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Specific probe substrate for cytochrome P450 3A4 enzyme and application of substrate

A technology of probe substrate and cytochrome, which is applied in the field of medical technology of the Ming Dynasty, can solve the problems of confusing the catalytic ability of cytochrome P4503A4 and 3A5, and the inability to clearly analyze the metabolic scavenging ability of cytochrome P4503A4, and achieve good ultraviolet absorption characteristics, detect sensitive effects

Active Publication Date: 2015-06-10
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, recent studies have found that the catalytic ability of cytochrome P4503A5 in some individuals is even comparable to that of cytochrome P4503A4 (Drug Metab Dispos 2002, 30:883-91). It will confuse the catalytic ability of cytochrome P4503A4 and 3A5, so that it is impossible to clearly analyze the metabolic clearance ability of cytochrome P4503A4 for a certain drug

Method used

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  • Specific probe substrate for cytochrome P450 3A4 enzyme and application of substrate
  • Specific probe substrate for cytochrome P450 3A4 enzyme and application of substrate
  • Specific probe substrate for cytochrome P450 3A4 enzyme and application of substrate

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Cinobufagin used to detect enzyme activity of human recombinant CYP3A4 and CYP3A4+cytochrome b5 system

[0032] Cinobufagin was used to detect the difference in the catalytic activity of two human recombinant single enzymes (the recombinant expression system contains cytochrome b5 and does not contain cytochrome b5), the specific steps are as follows:

[0033] (1) In 200 microliters of in vitro metabolic reaction system, 10 mM glucose-6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 4 mM MgCl 2 , the concentration of recombinant CYP3A4 containing cytochrome b5 was 0.05 mg / ml, the final concentration of cinobufagin was 30 μM, and pre-incubated at 38°C for 5 minutes;

[0034] (2) Add 20 μl NADP to the reaction system + (final concentration 1 mM) initial reaction;

[0035] (3) After 10 minutes, add 200 μl methanol and shake vigorously to terminate the reaction;

[0036] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 min...

Embodiment 2

[0040] Determination of CYP3A4 enzyme activity in 12 individual cases of human liver microsomes with bufagenin

[0041] Twelve commercial samples of human liver microsomal samples from different individuals were purchased, and the enzyme activity of CYP3A4 in human liver samples was determined by using bufagenin. The specific operation procedure is as follows:

[0042] (1) In 200 microliters of in vitro metabolic reaction system, 10 mM glucose-6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 4 mM MgCl 2 , the concentration of human liver microsomes was 0.2 mg / ml, the final concentration of bufagenin was 30 μM, and pre-incubated at 37°C for 3 minutes;

[0043] (2) Add 20 μl NADP to the reaction system + (final concentration 1 mM) initial reaction;

[0044] (3) After 10 minutes, add 200 μl methanol and shake vigorously to terminate the reaction;

[0045] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under the conditi...

Embodiment 3

[0048] Determination of Enzyme Activity of CYP3A4 in Human Hepatoma Cell BEL7402 by Bufalin

[0049] (1) Dilute BEL7402 cells with hepatocyte incubation solution, place in a 6-well culture plate, 4ml per well, put in a metal bath shaker, 80 r / min, 40 ℃ for 120 minutes with continuous shaking;

[0050] (2) Add bufalin to the culture plate, the final concentration is 50 μM;

[0051] (3) After 30 minutes, draw 200 μl of the incubation solution and place it in a -80°C ultra-low temperature refrigerator to stop the reaction;

[0052] (4) Add 200ul of methanol to the sample to precipitate protein, and use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under the condition of 20,000×g, then take the supernatant for UFLC-ESI-MS detection and analysis

[0053] UFLC-ESI-MS was used to detect bufalin and its metabolites, and the SIM mode was selected to detect the molecular ion peaks of bufalin and its metabolites [M+H] + ( m / z 387 & m / z 403)....

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Abstract

The invention provides a specific probe substrate for a cytochrome P450 3A4 enzyme and an application of the substrate in CYP3A4 enzyme activity measurement. The specific operation flow of the enzyme activity measurement comprises the following steps: carrying out the CYP catalytic reaction of the specific substrate by virtue of a CYP in-vitro incubation system by selecting any monomer in toad steroid series of compounds as a high-specificity probe substrate; and measuring the activity of the CYP3A4 enzyme in each biological sample and each cell by quantitatively detecting a product generated quantity or a substrate eliminated quantity in a unit time. The specific probe substrate provided by the invention can be used for quantitative evaluation of CYP3A4 enzyme activity in biological samples belonging to different species and deriving from different individual sources, and quantitative measurement of CYP3A4 enzyme activity in animal tissue cell culture fluids and cell products deriving from different sources.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a class of bufadenoid compounds, such as three compounds of cinobufagin, deacetylated cinobufagin, esterbufagenin and bufalin and their structural analogues, which can be As a specific probe substrate for cytochrome P450 3A4 enzyme, it is used for the quantitative determination of cytochrome P450 3A4 enzyme activity in biological samples from different sources, in order to realize the evaluation of the ability of important drug metabolizing enzyme CYP 3A4 to dispose of drugs. Background technique [0002] Cytochrome P450 enzymes (Cytochrome P450, CYP) superfamily includes the most important drug-metabolizing enzymes in the body, and participates in the metabolism of more than 80% of marketed drugs, of which drugs metabolized by the cytochrome P4503A subfamily account for about 50%. Four isoforms of cytochrome P4503A are expressed in the human body, namely cytochrome P...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07J71/00C12Q1/26
Inventor 杨凌葛广波宁静吴敬敬杜逊甫
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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