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Specific probe substrate composition of cytochrome P450 enzyme and application of specific probe substrate composition

A probe substrate, cytochrome technology, applied in the field of enzymology and biological analysis, can solve the problems of time-consuming, inefficient, sensitivity limitation, etc., and achieve the effect of saving cost and time, improving experimental throughput efficiency and strong specificity

Inactive Publication Date: 2014-12-10
广东中西达一新药开发有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] With the adoption of combinatorial chemistry and high-throughput screening methods, the number of compounds that need to be screened for CYP450 enzyme inhibition has increased dramatically, and currently due to sensitivity limitations of analytical methods (such as HPLC, UV or fluorescence), each analysis can only predict Inhibition of individual enzymes by compounds, time consuming and inefficient

Method used

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  • Specific probe substrate composition of cytochrome P450 enzyme and application of specific probe substrate composition
  • Specific probe substrate composition of cytochrome P450 enzyme and application of specific probe substrate composition
  • Specific probe substrate composition of cytochrome P450 enzyme and application of specific probe substrate composition

Examples

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Embodiment 1

[0039] Example 1 Screening of highly specific CYP450 enzyme probe substrates

[0040] (1) The enzyme kinetic parameters between CYP450 enzymes and their commonly used probe substrates were measured respectively. The results are shown in Table 1, which serve as a reference and basis for selecting probe substrates.

[0041] Table 1

[0042]

[0043]

[0044] Inhibition of CYP450 enzymes usually requires a constant reaction rate during the incubation period. It can be seen from Table 1 that because midazolam is metabolized too fast, the linear range is inconsistent with other substrates, so testosterone was used as the substrate of 3A4 instead; S-mephenytoin is metabolized too slowly and the signal is low. Therefore, it is excluded; 6-hydroxychlorzoxazone has only negative ions and is also excluded; amodiaquine has a strong inhibitory effect on other enzymes and cannot be incubated with other substrates. Therefore, the present invention uses phenacetin, coumarin, bupropio...

Embodiment 2

[0049] Example 2 Specific Probe Substrate Composition Detects the Inhibition of 6 Subtypes of CYP450 Enzymes

[0050] (1) The specific probe substrate composition is incubated with human liver microsomes in vitro:

[0051] a. Instruments and reagents

[0052] 8-channel or 12-channel pipette, 96-well deep-well plate, 1000× stock solutions of each substrate, commercial human liver microsomes, NAPDH coenzyme, and drugs to be tested.

[0053] b. Microsome in vitro incubation reaction system

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Abstract

The invention relates to a specific probe substrate composition of cytochrome P450 (CYP450) enzyme and an application of the specific probe substrate composition in subtype enzyme activity detection of the cytochrome P450 enzyme. The composition comprises at least two of phenacetin, coumarin, amfebutamone, dextromethorphan, diclofenac and testosterone. The characteristics of multi-ion channels can be simultaneously detected by the specific probe substrate composition and a liquid chromatography-mass spectrometry technology, and the inhibition conditions of six CYP450 enzymes are determined simultaneously, so that the experiment flux efficiency is greatly improved, and the experiment cost is reduced.

Description

technical field [0001] The present invention relates to the technical fields of enzymology and biological analysis, in particular to a specific probe substrate composition and its application, in particular to a specific probe substrate composition of cytochrome P450 enzymes and its function in cytochrome P450 Application in the detection of enzyme subtype enzyme activity. Background technique [0002] Cytochrome P450 (CYP450) enzymes are distributed in the liver, small intestine, and kidney, among which subtypes such as CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 are closely related to the metabolic clearance of exogenous substances (such as drugs). Participate in 90% of drug metabolism. In the process of new drug development, the rate of specific metabolites generated by the probe substrates of these CYP450 enzymes in in vitro metabolic systems (such as human liver microsomes, etc.) can be used to determine whether the drug will inhibit a specific CYP enzyme subty...

Claims

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Application Information

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IPC IPC(8): C12Q1/26G01N30/02
Inventor 张素行张飞鹏叶沛珍陈涛龙娜张科之黄晓勇
Owner 广东中西达一新药开发有限公司
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