Genetic variation in the CYP1A1 gene is related to circulating PCB118 levels in a population-based sample.

Several of the polychlorinated biphenyls (PCBs), i.e. the dioxin-like PCBs, are known to induce the P450 enzymes CYP1A1, CYP1A2 and CYP1B1 by activating the aryl hydrocarbon receptor (Ah)-receptor. We evaluated if circulating levels of PCBs in a population sample were related to genetic variation in the genes encoding these CYPs. In the population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (1016 subjects all aged 70), 21 SNPs in the CYP1A1, CYP1A2 and CYP1B1 genes were genotyped. Sixteen PCB congeners were analysed by high-resolution chromatography coupled to high-resolution mass spectrometry (HRGC/ HRMS). Of the investigated relationships between SNPs in the CYP1A1, CYP1A2 and CYP1B1 and six PCBs (congeners 118, 126, 156, 169, 170 and 206) that captures >80% of the variation of all PCBs measured, only the relationship between CYP1A1 rs2470893 was significantly related to PCB118 levels following strict adjustment for multiple testing (p=0.00011). However, there were several additional SNPs in the CYP1A2 and CYP1B1 that showed nominally significant associations with PCB118 levels (p-values in the 0.003-0.05 range). Further, several SNPs in the CYP1B1 gene were related to both PCB156 and PCB206 with p-values in the 0.005-0.05 range. Very few associations with p<0.05 were seen for PCB126, PCB169 or PCB170. Genetic variation in the CYP1A1 was related to circulating PCB118 levels in the general elderly population. Genetic variation in CYP1A2 and CYP1B1 might also be associated with other PCBs.

Effect of CYP2C9*3 mutant variants on meloxicam pharmacokinetics in a healthy Chinese population.

The aim of this study was to investigate the effect of the CYP2C9*3 (CYP2C9 1075 A>C) polymorphism on meloxicam pharmacokinetics in a Chinese population. Twenty-four healthy volunteers were enrolled in this study. The pyrosequencing technique was used to identify polymorphisms of CYP2C9. The concentration of meloxicam in plasma was determined by a high-performance liquid chromatography assay with mass spectrographic analysis. The Drug and Statistics Software (DAS, version 2.0) was used for curve fitting and calculations of pharmacokinetic parameters. The effects of CYP2C9*3 variant genotypes on meloxicam pharmacokinetics were compared with those of the wild type genotype. Among the 24 volunteers, two AC heterozygotes were observed in the multi-dose group. CYP2C9*3 was found to play an important role in the metabolism of meloxicam by reducing its enzymatic activity. Therefore, results of this study provide helpful information regarding inter-individual pharmacokinetic variability in the Chinese population.

Impact of the proton pump inhibitors and CYP2C19*2 polymorphism on platelet response to clopidogrel as assessed by four platelet function assays.

BACKGROUND: Previous studies suggested a possible negative interference of proton pump inhibitors (PPIs) on clopidogrel's antiplatelet effect because of the competitive inhibition of the CYP 2C19 isoenzyme. Moreover, carriers of the loss-of-function allele of CYP2C19 polymorphism (CYP2C19*2) display significantly lower responses to clopidogrel. In this study, we investigated the association between CYP2C19*2 genotype, PPI intake and clopidogrel resistance in patients with coronary artery disease (CAD) and their effect on clinical outcome. METHODS: We recruited 95 patients with CAD receiving chronic clopidogrel therapy in combination with aspirin. Platelet reactivity was simultaneously assessed by INNOVANCE PFA-100 P2Y, ADP-induced light transmission aggregometry (LTA), flow-cytometric vasodilator-stimulated phosphoprotein (VASP)-phosphorylation assay and multiple electrode aggregometry (Multiplate). Cardiovascular outcomes were recorded during 1-year follow-up period. RESULTS: Only platelet reactivity assessed by measuring platelet phosphorylated-VASP demonstrated a significant higher platelet reactivity in carriers of CYP2C19*2 (p=0.023). The other methods displayed higher - but not statistically significant - platelet reactivity in patients carrying the CYP2C19*2 variant as compared with non-carriers. Patients on PPIs demonstrated almost similar suppression of platelet reactivity in comparison with those not treated with PPIs by all platelet function assays. In logistic regression analysis none of the platelet function assays measurements were related with clinical outcomes. Similarly neither CYP2C19*2 genetic variant nor PPI treatment were associated with adverse clinical events. CONCLUSIONS: PPI co-administration did not influence clopidogrel's antiplatelet effect on laboratory testing by all platelet function assays used. On the contrary, patients carrying CYP2C19*2 genotype had significantly higher residual platelet reactivity as estimated by VASP-phosphorylation assay.

The role of CYP2C8 genotypes in dose requirement and levels of everolimus after heart transplantation.

Everolimus is an immunosuppressive drug metabolized by enzymes of the CYP family. A common variant of the CYP2C8 gene, CYP2C8*3, results in strongly decreased CYP2C8 activity, but its role for the pharmacogenetics of everolimus remains unclear. Aim of the present study was to examine the role of CYP2C8 variants in everolimus dose and drug levels after heart transplantation. The present study comprised 30 patients with everolimus based maintenance therapy after heart transplantation. CYP2C8 genotypes were determined and correlated with clinical data. In all, 21 subjects carried the CYP2C8 *1/*1 genotype and 9 subjects carried the CYP2C8 *1/*3 genotype. Neither everolimus dose nor everolimus levels were associated with CYP2C8 genotype at any point of time (p < 0.05). During follow-up, graft rejection reactions were observed in two patients and infections were observed in seven patients. In one patient, type 2 diabetes was diagnosed during follow-up. None of these adverse events were significantly associated with CYP2C8 genotypes. We conclude that in adult patients after heart transplantation, CYP2C8 genotypes are not associated with dose requirements or levels of everolimus.

[Electrochemical sensor systems based on one dimensional (1D) nanostructures for analysis of bioaffinity interactions].

It was shown that modification of screen printed graphite electrodes with gold nanoparticles (AuNPs) decorated Pb nanowires (PbNWs) demonstrates the enhancement of sensor's analytical characteristics such as effective surface area, electro catalytic properties and heterogeneous electron transfer kinetics. The reason for such improvement may be the synergistic effect ofAuNPs and PbNWs. Nanowires ensembles on electrode surface were employed for the detection of hemeproteins cytochrome P450 2B4, cytochrome c, and cardiac myoglobin in human plasma. Composite materials based on nanoparticles with different dimentions (3D three dimensional gold nanoparticles and 1D one dimensional Pb nanowires make it possible to construct biosensors with low detection limit of proteins.

CHRNA5-A3-B4 genetic variants alter nicotine intake and interact with tobacco use to influence body weight in Alaska Native tobacco users.

BACKGROUND AND AIMS: Gene variants in CHRNA5-A3-B4, which encode for the α5, α3 and ß4 nicotinic receptor subunits, are associated with altered smoking behaviors in European Americans. Little is known about CHRNA5-A3-B4 and its association with smoking behaviors and weight in Alaska Native people, which is a population with high prevalence but low levels of tobacco consumption, extensive smokeless tobacco use and high rates of obesity. We investigated CHRNA5-A3-B4 haplotype structure and its association with nicotine intake and obesity in Alaska Native people. DESIGN, SETTING AND PARTICIPANTS: A cross-sectional study of 400 Alaska Native individuals, including 290 tobacco users. MEASUREMENTS: CHRNA5-A3-B4 genotype, body weight and tobacco consumption biomarkers such as plasma cotinine and urinary total nicotine equivalents (TNE). FINDINGS: Alaska Native people have a distinct CHRNA5-A3-B4 haplotype structure compared with European/African Americans. In 290 Alaska Native tobacco users the 'G' allele of rs578776, which tagged a 30 kb haplotype in CHRNA5-A3-B4, was prevalent (16%) and associated significantly with nicotine intake (20% higher plasma cotinine, P < 0.001, 16% higher TNE, P = 0.076), while rs16969968 was not associated with nicotine intake. Rs578776 acted in combination with CYP2A6, the main nicotine-metabolizing enzyme, to increase nicotine intake by 1.8-fold compared with the low-risk group (P < 0.001). Furthermore, rs2869950, a single nucleotide polymorphism 5' to CHRNB4, was associated significantly with increased body mass index (P < 0.01) in the tobacco users even after controlling for differences in nicotine intake (P < 0.01). CONCLUSIONS: Genetic variants in CHRNA5-A3-B4 alter nicotine intake and body mass index in a population of Alaska Native people, who have a distinct haplotype structure, smoking behaviors and prevalence of obesity.

Establishment of a CYP2C19 genotyping assay for clinical use.

Conversion of clopidogrel (Plavix) to its active metabolite is catalyzed largely by the P450 enzyme 2C19 (CYP2C19). Numerous allelic variants of CYP2C19 exist. The *1 allele is considered wild type, whereas the *2 and *3 alleles have no in vivo enzymatic activity. Conversely, the *17 allele has increased expression, resulting in increased clopidogrel activation. Poor metabolizers (*2/*2 and *2/*3 genotypes) experience higher rates of therapeutic failure. For this reason, we have validated a CYP2C19 genotyping assay for the *1, *2, *3, and *17 alleles. Genomic DNA extracted from 30 deidentified EDTA whole-blood samples from patients was analyzed at 2 independent facilities using specific TaqMan realtime polymerase chain reaction primers and probes. Concordant genotypes were generated on all samples tested. Of the 30 samples, 15 were CYP2C19*1/*1, 8 were CYP2C19*1/*17, 5 were CYP2C19*1/*2, and 2 were CYP2C19*2/*17. There were no CYP2C19*3 alleles or *2/*2 homozygous genotypes detected. This CYP2C19 genotyping assay is appropriate for clinical testing, demonstrating excellent interlaboratory concordance, enabling the selection of the most effective clopidogrel treatment regimen for patients undergoing percutaneous coronary intervention.

Cytochrome P450 2A isoenzymes in freshly prepared blood lymphocytes isolated from rats and validation as a biomarker for clinical studies in humans.

1. The present study aimed to identify the expression of carcinogen metabolizing cytochrome P4502A (CYP2A) isoenzymes in freshly prepared rat peripheral blood lymphocytes (PBL) isolated from adult rats and investigate similarities in the regulation of lymphocyte CYP2A-isoenzymes with the tissue enzyme. 2. qRT-PCR studies demonstrated significant constitutive mRNA expression of CYP2A-isoenzymes in PBL isolated from male and female rats which further increases significantly after pretreatment with nicotine or 3-methylcholanthrene (MC) indicating responsiveness of CYP2A-isoenzymes in PBL. This increase in the CYP2A expression was associated with an increase in the protein expression and CYP2A3-dependent coumarin hydroxylase (COH) activity in PBL. 3. Clinical studies further demonstrated significant increase in the expression of CYP2A6 and associated enzyme activity in PBL isolated from lung cancer patients. Our data thus provided evidence for similarities in the regulation of carcinogen metabolizing CYP2A-isoenzymes in PBL with the tissue enzymes. Further, responsiveness of blood CYP2A6 in human blood lymphocytes isolated from lung cancer patients has led us to suggest that associating expression profiles of CYP2A6 and other polycyclic aromatic hydrocarbons (PAH)-responsive CYPs in PBL with the genotyping data could lead to the development of a possible screen to monitor and predict environment-induced diseases and toxicity in humans.

Genetic polymorphisms and novel allelic variants of CYP2C19 in the Chinese Han population.

AIM: This study aims to systematically investigate the genetic polymorphisms of the CYP2C19 gene and provide accurate data of the allele distribution pattern in the Chinese Han population. MATERIALS & METHODS: We amplified all nine exons of the CYP2C19 gene in 2127 unrelated healthy Chinese Han subjects from two geographical locations (Zhejiang province, n = 1127; Hebei province, n = 1000), using direct sequencing. RESULTS: In total, six previously reported alleles were found in our study, in which two alleles CYP2C19*6 and CYP2C19*18 were reported for the first time in Chinese Han subjects. In addition, 35 novel variants were detected in the present work, which included 11 new named alleles, 12 nonsynonymous mutations and one insert variant. CONCLUSION: This study provides important data on the pattern of CYP2C19 polymorphisms in Chinese Han subjects, using the largest group of individuals. Furthermore, the study also detects the largest number of novel alleles in one population. These findings are of potential benefit to the development of personalized medicine for the Chinese Han population.

Retrospective evidence for clinical validity of expanded genetic model in warfarin dose optimization in a South Indian population.

AIM: To optimize warfarin dose in patients at risk for thrombotic events, we have recently developed a pharmacogenomic algorithm, which explained 44.9% of the variability in warfarin dose requirements using age, gender, BMI, vitamin K intake, CYP2C9 (*2 and *3) and VKORC1 (*3, *4 and -1639 G>A) as predictors. The aim of the current study is to develop an expanded genetic model that can explain greater percentage of warfarin variability and that has clinical validity. PATIENTS & METHODS: CYP2C9*8, CYP4F2 V433M, GGCX G8016A and thyroid status were added to an expanded genetic model (n = 243). RESULTS: The expanded genetic model explained 61% of the variability in warfarin dose requirements, has a prediction accuracy of ±11 mg/week and can differentiate warfarin sensitive and warfarin resistant groups efficiently (areas under receiver operating characteristic curves: 0.93 and 0.998, respectively; p < 0.0001). Higher percentage of International Normalized Ratios in therapeutic range (52.68 ± 4.21 vs 43.80 ± 2.27; p = 0.04) and prolonged time in therapeutic range (61.74 ± 3.18 vs 47.75 ± 5.77; p = 0.03) were observed in subjects with a prediction accuracy of <1 mg/day compared with subjects with prediction accuracy >1 mg/day. In the warfarin-resistant group, primary hypothyroidism was found to induce more resistance while in the warfarin-sensitive group, hyperthyroidism was found to increase sensitivity. CONCLUSION: The expanded genetic model explains greater variability in warfarin dose requirements and it prolongs time in therapeutic range and minimizes out-of-range International Normalized Ratios. Thyroid status also influences warfarin dose adjustments.

High on-treatment platelet reactivity by ADP and increased risk of MACE in good clopidogrel metabolizers.

High on-treatment platelet reactivity (HPR) by ADP, which primarily reflects the effect of thienopyridines, has been found to be an independent predictor of ischemic events in patients with acute coronary syndrome (ACS) on dual antiplatelet therapy. CYP2C19*2 is associated with HPR by ADP. The aim of our study was to evaluate if high on-clopidogrel platelet reactivity (HPR) by ADP is associated with an increased risk of major adverse coronary events (MACE) after ACS independent of CYP2C19*2 allele, i.e. whether genotyping patients for CYP2C19*2 polymorphism is sufficient to identify those to be switched to novel antiplatelets. A total of 1187 patients were included (CYP2C19 *1/*1 n = 892; *1/*2 n = 264; *2/*2 n = 31); 76 MACE (CV death and non-fatal MI) were recorded in non-carriers of CYP2C19*2 (8.5%) and 39 in carriers of CYP2C19*2 (13.2%). At the landmark analysis in the first 6 months, HPR by ADP and CYP2C19*2 allele were both significantly and independently associated with MACE [HPR by ADP: HR = 2.0 (95% CI 1.2-3.4), p = 0.01; CYP2C19*2 allele: HR = 2.3 (95% CI 1.3-3.9), p = 0.003]. At the land mark analysis from 7 to 12 months, only HPR by ADP remained significantly associated with the risk of MACE [HPR by ADP: HR = 2.7 (95% CI 1.4-5.3), p = 0.003; CYP2C19*2: HR = 0.8 (95% CI 0.2-1.1), p = ns]. CYP2C19*2 allele and HPR by ADP are both independently associated with an increased risk of MACE in the first 6 months after ACS. HPR by ADP is associated with an increased risk until 12 months of follow-up. Therefore, both phenotype and genotype are clinically relevant for the evaluation of the antiplatelet effect of clopidogrel and for the prognostic stratification of ACS patients.

Effect of a traditional Chinese medicine Liu Wei Di Huang Wan on the activities of CYP2C19, CYP2D6 and CYP3A4 in healthy volunteers.

Liu Wei Di Huang Wan (LDW), a well-known traditional Chinese medicine, is widely used for the treatment of various diseases in China. This study was designed to investigate the potential herb-drug interactions of LDW in healthy volunteers and attempted to ascertain whether the interaction might be affected by genotypes. We assessed the effect of LDW on the activities of CYP2C19, CYP2D6 and CYP3A4 in 12 Chinese healthy subjects in a single-center, controlled, non-blinded, two-way crossover clinical trial. The subject pool consisted of six extensive metabolizers with CYP2C19*1/*1 and six poor metabolizers with CYP2C19*2/*2. Placebo or 4.8 g LDW (12 pills, 0.2 g/pill, twice daily) was given to each participant for 14 continuous days with a wash-out period of 2 weeks after an oral administration of 30 mg omeprazole, 30 mg dextromethorphan hydrobromide and 7.5 mg midazolam. The activities of CYP2C19, CYP2D6 and CYP3A4 were ascertained by their respective plasma or urinary metabolic ratios on day 14 post-treatment. There is no difference in the activities of the three tested enzymes before or after a 14-day administration of LDW. LDW had no effect on the pharmacokinetic parameters of the substrates and their metabolites. A 14-day administration of LDW did not affect the activities of CYP2C19, CYP2D6 and CYP3A4. LDW is unlikely to cause pharmacokinetic interaction when it is combined with other medications predominantly metabolized by these enzymes.

Current challenges in personalizing warfarin therapy.

In an exciting era where novel oral anticoagulants, such as the factor Xa and direct thrombin inhibitors, are beginning to emerge as therapeutic options, the vitamin K antagonists (VKAs) such as warfarin, which have been in clinical use for over half a century, will remain an important part of the therapeutic landscape for the foreseeable future. The optimal effectiveness and safety of the VKAs is limited by significant inter- and intra- patient variability in dose response. As such, routine laboratory monitoring with subsequent dose adjustment to achieve and maintain an international normalized ratio (INR) that falls within a narrow therapeutic range is necessary; even with frequent INR monitoring, time in therapeutic range of VKAs is generally <60% in usual care settings. Yet, personalized approaches to warfarin therapy, such as the routine incorporation of pharmacogenetic data into dose selection and adjustment, the selective use of prescribed doses of vitamin K for those patients with unstable INRs, and integration of patient self-testing /self-management, has the potential to improve the safety, efficacy and ease of use of warfarin. To date, no randomized trials have proven the benefits of routine pharmacogenetic testing for warfarin initiation; however, pivotal trials are ongoing. Through further investigative work, allowing these personalized strategies to realize their full potential, warfarin may remain a preferred therapeutic oral anticoagulant for years to come.

Evidence for cytochrome P450 2B1/2B2 isoenzymes in freshly prepared peripheral blood lymphocytes.

Cytochrome P450 2B1 and 2B2, the major hepatic drug metabolizing enzymes belonging to CYP2 family and associated constitutive androstane receptor (CAR) were found to be expressed in peripheral blood lymphocytes (PBL) isolated from rats. As observed in liver, pretreatment of phenobarbital (PB) or phenytoin were found to increase the expression of CYP2B1, CYP2B2 and associated enzyme activity in PBL. Like in liver, blood lymphocyte CYP2B1/2B2 catalyzed the activity of 7-pentoxyresorufin O-dealkylase (PROD). The present data, demonstrating similarities in the regulation of blood lymphocyte CYP2B-isoenzymes with the liver enzymes, suggests that blood lymphocyte CYP2B-isoenzymes could be used as a biomarker to monitor tissue levels.

Evaluation of a microarray-based genotyping assay for the rapid detection of cytochrome P450 2C19 *2 and *3 polymorphisms from whole blood using nanoparticle probes.

Numerous drugs such as clopidogrel have been developed to reduce coagulation or inhibit platelet function. The hepatic cytochrome P450 (CYP) pathway is involved in the conversion of clopidogrel to its active metabolite. A recent black-box warning was included in the clopidogrel package insert indicating a significant clinical link between specific CYP2C19 genetic variants and poor metabolism of clopidogrel. Of these variants, *2 and *3 are the most common and are associated with complete loss of enzyme activity. In patients who are carriers of a CYP2C19 *2 or *3 allele, the conversion of clopidogrel to its active metabolite may be reduced, which can lead to ischemic events and negative consequence for the patient. We examined the ability of the Verigene CLO assay (Nanosphere, Northbrook, IL) to identify CYP2C19 *2 and *3 polymorphisms in 1,286 unique whole blood samples. The Verigene CLO assay accurately identified homozygous and heterozygous *2 and *3 phenotypes with a specificity of 100% and a final call rate of 99.7%. The assay is fully automated and can produce a result in approximately 3.5 hours.

A Bayesian hierarchical nonlinear mixture model in the presence of artifactual outliers in a population pharmacokinetic study.

The purpose of this study is to develop a statistical methodology to handle a large proportion of artifactual outliers in a population pharmacokinetic (PK) modeling. The motivating PK data were obtained from a population PK study to examine associations between PK parameters such as clearance of dexmedetomidine (DEX) and cytochrome P450 2A6 phenotypes. The blood samples were sparsely sampled from patients in intensive care units (ICUs) while different doses of DEX were continuously infused. Conventional population PK analysis of these data revealed several challenges and intricacies. Especially, there was strong evidence that some plasma drug concentrations were artifactually high and likely contaminated with the infused drug due to blood sampling processes that are sometimes unavoidable in an ICU setting. If not addressed, or if arbitrarily excluded, these outlying values could lead to biased estimates of PK parameters and miss important relationships between PK parameters and covariates due to increased variability. We propose a novel population PK model, a Bayesian hierarchical nonlinear mixture model, to accommodate the artifactual outliers using a finite mixture as the residual error model. Our results showed that the proposed model handles the outliers well. We also conducted simulation studies with a varying proportion of the outliers. These simulation results showed that the proposed model can accommodate the outliers well so that the estimated PK parameters are less biased.

Drug interaction of (S)-warfarin, and not (R)-warfarin, with itraconazole in a hematopoietic stem cell transplant recipient.

BACKGROUND: Itraconazole is a potent inhibitor of CYP3A4 and P-glycoprotein, but not CYP2C9. Herein, we report a case study in which the plasma concentration of the CYP2C9 substrate (S)-warfarin, and not the CYP3A4 substrate (R)-warfarin, increased with itraconazole coadministration. CASE: A 67-y-old man received an allogenic bone marrow transplant for acute lymphoid leukemia. He was taking oral itraconazole (200mg/day) and was started on a warfarin dose of 2.0mg/day. The plasma concentrations of (S)- and (R)-warfarin 3 days after starting warfarin administration were 216 and 556 ng/mL, respectively (INR 0.98), and after 10 days, the concentrations were 763 and 545 ng/mL, respectively (INR 2.43). On day 11 after withdrawal of itraconazole, the concentrations of (S)- and (R)-warfarin were 341 and 605ng/mL, respectively (INR 1.38). The concentration of (R)-warfarin was not affected by itraconazole; however, the final (S)-warfarin concentration had increased 7.3-fold. The (S)-warfarin/(S)-7-hydroxywarfarin ratio decreased to 2.45 from 8.40 after discontinuation of itraconazole. The permeability of warfarin enantiomers across Caco-2 cells was not influenced by itraconazole and showed no difference between enantiomers. CONCLUSIONS: Careful INR monitoring is necessary for warfarin co-administration with itraconazole. Further examination is necessary to elucidate mechanisms of the interaction between warfarin and itraconazole.

Activity levels of tamoxifen metabolites at the estrogen receptor and the impact of genetic polymorphisms of phase I and II enzymes on their concentration levels in plasma.

The therapeutic effect of tamoxifen depends on active metabolites, e.g., cytochrome P450 2D6 (CYP2D6) mediated formation of endoxifen. To test for additional relationships, 236 breast cancer patients were genotyped for CYP2D6, CYP2C9, CYP2B6, CYP2C19, CYP3A5, UGT1A4, UGT2B7, and UGT2B15; also, plasma concentrations of tamoxifen and 22 of its metabolites, including the (E)-, (Z)-, 3-, and 4'-hydroxymetabolites as well as their glucuronides, were quantified using liquid chromatography-tandem mass spectrometry (MS). The activity levels of the metabolites were measured using an estrogen response element reporter assay; the strongest estrogen receptor inhibition was found for (Z)-endoxifen and (Z)-4-hydroxytamoxifen (inhibitory concentration 50 (IC50) 3 and 7 nmol/l, respectively). CYP2D6 genotypes explained 39 and 9% of the variability of steady-state concentrations of (Z)-endoxifen and (Z)-4-hydroxytamoxifen, respectively. Among the poor metabolizers, 93% had (Z)-endoxifen levels below IC90 values, underscoring the role of CYP2D6 deficiency in compromised tamoxifen bioactivation. For other enzymes tested, carriers of reduced-function CYP2C9 (*2, *3) alleles had lower plasma concentrations of active metabolites (P < 0.004), pointing to the role of additional pathways.

Failure of pharmacogenetic-based dosing algorithms to identify older patients requiring low daily doses of warfarin.

INTRODUCTION: Warfarin doses vary greatly among patients and warfarin administration is accompanied by risk of bleeding. Genes responsible for its metabolism (CYP2C9) and effect on clotting (VKORC1) have been identified. It has been suggested that genotyping for variants in these genes can improve warfarin dosing and decrease bleeding complications. We evaluated performance of pharmacogenetic-based warfarin dosing estimation algorithms in old and very old patients. METHODS: Cross-sectional study of stable patients older than 65 years receiving warfarin with therapeutic International Normalized Ratio (INR) anticoagulation. Medical and laboratory data were reviewed and genotyping for CYP2C9 and VKORC1 performed. Warfarin dose estimates with and without genotype information were compared to clinically established therapeutic doses. RESULTS: Sixty-nine patients (32 men, 37 women; 41 nursing home residents; 28 senior care community residents) aged 81.4 ± 8.3 (mean ± S.D) years; ethnicity: Caucasian in 53, Asian in 10, Hispanic in 4, and African American in 2, received 3.3 ± 1.7 mg/d (range 0.7-9) warfarin achieving target INRs of 2.5 ± 0.2. Pharmacogenetic-based dose estimates (in combination with age, weight, height, smoking history, ethnicity/race, history of liver disease, selected co-medications such as amiodarone and enzyme inducers, baseline INR, clinical indication, and target INR), explained 50% of variability (P < .0001) compared with 12% without genetic data (P = .003). However, doses were overestimated in 15 of 16 patients requiring less than 2 mg/d (2.6 ± 0.9 mg/d compared with observed 1.5 ± 0.3 mg/d, P = .0001). Renal disease was a potential variable contributing to low warfarin requirements. DISCUSSION: The role of pharmacogenetic testing in the management of warfarin administration in patients is undergoing evaluation. Currently available pharmacogenetic- based warfarin dose estimation algorithms reduce variability in estimates for groups of older patients but consistently overestimate doses for older patients requiring the lowest doses of warfarin. CONCLUSIONS: Pharmacogenetic data add to our understanding of variability in warfarin dosing requirements but do not accurately identify older patients requiring the lowest warfarin doses. Therefore, the most prudent approach to warfarin therapy in older patients should include low initial doses in the absence of genotype variants associated with very low warfarin sensitivity and careful monitoring of INR responses.

Quantitative plasma analysis using automated online solid-phase extraction with column switching LC-MS/MS for characterising cytochrome P450 2D6 and 2C19 metabolism.

In this study an easy and efficient assay for simultaneous quantitation of plasma concentrations of probe drugs and their metabolically relevant metabolites for the phenotypic analysis of cytochrome P450 2D6 and 2C19, respectively, has been established. This sensitive method makes use of a simple initial sample preparation, followed by a 6-min automated analysis that includes online solid-phase extraction (SPE), column switching and tandem mass spectrometry. Validation over a concentration range of 1.3-2500 ng/mL for dextromethorphan, omeprazole, dextrorphan and 5'-hydroxyomeprazole was performed with LOQ between 215 and 1145 pg/mL. Intra- and inter-day precision and accuracy over the calibration ranges were within 15% for all analytes with recoveries of greater than 85%. Advantages are small sample volumes required, a robust, sensitive and highly selective method suitable for pre-prescription metabolic screening. This method could compliment or offer an alternative to DNA mutation analysis for determining appropriate dosage regimens for personalised medicine.