In Vitro Approaches for Studying the Inhibition of Drug-Metabolizing Enzymes and Identifying the Drug-Metabolizing Enzymes Responsible for the Metabolism of Drugs (Reaction Phenotyping) with Emphasis on Cytochrome P450

Grandisin, a lignan isolated from many species of plants, such as , is a potential drug candidate due to its biological properties, highlighted by its antitumor and trypanocidal activities. In this study, the inhibitory effects of grandisin on the activities of human cytochrome P450 enzymes were investigated by using human liver microsomes. Results showed that grandisin is a competitive inhibitor of CYP2C9 and a competitive and mechanism-based inhibitor of CYP3A4/5. The apparent K value for CYP2C9 was 50.60 µM and those for CYP3A4/5 were 48.71 µM and 31.25 µM using two different probe substrates, nifedipine and midazolam, respectively. The apparent K, k, and k/K ratio for the mechanism-based inhibition of CYP3A4/5 were 6.40 µM, 0.037 min, and 5.78 mL · min µmol, respectively, by examining nifedipine oxidation, and 31.53 µM, 0.049 min, and 1.55 mL · min µmol, respectively, by examining midazolam 1'-hydroxylation. These apparent k/K values were comparable to or even higher than those for several therapeutic drugs that act as mechanism-based inhibitors of CYP3A4/5. CYP1A2 and CYP2D6 activities, in turn, were not substantially inhibited by grandisin (IC > 200 µM and 100 µM, respectively). In contrast, from a concentration of 4 µM, grandisin significantly stimulated CYP2E1 activity. These results improve the prediction of grandisin-drug interactions, suggesting that the risk of interactions with drugs metabolized by CYP3A4/5 and CYP2E1 cannot be overlooked.

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“…to formation of electrophilic reactive intermediates that can covalently bind to the enzyme [32], which reinforces our results.…”

Section: Resultssupporting

confidence: 91%

In Vitro Inhibition of Human CYP450s 1A2, 2C9, 3A4/5, 2D6 and 2E1 by Grandisin

et al. 2017

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“…Currently, several in vitro models are available for in vitro phenotyping assays. The most often used assays include: (1) recombinant CYPs (rhCYPs), (2) hepatocytes or human liver microsomes (HLMs) in the presence or absence of chemical CYP specific inhibitors or antibodies and (3) correlation analysis using multiple batches of HLMs (Ogilvie et al, 2008;Parmentier et al, 2007;Wienkers & Stevens, 2003). Despite the advantages of these models, in particular rhCYPs, they also suffer from many drawbacks that prevent them from being used alone such that more than one model must be conducted in order to derive a definitive fmCYP value.…”

Section: Introductionmentioning

confidence: 99%

Direct and quantitative evaluation of the major human CYP contribution (fmCYP) to drug clearance using thein vitroSilensomes™ model

et al. 2018

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1. We have applied the concept of using MBIs to produce CYP-Silensomes to quantify the contribution of the major CYPs to drug metabolism (fmCYP). 2. The target CYPs were extensively and selectivity inhibited by the selected MBIs, while non-target CYPs were inhibited by less than 20% of the homologous control activities. Only CYP2D6-Silensomes exhibited a CYP2B6 inhibition that could be easily and efficiently encountered by subtracting the fm measured using CYP2B6-Silensomes to adjust the fm. 3. To validate the use of a panel of 6 CYP-Silensomes, we showed that the fmCYP values of mono- and multi-CYP metabolised drugs were well predicted, with 70% within ± 15% accuracy. Moreover, the correlation with observed fmCYP values was higher than that for rhCYPs, which were run in parallel using the same drugs (<45% within ±15% accuracy). Moreover, the choice of the RAF substrate in rhCYP predictions was shown to affect the accuracy of the fmCYP measurement. 4. These results support the use of CYP1A2-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6 and CYP3A4-Silensomes to accurately predict fmCYP values during the in vitro enzyme phenotyping assays in early, as well as in development, phases of drug development.

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“…A inibição direta ocorre quando um determinado composto inibe a enzima, assim que a exposição ocorre e sem necessidade de biotransformação. Enquanto que na inibição tempo dependente, o potencial do inibidor aumenta com o tempo de contato entre o inibidor e a enzima, sendo quase irreversível ou irreversível (OGILVIE et. al., 2008).…”

Section: Cinética Enzimáticaunclassified

“…A primeira etapa do estudo de inibição de um praguicida sobre as enzimas do CYP450 é a determinação do seu valor de IC50, que corresponde a concentração do praguicida responsável pela inibição de 50% da atividade enzimática (OGILVIE et. al., 2008).…”

Section: Cinética Enzimáticaunclassified

“…Para a determinação do IC50 são utilizadas reações denominadas como marcadores, que são características e seletivas para uma determinada isoforma do CYP450 e recomendadas pelo FDA (Tabela 6) (ABASS; TURPEINEN; PELKONEN, 2009). A concentração de praguicida empregada deve ser a mesma obtida no estudo de cinética enzimática, sendo equivalente ou inferior ao valor de Km e as condições de incubação (tempo e concentração proteica) devem ser estabelecidas respeitando as condições de estado estacionário da reação enzimática do marcador (OGILVIE et. al., 2008).…”

Section: Cinética Enzimáticaunclassified

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Análise enantiosseletiva do praguicida miclobutanil após metabolismo in vitro por microssomas hepáticos de humanos

Fonseca¹

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Ao Prof. Dr. Anderson Rodrigo Moraes de Oliveira, que além de orientador, é também, amigo. Agradeço pela oportunidade e por todo aprendizado transmitido. Você é um exemplo, de profissional e humano, a ser seguido. Obrigada por todo apoio e por sempre me lembrar que as dificuldades fazem parte do caminho para alcançar o sucesso. "Quem tem um sonho, nunca cansa". Aos meus pais, Lucimara e Claudemar, que durante toda a minha vida, não mediram esforços, para que eu pudesse conquistar meus objetivos. Muitas vezes deixando seus sonhos de lado, para que eu pudesse realizar os meus. Agradeço por todo amor, carinho, dedicação, apoio e companheirismo. Vocês são a razão de tudo isso. Aos meus familiares, em especial, meus padrinhos Edmara e Luciano, meus

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In Vitro Approaches for Studying the Inhibition of Drug-Metabolizing Enzymes and Identifying the Drug-Metabolizing Enzymes Responsible for the Metabolism of Drugs (Reaction Phenotyping) with Emphasis on Cytochrome P450

Cited by 28 publications

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In Vitro Inhibition of Human CYP450s 1A2, 2C9, 3A4/5, 2D6 and 2E1 by Grandisin

In Vitro Inhibition of Human CYP450s 1A2, 2C9, 3A4/5, 2D6 and 2E1 by Grandisin

Direct and quantitative evaluation of the major human CYP contribution (fmCYP) to drug clearance using thein vitroSilensomes™ model

Análise enantiosseletiva do praguicida miclobutanil após metabolismo in vitro por microssomas hepáticos de humanos

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