Fernando De Andres

Losartan is metabolized to losartan carboxylic acid (E-3174) by the polymorphic cytochrome CYP2C9. The aim of the study was to develop a high-performance liquid chromatographic (HPLC) method with fluorescence detection for simultaneously measuring losartan and its metabolite E-3174 in urine to evaluate the losartan urinary metabolic ratio (MR: losartan/E-3174) for CYP2C9 phenotyping in humans. The compounds were separated in a reversed-phase chromatographic column and detected by fluorescence at a wavelength of 250 nm for excitation and of 370 nm for emission. No analytical interferences with endogenous compounds were found, and the extraction recoveries were over 88%. Limits of quantification of 2 ng mL-1 for losartan and 5 ng mL-1 for E-3174 were achieved, as well as good reproducibility with coefficients of variation of <9% in all cases. Analyses with the present HPLC method show significant differences (p<0.05) in losartan MRs between the four CYP2C9 genotype groups in 13 Spanish healthy volunteers. The method developed is simple and affordable, as well as sensitive and reliable to calculate the MR. Therefore, it appears to be useful for CYP2C9 phenotyping using losartan as a drug test in populations, such as Hispanics with different allele combinations.

Background: Interethnic differences in CYP2D6 allele frequency have been demonstrated across Latin–American countries. Only one previous study describing CYP2D6 genotypes in Colombian population has been performed. Thus, this study aimed to evaluate the CYP2D6 genetic variability in a mestizo Colombian population, as well as the similarities and differences concerning other Hispanic mestizo (HM) populations. Methodology: Two hundred and twelve unrelated healthy Colombian subjects were studied, in which different CYP2D6 polymorphisms were analyzed by extra long-PCR and real-time PCR. Results & discussion: A high percentage of ultrarapid metabolizers (18.4%) was found, representing the highest frequency calculated within the HM populations studied. However, the percentage of poor metabolizers (4.7%) was similar to those previously reported in HM populations.

Interethnic variability in the drug-metabolizing capacity of CYP450 enzymes may lead to discrepancies in the relationship between genotypes and phenotypes worldwide. The present study was aimed to analyze for the first time whether there is a relationship between clinically relevant CYP450 genetic polymorphisms and their drug oxidation capacity (metabolic phenotype) in a population of healthy Nicaraguan volunteers. Two hundred and twelve participants were genotyped for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and their actual metabolic phenotype (evaluated by the Metabolic Ratio, MR) was analyzed by using the CEIBA cocktail approach. The results showed the wide interindividual variability in all the studied enzymes and a significant difference (p < 0.004) in the activity of CYP1A2 between male and female subjects. The number of CYP2C19 (p < 0.0001) and CYP2D6 (p < 0.0001) active alleles were shown inversely correlated with their corresponding MR, although there were marked genotype-phenotype discrepancies. There was an actual enzyme capacity overlapping (MR) between genotypically Poor (gPMs) and Extensive Metabolizers (gEMs) of 3.14% subjects for CYP2D6 and 0.94% for CYP2C9. Similarly, there was an overlapping for metabolic phenotypes of 11.48% of genotypically ultrarapid metabolizers (gUMs) for CYP2C19 and 2.09% for CYP2D6 and gEMs. Therefore, the current approach for metabolic phenotype prediction based just on genotype does not predict properly for all individuals within this Nicaraguan Mestizo population, thus representing a potential barrier for the clinical implementation of personalized medicine in this region. However, it is necessary to improve the prediction of phenotype from genotype in order to improve the pharmacogenetic implementation in populations with specific ethnic backgrounds.

A novel analytical methodology for the extraction and determination of catecholamines (dopamine, epinephrine and norepinephrine) and their metabolites DL-3,4-dihydroxyphenyl glycol and DL-3,4-dihydroxymandelic acid by LC-MS is developed and validated for its application to human and animal urine and hair samples. The method is based on the preliminary extraction of the analytes by a magnetic multi-walled carbon nanotube poly(styrene-co-divinylbenzene) composite. This is followed by a <9 min chromatographic separation of the target compounds in an Onyx Monolithic C18 column using a mixture of 0.01% (v/v) heptafluorobutyric acid in water and methanol at 500 µL min-1 flow rate. Detection limits within range from 0.055 to 0.093 µg mL-1, and precision values of the response and retention times of analytes were >90%. Accuracy values comprised the range 79.5-109.5% when the analytes were extracted from deer urine samples using the selected MMWCNT-poly(STY-DVB) sorbent. This methodology was applied to real red deer urine and hair samples, and concentrations within range from 0.05 to 0.5 µg mL-1 for norepinephrine and from 1.0 to 44.5 µg mL-1 for its metabolite 3,4-dihydroxyphenyl glycol were calculated. Analyses of red deer hair resulted in high amounts of 3,4-dihydroxyphenyl glycol (0.9-266.9 µg mL-1).

Pharmacogenetic variation in Latin Americans is understudied, which sets a barrier for the goal of global precision medicine. The RIBEF-CEIBA Network Consortium was established to characterize interindividual and between population variations in CYP2D6, CYP2C9, and CYP2C19 drug metabolizing enzyme genotypes, which were subsequently utilized to catalog their "predicted drug metabolism phenotypes" across Native American and Ibero American populations. Importantly, we report in this study, a total of 6060 healthy individuals from Ibero-America who were classified according to their self-reported ancestry: 1395 Native Americans, 2571 Admixed Latin Americans, 96 Afro-Latin Americans, 287 white Latin Americans (from Cuba), 1537 Iberians, and 174 Argentinean Ashkenazi Jews. Moreover, Native Americans were grouped into North-, Central-, and South Amerindians (from Mexico, Costa Rica, and Peru, respectively). All subjects were studied for the most common and functional CYP2D6, CYP2...

A simple, rapid, selective and sensitive monitoring method for the simultaneous determination of the widely-prescribed antidepressants agomelatine, bupropion, citalopram, fluoxetine, mirtazapine, paroxetine, trazodone in just a human blood drop is here developed and validated. This methodology is based on the use of lab manufactured poly(styrene-co-divinylbenzene)-coated glass (PS-DVB) blood spot for the extraction of the analytes and their subsequent separation and detection by capillary liquid chromatography-mass spectrometry (CLC-MS). Briefly, 10 mm-side squares were punched out from blood spots collected on glass substrate coated by 10 µg of the PS-DVB polymer and eluted with 1.0 mL of 2.0% acetic acid in methanol. The analytes were then separated and detected in less than 20 min by capillary CLC-MS using a Jupiter 4 µm Proteo 90 Å column and water: acetonitrile (20:80 v/v) and ammonium acetate (5 mM, pH 3.0) as mobile phase. Limit of detection (LOD) ranged from 0.018 to 0.038 µ...

In a one-way cross-over study, we investigated the effect of Khat, a natural amphetamine-like psychostimulant plant, on catalytic activities of five major drug-metabolizing cytochrome P450 (CYP) enzymes. After a one-week Khat abstinence, 63 Ethiopian male volunteers were phenotyped using cocktail probe drugs (caffeine, losartan, dextromethorphan, omeprazole). Phenotyping was repeated after a one-week daily use of 400 g fresh Khat leaves. Genotyping for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A5 were done. Urinary cathinone and phenylpropanolamine, and plasma probe drugs and metabolites concentrations were quantified using LC-MS/MS. Effect of Khat on enzyme activities was evaluated by comparing caffeine/paraxanthine (CYP1A2), losartan/losartan carboxylic acid (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), dextromethorphan/dextrorphan (CYP2D6) and dextromethorphan/3-methoxymorphinan (CYP3A4) metabolic ratios (MR) before and after Khat use. Wilcoxon-matched-pair-test indicated a sign...

Poly(styrene-co-divinylbenzene)-coated magnetic multiwalled carbon nanotube composite synthesized by in-situ high temperature combination and precipitation polymerization of styrene-co-divinylbenzene has been employed as a magnetic sorbent for the solid phase extraction of antidepressants in human urine samples. Fluoxetine, venlafaxine, citalopram and sertraline were, afterwards, separated and determined by capillary electrophoresis with diode array detection. The presence of magnetic multiwalled carbon nanotubes in native poly(styrene-co-divinylbenzene) not only simplified sample treatment but also enhanced the adsorption efficiencies, obtaining extraction recoveries higher than 89.5% for all analytes. Moreover, this composite can be re-used at least 10 times without loss of efficiency and limits of detection ranging from 0.014 to 0.041 μg mL were calculated. Additionally, precision values ranging from 0.08 to 7.50% and from 0.21 to 3.05% were obtained for the responses and for the...

A sensitive and selective method for the separation and quantification of the three organic acids 3-hydroxy-3-methylglutaric acid, 3-methylglutaric acid, and glutaric acid in human urine samples by CE with mass spectrometry detection has been developed. This methodology is faster, simpler and less time-consuming, than other methodologies previously described, and requires of reduced amounts of reagents as well. Samples are first filtered and then diluted in water. For the electrophoretic separation, a 20mM ammonium acetate and 10% methanol solution at pH 9.1 was selected as the running electrolyte. With 5-s hydrodynamic injection, detection limits ranging from 15.5 to 39.3μM and linear responses ranging from the LOQ calculated for each analyte to more than 400μM were obtained for the analysis of the different organic acids in less than 13min. Remarkable selectivity is achieved by mass spectrometry detection using 0.25% of formic acid in 50% v/v 2-propanol-water solution as sheath li...

Depression is a common, severe, disabling mental disease that affects millions of people of all ages worldwide. Various studies have shown that neurotrophic/growth factors have a key role in depression and, more specifically, vascular endothelial growth factor (VEGF) is implicated in the pathogenesis of depression. The purpose of this study was to investigate the potential links between four VEGF-related single-nucleotide polymorphisms (SNPs), previously identified through a genome-wide association study (GWAS) and depression. The direct effects and epistatic interactions of the four VEGF-related SNPs (rs10738760, rs6921438, rs6993770 and rs4416670) on depression were investigated through a case-control study including 437 individuals diagnosed with depression and 477 healthy volunteers as controls. Gender, age and body mass index influence was additionally analyzed. The SNP rs4416670 was associated with increased risk for depression (OR: 1.60, P: 0.010). This result demonstrates th...

Aim: Several neuropsychopharmacological properties have been attributed to the 3α-reduced pregnane steroids, allopregnanolone and pregnanolone, as well as to dehydroepiandrosterone sulfate because of their ability to modulate γ-aminobutyric acid (GABAA) receptors in the CNS. In order to understand better their role in several mechanisms in CNS, a new methodology is proposed to monitor these compounds in human plasma. Methodology & results: The analytes were first derivatized with 2-hydrazinopyridine and extracted from plasma using SPE. Then, the compounds were separated and detected by LC–MS/MS. A mobile phase of formic acid (0.1%) in water and methanol through a gradient of composition and a flow rate of 0.3 ml min-1 resulted in good separations of the analytes. Linear responses in wide range of concentrations and LOQs ranging from 10 (dehydroepiandrosterone 3-sulfate) to 40 pg ml-1 (dehydroepiandrosterone) were obtained in <9 min. The method proposed has been validated and then...

Public policies to stop or reduce cigarette smoking and exposure to secondhand smoke and associated diseases have yielded successful results over the past decade. Yet, the growing worldwide popularity of another form of tobacco consumption, water pipe smoking, has received relatively less attention. To the best of our knowledge, no study to date has evaluated the effects of water pipe smoking on cytochrome P450 (CYP450) activities and drug interaction potential in humans, whereas only limited information is available on the impact of secondhand smoke on drug metabolism. In a sample of 99 healthy volunteers (28 water pipe smokers, 30 secondhand tobacco smoke exposed persons, and 41 controls), we systematically compared CYP1A2 and CYP2A6 enzyme activities in vivo using caffeine urine test. The median self-reported duration of water pipe smoking was 7.5 h/week and 3 years of exposure in total. The secondhand smoke group had a median of 14 h of self-reported weekly exposure to tobacco s...

Purpose - The CEIBA cocktail consisting of caffeine (CAF), omeprazole (OZ), dextromethorphan (DM) and losartan (LOS) was previously proposed for the clinical phenotyping of five major human cytochrome P450 (CYP) isoenzymes. This work aimed to assess the usefulness of CEIBA cocktail to study non-clinical drug interactions in the rat. Methods - Wistar rats were divided into five groups to receive a single-oral dose of each probe drug (CAF, OZ, LOS, DM), individually or in combination as a cocktail. Plasma concentrations of the probe drugs and their metabolites [paraxanthine (1,7-X), 5-hydroxyomeprazole (5-OZ), losartan carboxylic acid (E-3174), dextrorphan (DX) and 3-methoxymorphinan (3-MM)] were determined by LC-MS/MS, and the corresponding pharmacokinetic parameters were estimated by non-compartmental analysis. The AUC0-t and Cmax drug/metabolite ratios (phenotypic metrics) were calculated for each probe drug and compared (probe alone versus cocktail). Results - The primary analysis...

Migraine is a multifactorial, neurological and disabling disorder, also characterized by several autonomic symptoms. Triptans, selective serotonin 5-HT1B/1D agonists, are the first-line treatment option for moderate-to-severe headache attacks. In this paper, we review the recent data on eletriptan clinical efficacy, safety, and tolerability, and potential clinically relevant interactions with other drugs. Among triptans, eletriptan shows a consistent and significant clinical efficacy and a good tolerability profile in the treatment of migraine, especially for patients with cardiovascular risk factors without coronary artery disease. It shows the most favorable clinical response, together with sumatriptan injections, zolmitriptan and rizatriptan. Additionally, eletriptan shows the most complex pharmacokinetic/dynamic profile compared with the other triptans. It is metabolized primarily by the CYP3A4 hepatic enzyme and therefore the concomitant administration of CYP3A4-potent inhibito...

Bevacizumab was the first molecular-targeted antiangiogenic therapy approved for the treatment of metastatic colorectal cancer. Until now, there are no predictive biomarkers available to decide the prescription of bevacizumab in patients with colorectal cancer. The purposes of this review were to provide a critical appraisal of the evidence and to identify possible predictive genetic biomarkers. A literature search was performed to identify studies that determine different levels of treatment response between patients stratified according to defined biomarkers. Interesting findings were reported between patients stratified according to rs3025039 and rs833061 polymorphisms of the gene VEGFA, with statistically and clinically significant differences for progression-free survival and overall survival. However, another study conducted in a larger sample does not confirm these previous findings, suggesting that well-designed prospective studies are still needed to achieve conclusive resu...

The use of khat (Catha edulis) while on medication may alter treatment outcome. In particular, the influence of khat on the metabolic activities of drug-metabolizing enzymes is not known. We performed a comparative 1-way crossover study to evaluate the effect of khat on cytochrome P450 (CYP)2D6 and CYP3A4 enzyme activity. After 1 week of khat abstinence, baseline CYP2D6 and CYP3A4 metabolic activities were determined in 40 Ethiopian male volunteers using 30 mg dextromethorphan (DM) as a probe drug and then repeated after 1 week of daily use of 400 g fresh khat leaves. Urinary concentrations of cathinone and cathine were determined to monitor the subjects' compliance to the study protocol. Genotyping for CYP2D6*3 and CYP2D6*4 was done. Plasma DM, dextrorphan and 3-methoxymorphinan concentrations were quantified. CYP2D6 and CYP3A4 enzyme activities were assessed by comparing plasma log DM/dextrorphan and log DM/methoxymorphinan metabolic ratio (MR) respectively in the presence and absence of khat. Cytochrome 2D6 MR was significantly increased from baseline by concurrent khat use (paired t test, P = 0.003; geometric mean ratio, 1.38; 95% confidence interval [95% CI], 1.12-1.53). Moreover, the inhibition of CYP2D6 activity by khat was more pronounced in CYP2D6*1/*1 compared with CYP2D6*1/*4 genotypes (P = 0.01). A marginal inhibition of CYP3A4 activity in the presence of khat was observed (P = 0.24). The mean percentage increase of CYP2D6 and CYP3A4 MR from baseline by khat use was 46% (95% CI, 20-72) and 31% (95% CI, 8-54), respectively. This is the first report linking khat use with significant inhibition of CYP2D6 metabolic activity in humans.

e156 Volume 37 Number 8S Extremadura, Badajoz, Spain; Colegio de Ciencias de la Salud, Universidad San Francisco de Quito, Quito, Ecuador; and Servicio de Laboratorio, Hospital de los Valles, Quito, Ecuador Introduction: According to EMA recommendations, development of phenotyping procedures for drug interactions studies and clinical research are highly recommended, due to the discordances found between genotypes determined of the main CYP enzymes and their actual enzymatic activity. Recently, CEIBA cocktail approach to measure metabolic activity of the main CYPs in just one experiment has been designed. However, its optimal design remains to be elucidated, which is the main objective of this research, in order to appropriately select the sample to be analyzed and an optimal single time point for the analysis, to avoid diminish costs and discomfort to the volunteers without compromising the reliability of the results. Material and Methods: Thirteen healthy subjects were given low oral doses of 100mg caffeine, 25mg losartan, 20mg omeprazole and 30 mg dextromethorphan, and blood and urine samples were taken at different time points (predose, 0.5, 1, 2, 3, 4, 6, 8, and 12h) to assay the concentration and pharmacokinetic parameters (AUC). The MRs for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were calculated at each time point and their correlation with AUC ratios calculated. All subjects were genotyped. Results: The cocktail was well tolerated and single time point MRs at 4h or 6h (only for CYP2C9) after dosing showed high correlations with corresponding AUC ratios and can, therefore, be proposed as simple phenotyping metrics. On the other hand, metabolic ratios in urine samples were not comparable to plasma ratios. Conclusion: This cocktail was proven to reliably reflect the selected CYPs activities for the evaluation of CYPs hydroxylation capacity. The proposed simplified sampling scheme could facilitate clinical application of CYPs phenotyping with one blood sample collection, in a single analysis. Supported by AEXCID of GOBEX (11IA002) to Sociedad Iberoamericana de Farmacogenetica, Instituto de Salud Carlos IIISara Borrell program (CD13/00348), and Collaboration grant from USFQ (ET); coordinated by the RIBEF network. Disclosure of Interest: None Declared.

Aripiprazole (ARI) is an antipsychotic drug that is metabolized to dehydroaripiprazole (DARI) by CYP2D6. Because of the large interindividual variability in ARI and DARI plasma concentrations, therapeutic drug monitoring may be of use in psychiatric patients during treatment with ARI. The aim of the present study was to develop a simple and reliable method for the quantitative determination of ARI and DARI in plasma using liquid-liquid extraction and reverse-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The method was tested in psychiatric patients during regular treatment with ARI. Separation was by the liquid-liquid method, and UV detection at 254 nm. Linear responses for ARI and DARI were obtained between 2 and 1000 ng/mL, and precision assays were lower than 10.4 for both analytes. Lower limit of quantification and detection were 1 and 0.38 ng/mL for ARI and 0.78 and 0.44 ng/mL for DARI, respectively. The method was successfully applied to plasma samples drawn from 22 patients with concentrations ranging between 2 and 189 ng/mL for ARI and between 11 and 359 ng/mL for DARI. The chromatographic method developed has been demonstrated to be sensitive and reliable for the measurement of ARI and DARI simultaneously in human plasma, and the present method represents an alternative procedure to evaluate plasma concentration in patients during treatment with ARI.

The bioanalytical applications of supercritical fluid techniques, such as supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC), are of increasing interest. The main role of these techniques is in the sample preparation and separation of biologically active compounds, particularly drugs and their metabolites, as well as endogenous compounds. An insight is given into the different types of extracting fluids and modifiers, detectors, stationary phases, mobile phases and collection strategies. A critical discussion is presented on the existing state of the art concerning the applications of SFC and SFE with a specific focus on its advantages and limitations in the bioanalytical field. New developments and the possibilities for routine work in the near future are also covered.

A new rapid and sensitive high-performance liquid chromatography method has been developed for the screening and determination in human plasma of the 11 most commonly prescribed nontricyclic antidepressants and two metabolites: fluoxetine, norfluoxetine, sertraline, paroxetine, ...

Global precision medicine demands characterization of drug metabolism and phenotype variation in diverse populations, including the indigenous societies. A related question is the extent to which CYP450 drug metabolizing enzyme genotype and phenotype data are concordant and whether they can be used interchangeably. These issues are increasingly debated as precision medicine continues to expand as a popular research topic worldwide. We report here the first study in clinically relevant CYP450 drug metabolism phenotypes and genotypes in Mexican Amerindian indigenous subjects. In a large sample of 450 unrelated and medication free Mexican Amerindian indigenous healthy persons from four Mexican states (Chihuahua, Durango, Nayarit, and Sonora), we performed multiplexed phenotyping for the CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 drug metabolizing enzymes using the CEIBA cocktail and genotyped the same pathways for functional polymorphic variation. Remarkable interindividual variabilit...

A long-standing question and dilemma in precision medicine is whether and to what extent genotyping or phenotyping drug metabolizing enzymes such as CYP2D6 can be used in real-life global clinical and societal settings. Although in an ideal world using both genotype and phenotype biomarkers are desirable, this is not always feasible for economic and practical reasons. Moreover, an additional barrier for clinical implementation of precision medicine is the lack of correlation between genotype and phenotype, considering that most of the current methods include only genotyping. Thus, the present study evaluated, using dextromethorphan as a phenotyping probe, the relationship between CYP2D6 phenotype and CYP2D6 genotype, especially for the ultrarapid metabolizer (UM) phenotype. We report in this study, to the best of our knowledge, the first comparative clinical pharmacogenomics study in a Cuban population sample (N = 174 healthy volunteers) and show that the CYP2D6 genotype is not a ro...

Genetic variations within the cytochrome P450 (CYP450) superfamily of drug metabolizing enzymes confer substantial person-to-person and between-population differences in pharmacokinetics, and by extension, highly variable clinical effects of medicines. In this context, "personalized medicine," "precision medicine," and "stratified medicine" are related concepts attributed to what is essentially targeted therapeutics and companion diagnostics, aimed at improving safety and effectiveness of health interventions. We report here, to the best of our knowledge, the first comparative clinical pharmacogenomics study, in an Ecuadorian population sample, of five key CYP450s involved in drug…

The present study evaluates the worldwide frequency distribution of CYP2C19 alleles and CYP2C19 metabolic phenotypes ('predicted' from genotypes and 'measured' with a probe drug) among healthy volunteers from different ethnic groups and geographic regions, as well as the relationship between the 'predicted' and 'measured' CYP2C19 metabolic phenotypes. A total of 52 181 healthy volunteers were studied within 138 selected original research papers. CYP2C19*17 was 42- and 24-fold more frequent in Mediterranean-South Europeans and Middle Easterns than in East Asians (P<0.001, in both cases). Contrarily, CYP2C19*2 and CYP2C19*3 alleles were more frequent in East Asians (30.26% and 6.89%, respectively), and even a twofold higher frequency of these alleles was found in Native…

Background: Interethnic differences in CYP2D6 allele frequency have been demonstrated across Latin-American countries. Only one previous study describing CYP2D6 genotypes in Colombian population has been performed. Thus, this study aimed to evaluate the CYP2D6 genetic variability in a mestizo Colombian population, as well as the similarities and differences concerning other Hispanic mestizo (HM) populations. Methodology: Two hundred and twelve unrelated healthy Colombian subjects were studied, in which different CYP2D6 polymorphisms were analyzed by extra long-PCR and real-time PCR. Results/discussion: A high percentage of ultrarapid metabolizers (18.4%) was found, representing the highest frequency calculated within the HM populations studied. However, the percentage of poor metabolizers (4.7%) was similar to those previously reported in HM populations.

There is a growing demand for the clinical implementation of pharmacogenetics (PGx), personalized and precision medicine (PPM) for drug prescription to reduce adverse drug reactions (ADRs), drug failure, and ultimately health care costs. However, it is convenient to clarify the concept of clinical implementation to realize its benefits. Advances on PGx clinical implementation depend on the integration of genetic along with other relevant biomarkers and clinical information influencing variability in drug response for being interpreted and translated into clinical decision-making to optimize drug treatment choice during routine clinical practice. There are several initiatives related to PGx clinical implementation in Europe [1], using either a preventive (pre-emptive) measures approach or a reactive one (point of care) (https://www.icpermed.eu/). Some of them are integrated on different research platforms, that perform the genetic analyses using either single drug-gene combinations, gene-arrays, or next-generation sequencing (NGS) [1]. Nevertheless, many of them just aim to analyze the relevance of genetic information in the absence of other relevant data influencing drug response variation, without considering polypharmacy in the context of multi-morbidity. PGx applied to phar-macokinetic variability is so far the cornerstone of its clinical implementation [2]. Thus, determining actual phenotypes by analyzing metabolic ratios from actual drug treatments to evaluate enzyme activity (dose-dependent phenocopying [3]) as shown during treatment with thioridazine [4], risperidone [5] or fluoxetine [6], including additional influencing factors [7] such as liver or kidney function, and concomitant pharmacological treatments (to prevent drug-drug interactions [8]) becomes essential to develop a precise clinical implementation process. Therefore, despite the need of applying PPM into daily clinical practice, there are still barriers to overcome regarding the inclusion of all factors relevant for variation in drug response. Moreover, another important limitation is information management. Hence, there is a need for developing computational tools that may integrate the relevance of different factors influencing drug response in a context of polypharmacy to simplify the prescribers' decision-making. Currently, drug selection requires manual data entry, therefore a guided drug prescription e-tool integrated into the Electronic Medical Record (EMR) is needed for improving drug choices in the context of drug poly-therapy and poly-pathology. Moreover, the system needs to be evaluated to establish a cost/effectiveness analysis for its implementation in the Public Health Service.

Interethnic variability in the drug-metabolizing capacity of CYP450 enzymes may lead to discrepancies in the relationship between genotypes and phenotypes worldwide. The present study was aimed to analyze for the first time whether there is a relationship between clinically relevant CYP450 genetic polymorphisms and their drug oxidation capacity (metabolic phenotype) in a population of healthy Nicaraguan volunteers. Two hundred and twelve participants were genotyped for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and their actual metabolic phenotype (evaluated by the Metabolic Ratio, MR) was analyzed by using the CEIBA cocktail approach. The results showed the wide interindividual variability in all the studied enzymes and a significant difference (p < 0.004) in the activity of CYP1A2 between male and female subjects. The number of CYP2C19 (p < 0.0001) and CYP2D6 (p < 0.0001) active alleles were shown inversely correlated with their corresponding MR, although there were marked genotype-phenotype discrepancies. There was an actual enzyme capacity overlapping (MR) between genotypically Poor (gPMs) and Extensive Metabolizers (gEMs) of 3.14% subjects for CYP2D6 and 0.94% for CYP2C9. Similarly, there was an overlapping for metabolic phenotypes of 11.48% of genotypically ultrarapid metabolizers (gUMs) for CYP2C19 and 2.09% for CYP2D6 and gEMs. Therefore, the current approach for metabolic phenotype prediction based just on genotype does not predict properly for all individuals within this Nicaraguan Mestizo population, thus representing a potential barrier for the clinical implementation of personalized medicine in this region. However, it is necessary to improve the prediction of phenotype from genotype in order to improve the pharmacogenetic implementation in populations with specific ethnic backgrounds.

A simple and enantioselective method for the separation and determination of carnitine enantiomers in dietary supplements and pharmaceutical formulation samples is proposed. This method is based on achiral liquid chromatographic separation of carnitine enantiomers from interferences and direct circular dichroism (CD) detection. The calibration curve of the anisotropy factor (g) versus the enantiomeric excess was linear, with a correlation coefficient (R 2) of 0.996. The precision evaluated by UV peak area and CD peak area was suitable (RSD <5% in all cases). The usefulness of the proposed method was demonstrated by analysing natural dietary supplements and pharmaceutical formulation samples. This method has the advantages of being rapid and precise, without using an expensive chiral column. The method was suitable for the simultaneous determination of both enantiomers and for assessing the chemical purity of carnitine.

A novel analytical methodology for the extraction and determination of catecholamines (dopamine, epinephrine and norepinephrine) and their metabolites DL-3,4-dihydroxyphenyl glycol and DL-3,4-dihydroxymandelic acid by LC-MS is developed and validated for its application to human and animal urine and hair samples. The method is based on the preliminary extraction of the analytes by a magnetic multi-walled carbon nanotube poly (styrene-co-divinylbenzene) composite. This is followed by a < 9 min chromatographic separation of the target compounds in an Onyx Monolithic C 18 column using a mixture of 0.01% (v/v) heptafluorobutyric acid in water and methanol at 500 µL min −1 flow rate. Detection limits within range from 0.055 to 0.093 µg mL −1 , and precision values of the response and retention times of analytes were > 90%. Accuracy values comprised the range 79.5-109.5% when the analytes were extracted from deer urine samples using the selected MMWCNT-poly (STY-DVB) sorbent. This methodology was applied to real red deer urine and hair samples, and concentrations within range from 0.05 to 0.5 µg mL −1 for norepinephrine and from 1.0 to 44.5 µg mL −1 for its metabolite 3,4-dihydroxyphenyl glycol were calculated. Analyses of red deer hair resulted in high amounts of 3,4-dihydrox-yphenyl glycol (0.9-266.9 µg mL −1).

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