Variability In Drug Metabolism Research Articles

Genetic polymorphism of cynomolgus and rhesus macaque CYP2C9

Cynomolgus and rhesus macaques are non-human primate species widely used in drug metabolism studies. Cynomolgus CYP2C9 (formerly known as CYP2C43) is predominantly expressed in liver and encodes a drug-metabolizing enzyme that metabolizes human CYP2C substrates such as S-mephenytoin and progesterone. In addition, cynomolgus CYP2C9 also metabolizes caffeine, resulting in the formation of the metabolite that is not generated efficiently in humans. Genetic variants of human CYP2C genes account for the inter-individual variability in drug metabolism: however, CYP2C9 variants have not been found in macaques. To see if CYP2C9 is polymorphic in macaques, in this study, CYP2C9 was re-sequenced in 78 cynomolgus and 36 rhesus macaques. A total of 27 non-synonymous variants were found, among which 4 were located in substrate recognition sites, the domain important for protein function. Thirteen and seven variants were unique to cynomolgus and rhesus macaques, respectively. This study revealed the polymorphic nature of cynomolgus and rhesus CYP2C9, similar to human CYP2C genes, by identification of numerous genetic variants including non-synonymous variants.

Drug-related perinatal damage from the pathological point of view

Drug dosage in the perinatal period represents a continuous challenge for the neonatologist because of interindividual variability of drug metabolism. The human liver plays a central role in the uptake, transport, metabolism and excretion of the vast majority of xenobiotics and drugs. The protein products of human CYP3A account for the largest portion of CYP450 proteins in human liver. At least 50% of currently used drugs in neonatal intensive care units (NICUs) are substrates of CYP3A4 including antibiotics, antivirals, antifungals, immunomodulators, benzodiazepines, proton pump inhibitors, steroid hormones and acetaminophen. The variable CYP3A4 and CYP3A7 expression recently reported in neonatal liver suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine and neonatal lives and, as a consequence, the need of an individualized tailored therapeutic approach in NICUs. The increased risk for adverse effects reported for some drugs in neonates could be related to pharmacokinetic peculiarities of the newborn liver. The fetal and neonatal liver in infants undergoing drug-induced liver injury (DILI) is always characterized by the overlapping between developmental and pathological changes, the differential diagnosis between these changes representing often a challenge for the pathologist. Data here reported clearly evidence the peculiarity of the histological examination of the newborn liver, as compared to the adult liver. In conclusion, the role of the pathologist in the interpretation of liver reactions to drugs may be relevant, only when supported by the dialogue with neonatologists. A deep knowledge of the events taking place during liver development at different gestational ages is necessary for a dedicated neonatal pathologist, in order to avoid misinterpretation of the histological changes related to liver development, giving them a pathological significance. Proceedings of the International Course on Perinatal Pathology (part of the 10 th International Workshop on Neonatology · October 22 nd -25 th , 2014) · Cagliari (Italy) · October 25 th , 2014 · The role of the clinical pathological dialogue in problem solving Guest Editors: Gavino Faa, Vassilios Fanos, Peter Van Eyken

Genetic Analysis of CYP2D6 Polymorphism in Indian Populations and its Pharmacogenetic Implications

Despite the rapid development of pharmacogenetic testing and the frequencies of medication related emergencies increasing, pharmacogentic testing is not yet implemented extensively in clinical practice. Several studies document that polymorphic Cytochrome P450 isoenzymes CYP2C9, CYP2C19 and CYP2D6 are responsible for the metabolism of many clinically important drugs and xenobiotics. These polymorphisms contribute to inter-individual and interethnic variation in drug metabolism which may lead to differences in drug response, suggesting that common dose regimen will not always equivocate to efficacious dosing. The CYP2D6*2, CYP2D6*4 and CYP2D6*10 variants were studied in four Indian populations (Gujarati, Punjabi, Bengali and Koya tribe). Genotypes at individual alleles were identified using Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method. Differences in frequencies of CYP2D6 genotypes/ alleles were compared at regional and global level. CYP2D6*2 variant frequency was highest among Gujarati population (47%) Gujarati and Punjabi populations showed high levels of CYP2D6*4 (13% and 24% respectively), whilst high levels of CYP2D6*10 were found in Bengali and Koya populations (21% and 12% respectively). At genotypic level, CYP2D6 *4/*4 genotype was absent in all study populations, resulting in the absence of the PM (poor metaboliser) phenotype in these samples. Overall there are significant differences among Indian populations at this locus. Overall observed allele frequency spectrum fits well into other Indian and European populations. There are significant differences in CYP2D6 allele and genotype frequencies in 4 populations leading to differential estimates of EM and IM. These results suggest the importance of delivering pharmacotherapeutic regimes for patients on the basis of their genetic profile.

Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development

Objective: The cytochrome P450 (CYP450) superfamily is implicated in important life processes, including metabolism of many molecules. CYP3A account for the largest portion of CYP450 proteins in human, including CYP3A4, CYP3A5 and CYP3A7. The purpose of this study was to investigate the immunohistochemical expression of CYP3A4 and CYP3A7 in human liver at different post-conceptional (PC) ages.Methods: Human liver samples from 30 fetuses and newborns were, clustered according with the PC age, routinely processed for immunohistochemical analysis of CYP3A4 and CYP3A7.Results: CYP3A4 was positive in all but two cases, CYP3A7 was positive in all but one case, which was negative also for CYP3A4.Conclusions: Our data on immunohistochemical detection of CYP3A4 and CYP3A7 during development show that CYP3A4 expression is not restricted to the post-natal age, being the immunostaining for both CYP3A4 and CYP3A7 identical after 25 weeks of PC age, thus the relationship between these CYP450 isoforms should be considered much more complex than previous thought. A high interindividual variability was observed among subjects at all gestational age. The variable CYP3A expression suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine life and in perinatal period.

CYP2C19 polymorphisms account for inter-individual variability of drug metabolism in cynomolgus macaques

CYP2C19 (formerly known as CYP2C75), highly homologous to human CYP2C19, has been identified in cynomolgus and rhesus macaques, non-human primate species widely used in drug metabolism studies. CYP2C19 is predominantly expressed in liver and encodes a functional drug-metabolizing enzyme. Genetic variants in human CYP2C genes account for the inter-individual variability in drug metabolism; however, genetic variants have not been investigated in macaque CYP2C19. In the present study, re-sequencing of CYP2C19 in 78 cynomolgus and 36 rhesus macaques identified 34 non-synonymous variants. Among these, 6 were located in substrate recognition sites, the domains important for protein function. Eighteen and 6 variants were unique to cynomolgus and rhesus macaques, respectively. Four variants were characterized by site-directed mutagenesis and metabolic assays, and 3 variants (p.Phe100Asn, p.Ala103Val, and p.Ile112Leu) showed substantially reduced activity as compared with wild type in flurbiprofen 4′-hydroxylation, omeprazole 5-hydroxylation, and R-/S-warfarin 7-hydroxylation. These variants, co-segregating in the animals analyzed, influenced metabolic activities because the homozygotes and/or heterozygotes showed significantly reduced catalytic activities in liver toward flurbiprofen 4′-hydroxylation and omeprazole 5-hydroxylation as compared with wild type. Kinetic analysis for R-warfarin 7-hydroxylation and docking simulation indicated that CYP2C19 Ala103Val would change the function and conformation of this enzyme. Ala103Val variation diminished homotropic cooperativity of CYP2C19 with R-warfarin yielding low metabolic capacity. These results indicated that the interindividual variability of CYP2C-dependent drug metabolism is at least partly accounted for by CYP2C19 variants in cynomolgus macaques.

Cytochrome P450 interactions and clinical implication in rheumatology.

There are many potential drug interactions that involve the complex cytochromes P450 (CYP) enzyme system when treatments for chronic inflammatory rheumatic diseases are used. This iatrogenic risk is increased in patients taking multiple drugs such as those with rheumatoid arthritis or gout, whatever the type of CYP interaction (substrate, inducer, or inhibitor of one of the CYP isoenzymes). Some of these CYP interactions may have clinical consequences, sometimes serious (overdose or therapeutic failure) and are often unrecognized by clinicians. The aim of this article is first of all to act as a reminder of the metabolic role of membrane-bound CYP enzymes in the liver in the oxidation of drugs and the potential types of interaction (drug substrate, inducer, or inhibitor or indirectly by the modulation of CYP activity through its powerful antiinflammatory activity). Secondly, the different factors that modulate the enzymatic activity of CYP will be described that may contribute to variations in drug metabolism and therefore modify the benefit-risk ratio of the drug. Thirdly, an analysis based on a review of the literature will present the different known interactions via CYP for drugs used in clinical practice in rheumatic diseases: analgesics, antiinflammatory drugs, conventional disease-modifying antirheumatic drugs and biologic agents. To limit the clinical consequences of these CYP interactions, it is recommended to focus on drugs that are really essential, to systematically identify the rheumatic patients most at risk before prescribing, and thus to adopt therapeutic strategies that reduce iatrogenic risk.

Professor Folke Sjöqvist is the 2013 BCPT Nordic Prize Winner in Basic & Clinical Pharmacology & Toxicology

Professor Folke Sjöqvist is the 2013 <scp>BCPT</scp> Nordic Prize Winner in Basic &amp; Clinical Pharmacology &amp; Toxicology

Tu1942 Genetic Variants At FCGR2A and 13q12 Are Associated With Disease Couse in Ulcerative Colitis

G A A b st ra ct s loss of response to anti-TNFα therapy after one year. The cause of this limited efficacy is unclear, but past studies hypothesized that the individual variation of drug metabolism may play an important role. Thus, given the limited data available, the role of Fcγ IIIa receptor (i.e. one of the four receptors involved in the catabolic pathway of anti-TNF-α drugs) polymorphisms should be further explored. Aim: The aim of this prospective, long-term follow up study was to evaluate the correlation between Fcγ IIIa receptor polymorphisms and clinical outcome in IBD patients undergoing biologic therapy. Methods: We enrolled consecutive IBD patients who achieved clinical remission by anti-TNF-α therapy. Blood samples were collected at the beginning of biological therapy. The assessment of IBD activity was based on the Harvey-Bradshaw Index score (HBI, remission 16) for CD patients and on the Mayo score (Mayo<2 remission, mild disease 2-5, moderate/severe disease 6-12) for UC patients. Biochemical evaluation and clinical score were assessed every 8 weeks. For the genotyping analysis we used a Light Snips (Tib-Molbiol, Genova, Italy) and the Real-Time PCR Technique developed by Light Cycler 480 Instrument (Roche, Mannheim, Germany). Results: We prospectively included 39 patients (12UC/27 CD, 16F/23M) with a median follow-up of 66.8 weeks (10112). A total of 25 (64.1%) (10UC/15CD) patients kept in remission during the whole follow up period, while 14 (35.9%) (2UC/12CD) experienced disease relapse. As shown in the Table, four out of 14 (28.6%) (1UC/3CD) patients who experienced disease relapse, had FcγIIIa-158 V/V receptor polymorphism, while the remaining 10 (71.4%) (9CD/1UC) had FcγIIIa-158 F/V or F/F receptor polymorphisms. Out of 25 patients who kept in remission, 3 (12%) (1CD/2UC) had FcγIIIa-158 V/V receptor polymorphism, whereas the remaining 22 (88%) (14CD/8UC) showed FcγIIIa-158 F/V or F/F receptor polymorphisms. Patients in remission tended to have more often FcγIIIa-158 V/V receptor polymorphism compared to patients who relapsed, but statistical significance was not reached. Conclusion: The evaluation of Fcγ IIIa-158 V/V receptor polymorphism does not seem useful in identifying patients who are more likely to lose anti TNF-α response during a long term period. However, further larger studies are necessary to investigate the role of Fcγ IIIa receptor polymorphisms

The Pharmacogenetics of Cytochrome P450 2C19 Enzymes - Effects on Clopidogrel and Proton Pump Inhibitors

BACKGROUND: Cytochrome P450 (CYP) enzymes play important roles in human, including drug metabolism. CYP2 is the largest family of human CYP, with its sequence comprising almost one third of all CYP sequences, and responsible for the metabolism of approximately 2% of clinically administrated drugs. One of the most important enzymes in this family is the CYP2C19 enzyme. The CYP2C19 gene is polymorphic, and the variation is common especially in the Asian population.CONTENT: CYP2C19 is responsible for the metabolism of various drugs, including proton pump inhibitors (PPIs) such as omeprazole and lansoprazole, psychotropic drugs including diazepam and imipramine, anticonvulsants such as phenobarbital and mephenytoin. and the recently most studied the anti-platelet drug, clopidogrel, and many others. Drugs metabolized predominantly by this enzyme like clopidogrel and PPIs might be much affected by the genotype status of CYP2C19. Clopidogrel is a pro-drug requiring a group of enzymes to convert to its active form, particularly the CYP2C19. PPIs are metabolized to its inactive metabolites mainly by CYP2C19 in the liver. Some PPIs are inhibitor of CYP2C19 enzymes, and interaction of PPIs and clopidogrel has been widely studied.SUMMARY: The association of CYP2C19 genotypes with the plasma level of active clopidogrel and platelet reactivity in individual taking this drug is well-established. Although conflicting results still exist for the association of CYP2C19 genotypes to the clinical outcomes of clopidogrel therapy, this effect seems to be consistent in patients receiving clopidogrel for coronary stents. Due to the interaction of certain PPIs and clopidogrel, the use of PPIs other than omeprazole is recommended, especially for patients taking dual anti platelet therapy of clopidogrel and aspirin.KEYWORDS: pharmacogenetics, CYP2C19, proton pump inhibitors, clopidogrel

Mapping of hepatic expression quantitative trait loci (eQTLs) in a Han Chinese population

BackgroundElucidating the genetic basis underlying hepatic gene expression variability is of importance to understand the aetiology of the disease and variation in drug metabolism. To date, no genome-wide expression quantitative...

Allele and genotype frequencies of CYP2B6 in a Turkish population

Increasing interest in cytochrome P450 2B6 (CYP2B6) genetic polymorphism was stimulated by revelations of a specific CYP2B6 genotype significantly affecting the metabolism of various drugs in common clinical use in terms of increasing drug efficacy and avoiding adverse drug reactions. The present study aimed to determine the frequencies of CYP2B6*4 CYP2B6*5, CYP2B6*6, CYP2B6*7 and CYP2B6*9 alleles in healthy Turkish individuals (n = 172). Frequencies of three single nucleotide polymorphisms were 516G>T (28%), 785A>G (33%), and 1459C>T (12%). The frequencies of CYP2B6*1, *4, *5, *6, *7, and *9 alleles were 54.3 (95% CI 49.04-59.56), 6.4% (95% CI 3.81-8.99), 11% (95% CI 7.69-14.31), 25.3% (95% CI 20.71-29.89), 0.87% (95% CI -0.11-1.85) and 2.0% (95% CI 0.52-3.48), respectively. Allele *6 was more frequent (25.3%) than the other variant alleles in Turkish subjects. The frequencies of CYP2B6*4, *5, *6, *7, and *9 alleles were similar to European populations but significantly different from that reported for Asian populations. This is the first study to document the frequencies of the CYP2B6*4, *5, *6, *7, *9 alleles in the healthy Turkish individuals and our results could provide clinically useful information on drug metabolism by CYP2B6 in Turkish population.

A theoretical study on the mechanism of a superficial mutation inhibiting the enzymatic activity of CYP1A2

CYP1A2, one of the major members of cytochrome P450 in human liver, participates in the metabolism of various drugs. While most harmful mutations are located near the catalytic core of CYP1A2, a recently found loss-of-function mutation, F186L, is on the surface. By far, function of this superficial residue remains unclear. In this paper, 7-ethoxyresorufin, a widely used agent in benchmarking the O-deethylation activities of CYP1A subfamily enzymes, was employed as a substrate to investigate the impact of the F186L mutation through ensemble docking and molecular dynamics simulations. It was found that the F186L mutation altered the binding inclination of the substrate through a series of changes on the catalytic pocket, which are, actually, long-range effects. The activities of access channels in the enzyme are also affected by the F186L mutation and the substrate binding. Based on these findings, a detailed mechanism of how F186 regulates the functions of CYP1A2 was proposed, and it may shed light on the diverse effects of SNPs and the personalized drug design.

Distribution of CYP2C9 and VKORC1 Gene Polymorphisms in Healthy Macedonian Male Population

Background: Distribution of CYP2C9 and VKORC1 gene polymorphisms may vary significantly among different ethnic groups, and eventually influence the variation in drug metabolism or even failure. Objective: The aim of this study was to evaluate the prevalence of CYP2C9 and VKORC1 alleles in the healthy population of Republic of Macedonia compared to the global geographic data reported from different ethnic populations. Also, to genotype CYP2C9 and VKORC1 genes and eventually to divide individuals in poor, extensive, or intermediate metabolizer. Material and Methods: Blood samples were collected after signing written consent, DNA was isolated from peripheral blood, and CYP2C9 and VKORC1 genes were typed (n=124). Genotyping was performed by commercially available kits (GeneID GmbH, Strassberg, Germany, AID Diagnostica), based on the method of polymerase chain reaction with a subsequent hybridization. The population genetics analysis package, PyPop ver. 0.6.0, was used for analysis of the data. Results: The frequency of alleles varies from 0.931 for CYP2C9*3 to 0.109 for CYP2C9*2 indicating common wild type allele in those genes. The frequency ranges spanned ~50% for each allele of VKORC1 gene, indicating no common wild type allele in this gene. Test of neutrality showed significant negative value for VKORC1 polymorphism that indicates balancing selection operating on the alleles at that locus. All polymorphisms of CYP2C9*2, CYP2C9*3 and VKORC1 showed a good fit with Hardy-Weinberg expectations. Conclusion: The results of polymorphic alleles of CYP2C9 and VKORC1 genes in Macedonian population can be used for the variation in drug metabolism studies as well for adapting dosage regimes for oral anticoagulant therapies.

Distribution of CYP2C9 and VKORC1 Gene Polymorphisms in Healthy Macedonian Male Population

Background: Distribution of CYP2C9 and VKORC1 gene polymorphisms may vary significantly among different ethnic groups, and eventually influence the variation in drug metabolism or even failure.Objective: The aim of this study was to evaluate the prevalence of CYP2C9 and VKORC1 alleles in the healthy population of Republic of Macedonia compared to the global geographic data reported from different ethnic populations. Also, to genotype CYP2C9 and VKORC1 genes and eventually to divide individuals in poor, extensive, or intermediate metabolizer.Material and Methods: Blood samples were collected after signing written consent, DNA was isolated from peripheral blood, and CYP2C9 and VKORC1 genes were typed (n=124). Genotyping was performed by commercially available kits (GeneID GmbH, Strassberg, Germany, AID Diagnostica), based on the method of polymerase chain reaction with a subsequent hybridization. The population genetics analysis package, PyPop ver. 0.6.0, was used for analysis of the data.Results: The frequency of alleles varies from 0.931 for CYP2C9*3 to 0.109 for CYP2C9*2 indicating common “wild type” allele in those genes. The frequency ranges spanned ~50% for each allele of VKORC1 gene, indicating no common “wild type” allele in this gene. Test of neutrality showed significant negative value for VKORC1 polymorphism that indicates balancing selection operating on the alleles at that locus. All polymorphisms of CYP2C9*2, CYP2C9*3 and VKORC1 showed a good fit with Hardy-Weinberg expectations.Conclusion: The results of polymorphic alleles of CYP2C9 and VKORC1 genes in Macedonian population can be used for the variation in drug metabolism studies as well for adapting dosage regimes for oral anticoagulant therapies.

Pregnancy and Pharmacogenomics in the Context of Drug Metabolism and Response

It is well-known that profound physiological and biochemical changes occur throughout the course of pregnancy. At the same time, the role of pharmacogenomics in modulating the metabolism and response profile to numerous medications has been elucidated. Yet, the clinical impact of pharmacogenomics during pregnancy is less well understood. We present an overview of factors modulating the pharmacokinetics and pharmacodynamics of medications throughout the time span of pregnancy while providing insights on how pharmacogenomics may contribute to interindividual variability in drug metabolism and response amongst pregnant women.

Present status and perspective of pharmacogenetics in Mexico

Drug costs account for up to 24% of the country's health expenditure and there are 13,000 registered drugs being prescribed. Diabetes is the main cause of death in the country, with over 85% of diabetic patients currently under drug treatment. The importance of knowing interindividual variability in drug metabolism on Mexican populations is thus evident. The purpose of this article is to provide an overlook of the current situation of pharmacogenetic research in Mexico, focusing on drug-metabolizing enzymes, and the possibility of developing a phenotyping cocktail for Mexican populations. So far, 21 pharmacogenetic studies on Mexican population samples (Mestizos and Amerindian) have been published. These have reported interindividual variability through phenotyping and/or genotyping cytochromes: CYP2D6, 2C19, 2C9, 2E1, and phase II enzymes UGT and NAT2. Some cytochromes with important clinical implications have not yet been phenotyped in Mexican populations. The development of a cocktail adapted to them could be a significant contribution to a larger knowledge on drug response variability at a lower price and shorter time. There are validated phenotyping cocktails that present several practical advantages, being valuable, safe, and inexpensive tools in drug metabolism characterization, which require only a single experiment to provide information on several cytochrome activities.

Pharmacogenomic assessment of clopidogrel —/INS; A step toward personalized medicine for patients with ischemic stroke

Background: Stroke is a heterogeneous disease. The most common causes of cerebral infarction are: large artery atherosclerosis, small vessel disease and cardioembolism. Furthermore, there are several genetic variations in drug metabolism in regard to the most frequently used antiplatelet agents and anticoagulants that potentially underlie variability in drug efficacy and adverse drug reactions.

Epigenetic Regulation of ADME-Related Genes: Focus on Drug Metabolism and Transport

Epigenetic regulation of gene expression refers to heritable factors that are functionally relevant genomic modifications but that do not involve changes in DNA sequence. Examples of such modifications include DNA methylation, histone modifications, noncoding RNAs, and chromatin architecture. Epigenetic modifications are crucial for packaging and interpreting the genome, and they have fundamental functions in regulating gene expression and activity under the influence of physiologic and environmental factors. Recently, epigenetics has become one of the fastest-growing areas of science and has now become a central issue in biologic studies of development and disease pathogenesis. The interest in epigenetics is also true for studies of drug metabolism and transport. In this issue of Drug Metabolism and Disposition, a series of articles is presented to demonstrate the role of epigenetic factors in regulating the expression of genes involved in drug absorption, distribution, metabolism, and excretion in organ development, tissue-specific gene expression, sexual dimorphism, and in the adaptive response to xenobiotic exposure, both therapeutic and toxic. The articles also demonstrate that, in addition to genetic polymorphisms, epigenetics may also contribute to wide interindividual variations in drug metabolism and transport. Identification of functionally relevant epigenetic biomarkers in human specimens has the potential to improve prediction of drug responses based on patient's epigenetic profiles.

PP149— The Ceiba Cocktail for Drug Hydroxylation Phenotyping in Hispanic Populations

PP149— The Ceiba CoCkTail foR DRug hyDRoxylaTion PhenoTyPing in hisPaniC PoPulaTions F. De Andres; M.E.G. Naranjo; M. Sosa-Macias; B.P. Lazalde Ramos; E. Tarazona-Santos; A. LLerena; and CEIBA Consortium CICAB, Clinical Research Centre, Extremadura University Hospital, BADAJOZ, Spain; Centro Interdisciplinario de Investigacion para el Desarrollo Integral Regional del IPN Unidad Durango, CIIDIR-IPN, Durango; Laboratorio de Medicina Molecular, Unidad Academica de Medicina Humana y Ciencias de la Salud, Universidad Autonoma de Zacatecas, Zacatecas, Mexico; and Departamento de Biologia Geral, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Introduction: Interethnic variability of drug metabolism has been demonstrated across Iberoamerican populations by the CEIBA Consortium. Drug-metabolizing enzymes genotype just predicts actual drug metabolic activity (phenotype) in the cases of poor metabolizers. For example, only 30% of ultrarapid metabolizers can be predicted from genotyping (Llerena et al., 2012). Moreover, according to EMA recommendations, development of phenotyping procedures for drug interactions studies and clinical research recommended (EMA, 2013). Therefore, a novel cocktail approach to measure metabolic activity (metabolic ratios) of the main CYP enzymes in just one experiment is developed and validated to be used in the study of Latin-American populations. Patients (or Materials) and Methods: Subjects were given low oral doses of 100-mg caffeine, 25-mg losartan, 20-mg omeprazole, and 30-mg dextromethorphan. Blood samples were taken 4 hours after administering the drugs to assay the following metabolic ratios in plasma: CYP1A2 (caffeine/paraxanthine), CYP2C9 (losartan /E-3174), CYP2C19 (omeprazole/5-hydroxyomeprazole), CYP2D6 (dextromethorphan/ dextrorphan), and CYP3A4 (dextromethorphan/3-methoxymorphinan). Solid phase extraction was utilized for analyte extraction and LC-MS/MS to quantify the probe drugs and metabolites. Results: Recovery values > 80% were obtained for all analytes, and no carryover or relative matrix effects were observed. The analytes were separated and detected in 9 minutes and the method was fully validated, with lower limits of quantification ranging from 0.3 ng mL–1 for 5-hydroxyomeprazole to 3.2 ng mL–1 for paraxanthine. The correlation coefficient (r2) values obtained were over 0.995 for all analytes. According to the EMA guideline on bioanalytical method validation, precision and accuracy values below 15% were achieved. Conclusion: The method was proven to be useful to measure targeted analytes for the evaluation of CYPs hydroxylation capacity in just a single experiment. Funding Sources: Supported by AEXCID-Cooperacion Extremena of Junta de Extremadura (11IA002) to Sociedad Ibero-Americana de Farmacogenetica-SIFF, and coordinated by the RIBEF network (Red Iberoamericana de Farmacogenetica y Farmacogenomica; www. ribef.com). Disclosure of Interest: None declared. PP151—PRevalenCe of CaRRiage CyP2C9, vkoRC1 anD CyP4f2 PolymoRPhisms in Russian PaTienTs WiTh high ThRomboTiC Risk PResCRibeD WaRfaRin ComPaReD WiTh oTheR eThniC gRouPs D. Ivashchenko; D. Sychev; I. Rusin; A. Grachev; and T. Beloshickaya Clinical Pharmacology, I.M. Sechenov First Moscow State Medical University; Cardiology; and Medical Genetic, SM-Clinic, Moscow, Russian Federation Introduction: VKORC1, CYP2C9, and CYP4F2 responsible for the metabolism of warfarin. Personal and ethnic differences in the genetic profile reveals in determining the outcome of a drug therapy. Aim: to explore the frequencies of CYP2C9, VCORC1, and CYP4F2 genotypes in Russians, compare results with ones for other nations Patients (or Materials) and Methods: A total 91 Caucasian subjects were recruited into the study. Forty (48.2%) patients were male and age was 66.17 (10.9) years. All patients had indications to receive warfarin. Medical records for the patients group were reviewed for the relevant clinical data. 5 ml of blood was taken from each subject, and DNA was isolated and used for identification of the CYP2C9 allele *1, *2, *3; G-1639A VCORC1; CYP4F2 V433M rs2108622 C > T using real-time polymerase chain reaction-restriction fragment length polymorphism assay. Results were compared with other ethnic groups and statistically analyzed with chi-square test. Results: We described the prevalence of CYP2C9 polymorphisms *1/*1 (67%), *1/*2 (9,9%), *1/*3 (11%), *2/*2 (2,2%), *2/*3 (8,8%), same for VKORC1 GG (49,5%), GA (28,6%), AA (22,%) and CYP4F2 CC (57,1%), CT (34,1%), TT (7,7%). No significant deviantions from Hardy-Wainberg equilibrium were observed (p 0.05). Conclusion: Prevalence of carriage of CYP2C9, VKORC1 polymorphisms in Russian patients with high thrombotic risk is closed to Caucasians and distinguish from Asians but no for CYP4F2. Disclosure of Interest: None declared.

Evaluation of drug-metabolizing enzyme hydroxylation phenotypes in Hispanic populations: the CEIBA cocktail

Interindividual differences in response to drug treatments are mainly caused by differences in drug metabolism, in which cytochrome P450 (CYP450) enzymes are involved. Genetic polymorphisms of these enzymes have a key role in this variability. However, environmental factors, endogenous metabolism and disease states also have a great influence on the actual drug metabolism rate (metabolic phenotype). Consequently, the genotype does not always correlate with the actual drug hydroxylation phenotype. In this sense, in vivo phenotyping strategies represent an alternative to evaluate the interindividual variability in drug metabolism. Therefore, the 'cocktail' approach is considered as an advantageous strategy to obtain actual and reliable information on several CYP activities in just one experiment. As reviewed, phenotyping studies on Latin-American populations, which comprise about 400 million people, are scarce, and only selective phenotyping methods were applied. Therefore, a novel cocktail approach is here proposed as a phenotyping tool to evaluate the relationship between genotype and phenotype of major CYP enzymes in Hispanic populations. This determination will allow adaptation of drug therapies to these populations and consequently to benefit from the application of pharmacogenetics in the reduction of drug adverse effects and in the improvement of therapeutic responses.