Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes

A technology of inhibitory effect and screening method, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inclusion, false negatives, ignoring substrate interactions, etc., to improve screening throughput and improve accuracy Effect

Active Publication Date: 2015-09-23
CHINA PHARM UNIV
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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...

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  • Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes
  • Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes
  • Method for rapidly screening in-vitro inhibitory effect of nine human liver CYP450 enzymes

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Embodiment Construction

[0021] The present invention is explained in detail by the following examples, but it does not mean that the present invention is limited thereto.

[0022] 1 Substrate selection

[0023] As mentioned earlier, the mixed probe substrate method usually selects a specific probe substrate to characterize the enzyme activity, but for isoforms with multiple binding sites such as CYP3A4 and CYP2C9, multiple structurally unrelated The substrate characterizes the enzyme activity. In addition to the phenomenon of multiple binding sites, subtypes such as CYP2C8, CYP2C9, CYP2C19, and CYP2D6 also have a substrate-dependent inhibitory effect. Therefore, in the screening method established by the present invention, especially for CYP2C9, CYP2C19, CYP2D6 and CYP3A4, select two or more structurally unrelated probe substrates to characterize the metabolic enzyme activity (see Table 1 for details), to improve in vitro The reliability of the results as well as the accuracy of the in vivo predict...

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Abstract

The invention discloses a rapid screening method for comprehensively evaluating the in-vitro inhibitory effect of nine human liver CYP450 metabolic enzymes by utilizing 14 probe substrates and 16 probe reaction. The invention mainly relates to a method for monitoring metabolic activity variation of 9 human liver CYP450 enzymes and rapidly and comprehensively evaluating an inhibitory effect of a tested compound on the metabolic enzyme by adopting an in-vitro mixed probe incubation method and LC/MS/MS. According to the method, the exclusiveness and diversity of the probe substrate, interaction of the probe substrates, influence of different inoculation conditions (by charging organic solvent, buffer solution and BSA) in a warm inoculation system and the enzyme kinetics characteristics of 16 probe reactions under the selected inoculation condition are comprehensively considered, a brand new in-vitro system is established by integrating high-sensitive and high-selective LC-MS/MS technology, so that the inhibitory effect of the tested compounds on the nine human liver main metabolic enzymes can be more accurately and comprehensively predicted in the high-throughput screening of the novel drug development, and the predictability on the interaction of the later metabolism can be improved.

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...

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Application Information

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IPC IPC(8): C12Q1/26
Inventor 王广基彭英孙建国吴慧张雪媛冯冬祁欢欢张凤逸仲云熙肖亚楠尤国皎
Owner CHINA PHARM UNIV
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