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1.
Biomed Chromatogr ; 35(4): e5039, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33238041

RESUMO

Astilbin, neoastilbin and isoastilbin are three flavonoid isomers from Smilacis glabrae Roxb. (S. glabrae). Several studies have shown that consumption of flavonoids can increase the risk of food/drug-drug interaction by affecting the activities of human cytochrome CYP3A4 and 2D6. In the present study, an ultrahigh-performance liquid chromatography and triple quadrupole mass spectrometry method was developed for the determination of the interaction between three flavonoid isomers and two CYPs. Under the optimized reaction conditions, the Km values were 18.9 and 36.4 µM and the Vmax values were 0.02 and 0.20 µM/min for CYP3A4 and 2D6 in vitro, respectively. Astilbin showed the strongest inhibition on CYP3A4, followed by isoastilbin and neoastilbin with IC50 values of 2.63, 3.03 and 6.51 µM. Neoastilbin showed the strongest inhibition on CYP2D6, followed by isoastilbin and astilbin, with IC50 values of 1.48, 11.87 and 14.16 µM, respectively. The three isomers showed reversible inhibition on both enzymes. Neoastilbin and astilbin were noncompetitive type for CYP3A4 and 2D6, isoastilbin was a mixture and noncompetitive type for CYP3A4 and 2D6, respectively. Our study suggests that the three isomers may increase the risk of food/drug-drug interactions by affecting the activities of CYP3A4 and 2D6.


Assuntos
Citocromo P-450 CYP2D6 , Inibidores do Citocromo P-450 CYP3A/farmacologia , Citocromo P-450 CYP3A , Flavonoides/farmacologia , Flavonóis/farmacologia , Cromatografia Líquida de Alta Pressão/métodos , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/efeitos dos fármacos , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/análise , Citocromo P-450 CYP3A/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
2.
Farm. hosp ; 44(6): 243-253, nov.-dic. 2020. tab, graf
Artigo em Espanhol | IBECS | ID: ibc-197693

RESUMO

La rápida implantación clínica de las técnicas de secuenciación masiva en paralelo se debe a su capacidad para secuenciar un gran número de regiones genéticas con un coste menor a las técnicas convencionales. Sin embargo, su uso en el ámbito de la farmacogenética es, todavía, muy escaso. OBJETIVO: Diseño, desarrollo, implementación y validación de un panel de secuenciación masiva en paralelo de farmacogenética orientado a la práctica clínica. MÉTODO: Se desarrolló un panel de sondas de captura híbrida (Sure-Select(R)) para el análisis de las regiones genéticas de interés clínico recopiladas mediante búsqueda bibliográfica. Se empleó la plataforma de secuenciación Illumina HiSeq 1500(R). Se desarrolló un algoritmo de análisis bioinformático para la anotación de variantes puntuales, inferencia de haplotipos y determinación de variantes estructurales en los genes de interés. Los resultados obtenidos se validaron con materiales de referencia Coriell(R) de los repositorios de farmacogenética. RESULTADOS: El panel desarrollado permite el estudio de un total de 12.794 regiones comprendidas en 389 genes. Los resultados de validación mostraron una sensibilidad superior al 99% para variantes puntuales e inserciones y deleciones pequeñas. La imputación de haplotipos fue coherente con los resultados consenso de los materiales de referencia caracterizados. Además, la herramienta desarrollada pudo identificar correctamente diferentes tipos de variaciones de número de copias de CYP2D6, así como una gran variedad de alelos de HLA-B. CONCLUSIONES: Esta tecnología representa una alternativa adecuada para su empleo asistencial con ventajas frente a las técnicas convencionales en su rendimiento de producción y sus capacidades de estudio de genes complejos (CYP2D6, HLA-B)


The rapid clinical implementation of next generation sequencing techniques is due to its ability to sequence a large number of genetic regions at lower costs than conventional techniques. However, its use in the field of pharmacogenetics is still very limited. OBJECTIVE: Design, development, implementation and validation of a clinical pharmacogenetics next-generation sequencing panel. METHOD: We developed a panel of hybrid capture probes (SureSelect(R)) for the analysis of the genetic regions of clinical interest collected by literature search and using Illumina HiSeq 1500(R) sequencing platform. We developed a bioinformatic algorithm for variant annotation, haplotype inference and determination of structural variants in the genes of interest. The results obtained were validated with Coriell(R) reference material from the pharmacogenetic repositories. RESULTS: The developed panel allows the study of a total of 12,794 regions comprised in 389 genes. Validation results showed a sensitivity greater than 99% for single nucleotide variants and small INDELs. Haplotype imputation was consistent with the consensus results in the characterized reference materials. Furthermore, the developed tool was able to correctly identify different types of CYP2D6 copy number variations as well as a wide variety of HLA-B alleles. CONCLUSIONS: This technology represents an appropriate alternative for its clinical use with advantages over conventional techniques in its through-put and complex gene study capabilities (CYP2D6, HLA-B)


Assuntos
Humanos , Farmacogenética/métodos , Testes Farmacogenômicos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Medicina de Precisão/métodos , Algoritmos , Biologia Computacional , Citocromo P-450 CYP2D6/análise , Análise de Dados , Variantes Farmacogenômicos/genética
3.
JAMA Netw Open ; 3(12): e2027909, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33284338

RESUMO

Importance: Genetic polymorphism of genes encoding the drug metabolizing enzymes, cytochrome P450 2D6 and 2C19 (CYP2D6 and CYP2C19), is associated with treatment failure of and adverse reactions to psychotropic drugs. The clinical utility of routine CYP2D6 and CYP2C19 genotyping (CYP testing) is unclear. Objective: To estimate whether routine CYP testing effects the persistence of antipsychotic drug treatment. Design, Setting, and Participants: This single-masked, 3-group randomized clinical trial included patients aged 18 years or older who had been diagnosed within the schizophrenic spectrum (International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes, F20-F29) and not previously genotyped. A total of 669 of 1406 potentially eligible patients from 12 psychiatric outpatient clinics in Denmark were approached between July 2008 and December 2009. Overall, 528 patients were genotyped and randomly allocated to 1 of 3 study groups or exclusion in a sequence of 1:1:1:3 using a predictive enrichment design, aiming to double the proportion of poor or ultrarapid metabolizers for CYP2D6 or CYP2C19. Outcome measurements were recorded at baseline and 1-year follow-up. Data analysis was performed in December 2012 and updated March 2019. Interventions: The trial included 2 intervention groups, where antipsychotic drug treatment was guided by either CYP test (CYP test-guided [CTG]) or structured clinical monitoring (SCM), in which adverse effects and factors influencing compliance were systematically recorded at least once quarterly, and 1 control group. Main Outcomes and Measures: Primary outcome was antipsychotic drug persistence, ie, days to first modification of the initial treatment. Secondary outcomes were number of drug and dose changes, adverse effects, and psychotic symptoms, ie, hallucinations and delusions. Results: A total of 528 participants were genotyped, and 311 (median [interquartile range {IQR} age, 41 [30-50] years; 139 [45%] women; median [IQR] duration of illness, 6 [3-13] years) were randomly allocated to 1 of 3 study groups. Overall, 61 participants (20%) were extreme metabolizers. There was no difference in antipsychotic drug persistence between the CTG group and the control group (hazard ratio [HR], 1.02; 95% CI, 0.71-1.45) or SCM and the control group (HR, 0.88; 95% CI, 0.61-1.26). Subanalyses among extreme metabolizers showed similar results (CTG: HR, 0.99; 95% CI, 0.48-2.03; SCM: HR, 0.93; 95% CI, 0.44-1.96). Conclusions and Relevance: The results of this randomized clinical trial do not support routine CYP testing in patients with schizophrenia. Trial Registration: ClinicalTrials.gov Identifier: NCT00707382.


Assuntos
Citocromo P-450 CYP2C19/análise , Citocromo P-450 CYP2D6/análise , Técnicas de Genotipagem/métodos , Testes Farmacogenômicos/métodos , Esquizofrenia/genética , Adulto , Antipsicóticos/efeitos adversos , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Dinamarca , Resistência a Medicamentos/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Variantes Farmacogenômicos , Polimorfismo Genético , Esquizofrenia/tratamento farmacológico , Método Simples-Cego , Falha de Tratamento
4.
Medicina (Kaunas) ; 55(7)2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31284530

RESUMO

Tamoxifen is a drug that is often used in the clinical management of breast cancer. CYP2D6 is a key metabolizing enzyme that is involved in the conversion of tamoxifen to its active drug metabolites. CYP2D6 has several alleles that metabolize tamoxifen and other drugs at different rates that can alter therapeutic impact, a characteristic that renders it one of the most studied enzymes in the field of pharmacogenetics. Background and objectives: Portugal has no implemented measures based on pharmacogenomics analysis prior to therapy that might function as a cultural sample control when analyzing the individual and economic factors present in clinical practice paradigms. Therefore, we aim to investigate the impact of CYP2D6 genotyping of the tamoxifen metabolizing enzymes in the clinical management of breast cancer patients. Materials and Methods: Qualitative/quantitative studies regarding the impact of pharmacogenomics in breast cancer; personal interviews in different Portuguese laboratories within hospital setting using a survey. Analysis of data through interviews to management board and/or decision makers from major oncological centers. Results: Reasons for common adoption of pharmacogenomics practice are contradictory and based both in economic factors and cultural/clinical bias. Conclusions: This research study identifies specific cultural and/or clinical bias that act as obstacles to pharmacogenomic implementation and proposes viable courses of action that might bring about change in cultural/medical habits.


Assuntos
Citocromo P-450 CYP2D6/análise , Guias como Assunto/normas , Farmacogenética/normas , Tamoxifeno/normas , Adulto , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Citocromo P-450 CYP2D6/genética , Prova Pericial , Feminino , Humanos , Farmacogenética/métodos , Portugal , Sensibilidade e Especificidade , Inquéritos e Questionários , Tamoxifeno/uso terapêutico
5.
Drug Test Anal ; 8(10): 1005-1014, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26811026

RESUMO

Diphenidine is a new psychoactive substance (NPS) sold as a 'legal high' since 2013. Case reports from Sweden and Japan demonstrate its current use and the necessity of applying analytical procedures in clinical and forensic toxicology. Therefore, the phase I and II metabolites of diphenidine should be identified and based on these results, the detectability using standard urine screening approaches (SUSAs) be elucidated. Urine samples were collected after administration of diphenidine to rats and analyzed using different sample workup procedures with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-(high resolution)-mass spectrometry (LC-(HR)-MS). With the same approaches incubates of diphenidine with pooled human liver microsomes (pHLM) and cytosol (pHLC) were analyzed. According to the identified metabolites, the following biotransformation steps were proposed in rats: mono- and bis-hydroxylation at different positions, partly followed by dehydrogenation, N,N-bis-dealkylation, and combinations of them followed by glucuronidation and/or methylation of one of the bis-hydroxy-aryl groups. Mono- and bis-hydroxylation followed by dehydrogenation could also be detected in pHLM or pHLC. Cytochrome-P450 (CYP) isozymes CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were all capable of forming the three initial metabolites, namely hydroxy-aryl, hydroxy-piperidine, and bis-hydroxy-piperidine. In incubations with CYP2D6 hydroxy-aryl and hydroxy-piperidine metabolites were detected. After application of a common users' dose, diphenidine metabolites could be detected in rat urine by the authors' GC-MS as well as LC-MSn SUSA. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Citocromo P-450 CYP2D6/análise , Drogas Desenhadas/análise , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Piperidinas/análise , Urinálise/métodos , Animais , Cromatografia Líquida/métodos , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Drogas Desenhadas/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Japão , Fígado/química , Metilação , Microssomos Hepáticos/química , Piperidinas/química , Ratos , Ratos Wistar , Suécia
6.
Metabolism ; 64(3 Suppl 1): S16-21, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25468140

RESUMO

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells and oncology is a branch of medicine that deals with tumors. The last decade has seen significant advances in the development of biomarkers in oncology that play a critical role in understanding molecular and cellular mechanisms which drive tumor initiation, maintenance and progression. Clinical molecular diagnostics and biomarker discoveries in oncology are advancing rapidly as we begin to understand the complex mechanisms that transform a normal cell into an abnormal one. These discoveries have fueled the development of novel drug targets and new treatment strategies. The standard of care for patients with advanced-stage cancers has shifted away from an empirical treatment strategy based on the clinical-pathological profile to one where a biomarker driven treatment algorithm based on the molecular profile of the tumor is used. Recent advances in multiplex genotyping technologies and high-throughput genomic profiling by next-generation sequencing make possible the rapid and comprehensive analysis of the cancer genome of individual patients even from very little tumor biopsy material. Predictive (diagnostic) biomarkers are helpful in matching targeted therapies with patients and in preventing toxicity of standard (systemic) therapies. Prognostic biomarkers identify somatic germ line mutations, changes in DNA methylation, elevated levels of microRNA (miRNA) and circulating tumor cells (CTC) in blood. Predictive biomarkers using molecular diagnostics are currently in use in clinical practice of personalized oncotherapy for the treatment of five diseases: chronic myeloid leukemia, colon, breast, lung cancer and melanoma and these biomarkers are being used successfully to evaluate benefits that can be achieved through targeted therapy. Examples of these molecularly targeted biomarker therapies are: tyrosine kinase inhibitors in chronic myeloid leukemia and gastrointestinal tumors; anaplastic lymphoma kinase (ALK) inhibitors in lung cancer with EML4-ALk fusion; HER2/neu blockage in HER2/neu-positive breast cancer; and epidermal growth factor receptors (EGFR) inhibition in EGFR-mutated lung cancer. This review presents the current state of our knowledge of biomarkers in five selected cancers: chronic myeloid leukemia, colorectal cancer, breast cancer, non-small cell lung cancer and melanoma.


Assuntos
Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Oncologia , Neoplasias , Medicina de Precisão , Neoplasias da Mama , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Colorretais , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/genética , Di-Hidrouracila Desidrogenase (NADP)/análise , Di-Hidrouracila Desidrogenase (NADP)/genética , Receptores ErbB/análise , Receptores ErbB/genética , Feminino , Genes ras , Glucuronosiltransferase/análise , Glucuronosiltransferase/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Neoplasias Pulmonares , Oncologia/métodos , Oncologia/normas , Oncologia/tendências , Melanoma , Terapia de Alvo Molecular , Neoplasias/química , Medicina de Precisão/métodos , Medicina de Precisão/tendências , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas B-raf/análise , Proteínas Proto-Oncogênicas B-raf/genética , Receptor ErbB-2/análise , Receptor ErbB-2/genética , Receptores de Estrogênio/análise , Receptores de Estrogênio/genética , Receptores de Progesterona/análise , Receptores de Progesterona/genética , Neoplasias Cutâneas
7.
Biotechnol J ; 8(1): 146-52, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23070983

RESUMO

Cytochrome P450 (CYP) enzymes are useful biocatalysts for the pharmaceutical and biotechnological industries. A high-throughput method for quantification of CYP expression in yeast is needed in order to fully exploit the yeast expression system. Carbon monoxide (CO) difference spectra of whole cells have been successfully used for the quantification of heterologous CYP expressed in Escherichia coli in the 96-well format; however, very few researchers have shown whole-cell CO difference spectra with yeast cells using 1-cm path length. Spectral interference from the native hemoproteins often obscures the P450 peak, challenging functional CYP quantification in whole yeast cells. For the first time, we describe the high-throughput determination of CO difference spectra using whole cells in the 96-well format for the quantification of CYP genes expressed in Pichia pastoris. Very little interference from the hemoproteins of P. pastoris enabled CYP quantification even at relatively low expression levels. P. pastoris strains carrying a single copy or three copies of both hCPR and CYP2D6 integrated into the chromosomal DNA were used to establish the method in 96-well format, allowing to detect quantities of CYP2D6 as low as 6 nmol gCDW(-1 ) and 12 pmol per well. Finally, the established method was successfully demonstrated and used to screen P. pastoris clones expressing Candida CYP52A13.


Assuntos
Monóxido de Carbono/análise , Citocromo P-450 CYP2D6/análise , Ensaios de Triagem em Larga Escala/métodos , Pichia/química , Proteínas Recombinantes/análise , Monóxido de Carbono/metabolismo , Citocromo P-450 CYP2D6/biossíntese , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/genética , NADPH-Ferri-Hemoproteína Redutase/análise , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Pichia/citologia , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Fluorescência/métodos
9.
Oncologist ; 15 Suppl 5: 18-28, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21138952

RESUMO

Of more than one million women diagnosed with breast cancer each year, approximately 700,000 have hormone receptor (HR)(+) disease. Although endocrine therapy has revolutionized breast cancer management and substantially improved outcomes in these patients, the optimal management of these patients remains a significant challenge. For instance, the threshold for adding adjuvant chemotherapy is a topic of continuing debate, and the most effective regimens that include endocrine therapy and chemotherapy are still under debate as well. Tumor markers, such as Ki-67, and host markers, such as cytochrome P450 2D6, are being studied as potential tools to offer more tailored adjuvant endocrine therapy. Current research suggests that luminal A and luminal B cancers are two completely different diseases, and work is being performed to better distinguish between these two disease types and deliver more effective therapy to individual patients. This article addresses these important outstanding issues with respect to HR(+) disease.


Assuntos
Antineoplásicos Hormonais/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Quimioterapia Adjuvante/métodos , Tamoxifeno/uso terapêutico , Antineoplásicos Hormonais/administração & dosagem , Ensaios Clínicos como Assunto , Terapia Combinada , Citocromo P-450 CYP2D6/análise , Feminino , Perfilação da Expressão Gênica , Humanos , Antígeno Ki-67/análise , Estadiamento de Neoplasias , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Tamoxifeno/administração & dosagem , Resultado do Tratamento
10.
BMC Genomics ; 10 Suppl 3: S4, 2009 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19958502

RESUMO

BACKGROUND: Polymerase chain reaction (PCR) is very useful in many areas of molecular biology research. It is commonly observed that PCR success is critically dependent on design of an effective primer pair. Current tools for primer design do not adequately address the problem of PCR failure due to mis-priming on target-related sequences and structural variations in the genome. METHODS: We have developed an integrated graphical web-based application for primer design, called RExPrimer, which was written in Python language. The software uses Primer3 as the primer designing core algorithm. Locally stored sequence information and genomic variant information were hosted on MySQLv5.0 and were incorporated into RExPrimer. RESULTS: RExPrimer provides many functionalities for improved PCR primer design. Several databases, namely annotated human SNP databases, insertion/deletion (indel) polymorphisms database, pseudogene database, and structural genomic variation databases were integrated into RExPrimer, enabling an effective without-leaving-the-website validation of the resulting primers. By incorporating these databases, the primers reported by RExPrimer avoid mis-priming to related sequences (e.g. pseudogene, segmental duplication) as well as possible PCR failure because of structural polymorphisms (SNP, indel, and copy number variation (CNV)). To prevent mismatching caused by unexpected SNPs in the designed primers, in particular the 3' end (SNP-in-Primer), several SNP databases covering the broad range of population-specific SNP information are utilized to report SNPs present in the primer sequences. Population-specific SNP information also helps customize primer design for a specific population. Furthermore, RExPrimer offers a graphical user-friendly interface through the use of scalable vector graphic image that intuitively presents resulting primers along with the corresponding gene structure. In this study, we demonstrated the program effectiveness in successfully generating primers for strong homologous sequences. CONCLUSION: The improvements for primer design incorporated into RExPrimer were demonstrated to be effective in designing primers for challenging PCR experiments. Integration of SNP and structural variation databases allows for robust primer design for a variety of PCR applications, irrespective of the sequence complexity in the region of interest. This software is freely available at http://www4a.biotec.or.th/rexprimer.


Assuntos
Primers do DNA/análise , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Design de Software , Sequência de Bases , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/genética , Primers do DNA/química , Primers do DNA/genética , Bases de Dados de Ácidos Nucleicos , Humanos , Internet , Dados de Sequência Molecular
11.
Proteomics ; 9(9): 2313-23, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19402041

RESUMO

We describe the assay of the human cytochrome P450 2D6 in a set of 30 genotyped liver samples using the 'absolute quantification' (AQUA) technique. We found approximately 30 fmol CYP2D6 per microg of microsomal protein, with the values spanning from 0 to nearly 80 fmol/microg. This is greater by a factor of 5-10 from the compared to the currently accepted value, which was around 5 fmol/microg. Our results thus suggest that the amount of cytochrome P450 (CYP) enzymes in liver have to be reassessed. We used quantitative Western blotting, calibration standards and activity assays, to validate the results. Our results show, that using the AQUA technique a true assay of CYP2D6 in human liver was possible.


Assuntos
Citocromo P-450 CYP2D6/análise , Espectrometria de Massas/métodos , Microssomos Hepáticos/enzimologia , Western Blotting , Cromatografia Líquida , Citocromo P-450 CYP2D6/metabolismo , Humanos , Fígado/enzimologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Fenótipo , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Tripsina/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-18614408

RESUMO

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.


Assuntos
Cromatografia Líquida/métodos , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/genética , Espectrometria de Massas em Tandem/métodos , Animais , Citocromo P-450 CYP1A2/análise , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP3A/análise , Sistema Enzimático do Citocromo P-450/urina , Citocromos , Masculino , Mefenitoína/metabolismo , Midazolam/metabolismo , Fenacetina/metabolismo , Fenótipo , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tolbutamida/análogos & derivados , Tolbutamida/metabolismo
13.
J Pharm Biomed Anal ; 48(1): 92-9, 2008 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-18584988

RESUMO

The current study focused on the development of an automated IC50 cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC50 determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC50s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1 min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC50 assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.


Assuntos
Cromatografia Líquida/métodos , Sistema Enzimático do Citocromo P-450/análise , Espectrometria de Massas em Tandem/métodos , Hidrocarboneto de Aril Hidroxilases/análise , Hidrocarboneto de Aril Hidroxilases/metabolismo , Hidrocarboneto de Aril Hidroxilases/farmacologia , Bioensaio , Citocromo P-450 CYP1A2/análise , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/análise , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Dextrometorfano/metabolismo , Dextrometorfano/farmacologia , Diclofenaco/metabolismo , Diclofenaco/farmacologia , Interações Medicamentosas , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Midazolam/metabolismo , Midazolam/farmacologia , Miniaturização , Oxirredutases N-Desmetilantes/metabolismo , Oxirredutases N-Desmetilantes/farmacologia , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade por Substrato/efeitos dos fármacos , Tacrina/metabolismo , Tacrina/farmacologia , Fatores de Tempo
14.
Basic Clin Pharmacol Toxicol ; 101(5): 350-8, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17910620

RESUMO

The six commonly used trade herbal products, St. John's wort, common valerian, common sage, Ginkgo biloba, Echinacea purpurea and horse chestnut, and ethanol, were investigated for their in vitro inhibitory potential of cytochrome P450 2D6 (CYP2D6)-mediated metabolism. Herbal components were extracted from commercially available products in a way that ensured the same composition of constituents in the extract as in the original trade products. c-DNA baculovirus expressed CYP2D6 was used with dextromethorphan as substrate. Quinidine was included as a positive control inhibitor. A validated high performance liquid chromatography methodology was used to quantify the formation of dextrorphan (product of dextromethorphan O-demethylation). Ethanol showed a biphasic effect on CYP2D6 metabolism, increasing initially the CYP2D6 activity with 175% of control up to a concentration of 1.1%, where after ethanol linearly inhibited the CYP2D6 activity. All the investigated herbs inhibited CYP2D6 activity to some extent, but only St. John's wort, common sage and common valerian were considered possible candidates for in vivo clinically significant effects. They showed IC50 values of 0.07 +/- 7 x 10(-3) mg/ml, 0.8 +/- 0.05 mg/ml and 1.6 +/- 0.2 mg/ml, respectively. St. John's wort inhibited CYP2D6-mediated metabolism in an uncompetitive manner, while common valerian and common sage in a non-competitive manner demonstrated interherb differences in inhibition patterns and differences when compared to the more homogenous competitive inhibitor quinidine. Common valerian was the only herb that showed a mechanistic inhibition of CYP2D6 activity and attention should be paid to a possible toxicity of this herb.


Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Inibidores do Citocromo P-450 CYP2D6 , Inibidores Enzimáticos , Etanol/farmacologia , Preparações de Plantas/farmacologia , Biotransformação , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2D6/análise , Dextrometorfano/metabolismo , Humanos , Cinética , Extratos Vegetais/farmacologia , Quinidina/farmacologia
15.
J Chromatogr B Analyt Technol Biomed Life Sci ; 858(1-2): 49-58, 2007 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17826363

RESUMO

Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC-EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP2D6/química , Sistema Enzimático do Citocromo P-450/química , Citocromo P-450 CYP1A2/análise , Citocromo P-450 CYP2D6/análise , Sistema Enzimático do Citocromo P-450/análise , Humanos , Estereoisomerismo
16.
Med. clín (Ed. impr.) ; 128(20): 772-774, mayo 2007. ilus, tab
Artigo em Es | IBECS | ID: ibc-054286

RESUMO

Fundamento y objetivo: La capacidad metabólica del CYP2D6 presenta polimorfismo genético. A partir del cálculo del índice metabólico (IM) urinario de un fármaco test como la debrisoquina se ha demostrado la existencia de 2 fenotipos metabólicos: metabolizadores lentos y rápidos, entre los que hay un subgrupo de ultrarrápidos. El objetivo de este trabajo ha sido evaluar las diferencias interétnicas del polimorfismo metabólico de hidroxilación de debrisoquina en la población cubana, y compararla con la española. Pacientes y método: El IM se evaluó en 260 voluntarios sanos cubanos (divididos en blancos y mestizos) que se compararon con 925 voluntarios sanos españoles. Resultados: La frecuencia de metabolizadores lentos fue similar entre cubanos y españoles (un 4,6 y un 4,9%, respectivamente). Sin embargo, la prevalencia de metabolizadores ultrarrápidos fue menor en cubanos (3,8%) que en españoles (5,2%). El IM en metabolizadores rápidos fue mayor (p < 0,05) en los mestizos que en blancos cubanos. Conclusiones: Se observan diferencias interétnicas en el polimorfismo genético del CYP2D6 en la población cubana y en la frecuencia de metabolizadores ultrarrápidos respecto a los españoles. Estas diferencias podrían condicionar en la población cubana una variabilidad interindividual e interétnica en la respuesta a los fármacos sustratos del CYP2D6


Background and objective: CYP2D6 metabolic capacity shows genetic polymorphism. Two metabolic phenotypes, poor and extensive, can be determined by the ratio of debrisoquine to its metabolite in urine (MR). A subgroup of ultrarapids has been also described. We analyzed the inter-ethnic differences on the polymorphic hydroxylation of debrisoquine in a Cuban population in comparison to Spaniards. Patients and method: MR in a Cuban population of 260 white and mestizo healthy volunteers was studied and compared to 925 Spanish healthy volunteers. Results: The frequency of poor metabolizer in Cubans (4.6%) was almost identical to that found in Spaniards (4.9%). However, ultrarapids were lower in Cubans (3.8%) than in Spaniards (5.2%). MR in Cuban-mestizo extensive metabolizers was higher than in white (p < 0.05). Conclusions: Interethnic differences on debrisoquine hydroxylation have been demonstrated in a Cuban population. Furthermore, differences on the frequency of ultrarapids between Cubans and Spaniards have been shown. These results could explain inter-individual and interethnic differences on drug response such as side effects or therapeutic failures among Cuban patients receiving treatment with CYP2D6 substrates


Assuntos
Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Humanos , Farmacogenética/estatística & dados numéricos , Debrisoquina/metabolismo , Cuba/epidemiologia , Citocromo P-450 CYP2D6/análise , Polimorfismo Genético
17.
Basic Clin Pharmacol Toxicol ; 100(1): 23-30, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17214607

RESUMO

The aim of this study was to evaluate the in vitro inductive potential of six commonly used trade herbal products on CYP1A2, CYP2D6 and CYP3A4 metabolic activities. Herbal components were extracted from the trade products in a way that ensured a composition equal to that present in the original product. Primary human hepatocytes and specific CYP substrates were used. Classic inducers were used as positive controls and herbal extracts were added in in vivo-relevant concentrations. Metabolites were determined by high performance liquid chromatography (HPLC). St. John's wort and common valerian were the strongest inducing herbs. In addition to induction of CYP3A4 by St. John's wort, common valerian and Ginkgo biloba increased the activity of CYP3A4 and 2D6 and CYP1A2 and 2D6, respectively. A general inhibitory potential was observed for horse chestnut, Echinacea purpurea and common sage. St. John's wort inhibited CYP3A4 metabolism at the highest applied concentration. Horse chestnut might be a herb with high inhibition potentials in vivo and should be explored further at lower concentrations. We show for the first time that G. biloba may exert opposite and biphasic effects on CYP1A2 and CYP2D6 metabolism. Induction of CYP1A2 and inhibition of CYP2D6 were found at low concentrations; the opposite was observed at high concentrations. CYP2D6 activity, regarded generally as non-inducible, was increased by exposure to common valerian (linear to dose) and G. biloba (highest concentration). An allosteric activation is suggested. From the data obtained, G. biloba, common valerian and St. John's wort are suggested as candidates for clinically significant CYP interactions in vivo.


Assuntos
Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP2D6/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Hepatócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Adulto , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A2/análise , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/análise , Relação Dose-Resposta a Droga , Hepatócitos/enzimologia , Humanos , Masculino
19.
Eur J Clin Pharmacol ; 62(4): 277-84, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16525816

RESUMO

OBJECTIVE: The 5HT(1A) receptor agonist sarizotan is in clinical development for the treatment of dyskinesia, a potentially disabling complication in Parkinson's disease. We investigated the effect of sarizotan on the clinical pharmacokinetics of probe drugs for cytochrome P450 (CYP) to evaluate the risk of CYP-related drug-drug interactions. METHODS: This was a double-blind, randomised, two-period cross-over interaction study with repeated administration of 5 mg sarizotan HCl or placebo b.i.d. for 8 days in 18 healthy volunteers. On day 4, a single dose of 100 mg metoprolol (CYP2D6 probe) was administered. On day 8, single doses of 100 mg caffeine (CYP1A2 probe), 50 mg diclofenac (CYP2C9 probe), 100 mg mephenytoin (CYP2C19 probe) and 7.5 mg midazolam (CYP3A4 probe) were simultaneously applied. Pharmacokinetic parameters for probe drugs and their metabolites in plasma and urinary recovery were determined. RESULTS: Concentration-time profiles and pharmacokinetic parameters of all probes and their metabolites remained unchanged after co-administration of sarizotan, compared with placebo. Analysis of variance of the area under the plasma concentration-time curve for probe drugs/metabolites, metabolic ratios and urinary excretion resulted in 90% confidence intervals within the acceptance range (0.8-1.25), indicating the absence of drug-drug interactions. CONCLUSIONS: At a dose higher than that intended for clinical use (1 mg b.i.d.), sarizotan had no effect on the metabolism and pharmacokinetics of specific probe drugs for CYP isoenzymes 1A2, 2C19, 2C9, 2D6 and 3A4. Pharmacokinetic interactions with co-administered drugs metabolised by these CYP isoforms are not expected, and dose adjustment of co-administered CYP substrates is not necessary.


Assuntos
Sistema Enzimático do Citocromo P-450/análise , Sondas Moleculares/farmacocinética , Adolescente , Adulto , Antiparkinsonianos/administração & dosagem , Antiparkinsonianos/farmacologia , Hidrocarboneto de Aril Hidroxilases/análise , Estudos Cross-Over , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP3A , Método Duplo-Cego , Interações Medicamentosas , Humanos , Isoenzimas/análise , Masculino , Oxigenases de Função Mista/análise , Sondas Moleculares/administração & dosagem , Sondas Moleculares/metabolismo , Compostos Orgânicos/administração & dosagem , Compostos Orgânicos/farmacologia , Farmacocinética
20.
Anal Biochem ; 341(1): 77-82, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15866530

RESUMO

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.


Assuntos
Medições Luminescentes , Polimorfismo de Nucleotídeo Único , Adulto , Citocromo P-450 CYP2D6/análise , Citocromo P-450 CYP2D6/genética , Marcadores Genéticos , Humanos , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos
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