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A Specific Probe Substrate for Cytochrome p450 2A6 Enzyme and Its Application

A technology of probe substrate and cytochrome, which is applied to the specific probe substrate of a class of cytochrome P450 2A6 enzymes and its application field, can solve the problems of inability to establish detection methods, lack of specific probes, etc., and achieve easy The effect of detection, high sensitivity, and simple source

Active Publication Date: 2018-06-05
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the metabolic studies of CYP 2A6 are significantly less than those of CYP 3A4, and due to the lack of suitable CYP 2A6-specific probes, effective detection methods cannot be established

Method used

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  • A Specific Probe Substrate for Cytochrome p450 2A6 Enzyme and Its Application
  • A Specific Probe Substrate for Cytochrome p450 2A6 Enzyme and Its Application
  • A Specific Probe Substrate for Cytochrome p450 2A6 Enzyme and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Separation and purification of isoglycyrrhizin, licoricepyranocoumarin, isoglycyrrhizol and 7-O-methylluteone.

[0036] 1. Experimental materials and methods.

[0037] All chemical reagents mentioned in this method were purchased from Beijing Chemical Plant.

[0038] Get 35kg of licorice medicinal material (Glycyrrhiza branch of Inner Mongolia Yili Science and Technology Industrial Co., Ltd.), pulverize, extract twice with 10 times the amount of 95% ethanol and 70% ethanol, each time for 2 to 3 hours, reclaim the ethanol under reduced pressure, and the obtained The extract (10 L) was suspended in water and extracted 5 times with ethyl acetate to obtain 2500 g of the ethyl acetate fraction. Take 1280g of ethyl acetate, separate it by silica gel column chromatography (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.), and elute with petroleum ether-ethyl acetate gradient (1:0-1:1) to obtain fractions A-J . Fraction I (50g) was separated by silica gel column ch...

Embodiment 2

[0041] Example 2. The use of isolicyrrhizin coumarin in the determination of 2A6 subenzyme activity in human liver microsomes.

[0042] 1. Experimental materials and methods.

[0043] Materials: 1-aminobenzotriazole (ABT) and pilocarpine were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA) and Abcam Biochemicals (Cambridge, United Kingdom), respectively. Montelukast was purchased from Energy Chemical Ltd. (Shanghai, China). CYP3cide and clomethiazole were purchased from Toronto Research Chemical Inc. (Toronto, Ontario, Canada). Sulfafenpyrazole and furafylline were purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). 8-Methoxypsoralen, omeprazole, quinidine, ticlopidine, and ketoconazole were purchased from Sigma (St. Louis, MO, USA). Magnesium chloride and potassium phosphate were purchased from Aladdin Reagent Company. β-Nicotinamide adenine dinucleotide phosphate hydrate (β-NADP), glucose-6-phosphate (G-6-P) and glucose-6-phosphate de...

Embodiment 3

[0053] Example 3. Isoliquiritol and 7-O-Methylluteone are used for the determination of 2A6 subenzyme activity in human liver microsomes.

[0054] 1. Experimental materials and methods.

[0055] Materials: Isoglycyrrhizol and 7-O-Methylluteone were isolated from Glycyrrhiza uralensis Fisch., and the purity of the compounds reached 98% by HPLC / UV analysis. Other materials are consistent with Example 2.

[0056] Method: using the activity detection method of the probe substrate isolicyrrhizin coumarin in Example 1 after adding a selective inhibitor.

[0057] A. The concentration of selective inhibitor is IC 50 3-fold concentration of the substrate isoglycyrrhizol and 7-O-Methylluteone are both at 25 μM.

[0058] B. Add NADPH system (0.45mg / mL human liver microsomes, 2mM NADP, 8mM G-6-P, 3unit / mL G-6-P-DE, 6mM Magnesium chloride), the reaction temperature is 30-40°C, 200μL incubation system.

[0059] C. After the NADPH generating system was pre-incubated at 37°C for 30 minut...

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Abstract

The invention discloses specific probe substrates for a CYP450 (cytochrome P450) 2A6 enzyme and an application of the specific probe substrates in determination of the activity of the CYP450 2A6 enzyme. The specific method for determining the activity of the CYP450 2A6 enzyme comprises the following steps: isopentene phenolic compound monomers such as isoglycycoumarin, isoglycyrol and 7-O-methylluteone are taken as the high-specificity probe substrates for the CYP450 2A6 enzyme, CYP catalytic reactions of the specific substrates are carried out by the aid of a CYP in-vitro incubation system, the activity of the CYP 2A6 enzyme in each biological sample is determined by quantitatively detecting product production amount or substrate consumption in unit time, and evaluation of the activity of the important drug metabolic enzyme CYP 2A6 enzyme is expected to be realized.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for measuring cytochrome P450 2A6 enzyme activity by using isopentenyl phenolic compounds as specific probe substrates, including isoglycyrrhizin, isoglycyrrhizol and 7-O -Three compounds of Methylluteone and their structural analogues, which specifically undergo monohydroxylation under the action of cytochrome P450 2A6 enzymes, can be used as specific probe substrates of cytochrome P450 2A6 enzymes for human and mouse-derived organisms Quantitative evaluation of cytochrome P450 2A6 enzyme activity in samples, in order to realize the evaluation of the ability of important drug metabolizing enzyme CYP 2A6 to dispose of drugs. Background technique [0002] Cytochrome P450 (cytochromeP450 or CYP450, referred to as CYP450) is a superfamily of heme-thiolate proteins, which play an important role in prokaryotes and eukaryotes (Myasoedova N, Biochemistry.2008, 73, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D493/04C07D493/14C07D311/36C12Q1/26
CPCC07D311/36C07D493/04C07D493/14C12Q1/26
Inventor 叶敏王琦乔雪季帅匡易
Owner PEKING UNIV
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