2-fold increased risk of concurrent adenomas and hyperplastic polyps (odds ratio: 2.0 95% confidence interval: 1.1-3.6). Increased risk for concurrent adenomas and hyperplastic polyps was also observed for haplotype ACACAC. No other main associations were detected. Risk of adenomas associated with 2043G>A differed with nonsteroidal anti-inflammatory drug (NSAID) use. Among NSAID nonusers, there was a suggestion that the GA or AA genotypes were associated with decreased risk of adenomas; this was not seen among NSAID users (P interaction=0.03). We also observed interactions between UGT1A1 [TA](7) promoter repeat polymorphism and CRP tagSNPs -390C>T/A and 90A>T, in which only the homozygous variant CRP genotype was associated with increased risk of adenoma among those with the UGT1A1 6rpt/6rpt genotype (P interaction=0.02 and 0.04 for -390C>T/A and 90A>T, respectively).These results provide limited support for associations between genetic variation in CRP and colorectal polyp risk. The observed interactions should be evaluated further. [ABSTRACT FROM AUTHOR]

Oral anticoagulants (OA) are a leading cause of fatal haemorrhagic adverse events in relation with an important interindividual variability of response to these drugs. Besides several clinical factors, this interindividual variability of response to OA has a pharmacogenetic basis. Carriers of cytochrome P450 2C9 ( CYP2C9)-deficient alleles have a reduced clearance of warfarin and are exposed to dramatic overdoses in the first weeks of treatment. Genetic polymorphisms of vitamin K epoxide reductase ( VKORC1), the target of OA, identify patients with a high sensitivity to OA who are at risk of early overdose. Most pharmacogenetic evidence is presently restricted to warfarin. Several warfarin dosing algorithms have been constructed, adapted on CYP2C9 and VKORC1 genotypes and clinical factors, to predict the best dose for each patient. Carriers of one of allelic variant need a 20–30% reduction of warfarin dose. However, definite evidence concerning the usefulness of these algorithms in terms of reducing the frequency of major bleeding episodes is still lacking. Ongoing prospective randomised trials will ascertain definitive answer over the coming years. [ABSTRACT FROM AUTHOR]

Significant interest in the pharmacogenetics of warfarin therapy has been triggered with the recent package insert update that highlights the potential role of pharmacogenetics in improving the safety and effectiveness of warfarin. We review the evidence of the influence of the two key genes of interest, the cytochrome P450 2C9 gene, CYP2C9, and the vitamin K epoxide reductase complex 1 gene, VKORC1, on warfarin response and discuss the implications of current knowledge for clinical practice. The influence of CYP2C9 and VKORC1 genotypes on warfarin dose requirements has been consistently demonstrated in diverse racial and ethnic patient groups in observational studies and randomized clinical trials. Dosing algorithms have been developed that incorporate clinical, demographic, and genetic information to help select a warfarin starting dose. Furthermore, CYP2C9 variant genotypes have been associated with a significantly increased risk of serious bleeding events. However, evidence to date from prospective, controlled studies has not demonstrated an added benefit of incorporating genotype-guided therapy in improving anticoagulation control or in preventing or reducing the risk of hemorrhagic or thromboembolic complications. Research efforts designed to evaluate the effectiveness of genotype-guided therapy in improving outcomes are under way. However, the routine use of CYP2C9 and VKORC1 genotyping in the general patient population who begin warfarin therapy is not supported by evidence currently available. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome P450 2C9 (2C9) is one of the three major drug metabolizing cytochrome P450 enzymes in human liver. Although the crystal structure of 2C9 has been solved, the important physicochemical properties of substrate–enzyme interactions remain difficult to be determined. This is due in part to the conformational flexibility of mammalian P450 enzymes. Therefore, probing the active-site with high-affinity substrates is important in further understanding substrate–enzyme interactions. Three-dimensional quantitative structure–activity relationships (3D-QSAR) and docking experiments have been shown to be useful tools in correlating biological activity with structure. In particular we have previously reported that the very tight-binding inhibitor benzbromarone can provide important information about the active-site of 2C9. In this study we report the binding affinities and potential substrate–enzyme interactions of 4H-chromen-4-one analogs, which are structurally similar to benzbromarone. The chromenone structures are synthetically accessible inhibitors and give inhibition constants as low as 4.2nM, comparable with the very tightest-binding inhibitors of 2C9. Adding these compounds to our previous 2C9 libraries for CoMFA models reinforces the important electrostatic and hydrophobic features of substrate binding. These compounds have also been docked in the 2C9 crystal structure and the results indicate that Arg 108 plays significant roles in the binding of chromenone substrates. [Copyright &y& Elsevier]

Introduction: African-Americans are under-represented in studies assessing contributors to warfarin response. Our primary objective was to determine whether the genes for cytochrome P450 (CYP) 2C9, nicotinamide adenine dinucleotide phosphate, reduced, quinone oxidoreductase (NQO1) and vitamin K epoxide reductase complex subunit 1 (VKORC1) are associated with warfarin dose requirements in African-Americans. Patients and methods: The following factors were assessed: demographics; clinical data; the CYP2C9 Arg144Cys (*2), Ile358Leu (*3) and Asp360Glu (*5); NQO1 Pro187Ser (*1/*2); and VKORC1 G6853C genotypes were analyzed in 115 African-Americans on stable warfarin doses. Results: Allele frequencies were 0.05 for the CYP2C9 *2, *3 or *5 alleles; 0.20 for NQO1 *2; and 0.25 for VKORC1 6853C. Possession of a CYP2C9*2, *3 or *5 allele was associated with a 38% lower warfarin dose compared with the *1/*1 genotype (30 ± 13 vs 48 ± 18 mg/week; p = 0.003). Neither the NQO1 *1/*2 nor VKORC1 G6853C genotype was associated with warfarin dose requirements in the population as a whole or in CYP2C9*1 allele homozygotes. Multiple regression analysis revealed that CYP2C9 genotype (p = 0.015), age (p < 0.001) and body surface area (p < 0.001) were jointly associated with warfarin dose requirements, and together explained 33% of the variability in warfarin dose requirements among African-Americans. Discussion: Our data suggest that CYP2C9 genotype, age and body size are important determinants of warfarin dose requirements in African-Americans. Our data further suggest that the VKORC1 G6853C polymorphism alone may not be useful for predicting warfarin dose requirements in this racial group. [ABSTRACT FROM AUTHOR]

Introduction: Warfarin is a widely prescribed, efficacious oral anticoagulant. S-warfarin, the more active form, is metabolized by the cytochrome P450 (CYP)2C9 enzyme. The aim was to evaluate the influence of two CYP2C9 functional polymorphisms (*2 and *3) on warfarin dose in African-Americans, an unstudied population and Caucasians, and also to assess the effect of these polymorphisms on anticoagulation response after accounting for nongenetic factors and genetic factors that might also impact the dose-response relationship of warfarin. Patients and methods: A prospective cohort of 362 patients with a target international normalized ratio of between 2.0 and 3.0 were genotyped. Warfarin sensitivity stratified by genotype was investigated using univariate and multivariate analyses. Results: The maintenance dose of warfarin was significantly related to genotype (p < 0.01) (variant carriers: 31.25 mg/week; wild-type: 37.5 mg/week), even after adjustment for possible confounding factors (p = 0.046). However, the effect of genotype was restricted to Caucasians, in whom variant carriers had a significantly lower maintenance dose compared with wild-type homozygotes (unadjusted: p < 0.01; adjusted: p = 0.02). There was a greater risk of over-anticoagulation among Caucasian variant carriers, although this was only observed prior to reaching maintenance dose. For African-American variant carriers, there was no difference in warfarin response based on CYP2C9 genotype. Discussion: CYP2C9 *2 and *3 variants provide predictive information in anticoagulation response. However, these variants may not be useful in African-Americans or as a marker of long-term over-anticoagulation once a stable dose is reached. [ABSTRACT FROM AUTHOR]

Warfarin is the most commonly prescribed oral anticoagulant for the treatment and prevention of thromboembolic events. The correct maintenance dose of warfarin for a given patient is difficult to predict, the drug carries a high risk of toxicity, and variability among patients means that the safe dose range differs widely between individuals. Recent pharmacogenetic studies indicate that the routine incorporation of genetic testing into warfarin therapy protocols could substantially ease both the financial and health risks currently associated with this treatment. In particular, the variability in warfarin dose requirement is now recognized to be due, in large part, to polymorphisms in two genes: cytochrome P450 2C9 and the vitamin K epoxide reductase complex subunit 1. The development of algorithms that integrate all of the relevant genetic and physical factors into comprehensive, individualized predictive models for warfarin dose could be used to translate the results of pharmacogenetic testing into actionable clinical application. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome P450 2C9 (CYP2C9) plays a key role in the metabolism of clinical drugs. CYP2C9 is a genetically polymorphic enzyme and some of its allelic variants have less activity compared to the wild-type form. Drugs with a narrow therapeutic index may cause serious toxicity to the individuals who carry such allele. CYP2C9*13, firstly identified by some of the present authors in a Chinese poor metabolizer of lornoxicam, is characterized by mutation encoding Leu90Pro substitution. Kinetic experiments show that CYP2C9*13 has less catalytic activity in elimination of diclofenac and lornoxicam in vitro. In order to explore the structure–activity relationship of CYP2C9*13, the three-dimensional structure models of the substrate-free CYP2C9*1 and its variant CYP2C9*13 are constructed on the basis of the X-ray crystal structure of human CYP2C9*1 (PDB code 1R9O) by molecular dynamics simulations. The structure change caused by Leu90Pro replacement is revealed and used to explain the dramatic decrease of the enzymatic activity in clearance of the two CYP2C9 substrates: diclofenac and lornoxicam. The trans configuration of the bond between Pro90 and Asp89 in CYP2C9*13 is firstly identified. The backbone of residues 106–108 in CYP2C9*13 turns over and their side chains block the entrance for substrates accessing so that the entrance of *13 shrinks greatly than that in the wild-type, which is believed to be the dominant mechanism of the catalytic activity reduction. Consequent docking study which is consistent with the results of the kinetic experiments by Guo et al. identifies the most important residues for enzyme–substrate complexes. [Copyright &y& Elsevier]

Warfarin is the most commonly used oral anticoagulant for treatment of thromboembolism, but adjustment of the dose appropriate to each patient is not so easy because of the large inter-individual variation in dose requirement. We analyzed single nucleotide polymorphism (SNP) genotypes of the VKORC1 and CYP2C9 genes using DNA from 828 Japanese patients treated with warfarin, and investigated association between SNP genotype and warfarin-maintenance dose. Five SNPs in VKORC1, 5′ flanking−1413A>G, intron 1−136T>C, intron 2+124C>G, intron 2+837T>C and exon 3 343G>A, were in absolute linkage disequilibrium, and showed a significant association with daily warfarin dose of these patients. The median warfarin dose of patients with homozygosity for the minor allele was 4.0 mg/day, which is significantly higher than those heterozygous for the minor allele (3.5 mg/day) or those homozygous for the major allele (2.5 mg/day; P=5.1×10−11 in the case of intron 1−136T>C SNP). We then genotyped the CYP2C9 gene for the Japanese common genetic variant, CYP2C9*3 and, based on the genotype of these two genes, classified patients into three categories, which we call “warfarin-responsive index.” The median warfarin daily dose varied significantly in this classification according to the warfarin-responsive index (2.0 mg/day for index 0 group, 2.5 mg/day for index 1 group, and 3.5 mg/day for index 2 group; P=4.4×10−13). Thus, analysis of the combination of VKORC1 and CYP2C9 genotypes should identify warfarin-sensitive patients who require a lower dose of drug, allowing personalized warfarin treatment. [ABSTRACT FROM AUTHOR]

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 K m but no change in V max with tolbutamide as the substrate, a five-fold increase in K m and an 88.8% reduction in V max 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 ( V max / K m ) is greater than that associated with CYP2C9*3. [ABSTRACT FROM AUTHOR]

Patients on warfarin anticoagulant therapy demonstrate wide variation in maintenance dose. Patients possessing variants (∗2 and ∗3) of the cytochrome P450 2C9 gene require reduced maintenance doses compared to those having wild-type alleles (∗1). Many other clinical factors have been shown to affect warfarin dose as well. To determine the relative impact of CYP2C9 genotype, age, gender, body surface area, concomitant medication, treatment indication and comorbidity, we conducted a retrospective cohort study in 453 patients managed by the anticoagulation service of a large, horizontally integrated, multispecialty group practice. In this largely Caucasian patient population, the CYP2C9 gene frequencies for ∗1/∗1, ∗1/∗2, ∗1/∗3, ∗2/∗2, ∗2/∗3 and ∗3/∗3 were 65.1%, 19.0%, 12.1%, 1.6%, 1.8% and 0.4%, respectively, approximating Hardy-Weinberg equilibrium. Mean maintenance doses for these genotypes were 36.5, 29.1, 23.5, 28.0, 18.1 and 5.5 mg/week, respectively. In univariate analyses, genotype alone accounted for 19.8% of the variability in maintenance dose. Age, body surface area and male gender accounted for 14.6%, 7.5% and 4.7%, respectively, while cardiac valve replacement as the indication for warfarin accounted for 5.4% of the variability. Collectively, these factors accounted for 33.7% of all dosing variability according to multiple regression. These results will help strengthen the mathematical models that are currently being developed for prospective gene-based warfarin dosing. [ABSTRACT FROM AUTHOR]

Objective. This in-vitro study aimed at an identification of cytochrome P450 (CYP) enzymes catalysing the (S)- and (R)-hydroxylation of the widely used anticoagulant phenprocoumon (PPC) to its major, inactive metabolites. Methods. Relevant catalysts were identified by kinetic, correlation and inhibition experiments using human liver microsomes and recombinant enzymes. Results. Kinetics revealed (S)-7-hydroxylation as quantitatively most important. Biphasic Eadie-Hofstee plots indicated more than one catalyst for the 4′-, 6- and 7-hydroxylation of both enantiomers with mean Km1 and Km2 of 144.5±34.9 and 10.0±6.49 µM, respectively. PPC hydroxylation rates were significantly correlated with CYP2C9 and CYP3A4 activity and expression analysing 11 different CYP-specific probes. Complete inhibition of PPC hydroxylation was achieved by combined addition of the CYP3A4-specific inhibitor triacetyloleandomycin (TAO) and a monoclonal, inhibitory antibody (mAb) directed against CYP2C8, 9, 18 and 19, except for the (R)-4′-hydroxylation that was, however, inhibited by ~80% using TAO alone. (S)-PPC hydroxylation was reduced by ~2/3 and ~1/3 using mAb2C8–9-18–19 and TAO, respectively, but (R)-6- and 7-hydroxylation by ~50% each. Experiments with mAbs directed against single CYP2C enzymes clearly indicated CYP2C9 as a major catalyst of the 6- and 7-hydroxylation for both enantiomers. However, CYP2C8 was equally important regarding the (S)-4′-hydroxylation. Recombinant CYP2C8 and CYP2C9 were high-affinity catalysts (Km <5 µM), whereas CYP3A4 operated with low affinity (Km >100 µM). Conclusion. CYP2C9 and CYP3A4 are major catalysts of (S)- and (R)-PPC hydroxylation, while CYP2C8 partly catalysed the (S)-4′-hydroxylation. Increased vigilance is warranted when PPC treatment is combined with substrates, inhibitors, or inducers of these enzymes. [ABSTRACT FROM AUTHOR]

Objectives. The hypoglycaemic drug tolbutamide is used for assessment of CYP2C9 activity in vivo. However, therapeutically active doses of 500 mg bear the risk of hypoglycaemia, and a tolbutamide-derived parameter based on a single plasma or urine concentration reflecting CYP2C9 activity accurately is lacking. Methods. We examined tolbutamide and its metabolites 4′-hydroxy-tolbutamide and carboxytolbutamide in plasma and urine of 26 healthy, male volunteers up to 24 h after intake of 125 mg tolbutamide using liquid chromatography–tandem mass spectrometry. CYP2C9 genotypes were determined by sequencing of exons 3 and 7. Raw plasma and urine data were compared with pharmacokinetic parameters, CYP2C9 genotypes, and data from a study in 23 volunteers with all six CYP2C9*1–*3 combinations who received 500 mg tolbutamide. Results. Plasma clearance and tolbutamide plasma concentrations 24 h after drug intake reflected the genotypes: 0.85 l/h and 1.70 µg/ml (95% confidence interval, CI, 0.80–0.89 l/h and 1.50–1.90 µg/ml) for CYP2C9*1 homozygotes (n=15), 0.77 l/h and 2.14 µg/ml (95%CI, 0.67–0.88 l/h and 1.64–2.63 µg/ml) for *1/*2 genotypes (n=7), 0.60 l/h and 3.13 µg/ml (95%CI, 0.58–0.62 l/h and 2.68–3.58 µg/ml) for *1/*3 genotypes (n=3), and 0.57 l/h and 3.27 µg/ml in the single *2/*2 carrier. Natural logarithms of tolbutamide plasma concentrations 24 h after intake correlated to plasma clearance (r2=0.84, P<0.0000001). This correlation was confirmed in the comparison data set (r2=0.97, P<0.0000001). Conclusions. A low dose of 125 mg tolbutamide can safely and accurately be used for CYP2C9 phenotyping. As a simple metric for CYP2C9 activity, we propose to determine tolbutamide in plasma 24 h after drug intake. [ABSTRACT FROM AUTHOR]