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Application of sanguinarine in preparation of TRPA1 channel agonist

An agonist, sanguinarine technology, applied in the field of drug research and development, can solve the problems of irritating odor, poor water solubility, easy volatility, etc., and achieve the effect of relieving pain response

Active Publication Date: 2021-10-26
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research found that the existing classical agonists have many problems, including poor molecular selectivity, poor water solubility, volatile, pungent odor, etc.
For example, AITC is widely used as a specific agonist of TRPA1 in scientific experiments to induce acute pain and neurogenic inflammation, but recent studies have shown that TRPA1 is not the only target of AITC in vivo, and it produces The inflammatory and nociceptive pain responses may be partially mediated by TRPV1
At the same time, when using it as a positive agonist for TRPA1 inhibitor screening, its molecular instability and volatility cause a lot of inconvenience to compound screening.

Method used

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  • Application of sanguinarine in preparation of TRPA1 channel agonist
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  • Application of sanguinarine in preparation of TRPA1 channel agonist

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Sanguinarine potently stimulates TRPA1 channel current

[0023] 1. TRPA1 stable transfection cell culture

[0024] TRPA1-HEK293 stably transfected cells (Shanghai Institute of Materia Medica International Scientist Workstation)

[0025] Medium formula: DMEM basic (Gibco), adding 10% fetal bovine serum (FBS), 1% double antibody (penicillin-streptomycin), hygromycin B (Hygromycin B, 50μg / ml) and Blasticidin (BlasticidinS HCl, 5 μg / ml).

[0026] Cell culture at 37 °C, 5% CO 2 , carried out in a sterile environment with saturated humidity, using 0.25% trypsin for passage and plating. TRPA1 stably transfected cells were induced with Doxycycline hyclate (0.3 μg / ml) to express TRPA1, and manual patch clamp electrophysiological recording was performed 24 hours later.

[0027] According to 5×10 5 The cultured HEK293 cells were inoculated into 6-well plates at a density of cells / ml, and when the cell density grew to about 80%, the EGFP plasmid and the target plasmi...

Embodiment 2

[0045] Example 2 Sanguinarine Enhances Excitability of DRG Neurons and Induces Pain Model

[0046] At present, it is believed that a large influx of calcium ions (calcium load) and increased nerve excitability are two important causes of nerve damage in the body. Calcium imaging detection and manual patch clamp experiments have proved that sanguinarine has a strong agonistic activity on the heterologously expressed TRPA1 channel, suggesting that sanguinarine may increase the endogenous TRPA1 activity of neurons and enhance the excitability of neurons . The inventors isolated and cultured the dorsal root ganglion neurons (DRG) of C57 mice, and detected the effects of sanguinarine on the calcium influx of corresponding DRG neurons, as well as the effects on neuronal membrane potential and action potential firing.

[0047] Isolation and Culture of Dorsal Root Ganglion (DRG) Neurons:

[0048] (1) Reagents and instruments PBS buffer, DMEM: F12+10% FBS, Neurobasal+B27, Poly-D-lysi...

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Abstract

The invention provides application of sanguinarine in preparation of a TRPA1 channel agonist. The sanguinarine can be used as a tool molecule for constructing a pain animal model, and is applied to preclinical pharmacodynamic evaluation of analgesic drugs. The sanguinarine shows strong TRPA1 agonist activity and selectivity, and can be used as the TRPA1 channel agonist tool molecule to be applied to screening of a channel modulator taking a TRPA1 channel as a target spot; DRG neuron electrophysiological records show that the sanguinarine can enhance excitability of neurons by specifically activating TRPA1 channels on DRG neurons; and the pain reaction can be generated through metrological dependence induction by injecting sanguinarine on the sole of a C57 mouse, and the pain reaction is remarkably relieved through TRPA1 gene knockout and pharmacological blocking, so that the application of the sanguinarine in the preclinical pharmacodynamic evaluation of analgesic drugs taking TRPA1 as the target spot is realized.

Description

technical field [0001] The invention belongs to the field of drug research and development, and specifically relates to the application of sanguinarine in the preparation of TRPA1 channel agonists, wherein, sanguinarine can be used as a TRPA1 channel agonist tool molecule in the screening of channel regulators targeting TRPA1 channels . In addition, sanguinarine can be used as a tool molecule to construct animal pain models, which can be applied to the preclinical pharmacodynamic evaluation of analgesic drugs. Background technique [0002] Sanguinarine (Sanguinarine, SA) is a benzophenanthridine alkaloid mainly found in the Papaveraceae, Violaceae and Rutaceae plants, and its chemical name is 13-methyl[1.3]benzodioxolo[5,6, -c]-1,3-dioxolo[4,5-i]phenanthridinium, molecular formula C 20 h 14 NO 4 , the relative molecular mass is 332.33, and the chemical structural formula is: [0003] [0004] Clinically, sanguinarine plays an important role in the treatment of skin d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027A61K31/4741
CPCA01K67/0278A61K31/4741A01K2207/30A01K2207/15A01K2217/03A01K2227/105A01K2267/03
Inventor 高召兵池浩陈学勤方遂
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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