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Novel cytochrome CYP3A4 enzyme specific probe reaction and application thereof

A cytochrome and enzyme specific technology, applied in the field of medicine, can solve the problems of inability to clearly analyze the contribution rate of cytochrome CYP3A4 metabolic clearance, confusing the catalytic ability of cytochrome CYP3A4 and 3A5, etc., and achieve the effect of good ultraviolet absorption characteristics

Inactive Publication Date: 2015-09-09
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
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Problems solved by technology

However, recent studies have found that the catalytic ability of cytochrome CYP3A5 in some individuals is even comparable to that of cytochrome CYP3A4 (Drug Metab Dispos.2002,30:883-891). Substrates can confound the catalytic ability of cytochrome CYP3A4 and 3A5, making it impossible to clearly analyze the contribution of cytochrome CYP3A4 to the metabolic clearance of specific drugs

Method used

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  • Novel cytochrome CYP3A4 enzyme specific probe reaction and application thereof
  • Novel cytochrome CYP3A4 enzyme specific probe reaction and application thereof
  • Novel cytochrome CYP3A4 enzyme specific probe reaction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Gomisin A is used to detect the enzyme activity of human recombinant CYP3A4 and CYP3A4+cytochrome b5 system

[0043] Use gomisin A to detect the difference in catalytic activity of two human recombinant single enzymes (both containing cytochrome b5 and not containing cytochrome b5 in the recombinant expression system), the specific steps are as follows:

[0044] (1) In 200 microliters of in vitro metabolic reaction system, 10mM glucose-6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 4mM MgCl 2 , the concentration of recombinant CYP3A4 containing cytochrome b5 is 0.05mg / ml, the final concentration of gomisin A is 50μM, pre-incubated at 37°C for 5 minutes;

[0045] (2) Add 20μl NADP to the reaction system + (final concentration 1mM) initial reaction;

[0046] (3) After reacting for 10 minutes, add 200 μl of methanol and shake vigorously to terminate the reaction;

[0047] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 15 m...

Embodiment 2

[0051] Determination of CYP3A4 Enzyme Activity in Human Liver Microsomes of 12 Individual Cases with Gomisin A

[0052] 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 Gomisin A. The specific operation procedure is as follows:

[0053] (1) In 200 microliters of in vitro metabolic reaction system, 10mM glucose-6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, and 4mM MgCl 2 , the concentration of human liver microsomes is 0.2mg / ml, the final concentration of gomisin A is 50μM, pre-incubated at 37°C for 3 minutes;

[0054] (2) Add 20μl NADP to the reaction system + (final concentration 1mM) initial reaction;

[0055] (3) After 10 minutes, add 200 μl of methanol and shake vigorously to terminate the reaction;

[0056] (4) Use a high-speed refrigerated centrifuge to centrifuge the above system for 10 minutes under the condition of 20,000×g, a...

Embodiment 3

[0059] The specificity of gomisin A as an in vivo P4503A probe substrate was verified by perfusion experiments in isolated rat liver:

[0060] (1) Select 10 male Wistar rats with a body weight of 180-220 g, and establish a rat liver circulation perfusion model. Use perfusion buffer at a perfusion rate of 10ml / min to wash away the blood in the liver and balance the liver for about 10 minute;

[0061] (2) Then switch to a perfusate with a total volume of 200 ml, and add gomisin A to the perfusate at a dose of 5 mg / kg at one time;

[0062] (3) At 0, 5, 15, 25, 35, 50, 75, 100, 150, 210, and 300 minutes, collect about 200 microliters of perfusate samples, put them into centrifuge tubes, and store them at 4 degrees Celsius;

[0063] (4) Take out 100 microliters of the obtained perfusate sample, add an equal volume of methanol, mix thoroughly, use a high-speed refrigerated centrifuge, centrifuge at 12000g for 20 minutes, and take the supernatant for sample analysis;

[0064] (5) U...

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Abstract

The invention provides a novel cytochrome CYP3A4 enzyme specific probe reaction and an application thereof; gomisin A can be used as a probe substrate of a CYP3A4 enzyme for detecting the activity of the enzyme; the gomisin A or a pharmaceutical preparation thereof is used as the specific probe substrate, a reduction rate of the substrate gomisin A or a generation rate of a product 8-hydroxy gomisin A is quantitatively determined in unit time and is used as an evaluation index of the cytochrome CYP3A4 enzyme activity. The probe substrate also has high security and can be used as an overall probe; a to-be-tested mammal is allowed to take 0.1-500 mg / kg body weight of the gomisin A or the pharmaceutical preparation thereof by intravenous injection; time points are selected in 0 to 24 hours, and plasma samples of the to-be-tested animal are collected; the reduction rate of the substrate gomisin A or the generation rate of the product 8-hydroxy gomisin A is determined and is used as the evaluation index of the overall cytochrome CYP3A4 enzyme activity. The quantitative evaluation of different-source biological samples and in-vivo CYP3A4 enzyme activity can be realized.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a lignan compound gomisin A, which can be used as a specific probe substrate for cytochrome CYP3A4 enzymes, and is used for quantitative detection of biological samples from different sources and cytochrome CYP3A4 enzymes in vivo active. Background technique [0002] Cytochrome P450 enzymes (Cytochrome P450, CYP) is a superfamily of heme-thiolate proteins, and is the most important phase I drug metabolizing enzyme in the human body, catalyzing the metabolism of various endogenous and exogenous substances. This superfamily includes multiple subfamilies, such as CYP1A2, CYP2D6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP3A4, and CYP3A5, etc., which are involved in the metabolic transformation of more than 80% of marketed drugs, of which drugs metabolized by the CYP3A subfamily account for about 50% % (Nat Rev Drug Discov. 2005, 4(10):825-833). There are mainly four subtypes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D317/70C12Q1/26A61K31/36
Inventor 杨凌吴敬敬葛广波宁静杜逊甫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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