RESUMEN
Despite the importance of mice as a preclinical species in drug testing, their hepatic and extrahepatic drug-metabolising characteristics are poorly understood. Here, we compared the P450-dependent drug oxidation activity in tissue microsomes and distribution patterns of P450 protein/mRNA between humans and mice.The activities of midazolam 1'-/4-hydroxylation in the liver and intestine and chlorzoxazone 6-hydroxylation in the liver were similar in humans and mice. The activities of coumarin 7-hydroxylation, flurbiprofen 4'-hydroxylation, and S-mephenytoin 4'-hydroxylation in the liver were higher in humans than in mice. The activities of 7-ethoxyresorufin O-deethylation in the liver, 7-pentoxyresorufin O-depentylation in the lung/liver/intestine, bufuralol 1'-hydroxylation in the liver/intestine, propafenone 4'-hydroxylation in liver/intestine, and diazepam N-demethylation in the liver/intestine were higher in mice than in humans.CYP1A2/2E1 mRNAs were mainly expressed in the livers of humans and mice. Cyp2b9/2b10 mRNAs were abundant in the mouse lung/liver/intestine, but CYP2B6 was mainly expressed in the human liver. CYP2C/2D/3A mRNAs were expressed in the liver and intestine, with the respective proteins detected in tissue microsomes of both humans and mice.These information on P450-dependent drug-metabolising characteristics in hepatic and extrahepatic tissues is useful to understand the similarities and differences between humans and mice in drug metabolism.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Microsomas Hepáticos , Clorzoxazona/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Hígado/metabolismo , Mefenitoína/metabolismo , Microsomas/metabolismo , Microsomas Hepáticos/metabolismoRESUMEN
Inter-individual variability in pharmacokinetics and drug response is heavily influenced by single-nucleotide variants (SNVs) and copy-number variations (CNVs) in genes with importance for drug disposition. Nowadays, a plethora of studies implement next generation sequencing to capture rare and novel pharmacogenomic (PGx) variants that influence drug response. To address these issues, we present a comprehensive end-to-end analysis workflow, beginning from targeted PGx panel re-sequencing to in silico analysis pipelines and in vitro validation assays. Specifically, we show that novel pharmacogenetic missense variants that are predicted or putatively predicted to be functionally deleterious, significantly alter protein activity levels of CYP2D6 and CYP2C19 proteins. We further demonstrate that variant priorization pipelines tailored with functional in vitro validation assays provide supporting evidence for the deleterious effect of novel PGx variants. The proposed workflow could provide the basis for integrating next-generation sequencing for PGx testing into routine clinical practice.
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Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Variantes Farmacogenómicas , Algoritmos , Línea Celular , Simulación por Computador , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Citocromos b5/genética , Dextrometorfano/metabolismo , Humanos , Mefenitoína/metabolismo , Microsomas/metabolismo , Mutación Missense , Reproducibilidad de los ResultadosRESUMEN
High-performance liquid chromatography (HPLC) is the most common analytical method practiced in various fields and used for analysis of almost all drug compounds in the pharmaceutical industries. During drug development, an evaluation of potential drug interaction with cytochrome P450 (CYP) is essential. A "cocktail" approach is often used in drug development to evaluate the effect of a drug candidate on multiple CYP enzymes in a single experiment. So far, simultaneous analysis of multiple CYP substrates, which have greatly different structure and physicochemical properties, has required organic solvents and mobile phase gradient methods. However, despite the recent emphasis on environmental protection, analytical methods that use only aqueous solvents without the use of organic solvents for separation have not been studied well. This study sought to develop the simultaneous analysis of multiple CYP substrates by using poly(N-isopropylacrylamide) (PNIPAAm)-based temperature-responsive chromatography with only aqueous solvents and isocratic methods. Good separation of multiple CYP substrates was achieved without using organic solvents and any gradient methods by temperature-responsive chromatography utilizing a P(NIPAAm-co-n-butyl methacrylate (BMA))- and P(NIPAAm-co-N-acryloyl L-tryptophan methyl ester (L-Trp-OMe))-grafted silica column. Overall, PNIPAAm-based temperature-responsive chromatography represents a remarkably simple, versatile, and environmentally friendly bioanalytical method for CYP substrates and their metabolites.
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Resinas Acrílicas/química , Cromatografía Liquida/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Tecnología Química Verde/métodos , Clorzoxazona/metabolismo , Cumarinas/metabolismo , Desarrollo de Medicamentos , Mefenitoína/metabolismo , Estructura Molecular , Fenacetina/metabolismo , Solventes , Especificidad por Sustrato , Temperatura , Testosterona/metabolismo , Tolbutamida/metabolismo , AguaRESUMEN
There is evidence that the antipsychotic drug perazine is an inhibitor of CYP2D6. This study aimed at evaluating its effect on CYP2D6 and CYP2C19 activities in submitting psychiatric patients to phenotyping with dextromethorphan and mephenytoin, respectively, substrates of these enzymes, before and during a treatment with perazine. A total of 31 patients were phenotyped with dextromethorphan (CYP2D6) and mephenytoin (CYP2C19) before and after a 2-week treatment with 450 ± 51 mg/day (mean ± sd) perazine. At baseline, five patients appeared to be poor metabolizers (PM) of dextromethorphan and two patients of mephenytoin. The metabolic ratio (MR) of dextromethorphan/dextrorphan as determined in collected urine increased significantly (Wilcoxon; P < .0001) from baseline (0.39 ± 1.38 [mean ± sd]) till day 14 (1.46 ± 2.22). In 19 out of 26 extensive metabolizers (EM) of dextromethorphan, the phenotype changed from EM to PM. This suggests an almost complete inhibition of CYP2D6 by perazine and/or its metabolites. On the other hand, perazine (or some of its metabolites) did seemingly not inhibit CYP2C19. In conclusion, this study suggests that in patients treated with perazine and co-medicated with CYP2D6 substrates, there could be an increased risk of adverse effects as a consequence of a pharmacokinetic interaction.
Asunto(s)
Antipsicóticos/farmacología , Citocromo P-450 CYP2C19/metabolismo , Inhibidores del Citocromo P-450 CYP2D6/farmacología , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Mefenitoína/metabolismo , Perazina/farmacología , Esquizofrenia/tratamiento farmacológico , Adulto , Interacciones Farmacológicas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Esquizofrenia/enzimología , Adulto JovenRESUMEN
BACKGROUND: For estimation of fractions metabolized (fm) by different hepatic recombinant human CYP enzymes (rhCYP), calculation of inter-system extrapolation factors (ISEFs) has been proposed. METHODS: ISEF values for CYP1A2, CYP2C19 and CYP3A4/5 were measured. A CYP2C9 ISEF was taken from a previous report. Using a set of compounds, fractions metabolized by CYP enzymes (fm,CYP) values calculated with the ISEFs based on rhCYP data were compared with those from the chemical inhibition data. Oral pharmacokinetics (PK) profiles of midazolam were simulated using the physiologically based pharmacokinetics (PBPK) model with the CYP3A ISEF. For other CYPs, the in vitro fm,CYP values were compared with the reference fm,CYP data back-calculated with, e.g. modeling of test substrates by feeding clinical PK data. RESULTS: In vitro-in vitro fm,CYP3A4 relationship between the results from rhCYP incubation and chemical inhibition was drawn as an exponential correlation with R2=0.974. A midazolam PBPK model with the CYP3A4/5 ISEFs simulated the PK profiles within twofold error compared to the clinical observations. In a limited number of cases, the in vitro methods could not show good performance in predicting fm,CYP1A2, fm,CYP2C9 and fm,CYP2C19 values as reference data. CONCLUSIONS: The rhCYP data with the measured ISEFs provided reasonable calculation of fm,CYP3A4 values, showing slight over-estimation compared to chemical inhibition.
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Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Modelos Estadísticos , Farmacocinética , Simulación por Computador , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP3A/metabolismo , Humanos , Mefenitoína/metabolismo , Midazolam/metabolismo , Fenacetina/metabolismo , Testosterona/metabolismoRESUMEN
Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58.
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Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Mefenitoína/metabolismo , Variantes Farmacogenómicas/genética , Ticlopidina/análogos & derivados , Activación Metabólica , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Clopidogrel , Femenino , Regulación Enzimológica de la Expresión Génica , Frecuencia de los Genes , Genotipo , Humanos , Hidroxilación , Lactante , Recién Nacido , Cinética , Modelos Lineales , Masculino , Microsomas Hepáticos/enzimología , Persona de Mediana Edad , Análisis Multivariante , Oxidación-Reducción , Fenotipo , Especificidad por Sustrato , Ticlopidina/metabolismo , Adulto JovenRESUMEN
It is important to examine the cytochrome P450 2C19 (CYP2C19) genetic contribution to drug disposition and responses of CYP2C19 substrates during drug development. Design of such clinical trials requires projection of genotype-dependent in vivo clearance and associated variabilities of the investigational drug, which is not generally available during early stages of drug development, but is essential for CYP2C19 substrates with multiple clearance pathways. This study evaluated the utility of pharmacogenetics-based mechanistic modeling in predicting such parameters. Hepatic CYP2C19 activity and variability within genotypes were derived from in vitro S-mephenytoin metabolic activity in genotyped human liver microsomes (N = 128). These data were then used in mechanistic models to predict genotype-dependent disposition of CYP2C19 substrates (i.e., S-mephenytoin, citalopram, pantoprazole, and voriconazole) by incorporating in vivo clearance or pharmacokinetics of wild-type subjects and parameters of other clearance pathways. Relative to the wild-type, the CYP2C19 abundance (coefficient of variation percentage) in CYP2C19*17/*17, *1/*17, *1/*1, *17/null, *1/null, and null/null microsomes was estimated as 1.85 (117%), 1.79 (155%), 1.00 (138%), 0.83 (80%), 0.38 (130%), and 0 (0%), respectively. The subsequent modeling and simulations predicted, within 2-fold of the observed, the means and variabilities of urinary S/R-mephenytoin ratio (36 of 37 genetic groups), the oral clearance of citalopram (9 of 9 genetic groups) and pantoprazole (6 of 6 genetic groups), and voriconazole oral clearance (4 of 4 genetic groups). Thus, relative CYP2C19 genotype-dependent hepatic activity and variability were quantified in vitro and used in a mechanistic model to predict pharmacokinetic variability, thus allowing the design of pharmacogenetics and drug-drug interaction trials for CYP2C19 substrates.
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Anticonvulsivantes/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Variación Genética , Mefenitoína/metabolismo , Microsomas Hepáticos/enzimología , Modelos Biológicos , Farmacogenética/métodos , Administración Oral , Adolescente , Adulto , Anciano , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/análisis , Anticonvulsivantes/farmacocinética , Disponibilidad Biológica , Simulación por Computador , Citocromo P-450 CYP2C19/genética , Evaluación Preclínica de Medicamentos , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Mefenitoína/administración & dosificación , Mefenitoína/análisis , Mefenitoína/farmacocinética , Tasa de Depuración Metabólica , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
A sensitive and high-throughput LC-MS/MS method was established and validated for the simultaneous quantification of seven probe substrate-derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy-bupropion (CYP2B6), n-desethyl-amodiaquine (CYP2C8), 4'-hydroxy-diclofenac (CYP2C9), 4'-hydroxy-mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1'-hydroxy-midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well-known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC50 values of these inhibitors determined on this cocktail assay were highly correlated (R(2) > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89-113.35%) and between-day (RSD <13.95%) and within-day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze-thaw cycles. This seven-CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds' potential inhibition of the seven major CYPs in drug development settings.
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Inhibidores Enzimáticos del Citocromo P-450/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Microsomas Hepáticos/efectos de los fármacos , Espectrometría de Masas en Tándem/métodos , Bupropión/metabolismo , Bupropión/farmacología , Calibración , Cromatografía Liquida/métodos , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Humanos , Concentración 50 Inhibidora , Límite de Detección , Mefenitoína/metabolismo , Mefenitoína/farmacología , Microsomas Hepáticos/metabolismo , Midazolam/metabolismo , Midazolam/farmacología , Fenacetina/metabolismo , Fenacetina/farmacología , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Studies have suggested an increasing practice of concurrent herb-drug consumption. One of the major clinical risks of such concomitant herb-drug use is pharmacokinetic herb-drug interaction (HDI). This is brought about by the ability of phytochemicals to inhibit or induce the activity of metabolic enzymes. The aim of this study was to investigate the potential of the crude aqueous extracts of three popular medicinal herbs used in South Africa to inhibit major cytochrome P450 (CYP) enzymes. MATERIALS AND METHODS: The extracts of Bowiea volubilis, Spirostachys africana and Tulbaghia violacea were incubated with human liver microsomes (HLM) to monitor the phenacetin O-deethylation, diclofenac 4'-hydroxylation, S-mephenytoin 4'-hydroxylation and testosterone 6ß-hydroxylation as respective probe reactions for CYP1A2, CYP2C9, CYP2C19 and CYP3A4. The inhibitory activity, where observed, was profiled against the extract concentration. RESULTS: Extracts of Bowiea volubilis inhibited the metabolic activity of CYP1A2 and CYP3A4 with IC50 values of 92.3 ± 5.5 µg/mL and 8.1 ± 0.6 µg/mL respectively. Similar observation with Spirostachys africana showed inhibitory activity against CYP1A2 and CYP3A4 with respective IC50 values of 14.3 ± 0.6 µg/mL and 47.4 ± 2.4 µg/mL. Tulbaghia violacea demonstrated relatively weak inhibitory activity against CYP1A2 (767.4 ± 10.8 µg/mL) and CYP2C9 (921 ± 15.3 µg/mL). CONCLUSION: The results suggest the potential for HDI between the herbs and the substrates of the affected enzymes, if sufficient in vivo concentration is attained.
Asunto(s)
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interacciones de Hierba-Droga , Preparaciones Farmacéuticas/metabolismo , Extractos Vegetales/farmacología , Plantas Medicinales , Citocromo P-450 CYP2C19/metabolismo , Diclofenaco/metabolismo , Euphorbiaceae , Humanos , Hidroxilación , Liliaceae , Magnoliopsida , Mefenitoína/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Fenacetina/metabolismo , Sudáfrica , Testosterona/metabolismoRESUMEN
BACKGROUND/OBJECTIVES: The polymorphic hepatic enzyme CYP2C19 catalyzes the metabolism of clinically important drugs such as clopidogrel, proton-pump inhibitors, and others and clinical pharmacogenetic testing for clopidogrel is increasingly common. The CYP2C19*10 single-nucleotide polymorphism (SNP) is located 1 bp upstream the CYP2C19*2 SNP. Despite the low frequency of the CYP2C19*10 allele, its impact on metabolism of CYP2C19 substrates and CYP2C19*2 genotyping makes it an important SNP to consider for pharmacogenetic testing of CYP2C19. However, the effect of the CYP2C19*10 allele on clopidogrel metabolism has not been explored to date. METHODS: We measured the enzymatic activity of the CYP2C19.10 protein against clopidogrel. DNA samples from two clinical studies were genotyped for CYP2C19*2 and *10 by pyrosequencing genotyping method. RESULTS: The catalytic activity of CYP2C19.10 in the biotransformation of clopidogrel and 2-oxo-clopidogrel was significantly decreased relative to the wild-type CYP2C19.1B. We also reported that the CYP2C19*10 SNP interferes with the CYP2C19*2 TaqMan genotyping assay, resulting in miscalling of CYP2C19*10/*2 as CYP2C19*2/*2. CONCLUSIONS: Our data provide evidence that CYP2C19.10 variant partially metabolizes clopidogrel and 2-oxo-clopidogrel, and the presence of CYP2C19*10 allele affects the CY2C19*2 TaqMan genotyping assay and results in misclassification of CYP2C19*10/*2 as CYP2C19*2/*2.
Asunto(s)
Alelos , Hidrocarburo de Aril Hidroxilasas/genética , Ticlopidina/análogos & derivados , Catálisis , Cromatografía Liquida , Clopidogrel , Citocromo P-450 CYP2C19 , Genotipo , Humanos , Cinética , Mefenitoína/metabolismo , Omeprazol/metabolismo , Farmacogenética , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Espectrometría de Masas en Tándem , Ticlopidina/metabolismoRESUMEN
OBJECTIVE: We investigated whether human pharmacogenetic factors could be characterized using chimeric NOG mice expressing a thymidine kinase transgene (TK-NOG) with 'humanized' livers. MATERIALS AND METHODS: The rate of human-specific metabolism of two drugs was measured in chimeric mice reconstituted with human hepatocytes with different CYP2C19 and CYP2C9 genotypes. RESULTS: The rate of generation of human-predominant drug metabolites for S-mephenytoin and diclofenac in the chimeric mice was correlated with the CYP2C19 (n=9 donors, P=0.0005) or CYP2C9 (n=7 donors, P=0.0394) genotype, respectively, of the transplanted human hepatocytes. CONCLUSION: This study suggests that TK-NOG mice reconstituted with hepatocytes obtained from a relatively small number (3-10 per genotype) of human donors may be a promising model to identify human pharmacogenetic factors affecting the metabolism of clinically important drugs. For certain compounds, this innovative model system enables pharmacogenetic analyses to be efficiently performed in vivo within a human context and with control of all confounding environmental variables.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Diclofenaco/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Mefenitoína/metabolismo , Farmacogenética , Animales , Proteínas Portadoras/fisiología , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Hepatocitos/trasplante , Humanos , Hígado/citología , Ratones , Ratones Transgénicos , Timidina Quinasa/fisiologíaRESUMEN
The capacity of cytochrome b(5) (b(5)) to influence cytochrome P450 activities has been extensively studied and physiologically validated. Apo-b(5) enhances the activities of CYP3A4, CYP2A6, CYP2C19, and CYP17A1 but not that of CYP2E1 or CYP2D6, suggesting that the b(5) interaction varies among P450s. We previously showed that b(5) residues E48 and E49 are required to stimulate the 17,20-lyase activity of CYP17A1, but these same residues might not mediate b(5) activation of other P450 reactions, such as CYP2E1-catalyzed oxygenations, which are insensitive to apo-b(5). Using purified P450, b(5), and reductase (POR) in reconstituted assays, the D58G/D65G double mutation, of residues located in a hydrophilic α-helix of b(5), totally abolished the ability to stimulate CYP2E1-catalyzed chlorzoxazone 6-hydroxylation. In sharp contrast, the D58G/D65G double mutation retained the full ability to stimulate the 17,20-lyase activity of CYP17A1. The D58G/D65G double mutation competes poorly with wild-type b(5) for binding to the CYP2E1·POR complex yet accepts electrons from POR at a similar rate. Furthermore, the phospholipid composition markedly influences P450 turnover and b(5) stimulation and specificity, particularly for CYP17A1, in the following order: phosphatidylserine > phosphatidylethanolamine > phosphatidylcholine. The D58G/D65G double mutation also failed to stimulate CYP2C19-catalyzed (S)-mephenytoin 4-hydroxylation, whereas the E48G/E49G double mutation stimulated these activities of CYP2C19 and CYP2E1 equivalent to wild-type b(5). We conclude that b(5) residues D58 and D65 are essential for the stimulation of CYP2E1 and CYP2C19 activities and that the phospholipid composition significantly influences the b(5)-P450 interaction. At least two surfaces of b(5) differentially influence P450 activities, and the critical residues for individual P450 reactions cannot be predicted from sensitivity to apo-b(5) alone.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromos b5/química , Citocromos b5/metabolismo , Clorzoxazona/metabolismo , Citocromo P-450 CYP2C19 , Citocromos b5/genética , Humanos , Hidroxilación , Mefenitoína/metabolismo , Modelos Moleculares , Relajantes Musculares Centrales/metabolismo , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Fosfolípidos/metabolismo , Mutación Puntual , Progesterona/metabolismoRESUMEN
A series of omeprazole-based analogues was synthesized and assessed for inhibitory activity against CYP2C19. The data was used to build a CYP2C19 inhibition pharmacophore model for the series. The model was employed to design additional analogues with inhibitory potency against CYP2C19. Upon identifying inhibitors of CYP2C19, ligand-based design shifted to attenuating the rapid clearance observed for many of the inhibitors. While most analogues underwent metabolism on their aliphatic side chain, metabolite identification indicated that for analogues such as compound 30 which contain a heterocycle adjacent to the sulfur moiety, metabolism primarily occurred on the benzimidazole moiety. Compound 30 exhibited improved metabolic stability (Cl(int) = 12.4 mL/min/nmol) and was selective in regard to inhibition of CYP2C19-catalyzed (S)-mephenytoin hydroxylation in human liver microsomes. Finally, representative compounds were docked into a homology model of CYP2C19 in an effort to understand the enzyme-ligand interactions that may lead to favorable inhibition or metabolism properties.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Modelos Moleculares , Omeprazol/análogos & derivados , Omeprazol/síntesis química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Dominio Catalítico , Citocromo P-450 CYP2C19 , Diseño de Fármacos , Humanos , Hidroxilación , Técnicas In Vitro , Ligandos , Mefenitoína/metabolismo , Microsomas Hepáticos/metabolismo , Omeprazol/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/metabolismo , Receptores de Esteroides/metabolismo , Animales , Transporte Biológico , Biotransformación , Quimera , Interacciones Farmacológicas , Humanos , Inactivación Metabólica , Hígado/enzimología , Masculino , Mefenitoína/metabolismo , Mefenitoína/farmacocinética , Ratones , Microsomas Hepáticos/metabolismo , Modelos Animales , Pioglitazona , Receptor X de Pregnano , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rifampin/farmacología , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacocinética , Triazolam/metabolismo , Triazolam/farmacocinética , Warfarina/metabolismo , Warfarina/farmacocinéticaRESUMEN
CYP2C19 is a highly polymorphic enzyme that affects the metabolism of a wide range of therapeutic drugs. Almost all the identified alleles of CYP2C19 are derived from nonsynonymous single nucleotide polymorphisms (nsSNPs). The objective of this study was to functionally characterize 20 nsSNPs of CYP2C19, distributed throughout the entire coding region, most of which have not been thoroughly characterized. cDNAs of these variants were constructed and expressed in yeast cells. All variants had similar levels of apoprotein and holoprotein expression, except for CYP2C19.16 and D360N, which had significantly lower holoprotein levels than the wild-type (WT) CYP2C19 enzyme, and CYP2C19.5B, which showed only apoprotein. The activity of the CYP2C19 variants was investigated using two substrates, S-mephenytoin and omeprazole, and six different kinetic parameters were measured. CYP2C19.5B, CYP2C19.6, and CYP2C19.8 were found to be catalytically inactive. The entire dataset of the remaining 17 variants, together with the WT, was analyzed by multivariate analysis. This analysis indicated that CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.18, CYP2C19.19, A161P, W212C, and D360N were substantially altered in catalytic properties in comparison with the WT, with each of these variants exhibiting either dramatically decreased catalytic activities or higher K(m) values. These results not only generally confirmed the function of previously reported variants but also identified additional reduced-function variants. These findings will greatly extend our understanding of CYP2C19 genetic polymorphisms in humans as well as facilitate the structure-function study of the CYP2C19 protein.
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Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Mefenitoína/metabolismo , Microsomas Hepáticos/enzimología , Omeprazol/metabolismo , Polimorfismo de Nucleótido Simple , Programas Informáticos , Anticonvulsivantes/farmacocinética , Apoproteínas/análisis , Hidrocarburo de Aril Hidroxilasas/química , Simulación por Computador , Citocromo P-450 CYP2C19 , Inhibidores Enzimáticos/farmacocinética , Humanos , Hidroxilación , Mefenitoína/farmacocinética , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Omeprazol/farmacocinética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6ß-, 16α-, 16ß-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6ß-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.
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Sistema Enzimático del Citocromo P-450/metabolismo , Intestino Delgado/enzimología , Hígado/enzimología , Macaca fascicularis/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Biocatálisis , Clorzoxazona/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP2A6 , Familia 2 del Citocromo P450 , Diclofenaco/metabolismo , Duodeno/enzimología , Íleon/enzimología , Yeyuno/enzimología , Cinética , Masculino , Mefenitoína/metabolismo , Microsomas/enzimología , Microsomas Hepáticos/enzimología , Midazolam/metabolismo , Oxigenasas de Función Mixta/metabolismo , Paclitaxel/metabolismo , Esteroide 16-alfa-Hidroxilasa/metabolismo , Esteroide Hidroxilasas/metabolismo , Tolbutamida/metabolismoRESUMEN
BACKGROUND: Lichenoid drug eruptions (LDE) in the oral cavity are adverse drug reactions (ADR) that are impossible to differentiate from oral lichen planus (OLP) as no phenotypic criteria exist. Impaired function of polymorphic cytochrome 450-enzymes (CYPs) may cause increased plasma concentration of some drugs resulting in ADR/LDE. In an earlier study we did not find more patients with OLP (OLPs) with impaired CYP-genotype. OBJECTIVES: To test if more OLPs have an impaired CYP-phenotype than to be expected from the CYP-genotype and to find clinical criteria characterising oral LDE. METHODS: One hundred and twenty OLPs were genotyped for the most common polymorphisms of CYP2D6 and CYP2C19 that result in impaired function. One hundred and ten did a phenotype test of both enzymes. The exposure to drugs and polypharmacy and the CYP metabolism of the drugs were evaluated. The OLP manifestations were registered. RESULTS: The only difference in OLP manifestations was that patients with a CYP2D6 genotype with less than two fully functional alleles presented more asymmetrical OLP distribution in particular in non-medicated patients (P < 0.05). No more OLPs than expected from the genotype had a phenotype with reduced function. However, the established phenotypic categories could not differentiate between the genotypes with two or one fully functional allele. Nevertheless, among the patients with a phenotype with normal function the patients with only one functional allele had a statistically significant higher metabolic ratio compared to patients with two fully functional alleles (P < 0.05). CONCLUSION: It was not possible to identify LDE by impaired function of polymorphic CYPs.
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Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP2D6/genética , Liquen Plano Oral/inducido químicamente , Liquen Plano Oral/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Hidrocarburo de Aril Hidroxilasas/metabolismo , Distribución de Chi-Cuadrado , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6/metabolismo , Diagnóstico Diferencial , Interacciones Farmacológicas , Femenino , Genotipo , Humanos , Liquen Plano Oral/genética , Liquen Plano Oral/patología , Masculino , Mefenitoína/metabolismo , Mefenitoína/orina , Persona de Mediana Edad , Fenotipo , Polimorfismo Genético , Polifarmacia , Esparteína/metabolismo , Esparteína/orina , Estadísticas no Paramétricas , Encuestas y CuestionariosRESUMEN
The macaque is widely used for investigation of drug metabolism due to its evolutionary closeness to the human. However, the genetic backgrounds of drug-metabolizing enzymes have not been fully investigated; therefore, identification and characterization of drug-metabolizing enzyme genes are important for understanding drug metabolism in this species. In this study, we isolated and characterized a novel cytochrome P450 2C18 (CYP2C18) cDNA in cynomolgus macaques. This cDNA was highly homologous (96%) to human CYP2C18 cDNA. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney. Moreover, a metabolic assay using cynomolgus CYP2C18 protein heterologously expressed in Escherichia coli revealed its activity toward S-mephenytoin 4'-hydroxylation. These results suggest that cynomolgus CYP2C18 could function as a drug-metabolizing enzyme in the liver.
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Hidrocarburo de Aril Hidroxilasas/metabolismo , Hígado/enzimología , Macaca fascicularis/metabolismo , Mefenitoína/metabolismo , Filogenia , Secuencia de Aminoácidos , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Secuencia de Bases , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Although many cases of interindividual variation in the metabolism of CYP2C19 drugs are explained by the CYP2C19*2, *3, and *17, a wide range of metabolic variation still occurs in people who do not carry these genetic variants. The objectives of this study were to identify new genetic variants and to characterize functional consequences of these variants in metabolism of CYP2C19 substrates. In total, 21 single-nucleotide polymorphisms including three new coding variants, V394M, E405K, and D256N, were identified by direct DNA sequencing in 50 randomly selected subjects and in individuals who exhibited an outlier phenotype response in the omeprazole study. Recombinant proteins produced from the coding variants V394M, E405K, and D256N were prepared by using an Escherichia coli expression system and purified. Metabolism of S-mephenytoin and omeprazole by V394M was comparable with that of the wild-type protein. E405K showed a moderate decrease in metabolism of the substrates. However, D256N exhibited a significantly decreased activity in S-mephenytoin metabolism, resulting in 50 and 76% decreases in V(max) and intrinsic clearance, respectively, compared with the wild type. This variant also exhibited a significant decrease in omeprazole metabolism in vivo. CYP2C19 D256N and E405K were assigned as CYP2C19*26 and *2D, respectively, by the Cytochrome P450 Nomenclature Committee. In summary, this report characterizes the allele frequency and haplotype distribution of CYP2C19 in a Korean population and provides functional analysis of new coding variants of the CYP2C19 gene. Our findings suggest that individuals carrying CYP2C19*26 would have lower activity for metabolizing CYP2C19 substrate drugs.
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Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Variación Genética/fisiología , Mefenitoína/metabolismo , Omeprazol/metabolismo , Adulto , Animales , Bovinos , Citocromo P-450 CYP2C19 , Activación Enzimática/genética , Frecuencia de los Genes , Haplotipos , Humanos , Mefenitoína/química , Distribución Aleatoria , Especificidad por Sustrato/genética , Adulto JovenRESUMEN
INTRODUCTION: Inhibition of cytochrome P450 (CYP) is a principal mechanism for metabolism-based drug-drug interactions (DDIs). This article describes a robust, high-throughput CYP-mediated DDI assay using a cocktail of 5 clinically relevant probe substrates with quantification by liquid chromatography/tandem mass spectrometry (LC/MS-MS). METHODS: The assay consisted of human liver microsomes and a cocktail of probe substrates metabolized by the five major CYP isoforms (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). The assay was fully automated in both 96- and 384-well formats. RESULTS: A series of experiments were conducted to define the optimal kinetic parameters and solvent concentrations, as well as, to assess potential reactant and product interference. The assay was validated against known CYP inhibitors (miconazole, sulfaphenazole, ticlopidine, quinidine, ketoconazole, itraconazole, fluoxetine) and evaluated in a screening environment by testing 9494 compounds. DISCUSSION: Our findings show that this assay has application in early stage drug discovery to economically, reliably and accurately assess compounds for DDIs.