An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes - application to establishing CYP2C8 inhibitor selectivity.
Eur J Pharm Sci
; 162: 105810, 2021 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-33753217
ABSTRACT
We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-ß-glucuronide and clopidogrel acyl-ß-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-ß-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-ß-glucuronide was a strong (>90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 µM, while the selectivity of clopidogrel acyl-ß-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 µM, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting time-dependent inhibition. Moreover, gemfibrozil 1-O-ß-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas em Tandem
/
Inibidores das Enzimas do Citocromo P-450
Limite:
Humans
Idioma:
En
Revista:
Eur J Pharm Sci
Assunto da revista:
FARMACIA
/
FARMACOLOGIA
/
QUIMICA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Finlândia