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1.
Xenobiotica ; 52(3): 229-239, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35416115

RESUMO

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.


Assuntos
Sistema Enzimático do Citocromo P-450 , Microssomos Hepáticos , Clorzoxazona/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Fígado/metabolismo , Mefenitoína/metabolismo , Microssomos/metabolismo , Microssomos Hepáticos/metabolismo
2.
Pharmacol Res ; 176: 106087, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35033648

RESUMO

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.


Assuntos
Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Sequenciamento de Nucleotídeos em Larga Escala , Variantes Farmacogenômicos , Algoritmos , Linhagem Celular , Simulação por Computador , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Citocromos b5/genética , Dextrometorfano/metabolismo , Humanos , Mefenitoína/metabolismo , Microssomos/metabolismo , Mutação de Sentido Incorreto , Reprodutibilidade dos Testes
3.
Chem Biol Interact ; 352: 109775, 2022 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-34910929

RESUMO

Vicagrel, an antiplatelet drug candidate targeting platelet P2Y12 receptor and has finished its phase II clinical trial. The inhibition of six major cytochrome P450 enzymes (P450) (CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six UDP-glucuronosyltransferases (UGT) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) by vicagrel was evaluated using pooled human liver microsomes and specific probe substrates. Physiology-based pharmacokinetic (PBPK) simulation was further applied to predict the in vivo drug-drug interaction (DDI) potential between vicagrel and bupropion as well as S-mephenytoin. The results suggested that vicagrel inhibited CYP2B6 and CYP2C19 potently with apparent IC50 values of 1.6 and 2.0 µM, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of CYP2B6-catalyzed bupropion hydroxylation and noncompetitive inhibition of CYP2C19-mediated S-mephenytoin 4'-hydroxylation with Ki values of 0.19 µM and 1.2 µM, respectively. Vicagrel displayed profound time-dependent inhibition towards CYP2B6 with maximal rate constant of inactivation (kinact) and half-maximal inactivator concentration (KI) values of 0.062 min-1 and 1.52 µM, respectively. No time-dependent inhibition by vicagrel was noted for CYP2C19. For UGT, negligible to moderate inhibition by vicagrel was observed with IC50 values of >50.0, >50.0, 28.2, 8.7, >50.0 and 28.2 µM for UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of UGT1A6-catalyzed N-Acetylserotonin ß-D-glucuronidation with a Ki value of 5.6 µM. No time-dependent inhibition by vicagrel was noted for UGT1A6. PBPK simulation indicated that neither altered AUC nor Cmax of bupropion and S-mephenytoin was observed in the presence of vicagrel. Our study provides inhibitory constants for future DDI prediction between vicagrel and drug substrates of CYP2B6, CYP2C19 and UGT1A6. In addition, our simulation suggests the lack of clinically important DDI between vicagrel and bupropion or S-mephenytoin.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Fenilacetatos/farmacologia , Tiofenos/farmacologia , Bupropiona/administração & dosagem , Bupropiona/farmacocinética , Simulação por Computador , Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/administração & dosagem , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Citocromo P-450 CYP2C19/metabolismo , Inibidores do Citocromo P-450 CYP2C19/administração & dosagem , Inibidores do Citocromo P-450 CYP2C19/farmacologia , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Glucuronosiltransferase/metabolismo , Humanos , Técnicas In Vitro , Cinética , Mefenitoína/administração & dosagem , Mefenitoína/farmacocinética , Fenilacetatos/administração & dosagem , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/farmacologia , Tiofenos/administração & dosagem
4.
Ther Drug Monit ; 43(3): 436-442, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33060488

RESUMO

BACKGROUND: Authors compared plasma concentrations of citalopram (CIT) enantiomers and their metabolites in patients with depression administered either intravenously (IV) or as oral racemic CIT. Then, plasma concentrations were related to the metabolism of probes used for phenotyping patients with depression for CYP2C19 and CYP2D6 activity and cardiovascular functions. METHODS: Dextromethorphan-mephenytoin-phenotyped patients with depression were administered racemic CIT (days 1 and 2: 20 mg/d; days 3-10: 40 mg/d) either orally or as a slow-drop infusion for 10 days and were then orally administered the drug for another 32 days. Blood probes were collected at the time of minimal and maximal concentrations on day 10, immediately before and 2 hours after drug administration, and on days 21 and 42. Plasma CIT and its metabolites were assayed by stereoselective high-performance liquid chromatography. RESULTS: The following concentrations (ng/mL) were noted in the group receiving active IV infusion (IV-POS group, n = 27) of racemic CIT on day 10, before drug administration: escitalopram (S-CIT): 24 ± 10.2; R-citalopram (R-CIT): 45 ± 14.5; S-desmethyl-CIT: 13 ± 4.4; and R-desmethyl-CIT: 17 ± 8.2. In patients receiving oral administration (POS-POS group, n = 25), the values were 30 ± 12.7, 51 ± 17.4, 13 ± 4.6, and 17 ± 7.9 ng/mL, respectively. In the IV-POS group, 3 patients were poor dextromethorphan (CYP2D6) metabolizers; in the POS-POS group, one was a poor mephenytoin (CYP2C19) metabolizer. On day 10, before CIT treatment, S/R-CIT and S/R-mephenytoin ratios were significantly correlated, determined at baseline. Overall, CIT reduced the heart rate but did not significantly modify QTc. No relationship was found between any cardiovascular parameters and pharmacokinetic and pharmacogenetic data. CONCLUSIONS: Owing to CIT's high bioavailability, the plasma concentrations of its enantiomers remained largely independent on the administration route. CYP2C19 preferentially demethylated S-CIT after CIT therapy.


Assuntos
Citalopram , Transtorno Depressivo Maior , Citalopram/sangue , Citalopram/farmacocinética , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Transtorno Depressivo Maior/tratamento farmacológico , Dextrometorfano , Humanos , Mefenitoína
5.
Drug Metab Dispos ; 48(11): 1121-1128, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32839278

RESUMO

Early assessment of metabolism pathways of new chemical entities guides the understanding of drug-drug interactions. Selective enzyme inhibitors are indispensable in CYP reaction phenotyping. The most commonly applied CYP2C19 inhibitor, omeprazole, lacks selectivity. Two promising alternatives, (+)-N-3-benzylnirvanol and (-)-N-3-benzylphenobarbital, are already used as CYP2C19 inhibitors in some in vitro studies with suspended human hepatocytes. However, a full validation proving their suitability in terms of CYP and non-CYP selectivity has not been presented in literature. The present study provides a thorough comparison between omeprazole, (+)-N-3-benzylnirvanol, and (-)-N-3-benzylphenobarbital in terms of potency and selectivity and shows the superiority of (-)-N-3-benzylphenobarbital as a CYP2C19 inhibitor in suspended human hepatocytes. Furthermore, we evaluated the application of (-)-N-3-benzylphenobarbital to predict the in vivo contribution of CYP2C19 to drug metabolism [fraction metabolized (fm) of CYP2C19, fmCYP2C19]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fmCYP2C19 data was evaluated. fmCYP2C19, which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fmCYP2C19 The use of a different hepatocyte batch with a different CYP3A4/CYP2C19 activity ratio showed the impact of intrinsic CYP activities on the determination of fmCYP2C19 Overall, this study confirms the selective CYP2C19 inhibition by (-)-N-3-benzylphenobarbital over other CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4) and clinically relevant non-CYP enzymes [aldehyde oxidase, flavin-containing monooxygenase 3, N-acetyltransferase 2, uridine diphosphate glucuronosyltransferase (UGT) 1A1, UGT1A4, UGT2B7, UGT2B15] in suspended human hepatocytes. (-)-N-3-benzylphenobarbital is therefore the preferred CYP2C19 inhibitor to assess fmCYP2C19 in suspended human hepatocytes in comparison with omeprazole and (+)-N-3-benzylnirvanol. SIGNIFICANCE STATEMENT: (-)-N-3-Benzylphenobarbital is a more potent and selective inhibitor of CYP2C19 in suspended human hepatocytes than omeprazole and (+)-N-3-benzylnirvanol. (-)-N-3-Benzylphenobarbital can be used to predict the fraction metabolized by CYP2C19 in suspended human hepatocytes.


Assuntos
Inibidores do Citocromo P-450 CYP2C19/farmacologia , Citocromo P-450 CYP2C19/metabolismo , Mefenitoína/análogos & derivados , Omeprazol/farmacologia , Fenobarbital/análogos & derivados , Técnicas de Cultura de Células , Células Cultivadas , Hepatócitos , Humanos , Concentração Inibidora 50 , Mefenitoína/farmacologia , Fenobarbital/farmacologia
6.
Sci Rep ; 10(1): 8828, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483226

RESUMO

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.


Assuntos
Resinas Acrílicas/química , Cromatografia Líquida/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Química Verde/métodos , Clorzoxazona/metabolismo , Cumarínicos/metabolismo , Desenvolvimento de Medicamentos , Mefenitoína/metabolismo , Estrutura Molecular , Fenacetina/metabolismo , Solventes , Especificidade por Substrato , Temperatura , Testosterona/metabolismo , Tolbutamida/metabolismo , Água
7.
Xenobiotica ; 50(2): 209-222, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30902024

RESUMO

1. Meperidine is an opioid analgesic that undergoes N-demethylation to form the neurotoxic metabolite normeperidine. Previous studies indicate that meperidine N-demethylation is catalyzed by cytochrome P450 2B6 (CYP2B6), CYP3A4, and CYP2C19.2. The purpose of this study was to examine the relative P450 contributions to meperidine N-demethylation and to evaluate the effect of CYP2C19 polymorphism on normeperidine generation. Experiments were performed using recombinant P450 enzymes, selective chemical inhibitors, enzyme kinetic assays, and correlation analysis with individual CYP2C19-genotyped human liver microsomes.3. The catalytic efficiency (kcat/Km) for meperidine N-demethylation was similar between recombinant CYP2B6 and CYP2C19, but markedly lower by CYP3A4.4. In CYP2C19-genotyped human liver microsomes, normeperidine formation was significantly correlated with CYP2C19 activity (S-mephenytoin 4´-hydroxylation).5. CYP2C19 inhibitor (+)-N-3-benzylnirvanol and CYP3A inhibitor ketoconazole significantly reduced microsomal normeperidine generation by an individual donor with high CYP2C19 activity, whereas donors with lower CYP2C19 activity were sensitive to inhibition by ketoconazole but not benzylnirvanol.6. These findings demonstrate that the relative CYP3A4, CYP2B6, and CYP2C19 involvement in meperidine N-demethylation depends on the enzyme activities in individual human liver microsomal samples. CYP2C19 is likely an important contributor to normeperidine generation in individuals with high CYP2C19 activity, but additional factors influence inter-individual metabolite accumulation.


Assuntos
Inibidores da Colinesterase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Meperidina/análogos & derivados , Citocromo P-450 CYP2C19/metabolismo , Desmetilação , Humanos , Meperidina/metabolismo , Mefenitoína
8.
Basic Clin Pharmacol Toxicol ; 126(5): 444-447, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31814297

RESUMO

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.


Assuntos
Antipsicóticos/farmacologia , Citocromo P-450 CYP2C19/metabolismo , Inibidores do Citocromo P-450 CYP2D6/farmacologia , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/metabolismo , Mefenitoína/metabolismo , Perazina/farmacologia , Esquizofrenia/tratamento farmacológico , Adulto , Interações Medicamentosas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Esquizofrenia/enzimologia , Adulto Jovem
9.
Drug Metab Pers Ther ; 32(4): 191-200, 2017 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-29176011

RESUMO

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.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Modelos Estatísticos , Farmacocinética , Simulação 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/metabolismo
10.
Bull Exp Biol Med ; 162(1): 170-174, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27882460

RESUMO

We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.


Assuntos
Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Dispositivos Lab-On-A-Chip , Bupropiona/metabolismo , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2B6/análise , Citocromo P-450 CYP2C19/análise , Citocromo P-450 CYP2C9/análise , Citocromo P-450 CYP3A/análise , Inibidores das Enzimas do Citocromo P-450/farmacologia , Humanos , Cetoconazol/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Espectrometria de Massas , Mefenitoína/análogos & derivados , Mefenitoína/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Omeprazol/metabolismo , Fenciclidina/análogos & derivados , Fenciclidina/farmacologia , Especificidade por Substrato , Sulfafenazol/farmacologia , Testosterona/metabolismo , Tolbutamida/metabolismo
11.
Pharmacogenomics J ; 16(4): 375-87, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26323597

RESUMO

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.


Assuntos
Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/enzimologia , Mefenitoína/metabolismo , Variantes Farmacogenômicos/genética , Ticlopidina/análogos & derivados , Ativação Metabólica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Clopidogrel , Feminino , Regulação Enzimológica da Expressão Gênica , Frequência do Gene , Genótipo , Humanos , Hidroxilação , Lactente , Recém-Nascido , Cinética , Modelos Lineares , Masculino , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Análise Multivariada , Oxirredução , Fenótipo , Especificidade por Substrato , Ticlopidina/metabolismo , Adulto Jovem
12.
Drug Metab Dispos ; 43(8): 1226-35, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26021325

RESUMO

CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 Genomes project (http://browser.1000genomes.org/index.html) indicated rs12769205 is in linkage disequilibrium with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this single nucleotide polymorphism (SNP) alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream from rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205, demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 and rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3, or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the HapMap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher relative extended haplotype homozygosity score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later, creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.


Assuntos
Citocromo P-450 CYP2C19/genética , Fígado/enzimologia , Acetilação , Adulto , Alelos , Citocromo P-450 CYP2C19/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Etiópia/epidemiologia , Éxons , Feminino , Frequência do Gene , Haplótipos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Mefenitoína/farmacocinética , Fenótipo , Filogenia , Polimorfismo de Nucleotídeo Único , Splicing de RNA , Espectrometria de Massas em Tandem
13.
Drug Metab Dispos ; 43(6): 870-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25845826

RESUMO

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.


Assuntos
Anticonvulsivantes/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Variação Genética , Mefenitoína/metabolismo , Microssomos Hepáticos/enzimologia , Modelos Biológicos , Farmacogenética/métodos , Administração Oral , Adolescente , Adulto , Idoso , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/análise , Anticonvulsivantes/farmacocinética , Disponibilidade Biológica , Simulação por Computador , Citocromo P-450 CYP2C19/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , Estudos de Associação Genética , Humanos , Masculino , Mefenitoína/administração & dosagem , Mefenitoína/análise , Mefenitoína/farmacocinética , Taxa de Depuração Metabólica , Microssomos Hepáticos/metabolismo , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto Jovem
14.
Artigo em Inglês | MEDLINE | ID: mdl-25797720

RESUMO

A rapid selective and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the quantitative determination of derivatised cytochrome P450-2C19 oxidation product (dansyl-4-OH mephenytoin) and its underivatised form (4-OH mephenytoin). Samples were anaysed on C18 column (Waters Xbridge, 50 mm×4.6 mm, 3.5 µm particle size) with the mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. A gradient method with a short run time of 2.5 min and 3.5 min was developed for the analysis of dansyl-4-OH mephenytoin and 4-OH mephenytoin, respectively. The standard curve was linear (r(2)=0.9972 for 4-OH mephenytoin; r(2)=0.9946 for dansyl-4-OH mephenytoin) over the concentration range of 0.16 to 40 ng/mL for both derivatised and underivatised forms. The CV (%) and relative error (RE) for inter and intraassay at three QC levels for dansyl-4-OH mephenytoin was 0.97-5.85% and -9.80 to 2.51%, respectively. Whereas, for 4-OH mephenytoin the CV (%) and RE (%) at three QC levels was 0.82-3.47% and -6.69 to -0.01%, respectively. The developed method was validated for various parameters such as linearity, precision & accuracy, extraction recovery, matrix effect, autosampler stability and was proved to be consistent across three QC levels with overall CV (%) less than 15. Dansylation helped in increasing the sensitivity of hydroxy mephenytoin by 100-200 fold. Given the simplicity involved in derivatisation process, we believe that this novel methodology will change the current approaches used for the enhancing the detection sensitivity of 4-OH mephenytoin.


Assuntos
Citocromo P-450 CYP2C19/química , Compostos de Dansil/química , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/química , Mefenitoína/química , Cinética , Oxirredução
15.
Biomed Chromatogr ; 29(3): 437-44, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25098274

RESUMO

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.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Microssomos Hepáticos/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos , Bupropiona/metabolismo , Bupropiona/farmacologia , Calibragem , Cromatografia Líquida/métodos , Inibidores das Enzimas do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Humanos , Concentração Inibidora 50 , Limite de Detecção , Mefenitoína/metabolismo , Mefenitoína/farmacologia , Microssomos Hepáticos/metabolismo , Midazolam/metabolismo , Midazolam/farmacologia , Fenacetina/metabolismo , Fenacetina/farmacologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
Artigo em Inglês | MEDLINE | ID: mdl-25392581

RESUMO

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.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interações Ervas-Drogas , Preparações Farmacêuticas/metabolismo , Extratos Vegetais/farmacologia , Plantas Medicinais , Citocromo P-450 CYP2C19/metabolismo , Diclofenaco/metabolismo , Euphorbiaceae , Humanos , Hidroxilação , Liliaceae , Magnoliopsida , Mefenitoína/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Fenacetina/metabolismo , África do Sul , Testosterona/metabolismo
17.
Pharmacogenet Genomics ; 24(8): 381-6, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24945780

RESUMO

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.


Assuntos
Alelos , Hidrocarboneto de Aril Hidroxilases/genética , Ticlopidina/análogos & derivados , Catálise , Cromatografia Líquida , Clopidogrel , Citocromo P-450 CYP2C19 , Genótipo , Humanos , Cinética , Mefenitoína/metabolismo , Omeprazol/metabolismo , Farmacogenética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Espectrometria de Massas em Tandem , Ticlopidina/metabolismo
18.
Br J Clin Pharmacol ; 75(4): 1019-28, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22924488

RESUMO

AIM: To determine the effect of increasing adult age on predicted metabolic drug clearance. METHOD: Predicted metabolic drug clearances (CLPT ) were determined using in vitro-in vivo extrapolation coupled with physiological-based pharmacokinetic modelling and simulation (IVIVE-PBPK) in Simcyp®. Simulations were conducted using CYP-selective 'probe' drugs with subjects in 5 year age groups (20-25 to 90-95 years). CLPT values were compared with human pharmacokinetic data stratified according to age (young = 20-40 years and elderly = 65-85 years) and gender. Age-related changes in the physiological parameters used for IVIVE of CLPT were described. RESULTS: Predicted metabolic drug clearances decreased with increasing adult age to approximately 65-70 years: caffeine from 1.5 to 1.0 ml min(-1) kg(-1) (a 33% decrease), S-warfarin from 0.100 to 0.064 ml min(-1) kg(-1) (36%), S-mephenytoin from 4.1 to 2.5 ml min(-1) kg(-1) (39%), desipramine from 10.6 to 7.3 ml min(-1) kg(-1) (31%) and midazolam from 5.4 to 3.9 ml min(-1) kg(-1) (27%). Except for S-mephenytoin, predictions were within 3.5-fold of clearances from clinical studies when stratified by age and gender. A trend towards higher CLPT was observed in females, but this was only statistically significant in larger virtual trials. Physiological parameters that determine CLPT decreased with increasing adult age: mean microsomal protein g(-1) of liver, liver weight, hepatic blood flow and human serum albumin concentration. CONCLUSION: Decreased metabolic clearance in the elderly was predicted by Simcyp® and was generally consistent with limited clinical data for four out of five drugs studied and the broader literature for drugs metabolized by CYP enzymes. IVIVE-PBPK may be increasingly useful in predicting metabolic drug clearance in the elderly.


Assuntos
Envelhecimento/metabolismo , Cafeína/farmacocinética , Desipramina/farmacocinética , Mefenitoína/farmacocinética , Midazolam/farmacocinética , Varfarina/farmacocinética , Adulto , Idoso , Idoso de 80 Anos ou mais , Simulação por Computador , Feminino , Humanos , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Modelos Biológicos
19.
Biochemistry ; 52(1): 210-20, 2013 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-23193974

RESUMO

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.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromos b5/química , Citocromos b5/metabolismo , Clorzoxazona/metabolismo , Citocromo P-450 CYP2C19 , Citocromos b5/genética , Humanos , Hidroxilação , Mefenitoína/metabolismo , Modelos Moleculares , Relaxantes Musculares Centrais/metabolismo , NADP/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosfolipídeos/metabolismo , Mutação Puntual , Progesterona/metabolismo
20.
Pharmacogenet Genomics ; 23(2): 78-83, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23241944

RESUMO

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.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Diclofenaco/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Mefenitoína/metabolismo , Farmacogenética , Animais , Proteínas de Transporte/fisiologia , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Hepatócitos/transplante , Humanos , Fígado/citologia , Camundongos , Camundongos Transgênicos , Timidina Quinase/fisiologia
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