A rapid screening method for the in vitro inhibitory effect of nine human liver cyp450 enzymes

Abstract

The invention discloses a rapid screening method for comprehensively evaluating the in vitro inhibitory effects of 9 human liver CYP450 metabolic enzymes by using 14 kinds of probe substrates and 16 kinds of probe reactions. The invention mainly relates to monitoring the metabolic activity changes of 9 kinds of human liver CYP450 enzymes by using an in vitro mixed probe incubation method combined with LC / MS / MS, and rapidly and comprehensively evaluating the inhibitory effect of test compounds on metabolic enzymes. This method comprehensively considers the specificity and diversity of probe substrates, the interaction between probe substrates, and the reaction of different incubation conditions (organic solvent, buffer, BSA, etc.) in the incubation system to the probe reaction. The influence of various factors such as the influence on the activity of the enzyme, and the enzyme kinetics characteristics of the 16 probe reactions under the selected incubation conditions, combined with the high sensitivity and high selectivity LC-MS / MS technology, established a new in vitro evaluation System, in order to more accurately and comprehensively predict the possible inhibitory effects of test compounds on 9 major metabolic enzymes of human liver in the high-throughput screening of new drug development, and improve the predictability of later metabolic interactions.

Description

technical field [0001] The invention relates to a method for rapid screening of the in vitro inhibitory effect of human liver CYP450 enzymes, specifically relates to the use of specific probe substrates to characterize enzyme activity, and the use of in vitro mixed probe substrate method (N-in-one) combined with LC-MS / MS Simultaneous determination of a variety of specific metabolites was used to identify the changes in the activity of nine human liver CYP450 enzymes, which were used as the basis for judging the inhibitory effect of metabolic enzymes in vitro. Background technique [0002] For a long time, cytochrome P450s (CYP450) enzymes have played an important role in drug metabolism and drug metabolism-inhibitory interactions in drug safety evaluation. In the early stage of new drug development, the use of in vitro experimental data to predict the potential drug interactions of new chemical entities (NCEs) in vivo as early as possible and obtain relevant metabolic inform...

AI Technical Summary

Problems solved by technology

However, the methods reported so far still have defects to varying degrees, such as ignoring the interaction between substrates and incubating different subtypes of substrates in the same system, resulting in false negative results.

However, there is no report on the inclusion of BSA as a cofactor in the mixed probe substrate system.

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