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A Novel Cytochrome p450 3A5 Enzyme Specific Probe Substrate and Its Application

A technology of probe substrate and cytochrome, which is applied in the field of medicine and achieves the effects of easy preparation, good ultraviolet absorption characteristics and high conversion rate characteristics

Active Publication Date: 2019-07-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is still a lack of CYP3A5 highly selective probes with high conversion efficiency or high sensitivity and high safety

Method used

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  • A Novel Cytochrome p450 3A5 Enzyme Specific Probe Substrate and Its Application
  • A Novel Cytochrome p450 3A5 Enzyme Specific Probe Substrate and Its Application
  • A Novel Cytochrome p450 3A5 Enzyme Specific Probe Substrate and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Schizandra pentamide used to detect the enzyme activity of human recombinant CYP3A5 and CYP3A5+cytochrome b5 system

[0042] The difference in catalytic activity of two human recombinant single enzymes (both containing cytochrome b5 and non-cytochrome b5 in the recombinant expression system) was detected by Schisandra pentamethylene, and the specific steps were as follows:

[0043] (1) In 100 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 CYP3A5 containing cytochrome b5 is 10nM, the final concentration of schisandra pentamyl is 1-200μM, pre-incubated at 37°C for 5 minutes;

[0044] (2) Add 10μl NADP to the reaction system + (final concentration 1mM) initial reaction;

[0045] (3) After reacting for 5 minutes, add 100 μl of methanol, shake vigorously, and terminate the reaction;

[0046] (4) Using a high-speed refrigerated centrifuge, under the ...

Embodiment 2

[0050] Schisandra pentamethylene for detection of CYP3A5 activity in human liver microsomes with different CYP3A5 genotypes

[0051] The differences in the catalytic activity of CYP3A5 in human liver microsomes of different CYP3A5 genotypes were detected by Schisandra pentamethylene, and the specific steps were as follows:

[0052] (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 microparticles is 0.4mg / ml, the final concentration of schisandra pentamyl is 1-200μM, pre-incubated at 37°C for 5 minutes;

[0053] (2) Add 20μl NADP to the reaction system + (final concentration 1mM) initial reaction;

[0054] (3) After reacting for 20 minutes, add 200 μl of methanol, shake vigorously, and terminate the reaction;

[0055] (4) Using a high-speed refrigerated centrifuge, under the condition of 20,000×g, after high-speed centrifugation of the above sy...

Embodiment 3

[0058] Determination of CYP3A5 Enzyme Activity in Human Liver Microsomes of 14 Cases of Schizandra Esterpenta

[0059] Fourteen commercial samples of human liver microsomal samples from different individuals were purchased, and the enzymatic activity of CYP3A5 in human liver samples was determined by Schizandra pentamethylene. The specific operation procedure is as follows:

[0060] (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 0.4mg / ml, the final concentration of schisandra pentamyl is 20μM, pre-incubated at 37°C for 3 minutes;

[0061] (2) Add 20μl NADP to the reaction system + (final concentration 1mM) initial reaction;

[0062] (3) After reacting for 20 minutes, add 200 μl of methanol, shake vigorously, and terminate the reaction;

[0063] (4) Using a high-speed refrigerated centrifuge, under the condition of 20,000×g, af...

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Abstract

The invention provides a novel cytochrome P4503A5 enzyme-specific probe substrate and application thereof, belonging to the technical field of medicine. The substrate is schisandra pentyl, which can be used for the quantitative determination of CYP3A5 enzyme activity. The specific operation process is as follows: use Schisandra pentamethylene as the specific probe substrate, carry out the CYP catalyzed reaction of the specific substrate with the help of the CYP in vitro incubation system or human cells, and quantitatively detect the reduction of the substrate per unit time or its 2 ‑O‑demethylation products were used to determine the activity of CYP3A5 enzymes in various human biological samples or human cells. The invention can realize quantitative evaluation of CYP3A5 enzyme activity in biological samples from different sources.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a novel cytochrome P4503A5 enzyme-specific probe substrate and application thereof. Background technique [0002] Cytochrome P450 (CYP) 3A subfamily is a family of drug-metabolizing enzymes with the most content in the human body, the most metabolized drugs, and many subtypes (Science 1999; 286:487), participating in the in vivo metabolism of nearly 50% of clinical drugs , directly affect the clinical effectiveness and safety of the drug. The human CYP3A subfamily mainly includes four members: CYP3A4, CYP3A5, CYP3A7 and CYP3A43 (Clin Pharmacokinet 2006; 45:13). Among them, CYP3A7 is the main CYP protein detected in human fetal liver, and its content decreases rapidly after birth; CYP3A43 is the newly discovered CYP3A subtype, and its expression level in the liver is very low. CYP3A4 and CYP3A5 are the two main members of the adult CYP3A subfamily. CYP3A4 is the mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C69/78C12Q1/26
CPCC07C69/78C12Q1/26
Inventor 杨凌吴敬敬葛广波王平
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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