Receptor antagonist long-term effectiveness analysis method

A receptor antagonist and analysis method technology, applied in the field of long-term analysis of receptor antagonists, can solve the problems of low screening throughput and long test period, achieve high screening throughput, reliable detection results, and improve test efficiency Effect

Pending Publication Date: 2021-04-30
TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The existing methods for evaluating the long-acting effect of receptor M3 antagonists have the following problems: at present, they are mainly confirmed by competitive binding tests of radioligands, but this method has certain limitations, such as the need for washing and filtration, and the test cycle Shortcomings such as length and low screening throughput

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  • Receptor antagonist long-term effectiveness analysis method
  • Receptor antagonist long-term effectiveness analysis method
  • Receptor antagonist long-term effectiveness analysis method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] First, the first step is to obtain the agonist DMR characteristic signal spectrum of the agonist carbachol on HEK-293T-M3 cells:

[0059] Materials Carbachol and tiotropium bromide were purchased from Sigma; refenacin and glycopyrrolate were purchased from Abmole; ipratropium bromide was purchased from Coolaber; human embryonic kidney cells HEK-293T-M3 cells were derived from University of California, Irvine. The detection platform is the third generation of Corning The signal detected by the imager is the wavelength shift caused by the dynamic mass reset (DMR) of the cell and the change value of the resonance wavelength caused by the drug acting on the cell.

[0060] HEK-293T-M3 cells in the logarithmic growth phase were inoculated in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells is 2.0 x 10 4 Firstly, place the inoculated cell p...

Embodiment 2

[0066] Based on the antagonistic DMR characteristic signal spectrum of the antagonist refenacin and ipratropium bromide obtained in Example 1 on HEK-293T-M3 cells, the calculated IC50 values ​​are 101.39±30.24nM and 10.81±2.09nM respectively .

[0067] Then the long-term analysis of the four M3 receptor antagonists:

[0068]HEK-293T-M3 cells in the logarithmic growth phase were inoculated in a cell-compatible 384 microwell plate, the medium used was DMEM (#SH30022.01B, Thermo), the inoculation volume of each well was 40 μL, and each well was inoculated The number of cells was 2.0×104, and the inoculated cell plate was placed in a cell incubator and cultured for 20-22 hours until the cell confluence reached about 95%, and the activity experiment was carried out. Replace the cell culture solution in the microwell plate with Hank's balanced salt solution (containing 10mM HEPES), and add a volume of 30 μL to each well. After adding, place in Equilibrate on the imager for 1 hour...

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Abstract

The invention discloses a receptor antagonist long-term effect analysis method, and belongs to the field of receptor antagonist long-term effect analysis methods. Previously, receptor antagonist long-term effectiveness evaluation is mainly confirmed by competitive binding tests of radioactive ligands, but the method has certain limitations, such as washing and filtering, long experimental period and low flux. The method is based on a label-free cell integration pharmacology technology, does not need radioactive labeling, reduces the test difficulty, effectively shortens the test period, and improves the test efficiency. The compounds tiotropium bromide and rafenacin evaluated by the method are both M3 receptor long-acting antagonists, glycopyrronium bromide is an M3 receptor medium-long-acting antagonist, isopropylammonium bromide is an M3 receptor short-acting antagonist, and the result is consistent with the conclusion of a radiolabeling method. The novel method for analyzing the long-acting property of the receptor antagonist has the advantages of reliable detection result, high sensitivity, high screening flux, simplicity and convenience in operation and the like.

Description

technical field [0001] The invention relates to the technical field of long-acting analysis methods, in particular to the field of long-acting analysis methods of receptor antagonists. Background technique [0002] G protein-coupled receptor (GPCR) is the most important class of membrane receptors in cell signal transduction, and it is also one of the most concerned drug targets in the development of small molecule drugs, with about 34% of Modern drugs directly target this family of receptors. There are currently five subtypes of M receptors identified, namely M1-M5, among which M1, M2 and M3 receptors exist in human lungs and are closely related to the occurrence and development of COPD. Among them, M3 is mainly expressed in airway smooth muscle cells, submucosal glands, and pulmonary blood vessels. Although the amount of M3 in the airway smooth muscle of the lung is very small, M3 can combine with M1 to activate phospholipase C mainly through the Gq protein to act on the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50
CPCG01N33/5008G01N33/5044
Inventor 梁鑫淼单彩龙王纪霞王志伟于广璞薛珍珍
Owner TAIZHOU GUOKEHUAWU BIOMEDICAL TECH CO LTD
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