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Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application

A technology of glucuronic acid and probe substrates, applied in the field of medicine, can solve the problems of use, cytotoxicity, and inability to probe in vivo, and achieve high safety, sensitive detection, and good safety effects

Active Publication Date: 2013-06-12
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

AI Technical Summary

Problems solved by technology

[0005] Currently, there are three reported probe substrates most likely to be UGT1A1, namely bilirubin, estradiol and etoposide. However, since bilirubin and estradiol are endogenous compounds, they cannot be used as Probe use in vivo, and etoposide is cytotoxic, which also limits its use as a substrate for 1A1 probes in vivo

Method used

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  • Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application
  • Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application
  • Specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1. Psoralen dihydroflavone methyl ether is used for the quantitative determination of the enzyme activity of UGT1A1 in the recombinant single enzyme

[0032] (1) Prepare 190 μl UGT metabolic reaction system in advance, including Tris-HCl buffer (50mM) at pH 7.4, 5mM MgCl 2 , recombinant human UGT1A1 (0.05mg / ml), the final concentration of psoralen flavone methyl ether is 25μM, pre-incubated at 37°C for 3 minutes;

[0033] (2) Add 10 μl of 40 mM UDPGA (final concentration 2 mM) to the reaction system to initiate the reaction;

[0034] (3) After 30 minutes, add 200 μl of ice-cold acetonitrile and shake vigorously to terminate the reaction;

[0035] (4) Use a high-speed refrigerated centrifuge at 4°C, 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant for UFLC-UV detection;

[0036] (5) Quantitative detection of psoralen dihydroflavone methyl ether and its glucuronidation products at 320nm. The maximum catalytic rate of recombinant hu...

Embodiment 2

[0037] Example 2. Psoralen dihydroflavone methyl ether is used for quantitative determination of the enzyme activity of UGT1A1 in human liver microsomes

[0038] (1) The reaction system is the same as above. The UGT1A1 in the system was replaced with human liver microsomes, the protein concentration of the microsomes was 0.25 mg / ml, and the reaction time was 30 minutes.

[0039] (2) After high-speed centrifugation at 4°C and 20,000×g for 20 minutes, take the supernatant for UFLC-UV detection;

[0040] (3) Quantitative detection of psoralen dihydroflavone methyl ether and its glucuronidation products at 320nm. The maximum catalytic rate of UGT1A1 was measured to be 8.3 pmol / min / mg in human liver microsomes.

Embodiment 3

[0041] Example 3. Correlation analysis between psoralen dihydroflavone methyl ether and bilirubin metabolism

[0042] Using psoralen dihydroflavone methyl ether and bilirubin as substrates, 12 human liver individual samples were incubated for UGT metabolic reaction, and the reaction incubation conditions were the same as those in Examples 1 and 2. The generation rate of psoralen dihydroflavone methyl ether / bilirubin UGT metabolites in each case of human liver microsome incubation system was measured, and the generation rate of psoralen dihydroflavone methyl ether / bilirubin UGT metabolites was The horizontal and vertical coordinates were plotted, and linear regression analysis was performed on the formed scatter points. The r value obtained was 0.92, suggesting that psoralen flavone methyl ether / bilirubin has similar liver metabolic enzymes and similar contribution to UGT1A1 metabolism Rate.

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Abstract

The invention provides specificity probe zymolyte of glucuronic acid transferase UGT1A1 and application. The specificity probe zymolyte includes dihydro flavonoids compounds of C-4' hydroxyl. Procedures of enzymatic determination are that C-4' hydroxyl-glucose aldehyde acid of bavachinin is selected to be metabolized to a probe reaction. Activity of UGT1A1 enzyme of each biological sample, cell, in vivo and whole organ is measured by quantitative determining of removing quantity of fructus psoraleae flavanone methyl ether in unit time or generation quantity of glucose aldehyde acidize metabolite of the fructus psoraleae flavanone methyl ether. The specificity probe zymolyte of glucuronic acid transferase UGT1A1 can be used for quantitative evaluating of the activity of the UGT1A1 enzyme of different species, different individual source biology samples and quantitative evaluating of the activity of the UGT1A1 enzyme of animal tissue cell culture fluid of different sources and cell prepared products so as to assess ability of handling drugs on important drug metabolic enzyme UGT1A1.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a specific probe substrate of glucuronosyltransferase UGT1A1 and an application thereof. Background technique [0002] The glucuronosyltransferase (Uridine diphosphate-glucuronosyltransferase, UGT) superfamily is an important phase II metabolic enzyme in the body, which catalyzes the combination of the compound with the cofactor uridine diphosphate glucuronic acid (UDPGA), thereby increasing the hydrophilicity of the substrate Sex, so that it can be more effectively excreted from the body in urine or bile, which is an important detoxification process of the body. A variety of exogenous substances, such as SN-38 and nitrosamine compounds, and endogenous substances, such as bilirubin and estradiol, are detoxified through the glucuronidation pathway. [0003] Human UGTs can be divided into 4 families: UGT1, UGT2, UGT3 and UGT8. The most important enzymes involved in d...

Claims

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Application Information

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IPC IPC(8): C12Q1/48A61K49/00
Inventor 杨凌葛广波夏杨柳朱亮亮吕侠何桂元宁静
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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