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Application of oxymetazoline as special substrate for glucuronyl transferase UGT1A9

A technology of glucuronic acid and oxymetazoline, applied in the field of medicine, can solve the problem that the substrate does not have enzyme specificity, and achieve the effects of remarkable technical performance, good industrialization prospect and easy detection.

Inactive Publication Date: 2014-12-03
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a distinctive feature of human glucuronosyltransferases is that they often have no enzyme specificity for their substrates

Method used

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  • Application of oxymetazoline as special substrate for glucuronyl transferase UGT1A9
  • Application of oxymetazoline as special substrate for glucuronyl transferase UGT1A9
  • Application of oxymetazoline as special substrate for glucuronyl transferase UGT1A9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Oxymetazoline is used for measuring the enzymatic activity of single enzyme UGT1A9

[0037] (1) Prepare 300μL incubation system in advance, including 50mM phosphate buffer, 1mM MgCl 2 , 5mM Saccharolactone (glucaric acid monolactone), human UGT1A9 single enzyme (BD company, the United States) (final concentration is 0.05mg / mL), a series of concentrations of oxymetazoline (10mM, 5mM, 2.5mM, 1.25 mM, 0.625mM, 0.3125mM, 0.15625mM) and UDPGA with a final concentration of 2mM; pH 7.2-7.5. The preparation process of the system must be operated on ice, and finally UDPGA is added to the system to start the reaction;

[0038] 50mM phosphate buffer was prepared by KH 2 PO 4 and K 2 HPO 4 Prepared with a pH of 7.4;

[0039] (2) Transfer the incubation system to a constant temperature environment of 37°C for reaction. After 2 hours, add 100 μL of acetonitrile into the system and mix thoroughly to terminate the reaction;

[0040] (3) Under the centrifugation cond...

Embodiment 2

[0043] Example 2 Oxymetazoline Determination of UGT1A9 Enzyme Activity in Human Liver Microsomes

[0044] (1) The incubation system was as in Example 1, the enzyme in the system was replaced with human liver microsomes (BD Company, the United States), the final protein concentration of human liver microsomes in the system was 0.5 mg / mL, and the reaction time was 1 h;

[0045] (2) Sample processing and analysis As described in Example 1, the enzyme activity of UGT1A9 in liver microsomes was measured by the amount of glucuronidation product produced per unit time. The maximum catalytic rate of UGT1A9 enzyme in human liver microsomes was measured to be 7.53±0.52 pmol / min / mg.

[0046] The more metabolites generated per unit time, the stronger the UGT1A9 enzyme activity in human liver microsomes.

Embodiment 3

[0047] Example 3 Utilizing the correlation between the metabolic rate of oxymetazoline and propofol in individual human liver microsomes to evaluate the ability of UGT1A9 enzymes in human liver microsomes to dispose of glucuronidation of propofol

[0048] Using oxymetazoline and propofol (purchased from Sigma) as substrates, incubation reactions were carried out in 10 cases of individual human liver microsomes (BD Company, USA) with different UGT1A9 enzyme activities, incubation conditions and sample processing Analysis is the same as described in Example 1. The generation rate of the glucuronidation product of oxymetazoline / propofol in each case of human liver microsome reaction system was measured, and then statistical software (GraphPad Prism V5 software (SanDiego, CA)) was used to determine the rate of glucuronidation products of oxymetazoline and propofol, respectively. The generation rate of the glucuronidation product of pophenol is taken as the abscissa and ordinate, a...

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Abstract

The invention discloses application of oxymetazoline as a special substrate for glucuronyl transferase UGT1A9. The oxymetazoline used as the substrate is subjected to glucuronatation reaction in a biological system, and the elimination quantity of the substrate or the generation quantity of the glucuronatation product in unit time is quantitatively detected to determine the UGT1A9 enzyme activity in the biological system. The oxymetazoline can also be used for evaluating the disposition capacity of the UGT1A9 enzyme for the drugs in the human liver microsome. The oxymetazoline has high UGT metabolizing enzyme selectivity (only metabolized by UGT1A9), thereby preventing the oxymetazoline from being influenced by other metabolizing enzymes when being used as the special substrate. Besides, the glucuronatation of the oxymetazoline has the characteristics of metabolite singleness, and easy detection of the metabolites and the like.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to the application of a compound oxymetazoline as a specific substrate of glucuronosyltransferase UGT1A9. Background technique [0002] The glucuronidation reaction catalyzed by glucuronosyltransferases (UDP-glucuronosyltransferases, UGTs) is the main way of metabolism of exogenous and endogenous substances in the human body. After the glucuronidation reaction of the substrate, the polarity will generally increase, the activity will decrease, and it will be excreted from the body. Therefore, the glucuronidation reaction is an important detoxification method for the human body. The human glucuronosyltransferase family is systematically divided into four families, including UGT1, UGT2, UGT3 and UGT8. Among them, the most important UGT families related to drugs are UGT1 and UGT2. [0003] The glucuronidation reaction is catalyzed by glucuronosyltransferase to transfer the...

Claims

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Application Information

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IPC IPC(8): C12Q1/48A61K49/00
Inventor 吴宝剑吴祝峰刘红明张兴旺
Owner JINAN UNIVERSITY
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