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Method for efficiently evaluating safety of medicine in hERG channel

An evaluation method and drug technology, applied in the medical field, can solve problems such as high requirements for technicians, expensive experimental equipment, and low efficiency of hERG channel safety evaluation, and achieve the effects of improved efficiency, faster screening, and simple methods

Inactive Publication Date: 2012-10-24
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The safety evaluation of hERG channel is a very important part of the preclinical test of new drugs. The classic detection method uses the patch clamp technique for evaluation. Self-deficiency makes the safety evaluation of hERG channel very inefficient

Method used

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  • Method for efficiently evaluating safety of medicine in hERG channel
  • Method for efficiently evaluating safety of medicine in hERG channel
  • Method for efficiently evaluating safety of medicine in hERG channel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) Cell culture: CHO cell lines stably expressing hERG channels (existing in the prior art) were inoculated in Hams F-12 medium, at 37°C, saturated air humidity, 95% air + 5% CO 2 under the condition of CO 2 Routine cultivation in an incubator.

[0033] (2) Seed plate: Take cell lines in good condition and the degree of polymerization reaches 90% and plant them in a 96-well plate at a density of 50,000 per well. 2 under the condition of CO 2 Routine cultivation in an incubator.

[0034] (3) Fixed Rb + : When the cells grow to cover about 90% of the bottom of the well, discard the culture medium in the well, add 200 μL of fixation buffer, and store at 37°C, saturated air humidity, 95% air + 5% CO 2 under the condition of CO 2 Incubate in the incubator for 2.5h.

[0035] (4) Drug treatment: Discard the immobilization buffer in each well, add the drug to be tested prepared with the immobilization buffer, and incubate for 30 min.

[0036] (5) Washing: discard the im...

Embodiment 2

[0049] (1) Cell culture: CHO cell lines stably expressing hERG channels (existing in the prior art) were inoculated in Hams F-12 medium, at 37°C, saturated air humidity, 95% air + 5% CO 2 under the condition of CO 2 Routine cultivation in an incubator.

[0050] (2) Seed plate: Take cell lines in good condition and the degree of polymerization reaches 90% and plant them in a 96-well plate at a density of 50,000 per well. 2 under the condition of CO 2 Routine cultivation in an incubator.

[0051] (3) Fixed Rb + : When the cells grow to cover about 90% of the bottom of the well, discard the culture medium in the well, add 200 μL of fixation buffer, and store at 37°C, saturated air humidity, 95% air + 5% CO 2 under the condition of CO 2 Incubate for 1 h in the incubator.

[0052] (4) Drug treatment: discard the immobilization buffer in each well, add the drug to be tested prepared with the immobilization buffer, and incubate for 40 min.

[0053] (5) Washing: discard the imm...

Embodiment 3

[0066] (1) Cell culture: CHO cell lines stably expressing hERG channels (existing in the prior art) were inoculated in Hams F-12 medium, and incubated at 37°C, saturated air humidity, 95% air + 5% CO 2 under the condition of CO 2 Routine cultivation in an incubator.

[0067] (2) Seed plate: Take cell lines in good condition and the degree of polymerization reaches 90% and plant them in a 96-well plate at a density of 50,000 per well. 2 under the condition of CO 2 Routine cultivation in an incubator.

[0068] (3) Fixed Rb + : When the cells grow to cover about 90% of the bottom of the well, discard the medium in the well, add 200 μL of fixation buffer, and store at 37°C, saturated air humidity, 95% air + 5% CO 2 under the condition of CO 2 Incubate for 4 hours in the incubator.

[0069] (4) Drug treatment: Discard the immobilization buffer in each well, add the drug to be tested prepared with the immobilization buffer, and incubate for 5 min.

[0070] (5) Washing: Discar...

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Abstract

The invention discloses a method for efficiently evaluating the safety of a medicine in an hERG channel, characterized by firstly conducting routine culture of a cell strain expressing the hERG channel, then removing the culture medium and adding the cell strain into a fixed buffer, incubating for 1-4h under the routine culture conditions of the cell strain, removing the fixed buffer, then adding a fixed buffer containing the medicine to be evaluated and incubating for 5-40min, and then removing the fixed buffer containing the medicine to be evaluated, washing the cell strain by using a washing buffer to remove residual Rb+, adding an ion channel opening buffer and incubating for 5-20 min, removing the cell supernatant, cracking the cell strain into a cell lysate, and respectively determining the Rb+ concentrations in the cell supernatant and cell lysate; setting different concentrations of the medicine to be evaluated in the fixed buffer containing the medicine to be evaluated to repeatedly determine, acquiring an IC50 value according to obtained data of the determination of the medicine to be evaluated with different concentrations, wherein if the IC50 value is no larger than 1000 mumol / L, the medicine to be evaluated has a certain inhibition on the hERG channel, and the lower the IC50 value is, the higher the inhibition is and the larger the risk of cardiotoxicity is.

Description

Technical field: [0001] The invention belongs to the medical field, and in particular relates to an evaluation method for efficiently evaluating the safety of drugs on hERG channels. Background technique: [0002] The QT interval refers to the period of Q wave and T wave in the heartbeat electrical cycle measured on the electrocardiogram, which represents the whole process of cardiomyocytes from depolarization to repolarization. Long QT syndrome (long QT syndrome, LQTs) is divided into congenital long QT syndrome (congenital long QT syndrome) and acquired long QT syndrome (acquired long QT syndrome). Congenital long QT syndrome is caused by ion channel dysfunction due to mutations in genes encoding ion channel proteins in the membranes of cardiomyocytes. Drugs are an important cause of acquired long QT syndrome. When the QT interval is significantly prolonged, it may lead to torsades de pointes (TdP), which is self-limiting, and may further develop into ventricular fibrill...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/31
Inventor 程娜李志远徐慧娟梁洞泉
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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