Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of cytochrome cyp3a4 enzyme specific probe reaction and its application

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

Inactive Publication Date: 2017-09-22
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 MetabDispos. 2002, 30:883-891). Drugs will confuse 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of cytochrome cyp3a4 enzyme specific probe reaction and its application
  • A kind of cytochrome cyp3a4 enzyme specific probe reaction and its application
  • A kind of cytochrome cyp3a4 enzyme specific probe reaction and its application

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a specific probe reaction of novel cytochrome CYP3A4 enzyme and its application: gomisin A can be used as the probe substrate of CYP3A4 enzyme to detect the activity of the enzyme, and gomisin A or its pharmaceutical preparation As a specific probe substrate, the reduction rate of the substrate gomisin A or the production rate of the product 8-hydroxygomisin A is quantitatively measured per unit time as an evaluation index of the cytochrome CYP3A4 enzyme activity. The probe substrate also has high safety and can be used as an overall probe, and the mammal to be tested is administered 0.1 to 500 mg / kg body weight of gomisin A or its pharmaceutical preparations by intravenous injection; selected within 0 to 24 hours At the time point, the plasma samples of the animals to be tested were collected; the reduction rate of the substrate gomisin A or the production rate of the product 8-hydroxygomisin A was measured as an evaluation index of the overall cytochrome CYP3A4 enzyme activity. The invention can realize quantitative evaluation of biological samples from different sources and CYP3A4 enzyme activity in vivo.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com