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The majority of Mexican patients with diabetes mellitus type 2 (DMT2) (67.9-85.0%) are prescribed sulpho nylureas (SUs), which are metabolized by cytochrome P450 2C9 (abbreviated as CYP2C9). SUs are a type of oral anti-diabetic compound which inhibit ATP-sensitive potas sium 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 vari ability. 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 transcrip tion-quantitative polymerase chain reaction (PCR), end-point allelic discrimination and PCR amplifying enzymatic restric tion 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 (PT 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

Purpose Warfarin is an oral anticoagulant associated with adverse reaction to drugs due to wide inter- and intra-individual dosage variability. Warfarin dosage has been related to non-genetic and genetic factors. CYP2C9 and VKORC1 gene polymorphisms affect warfarin metabolism and dosage. Due to the central role of populations’ ethnical and genetic origin on warfarin dosage variability, novel algorithms for Latin American subgroups are necessary to establish safe anticoagulation therapy. Patients and methods We genotyped CYP2C9*2 (c.430C > T), CYP2C9*3 (c.1075A > C), CYP4F2 (c.1297G > A), and VKORC1 (−1639 G > A) polymorphisms in 152 Colombian patients who received warfarin. We evaluated the impact on the variability of patients’ warfarin dose requirements. Multiple linear regression analysis, using genetic and non-genetic variables, was used for creating an algorithm for optimal warfarin maintenance dose. Results Median weekly prescribed warfarin dosage was significantly lower in patients having the VKORC1-1639 AA genotype and poor CYP2C9*2/*2,*2/*3 metabolizers than their wild-type counterparts. We found a 2.3-fold increase in mean dose for normal sensitivity patients (wild-type VKORC1/CYP2C9 genotypes) compared to the other groups (moderate and high sensitivity); 31.5% of the patients in our study group had warfarin sensitivity-related genotypes. The estimated regression equation accounted for 44.4% of overall variability in regard to warfarin maintenance dose. The algorithm was validated, giving 45.9% correlation (R2=0.459). Conclusion Our results describe and validate the first algorithm for predicting warfarin maintenance in a Colombian mestizo population and have contributed toward the understanding of pharmacogenetics in a Latin American population subgroup., Video abstract

Essentials The EU-PACT trial was used to investigate age on the interaction between coumarins and genotype. The results support the use of genotype-guided dosing for phenprocoumon in patients < 75 years. For patients ≥ 75 years the phenprocoumon algorithm should be revised and further tested. No influence of comorbidities and co-current drug use was found that could explain the differences. SUMMARY Background Age seemed to affect the interaction between coumarins and genotype in the acenocoumarol and phenprocoumon arm of the European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) trial. Objectives To investigate the effect of genotype-guided dosing stratified by age and the potential factors causing a difference. Patients/Methods Data from the acenocoumarol/phenprocoumon arm of the EU-PACT trial were used. The percentages of time below the therapeutic range, time above the therapeutic range and time in the therapeutic range (TTR) during the initial 12 weeks of therapy were compared between the genotype-guided group and the control group among younger (< 75 years) and older (≥ 75 years) patients by the use of independent t-tests, and adjusted for sex, height, weight and co-medications by the use of linear regression. Results Among younger phenprocoumon users, TTR during the first 12 weeks in the genotype-guided group (n = 55) was 9.5% (95% confidence interval [CI] 1.3 to 17.8) higher than in the control group (n = 63), with a remarkably lower percentage of time above this range (difference: - 9.6%, 95% CI - 19.0 to - 0.2) and a similar time below this range. Older patients dosed by the genotype-guided algorithm (n = 24) spent more time above the range (difference: 27.5%, 95% CI 12.9 to 42.0). For acenocoumarol users, there were no significant differences between the genotype-guided and control groups for most outcomes, except for a lower percentage of time below the range among older patients. Conclusions The genotype-guided algorithm for phenprocoumon in the EU-PACT trial benefitted younger patients more, but for older patients the algorithm needs to be revised and tested in further research.

BACKGROUND: Age seemed to affect the interaction between coumarins and genotype in the acenocoumarol and phenprocoumon arm of the European pharmacogenetics of anticoagulant therapy (EU-PACT) trial. OBJECTIVES: To investigate the effect of genotype-guided dosing stratified by age and the potential factors causing a difference. PATIENTS/METHODS: Data from the acenocoumarol/phenprocoumon arm of the EU-PACT trial was used. The percentage of time below, above and in the therapeutic range (TTR) during the initial 12 weeks of therapy were compared between the genotype-guided group and the control group among younger (

It is well known that drug responses differ among patients with regard to dose requirements, efficacy, and adverse drug reactions (ADRs). The differences in drug responses are partially explained by genetic variation. This paper highlights some examples of areas in which the different responses (dose, efficacy, and ADRs) are studied in children, including cancer (cisplatin), thrombosis (vitamin K antagonists), and asthma (long-acting β2 agonists). For childhood cancer, the replication of data is challenging due to a high heterogeneity in study populations, which is mostly due to all the different treatment protocols. For example, the replication cohorts of the association of variants in TPMT and COMT with cisplatin-induced ototoxicity gave conflicting results, possibly as a result of this heterogeneity. For the vitamin K antagonists, the evidence of the association between variants in VKORC1 and CYP2C9 and the dose is clear. Genetic dosing models have been developed, but the implementation is held back by the impossibility of conducting a randomized controlled trial with such a small and diverse population. For the long-acting β2 agonists, there is enough evidence for the association between variant ADRB2 Arg16 and treatment response to start clinical trials to assess clinical value and cost effectiveness of genotyping. However, further research is still needed to define the different asthma phenotypes to study associations in comparable cohorts. These examples show the challenges which are encountered in pediatric pharmacogenomic studies. They also display the importance of collaborations to obtain good quality evidence for the implementation of genetic testing in clinical practice to optimize and personalize treatment.

SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed >99% binding to plasma from mice, dog and humans. It was metabolically stable in human and dog liver microsomes relative to mouse and rat. SB1317 was mainly metabolized by CYP3A4 and CY1A2 in vitro. SB1317 did not inhibit any of the major human CYPs in vitro except CYP2D6 (IC50=1 μM). SB1317 did not significantly induce CYP1A and CYP3A4 in human hepatocytes in vitro. The metabolic profiles in liver microsomes from preclinical species were qualitatively similar to humans. In pharmacokinetic studies SB1317 showed moderate to high systemic clearance (relative to liver blood flow), high volume of distribution (>0.6 L/kg), oral bioavailability of 24%, ~ 4 and 37% in mice, rats and dogs, respectively; and extensive tissue distribution in mice. The favorable ADME of SB1317 supported its preclinical development as an oral drug candidate. © 2012 Bentham Science Publishers.

Summary. Background: The required acenocoumarol dose and the risk of underanticoagulation and overanticoagulation are associated with the CYP2C9 and VKORC1 genotypes. However, the duration of the effects of these genes on anticoagulation is not yet known. Objectives: In the present study, the effects of these polymorphisms on the risk of underanticoagulation and overanticoagulation over time after the start of acenocoumarol were investigated. Patients/methods: In three cohorts, we analyzed the relationship between the CYP2C9 and VKORC1 genotypes and the incidence of subtherapeutic or supratherapeutic International Normalized Ratio (INR) values ( 3.5) or severe overanticoagulation (INR > 6) for different time periods after treatment initiation. Results: Patients with polymorphisms in CYP2C9 and VKORC1 had a higher risk of overanticoagulation (up to 74%) and a lower risk of underanticoagulation (down to 45%) in the first month of treatment with acenocoumarol, but this effect diminished after 1–6 months. Conclusions: Knowledge of the patient’s genotype therefore might assist physicians to adjust doses in the first month(s) of therapy.

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.

Acknowledgments: The Euro-PGx project was partly funded by European grant (RD-Connect; FP7-305444) and the Golden Helix Foundation and encouraged by the Genomic Medicine Alliance Pharmacogenomics Working group. The Lithuanian segment of the project was supported by the LITGEN project (VP1-3.1-ŠMM-07-K-01-013), funded by the European Social Fund under the Global Grant Measure. The Czech segment of the project was supported by 00064203, LN14073, LM2015091, NF-CZ11-PDP-3-003-2014 and CZ.2.16/3.1.00/24022OPPK grants to MM. JK was funded by the National Institutes of Health Common Fund Award NHGRI Grant Number U41HG006941. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflict of interests., Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant interpopulation pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective., The Euro-PGx project was partly funded by European grant (RD-Connect; FP7-305444) and the Golden Helix Foundation and encouraged by the Genomic Medicine Alliance Pharmacogenomics Working group. The Lithuanian segment of the project was supported by the LITGEN project (VP1- 3.1-ŠMM-07-K-01-013), funded by the European Social Fund under the Global Grant Measure. The Czech segment of the project was supported by 00064203, LN14073, LM2015091, NF-CZ11-PDP-3-003-2014 and CZ.2.16/3.1.00/24022OPPK grants to MM. JK was funded by the National Institutes of Health Common Fund Award NHGRI Grant Number U41HG006941. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., peer-reviewed

Introduction: A large proportion of the coumarin dose variability is explained by environmental factors and by common genetic variants in the VKORC1 and CYP2C9 genes. Genotype-guided coumarin dosing has been proposed for a more accurate prediction of the coumarin dose in order to reduce the incidence of coumarin-related complications.Areas covered: This review discusses the current state of coumarin pharmacogenetics, the evidence from recent randomized controlled trials and economic evaluations regarding the possible clinical implementation of genotype-guided coumarin dosing.Expert opinion: When the VKORC1 and CYP2C9 genotypes are available before the start of coumarin therapy in individuals of European ancestry, a genetic-guided algorithm should be used for dose determination. Ethnicity-specific pharmacogenetic algorithms should be tested in other populations. At this moment the evidence is not sufficient to support genotyping before coumarin therapy initiation. Based on results from recent randomized controlled trials, a clinical dosing algorithm could be considered in the initial phase of coumarin treatment. Current economic studies indicate that genotype-guided dosing could be cost-effective, but the clinical implementation of genetic-guided coumarin therapy will depend on the cost of pharmacogenetic tests and the availability of novel oral anticoagulants.

PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 μm) incubated for 0-60 min with S9 fractions and NADPH was determined by HPLC, using the Waters AccQ.Tag method after derivatization of amino acids to stable, fluorescent derivatives. CYP assays were performed using pooled human liver microsomes with substrates, selective towards human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, incubated at concentrations around the Km. Incubation mixtures were preincubated with PLD-118 (0.1-100 μM) or control inhibitor for 5 min. No metabolism of PLD-118 was detected with rat and dog S9 fractions. A small (8%) decrease in PLD-118 at 100 μM (not detected at 10 μM) with human microsomes was considered to be biologically irrelevant. PLD-118 did not inhibit any of the tested CYPs. PLD-118, at concentrations up to 100 μM, is not metabolized by rat, dog or human liver S9 homogenates and does not inhibit human CYPs in vitro, suggesting little likelihood for interaction of PLD-118 with drugs metabolized by these enzymes. Copyright © 2004 John Wiley & Sons, Ltd.

V-PYRRO/NO [O(2)-vinyl-1-(pyrrolidin-1-yl) diazen-1-ium-1,2-diolate] and V-PROLI/NO (O2-vinyl-[2-(carboxylato) pyrrolidin-1-yl]diazen-1-ium-1,2-diolate), two structurally similar diazeniumdiolate derivatives, were designed as liver-selective prodrugs that are metabolized by cytochrome P450 isoenzymes, with subsequent release of nitric oxide (NO). Yet, their efficacy in the treatment of nonalcoholic fatty liver disease (NAFLD) and their comparative pharmacokinetic and metabolic profiles have not been characterized. The aim of the present work was to compare the effects of V-PYRRO/NO and V-PROLI/NO on liver steatosis, glucose tolerance, and liver fatty acid composition in C57BL/6J mice fed a high-fat diet, as well as to comprehensively characterize the ADME (absorption, distribution, metabolism and excretion) profiles of both NO donors. Despite their similar structure, V-PYRRO/NO and V-PROLI/NO showed differences in pharmacological efficacy in the murine model of NAFLD. V-PYRRO/NO, but not V-PROLI/NO, attenuated liver steatosis, improved glucose tolerance, and favorably modified fatty acid composition in the liver. Both compounds were characterized by rapid absorption following i.p. administration, rapid elimination from the body, and incomplete bioavailability. However, V-PYRRO/NO was eliminated mainly by the liver, whereas V-PROLI/NO was excreted mostly in unchanged form by the kidney. V-PYRRO/NO was metabolized by CYP2E1, CYP2C9, CYP1A2, and CYP3A4, whereas V-PROLI/NO was metabolized mainly by CYP1A2. Importantly, V-PYRRO/NO was a better NO releaser in vivo and in the isolated, perfused liver than V-PROLI/NO, an effect compatible with the superior antisteatotic activity of V-PYRRO/NO. In conclusion, V-PYRRO/NO displayed a pronounced antisteatotic effect associated with liver-targeted NO release, whereas V-PROLI/NO showed low effectiveness, was not taken up by the liver, and was eliminated mostly in unchanged form by the kidney.

Context: Although humans are exposed to o-coumaric acid (OCA) in their diet, there is no available literature related to drug interaction and the carcinogen-activating potential of OCA in the HepG2 cell line. Objective: This study was undertaken to determine the effects of OCA on the cytochrome P450 (CYP) 1A2, CYP2E1, CYP2C9, and CYP3A4 enzymes, which are primarily involved in carcinogen and drug metabolism. Materials and methods: The cytotoxicity of OCA in HepG2 cells was investigated by measuring the cleavage of WST-1. The protein and mRNA levels of CYPs were determined by western blotting and RT-PCR, respectively. Results: The EC10, EC25, and EC50 values of OCA were calculated to be 1.84, 3.91 and 7.39mM, respectively. A sublethal dose of 5mM was used throughout this study. The CYP1A2 protein and mRNA levels were increased by 52 and 40% (p

Background: Dosing of vitamin K antagonists (VKA) in paediatric patients is complex. The large variability in VKA dose requirement asks for elucidating the factors associated with this variability and taking these into account when defining the dose for a patient. For warfarin, paediatric dosing algorithms have been developed, but not for acenocoumarol. Objectives: To develop a dosing algorithm for acenocoumarol in pediatric patients with and without genetic information. Methods: This multicentre retrospective follow-up study was carried out in Dutch anticoagulation clinics and children's hospitals. Patients were selected when they used acenocoumarol for >1 month between January 1995 and December 2014 and were ≤18 years of age. The primary outcome was the mean daily dose during a stable period. A stable period was defined as ≥3 consecutive international normalized ratio measurements within therapeutic range over a period of ≥3 weeks. Clinical information (including height, weight and indication) and saliva samples for genotyping of CYP2C9 (∗2 and ∗3), VKORC1, CYP4F2, CYP2C18 and CYP3A4 (∗1B and ∗22) were collected. Linear regression was used to analyse their association with the log mean stable dose. Results: In total, 175 patients were included of whom 86 patients had a stable period and no missing clinical information (clinical algorithm cohort) and of 80 also genetic information was available (genetic algorithm cohort). The mean age at the stable period was 9 years. The most common indications were Fontan circulation, prosthetic heart valve, deep venous thrombosis and dilated cardiomyopathy. The clinical algorithm, containing body surface area and indication, explained 45.0% of the variability in dose requirement of acenocoumarol. By adding the genotypes of VKORC1, CYP2C18, and CYP2C9∗2/∗3, 61.8% of the variability was explained (genetic algorithm). Conclusions: Clinical factors had the largest impact on the required dose of acenocoumarol in pediatric patients. Including genetic factors in the algorithm, and especially VKORC1, increased this with 16.8%.

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.

Among hydroxycinnamic acids, caffeic, ferulic and p-coumaric acids have received considerable attention due to their biological activities. However, studies related to the biological activities of o-coumaric acid (OCA) are limited. In this regard, this study was designed to determine the chemopreventive potential of OCA in human breast cancer cells (MCF-7). The EC50 value of OCA was found to be 4.95 mM and was used throughout the study. Caspase-3 protein and mRNA levels increased by 59% and 72%. Similarly, protein and mRNA levels of Bax were increased by 115% and 152%. However, OCA treatment caused 48% and 35% decreases in Bcl-2 protein and mRNA levels. Cyclin D1 and cyclin dependent kinase-2 protein and mRNA levels decreased significantly. Moreover, p53 protein and mRNA levels increased by 178% and 245%, respectively. In addition to p53, PTEN protein and mRNA levels were induced. Although, CYP1A1, CYP1A2 and CY2E1 mRNA levels increased, CYP3A4 and CYP2C9 mRNA levels decreased in response to OCA treatment. These results suggest that OCA demonstrates anticarcinogenic activity on MCF-7 cells by activating multiple pathways. However, it also has high carcinogen activating and drug interaction potential. Therefore, serious precautions must be taken before using OCA.

SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed >99% binding to plasma from mice, dog and humans. It was metabolically stable in human and dog liver microsomes relative to mouse and rat. SB1317 was mainly metabolized by CYP3A4 and CY1A2 in vitro. SB1317 did not inhibit any of the major human CYPs in vitro except CYP2D6 (IC50=1 μM). SB1317 did not significantly induce CYP1A and CYP3A4 in human hepatocytes in vitro. The metabolic profiles in liver microsomes from preclinical species were qualitatively similar to humans. In pharmacokinetic studies SB1317 showed moderate to high systemic clearance (relative to liver blood flow), high volume of distribution (>0.6 L/kg), oral bioavailability of 24%, ~ 4 and 37% in mice, rats and dogs, respectively; and extensive tissue distribution in mice. The favorable ADME of SB1317 supported its preclinical development as an oral drug candidate. © 2012 Bentham Science Publishers.

A case report of therapeutic efficacy of acenocoumarol in a warfarin-resistant patient with deep venous thrombosis.

The fields of pharmacogenetics and pharmacogenomics have become important practical tools to progress goals in medical and pharmaceutical research and development. As more screening tests are being developed, with some already used in clinical practice, consideration of cost-effectiveness implications is important. A systematic review was performed on the content of and adherence to pharmacoeconomic guidelines of recent pharmacoeconomic analyses performed in the field of pharmacogenetics and pharmacogenomics. Economic analyses of screening strategies for genetic variations, which were evidence-based and assumed to be associated with drug efficacy or safety, were included in the review. The 20 papers included cover a variety of healthcare issues, including screening tests on several cytochrome P450 (CYP) enzyme genes, thiopurine S-methyltransferase (TMPT) and angiotensin-converting enzyme (ACE) insertion deletion (ACE I/D) polymorphisms. Most economic analyses reported that genetic screening was cost effective and often even clearly dominated existing non-screening strategies. However, we found a lack of standardization regarding aspects such as the perspective of the analysis, factors included in the sensitivity analysis and the applied discount rates. In particular, an important limitation of several studies related to the failure to provide a sufficient evidence-based rationale for an association between genotype and phenotype. Future economic analyses should be conducted utilizing correct methods, with adherence to guidelines and including extensive sensitivity analyses. Most importantly, genetic screening strategies should be based on good evidence-based rationales. For these goals, we provide a list of recommendations for good pharmacoeconomic practice deemed useful in the fields of pharmacogenetics and pharmacogenomics, regardless of country and origin of the economic analysis. © 2008 Adis Data Information BV. All rights reserved.

The degree of anticoagulation obtained during oral anticoagulation therapy with vitamin K antagonists (VKA) varies among patients due to individual and environmental factors. The rate of anticoagulation influences the hemorrhagic risk. Therefore, it is plausible that patients specially sensitive to oral anticoagulants are at higher hemorrhagic risk, specially during the first weeks. The role of a series of polymorphisms of the enzymes involved in the metabolism of VKA or in the vitamin K cycle are reviewed. Three polymorphisms, two in the cytochrome P450 2C9 and one in the VKORC1 enzyme, are responsible for a high portion of the variability observed in the sensitivity to AVK. Although the available literature suggests that these genetic variants could increase the risk of severe hemorrhage, larger, well designed studies are needed to confirm this notion.

Introduction: A large proportion of the coumarin dose variability is explained by environmental factors and by common genetic variants in the VKORC1 and CYP2C9 genes. Genotype-guided coumarin dosing has been proposed for a more accurate prediction of the coumarin dose in order to reduce the incidence of coumarin-related complications.Areas covered: This review discusses the current state of coumarin pharmacogenetics, the evidence from recent randomized controlled trials and economic evaluations regarding the possible clinical implementation of genotype-guided coumarin dosing.Expert opinion: When the VKORC1 and CYP2C9 genotypes are available before the start of coumarin therapy in individuals of European ancestry, a genetic-guided algorithm should be used for dose determination. Ethnicity-specific pharmacogenetic algorithms should be tested in other populations. At this moment the evidence is not sufficient to support genotyping before coumarin therapy initiation. Based on results from recent randomized controlled trials, a clinical dosing algorithm could be considered in the initial phase of coumarin treatment. Current economic studies indicate that genotype-guided dosing could be cost-effective, but the clinical implementation of genetic-guided coumarin therapy will depend on the cost of pharmacogenetic tests and the availability of novel oral anticoagulants.

In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1′-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19, and 2D6 are intrinsically able to 1′-hydroxylate methyleugenol. An additional experiment with Gentest microsomes, expressing the same individual enzymes to roughly average liver levels, indicated that P450 1A2, 2C9, 2C19, and 2D6 contribute to methyleugenol 1′-hydroxylation in the human liver. A study, in which correlations between methyleugenol 1′-hydroxylation in human liver microsomes from 15 individuals and the conversion of enzyme specific substrates by the same microsomes were investigated, showed that P450 1A2 and P450 2C9 are important enzymes in the bioactivation of methyleugenol. This was confirmed in an inhibition study in which pooled human liver microsomes were incubated with methyleugenol in the presence and absence of enzyme specific inhibitors. Kinetic studies revealed that at physiologically relevant concentrations of methyleugenol P450 1A2 is the most important enzyme for bioactivation of methyleugenol in the human liver showing an enzyme efficiency (kcat/Km) that is ∼30, 50, and >50 times higher than the enzyme efficiencies of, respectively, P450 2C9, 2C19, and 2D6. Only when relatively higher methyleugenol concentrations are present P450 2C9 and P450 2C19 might contribute as well to the bioactivation of methyleugenol in the human liver. A 5-fold difference in activities was found between the 15 human liver microsomes of the correlation study (range, 0.89-4.30 nmol min-1 nmol P450-1). Therefore, interindividual differences might cause variation in sensitivity toward methyleugenol. Especially lifestyle factors such as smoking (induces P450 1A) and the use of barbiturates (induces P450 2C) can increase the susceptibility for adverse effects of methyleugenol. © 2006 American Chemical Society.

PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 μm) incubated for 0-60 min with S9 fractions and NADPH was determined by HPLC, using the Waters AccQ.Tag method after derivatization of amino acids to stable, fluorescent derivatives. CYP assays were performed using pooled human liver microsomes with substrates, selective towards human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, incubated at concentrations around the Km. Incubation mixtures were preincubated with PLD-118 (0.1-100 μM) or control inhibitor for 5 min. No metabolism of PLD-118 was detected with rat and dog S9 fractions. A small (8%) decrease in PLD-118 at 100 μM (not detected at 10 μM) with human microsomes was considered to be biologically irrelevant. PLD-118 did not inhibit any of the tested CYPs. PLD-118, at concentrations up to 100 μM, is not metabolized by rat, dog or human liver S9 homogenates and does not inhibit human CYPs in vitro, suggesting little likelihood for interaction of PLD-118 with drugs metabolized by these enzymes. Copyright © 2004 John Wiley & Sons, Ltd.

In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1′-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1′-hydroxylation of safrole was characterized in a variety of in vitro test systems, including Supersomes, expressing individual human P450 enzymes to a high level, and microsomes derived from cell lines expressing individual human P450 enzymes to a lower, average human liver level. Additionally, a correlation study was performed, in which safrole was incubated with a series of 15 human liver microsomes, and the 1′-hydroxylation rates obtained were correlated with the activities of these microsomes toward specific substrates for nine different isoenzymes. To complete the study, a final experiment was performed in which pooled human liver microsomes were incubated with safrole in the presence and absence of coumarin, a selective P450 2A6 substrate. On the basis of the results of these experiments, important roles for P450 2C9*1, P450 2A6, P450 2D6*1, and P450 2E1 were elucidated. The possible consequences of these results for the effects of genetic polymorphisms and life style factors on the bioactivation of safrole are discussed. Polymorphisms in P450 2C9, P450 2A6, and P450 2D6, leading to poor metabolizer phenotypes, may reduce the relative risk on the harmful effects of safrole, whereas life style factors, such as the use of alcohol, an inducer of P450 2E1, and barbiturates, inducers of P450 2C9, and polymorphisms in P450 2D6 and P450 2A6, leading to ultraextensive metabolizer phenotypes, may increase the relative risk.

In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1′-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1′-hydroxylation of safrole was characterized in a variety of in vitro test systems, including Supersomes, expressing individual human P450 enzymes to a high level, and microsomes derived from cell lines expressing individual human P450 enzymes to a lower, average human liver level. Additionally, a correlation study was performed, in which safrole was incubated with a series of 15 human liver microsomes, and the 1′-hydroxylation rates obtained were correlated with the activities of these microsomes toward specific substrates for nine different isoenzymes. To complete the study, a final experiment was performed in which pooled human liver microsomes were incubated with safrole in the presence and absence of coumarin, a selective P450 2A6 substrate. On the basis of the results of these experiments, important roles for P450 2C9*1, P450 2A6, P450 2D6*1, and P450 2E1 were elucidated. The possible consequences of these results for the effects of genetic polymorphisms and life style factors on the bioactivation of safrole are discussed. Polymorphisms in P450 2C9, P450 2A6, and P450 2D6, leading to poor metabolizer phenotypes, may reduce the relative risk on the harmful effects of safrole, whereas life style factors, such as the use of alcohol, an inducer of P450 2E1, and barbiturates, inducers of P450 2C9, and polymorphisms in P450 2D6 and P450 2A6, leading to ultraextensive metabolizer phenotypes, may increase the relative risk.

The maintenance of the major hepatic cytochrome P450 (CYP) enzymes has been studied in precision-cut human liver slices cultured for up to 72 h in supplemented RPMI 1640 medium. The relative apoprotein levels of 11 CYP enzymes were determined using a panel of antipeptide antibodes. In addition, 7-ethoxyresorufin O-deethylase, tolbutamide methylhydroxylase, debrisoquine 4-hydroxylase, and testosterone 6β-hydroxylase activities were determined as enzymatic markers for CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. There was a large variation in the rate of decline of different CYP levels with time in culture. Based on the rate of decrease, CYP enzymes could be separated into two groups, with CYP2C9, CYP2D6, CYP3A4, and CYP4A11 being relatively stable (half-lives between 70 and 104 h), compared with CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, and CYP3A5, which were relatively unstable (half-lives between 23 and 36 h). Enzyme activities decreased at rates similar to those of their corresponding apoproteins. There was also a large difference in the stability of individual CYP enzymes from different liver donors, particularly for the most rapidly declining CYP enzymes. Similar losses of CYP enzymes were found when human river slices were cultured in supplemented Williams' medium E for 72 h, except that CYP2E1 apoprotein levels were better maintained. Because of the variable decreases of CYP enzymes, xenobiotic metabolism studies are best performed with freshly cut rather than cultured human liver slices. Chemicals/CAS: Culture Media; Cytochrome P-450 Enzyme System, 9035-51-2; Isoenzymes

The maintenance of the major hepatic cytochrome P450 (CYP) enzymes has been studied in precision-cut human liver slices cultured for up to 72 h in supplemented RPMI 1640 medium. The relative apoprotein levels of 11 CYP enzymes were determined using a panel of antipeptide antibodes. In addition, 7-ethoxyresorufin O-deethylase, tolbutamide methylhydroxylase, debrisoquine 4-hydroxylase, and testosterone 6β-hydroxylase activities were determined as enzymatic markers for CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. There was a large variation in the rate of decline of different CYP levels with time in culture. Based on the rate of decrease, CYP enzymes could be separated into two groups, with CYP2C9, CYP2D6, CYP3A4, and CYP4A11 being relatively stable (half-lives between 70 and 104 h), compared with CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, and CYP3A5, which were relatively unstable (half-lives between 23 and 36 h). Enzyme activities decreased at rates similar to those of their corresponding apoproteins. There was also a large difference in the stability of individual CYP enzymes from different liver donors, particularly for the most rapidly declining CYP enzymes. Similar losses of CYP enzymes were found when human river slices were cultured in supplemented Williams' medium E for 72 h, except that CYP2E1 apoprotein levels were better maintained. Because of the variable decreases of CYP enzymes, xenobiotic metabolism studies are best performed with freshly cut rather than cultured human liver slices. Chemicals/CAS: Culture Media; Cytochrome P-450 Enzyme System, 9035-51-2; Isoenzymes