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3-Hydroxy-V-coumarin sulfonation and LC-MS analysis
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Resumo(s)
O estudo do metabolismo é uma parte crucial no desenvolvimento de fármacos. A sulfonação
é um processo fundamental no metabolismo, sendo que esta reação de conjugação de fase II é
catalisada por sulfotransferases (SULTs), formando metabolitos inativos que são mais solúveis
em água e mais fáceis de remover do organismo.
A cumarina e os seus derivados têm desempenhado um papel essencial na descoberta de novos
fármacos com propriedades anticoagulantes, anti-inflamatórias, antioxidantes e antitumorais. O
metabolismo de um novo derivado, a 3-hidroxi-V-cumarina, é estudado neste trabalho. Este
estudo é de enorme importância de modo que se consiga minimizar os efeitos de toxicidade
devido à exposição a xenobióticos.
A 3-hidroxi-V-cumarina, uma bis-cumarina em forma de V, forma uma estrutura eletrónica
push-pull e emite fluorescência, que pode ser medida para quantificar a sua concentração ao
longo da reação.
Em estudos de metabolismo de fase inicial, a análise de amostras obtidas de incubações in vitro
e/ou experiências in vivo é habitualmente feita por cromatografia líquida e espetrometria de
massa (LC-MS). Com esse propósito, foram realizadas análises dos metabolitos formados na
reação de sulfonação, recorrendo a LC-MS, bem como avaliação dos parâmetros cinéticos.
Os resultados obtidos sugerem que a diminuição da fluorescência da 3-hidroxi-V-cumarina
indica a sua sulfonação, sendo esta reação catalisada pela SULT1A1, SULT1A2 e SULT1E1.
A reação de sulfonação da 3-hidroxi-V-cumarina com SULT1A1 é a mais rápida e requer uma
concentração mais baixa de substrato para produzir metade da Vmax. Também foi possível
concluir que a maior clearance intrínseca ocorre quando a SULT1A1 catalisa a reação. Além
disso, a concentração do metabolito sulfonado aumenta ao longo do tempo de reação, até aos
30 minutos, e a partir desse tempo de incubação diminui, devido ao facto de o metabolito
começar a ser hidrolisado.
The study of metabolism is a crucial part in drug development. Sulfonation is an important process of drug metabolism, this phase II conjugation reaction is catalyzed by sulfotransferases (SULTs), forming inactive sulfate metabolites that are more water-soluble and easier to remove from the body. Coumarin and its derivatives have played their fundamental role in the discovery of new drugs with anticoagulant, anti-inflammatory, antioxidant, and antitumor properties. The metabolism of a new derivative, 3-hydroxy-V-coumarin, is studied in this work. This study is of enormous importance in order to minimize the toxicity events due to exposure to xenobiotics. 3-hydroxy-V-coumarin, a V-shaped bis-coumarin, form a push-pull electron structure and release fluorescent light that can be measured to quantify its concentration throughout the reaction. In early phase metabolism studies, the analysis of samples obtained from in vitro incubations and/or in vivo experiments is commonly done by liquid chromatography and mass spectrometry (LC-MS). With this purpose, LC-MS analysis of the metabolites formed in the sulfonation reaction was carried out, as well as evaluation of kinetic parameters. The results obtained suggest that the decrease in 3-hydroxy-V-coumarin fluorescence indicates its sulfonation, and this reaction is catalyzed by SULT1A1, SULT1A2 and SULT1E1. The sulfonation reaction of 3-hydroxy-V-coumarin with SULT1A1 is the fastest and it requires a lower substrate concentration to produce half of Vmax. It was also possible to conclude that the highest intrinsic clearance occurs when SULT1A1 catalyze the sulfonation of 3-hydroxy-V coumarin. Furthermore, sulfate metabolite concentration increases over the reaction time up to 30 minutes and then decreases because the metabolite starts to be hydrolyzed.
The study of metabolism is a crucial part in drug development. Sulfonation is an important process of drug metabolism, this phase II conjugation reaction is catalyzed by sulfotransferases (SULTs), forming inactive sulfate metabolites that are more water-soluble and easier to remove from the body. Coumarin and its derivatives have played their fundamental role in the discovery of new drugs with anticoagulant, anti-inflammatory, antioxidant, and antitumor properties. The metabolism of a new derivative, 3-hydroxy-V-coumarin, is studied in this work. This study is of enormous importance in order to minimize the toxicity events due to exposure to xenobiotics. 3-hydroxy-V-coumarin, a V-shaped bis-coumarin, form a push-pull electron structure and release fluorescent light that can be measured to quantify its concentration throughout the reaction. In early phase metabolism studies, the analysis of samples obtained from in vitro incubations and/or in vivo experiments is commonly done by liquid chromatography and mass spectrometry (LC-MS). With this purpose, LC-MS analysis of the metabolites formed in the sulfonation reaction was carried out, as well as evaluation of kinetic parameters. The results obtained suggest that the decrease in 3-hydroxy-V-coumarin fluorescence indicates its sulfonation, and this reaction is catalyzed by SULT1A1, SULT1A2 and SULT1E1. The sulfonation reaction of 3-hydroxy-V-coumarin with SULT1A1 is the fastest and it requires a lower substrate concentration to produce half of Vmax. It was also possible to conclude that the highest intrinsic clearance occurs when SULT1A1 catalyze the sulfonation of 3-hydroxy-V coumarin. Furthermore, sulfate metabolite concentration increases over the reaction time up to 30 minutes and then decreases because the metabolite starts to be hydrolyzed.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
3-hydroxy-V-coumarin Liquid chromatography Mass spectrometry Metabolism Sulfonation Mestrado Integrado - 2023