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1.
Drug Metab Pharmacokinet ; 40: 100412, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34352706

RESUMO

The predicted contributions of flavin-containing monooxygenase 3 (FMO3) to drug candidate N-oxygenations can be estimated using classic base dissociation constants of the N-containing moiety. In this study, metabolic clearance values in human liver microsomes were experimentally determined for available model drugs. Typical metabolic clearance values (34-96 µL/min/mg protein) at pH 8.4 of trimethylamine, benzydamine, and itopride were two-to fourfold higher than those at pH 7.4. In contrast, the metabolic clearance of control drug midazolam at pH 8.4 was half that at pH 7.4. The ratios of clearance values at pH 8.4 to those at pH 7.4 and the substrate pKa (base) values of reported metabolic N-oxygenation sites of trimethylamine, benzydamine, clomipramine, chlorpromazine, tamoxifen, itopride, loxapine, xanomeline, tozasertib, dasatinib, and clozapine were significantly correlated (r = 0.60, p < 0.05, n = 11). These results suggested that the simple comparison of metabolic clearance values at pH 8.4 and at pH 7.4 could be useful for predicting the contributions of FMO3 to the N-oxygenations of new drug candidates. This method, along with in silico pKa (base) values > 8.4, could prove useful for predicting the contributions of FMO3 to N-oxygenations as part of drug development.


Assuntos
Microssomos Hepáticos , Preparações Farmacêuticas , Sistema Enzimático do Citocromo P-450 , Humanos , Concentração de Íons de Hidrogênio , Oxigenases
2.
Bioanalysis ; 10(17): 1349-1360, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30182726

RESUMO

It is important to select an appropriate surrogate matrix for preparing calibration standards and quality control samples while quantitatively assaying for endogenous substances, because a blank matrix that does not contain the endogenous substance cannot be derived from the species from which the target study samples are collected. This is because the assay results might be affected, depending on the characteristics of the analyte in the surrogate matrix. Our discussion group that participated in the Japan Bioanalysis Forum discussed the recommended selection strategies, focusing on large and small molecules in ligand binding assays and LC-MS, respectively. We established an efficient selection strategy for a surrogate matrix, with simple compositions as the first candidates stated in this article.


Assuntos
Técnicas de Química Analítica/métodos , Calibragem , Técnicas de Química Analítica/normas , Cromatografia Líquida , Japão , Padrões de Referência , Espectrometria de Massas em Tandem
3.
Drug Metab Lett ; 12(1): 14-23, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29357810

RESUMO

BACKGROUND: The HepaRG cells have key drug metabolism functionalities comparable to those of primary human hepatocytes. Many studies have reported that this cell line can be used as a reliable in vitro model for human drug metabolism studies, including the assessment of cytochrome P450 (CYP) induction. OBJECTIVES: The objective of this study is to determine whether CYP mRNA level measurement is superior to the CYP enzyme activity measurement as a convenient high-throughput method for evaluating CYP induction potential using HepaRG cells. METHODS: QuantiGene Plex 2.0 Assay and LC/MS/MS. mRNA expression levels and enzyme activities of CYP1A2, CYP2B6, and CYP3A in HepaRG cells treated with prototypical inducers of each CYP isoform [omeprazole (OME) for CYP1A2, phenobarbital (PB) for CYP2B6, and rifampicin (RIF) for CYP3A] were evaluated. RESULTS: Although the activities of CYP2B6 and CYP3A were induced by treatment with PB and RIF, we found that the activity of phenacetin O-deethylase (PHOD), which is known as a marker of the activity of CYP1A2, was also enhanced by treatment with these non-CYP1A2 inducers in HepaRG cells. Based on previously published reports, we hypothesized that the expression ratio of CYP3A to CYP1A2 is much higher in HepaRG cells than in human hepatocytes; this may result in a nonnegligible contribution of CYP3A to the PHOD reaction in HepaRG cells. Studies using CYP3A inhibitor and pregnane X receptor-knockout HepaRG cells supported this hypothesis. CONCLUSION: The measurement of mRNA serves as a higher reliable indicator for the evaluation of CYP induction potential when using HepaRG cells.


Assuntos
Citocromo P-450 CYP1A2/metabolismo , Indutores das Enzimas do Citocromo P-450/farmacologia , Taxa de Depuração Metabólica/efeitos dos fármacos , RNA Mensageiro/análise , Biomarcadores/análise , Linhagem Celular , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Indução Enzimática/efeitos dos fármacos , Hepatócitos , Humanos , Omeprazol/farmacologia , Fenacetina/metabolismo , Fenobarbital/farmacologia , Reprodutibilidade dos Testes , Rifampina/farmacologia
4.
Kobe J Med Sci ; 60(4): E78-85, 2015 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-25791416

RESUMO

BACKGROUND: Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletions. Thus, the SMN1 deletion test should be performed initially as part of the diagnostic process. However, SMN2, a highly homologous gene, hampers detection of SMN1 deletion. To differentiate between SMN1 and SMN2, many analysis methods have been developed yet they are not all available worldwide. AIM: To establish a simple but accurate SMN1-deletion detection system that can be used worldwide. METHODS: Fifty DNA samples (29 SMA patients and 21 controls) from dried blood spots (DBS) on filter paper were assayed. All participants had previously been screened for SMA by PCR-restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25℃) for between 1 and 8 years. Competitive oligonucleotide priming-PCR (COP-PCR) was performed to distinguish SMN1 and SMN2 exon7. RESULTS: DNA yield from an 11-mm diameter DBS circle was 21,171 ± 7,485 ng (mean ± SD), with an 260/280 OD ratio from 1.49 to 2.1(mean ± SD; 1.67 ±0.13). Nucleotide sequencing confirmed gene-specific amplification of SMN1 and SMN2 by COP-PCR. SMN1 and SMN2 COP-PCR results are completely consistent with those obtained by PCR-RFLP. CONCLUSION: We have combined DNA extraction from DBS on filter paper with COP-PCR that specifically detects SMN1 and SMN2, establishing a new SMN1-deletion detection system with practical application worldwide.


Assuntos
Teste em Amostras de Sangue Seco , Deleção de Genes , Atrofia Muscular Espinal/diagnóstico , Reação em Cadeia da Polimerase/métodos , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Primers do DNA , Humanos , Atrofia Muscular Espinal/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética
5.
Kobe J Med Sci ; 54(5): E227-36, 2009 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-19628962

RESUMO

Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical gene, SMN2, fails to compensate for the loss of SMN1 because SMN2 produces mainly an exon 7-skipped product. The +6C in SMN1 exon 7 proceeds to include exon 7 into mRNA, while the +6U in SMN2 causes skipping of exon 7. Here, approximately 45kD proteins bound to the SMN exon 7 RNA probe was found, and identified as hnRNP C1/C2. In gel-shift assay, hnRNP C1/C2 had a greater affinity for the RNA probe with +6C than for the RNA probe with +6U. In vitro splicing assay showed that anti-hnRNP C1/C2 antibody hampered splicing of SMN1 exon 7, but did not affect splicing of SMN2 exon 7. In conclusion, we showed the possibility that hnRNP C1/C2 enhanced SMN1 exon 7 splicing specifically.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Atrofia Muscular Espinal/genética , Splicing de RNA , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Sequência de Aminoácidos , Anticorpos , Éxons , Inativação Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/imunologia , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Sondas RNA , Proteína 2 de Sobrevivência do Neurônio Motor/genética
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