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Application of benzbromarone as FXR agonist

A technology of benzbromarone and agonist, applied in the field of new drug use, can solve the problems of poor selectivity, limited clinical development, toxic and side effects, etc.

Inactive Publication Date: 2020-06-12
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, steroidal compounds such as CDCA and OCA have poor selectivity to nuclear receptors and are likely to cause toxic and side effects; the distyryl group in the structure of GW-4064 has potential toxicity, and the availability of this compound is low, which limits clinical development

Method used

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  • Application of benzbromarone as FXR agonist
  • Application of benzbromarone as FXR agonist
  • Application of benzbromarone as FXR agonist

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: HTRF homogeneous time-resolved fluorescence technique.

[0017] The test system in the 384-well plate contains 10nM GST-FXRαLBD, 100nM biotin-SRC1, 0.83nMEu-labeled anti-GST (Cisbio) and 41.75nM Streptavidin-XL665 (Cisbio), and the test buffer consists of 50mM Hepes pH 7.0, 125mM KF, 0.125 % CHAPS and 0.05% milk powder, adding different concentrations of the test compound, incubating at room temperature for 1 hour, using a multi-functional microplate reader to detect the fluorescence value at 620nm and 665nm, the measured value is represented by (665nm / 620nm*10000), Three replicate wells were set up for each compound, 50 μM CDCA was used as the positive control drug, DMSO was used as the blank control, and the experimental results of homogeneous time-resolved fluorescence (HTRF) were shown in figure 1 .

Embodiment 2

[0018] Example 2: Transcription activation assay.

[0019] HEK293T cells were seeded in 24-well plates, and when they grew overnight to a density of 50%-60%, the medium was replaced with serum-free DMEM medium. Firstly, the reporter gene plasmids pCDNA3.1-FXR (400ng / well), pCDNA3.1-RXRα (400ng / well), pGL3-FXRE-luc (400ng / well) and PRL-SV40 (100ng / well) were mixed with CaCl 2 (20 μL / well) and mix well, then add the mixture to BBS (20 μL / well) and mix well for 15-30 minutes, and finally 40 μL / well are co-transfected into the cells. After 6 hours, the medium was changed, and 20 μM CDCA or 2 μM benzbromarone were given for 24 hours at the same time. After 24 hours, the medium was removed, the cells were gently washed twice with PBS, and then the cells were lysed with diluted lysis buffer for 15-20 minutes, and the luciferase activity was measured with Dual Luciferase Assay System Kit (Promega). Such as figure 2 The results showed that the agonistic activity of benzbromarone on...

Embodiment 3

[0020] Example 3: qRT-PCR experiment detects the expression level of FXR-related genes.

[0021] HepG2 cells were incubated for 8 h in the presence of the test compound or control drug, the cells were collected, washed with PBS buffer, and then used for RNA extraction. RNA was extracted from HepG2 cells using a total RNA extraction kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA), and then reverse-transcribed into cDNA according to the instructions of the reverse transcription kit (Applied Biosystems, FosterCity, CA). Finally, the FXR-related genes were analyzed by real-time quantitative PCR instrument. The results show image 3 , benzbromarone can significantly increase the expression of SHP and reduce the content of CYP7A1, which is consistent with the results of the positive control drug CDCA. The primer sequences used in the quantitative PCR experiment are shown in Table 1.

[0022] Table 1. Primer sequences used in quantitative PCR experiments.

[0023]

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Abstract

The invention belongs to the technical field of new medical application, and particularly relates to an application of benzbromarone as an FXR agonist. A homogeneous time-resolved fluorescence experiment, a transcriptional activation experiment and a quantitative PCR experiment prove that the benzbromarone can effectively excite a FXR receptor, and can be applied to medicines for treating FXR receptor mediated bile blockage, gall-stone, diabetes, non-alcoholic fatty liver disease, atherosclerosis and inflammation.

Description

technical field [0001] The invention belongs to the field of new application of medicine, in particular to the application of benzbromarone as an FXR agonist. Background technique [0002] The bile acid receptor farnesoid X receptor (FXR, NR1H4) was discovered in 1995 as an orphan nuclear receptor, which is mainly expressed in the organs of liver, intestine, kidney and adrenal gland. Currently, FXR has been shown to play a regulatory role in a variety of metabolic pathways, including cholesterol, bile acids, lipids, and glucose metabolism. Typically, FXR regulates the expression of a range of target genes either as a monomer or as a heterodimer with the retinoic acid X receptor (RXR). For example: FXR on bile acid binding protein (humanintestinal bile acid binding protein, IBABP), small heterodimer partner molecule (small heterodimer partner, SHP), bile salt export pump (bile salt export pump, BSEP) and phospholipid transfer protein ( Phospholipid transfer protein, PLTP) a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/343A61P3/00A61P1/16A61P3/10A61P9/10A61P29/00
CPCA61K31/343A61P3/00A61P1/16A61P3/10A61P9/10A61P29/00
Inventor 陈卫东赵世振王艳东聂小博周云徐晓李新萍
Owner HENAN UNIVERSITY
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