CN112826820B

CN112826820B - NLRP3 inhibitor and application thereof

Info

Publication number: CN112826820B
Application number: CN202011372536.8A
Authority: CN
Inventors: 田力; 赵锴; 龙秀艳; 罗睿恒; 李静; 许学明
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2022-06-14
Anticipated expiration: 2040-11-30
Also published as: CN112826820A

Abstract

The invention discloses a novel application of a compound shown in a formula 1, namely the compound is used for preparing a medicament for treating Inflammatory Bowel Disease (IBD) by inhibiting NLRP3 inflammasome activation and IL-1 beta maturation and release. The research finds that the compound shown in the formula 1 can inhibit the mouse colitis induced by Dextran Sodium Sulfate (DSS), and the application mechanism of the compound is mainly to inhibit the inflammation by inhibiting the activation of NLRP3 inflammatory bodies so as to reduce the release of IL-1 beta. The inhibitory effect of the compound of formula 1 on the colon inflammation in the DSS-induced colitis model was evaluated by analyzing the weight loss, Disease Activity Index (DAI) score, colon shortening, and IL-1 β expression in colon tissue of mice in the DSS-induced colitis model.

Description

NLRP3 inhibitor and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to an NLRP3 inhibitor and application thereof.

Background

Clinically, Inflammatory Bowel Diseases (IBD) including ulcerative colitis and crohn's disease are a group of chronic recurrent non-specific Inflammatory diseases of the intestinal tract, which are better developed in young and strong years, and have the characteristics of rapid progress, great destructiveness and need of life-long treatment. IBD has limited medicine and poor curative effect, patients often have complications such as intestinal obstruction, bleeding and canceration, 70-80% of patients need surgical treatment, the disability rate is high, and the prognosis and the quality of life are very poor, so IBD is called green tumor. In recent years, with the change of living environment and dietary structure, the incidence rate of IBD in China is exponentially increased. The traditional medicaments for treating IBD at home and abroad comprise: the drugs such as aminosalicylic acid preparation, glucocorticoid, immunosuppressant and the like have the defects of poor effect, large side effect and the like, and in recent years, biological preparations and small molecular drugs are emerging continuously, but the wide clinical application of the drugs is limited due to the limitations of high cost, easy generation of drug resistance and the like. Therefore, exploring the pathogenesis of IBD, and developing new effective therapeutic drugs is a key scientific problem in the field of IBD research, and has significant economic and social values.

Disclosure of Invention

The first purpose of the invention is to provide a compound shown in formula 1 as an NLRP3(NOD-like receptor protein 3; NOD-like receptor protein 3) inhibitor and a brand-new application thereof in treating IBD based on an NLRP3 inhibition mechanism.

It is a second object of the present invention to provide a medicament for treating IBD comprising an active compound of formula 1.

The technical scheme of the invention is as follows:

use of an NLRP3 inhibitor for the manufacture of a medicament for inhibiting activation of NLRP3 inflammasome;

the NLRP3 inhibitor is a compound with a structural formula of formula 1;

formula 1.

The research of the invention finds that the compound shown in the formula 1 can be used as an NLRP3 inhibitor, can effectively inhibit the activation of NLRP3 inflammasome, and can obtain related pharmacological effects based on the inhibition of NLRP 3.

A preferred application is that the compound shown in the formula 1 is used for preparing a medicament for inhibiting ASC (apoptosis-associated spot-like protein) oligomerization and further inhibiting activation of NLRP3 inflammasome.

In a further preferred use, the compound of formula 1 is used for the manufacture of a medicament for inhibiting NLRP3 inflammasome activation and IL-1 β maturation and release.

The research of the invention finds the new application of the compound shown in the formula 1 in inhibiting the activation of the mouse abdominal cavity macrophage NLRP3 inflammasome and the maturation and release of IL-1 beta, and finds the dose dependence of the compound shown in the formula 1 in inhibiting the activation of the mouse abdominal cavity macrophage NLRP3 inflammasome.

A further preferred use is the use of a compound of formula 1 for the manufacture of a medicament for the treatment of IBD by inhibiting NLRP3 inflammasome activation and IL-1 β maturation and release.

The research of the invention finds that the compound of the formula 1 can inhibit DSS (Dextran sulfate sodium salt)) induced mouse colitis by inhibiting activation of NLRP3 inflammasome. The inhibition effect of the compound shown in the formula 1 on the colon inflammation of a DSS-induced colitis model mouse is evaluated by recording and analyzing the weight loss, DAI (disease activity index) score, colon shortening condition, IL-1 beta expression condition and the like in colon tissues of the mouse of the DSS-induced colitis model mouse.

The compound shown in the formula 1 and other active ingredients for treating IBD can be combined to prepare a compound medicine for treating IBD.

According to the application, the compound shown in the formula 1 and pharmaceutically acceptable auxiliary materials are combined to prepare a pharmaceutically acceptable preparation with any administration route.

The invention also provides a medicament for treating IBD, comprising a pharmaceutically effective amount of a compound of formula 1.

The medicament for treating IBD further comprises pharmaceutically acceptable auxiliary materials.

The medicament for treating IBD is preferably an injection preparation.

Advantageous effects

1. The invention finds a novel application of the compound shown in the formula 1 in inhibiting the activation of NLRP3 inflammasome;

2. the research of the invention finds that the compound shown in the formula 1 can treat IBD based on a completely new mechanism for inhibiting the activation of NLRP3 inflammasome, and finds that the compound has a good treatment effect.

For example, the present study found that the compound of formula 1 is capable of inhibiting DSS-induced colitis in mice by inhibiting the activation of NLRP3 inflammasome and the maturation and release of IL-1 β. In addition, the invention also evaluates the therapeutic effect of the compound shown in the formula 1 on the DSS-induced colitis of the mice through the maturation and release level of the IL-1 beta, the weight loss of the mice with DSS-induced colitis models, the DAI score, the colon shortening condition and the expression condition of the IL-1 beta and the like in colon tissues of the mice, and as a result, the compound shown in the formula 1 can realize the effective treatment of IBD based on the NLRP3 inflammasome inhibition mechanism.

Drawings

Figure 1 is a graph of the inhibition of DSS-induced colitis in mice by compounds of formula 1. Panel a shows the change in body weight of mice. Panel B shows mouse DAI scores. Panel C shows HE staining (200 x) of mouse colon tissue and histopathological scoring of colon tissue. Panel D shows changes in gross morphology and colon length of the mouse colon. The results demonstrate that compound intervention of formula 1 can significantly improve the weight loss of mice in DSS-induced colitis model (fig. 1A, p < 0.05) and reduce the DAI score of mice (fig. 1B, p < 0.05), protect the intestinal mucosal integrity and normal crypt structure of mice, reduce infiltration of mucosal lymphocytes and reduce histopathological score (fig. 1C, p < 0.05), and significantly reduce the degree of shortening of colon (fig. 1D, p < 0.05), suggesting that compound of formula 1 plays an anti-inflammatory role in colonic inflammation.

FIG. 2 is a graph showing that the compound of formula 1 inhibits the expression of IL-1 β and IL-6 in colon tissue of mice in a DSS-induced mouse colitis model. The results show that the compound shown in the formula 1 inhibits the increase of IL-1 beta and IL-6 of colon tissues of mice in a DSS-induced mouse colitis model, and the inhibition effect on IL-1 beta is more obvious, which suggests that the compound shown in the formula 1 can inhibit intestinal inflammation mainly by inhibiting the activation of NLRP3 inflammatory corpuscles in the colon tissues of the mice.

FIG. 3 shows that the compound of formula 1 inhibits the activation of mouse peritoneal macrophage NLRP3 inflammasome and the mature release of IL-1 beta. FIG. 3 shows the inhibition of the expression of mouse peritoneal macrophage NLRP3 and IL-1 beta maturation and release by the compound of formula 1. FIG. 3A shows that the release of mouse peritoneal macrophage IL-1 beta is increased after LPS + Nigericin (lipopolysaccharide + Nigericin), the increase of IL-1 beta is inhibited after the compound of formula 1 is added, and the content of IL-1 beta is gradually reduced along with the increase of the dose of the compound of formula 1, which indicates that the compound of formula 1 has dose-dependent inhibition effect on the activation of mouse peritoneal macrophage NLRP3 inflammasome; FIG. 3B shows that the expression of NLRP3 and pro-IL-1 beta of mouse abdominal macrophages is increased, the released caspae-1 spliceosome (p10) and pro-IL-1 beta spliceosome (p17) are increased after ATP, Nigericin and MSU (Monosodium urate) are added, and the expression of NLRP3 and pro-IL-1 beta is not obviously changed and p10 and p17 are inhibited after the compound of formula 1 is added, namely the compound of formula 1 has no obvious influence on the expression of NLRP3 protein, caspase-1 precursor (pro-caspase-1), pro-IL-1 beta and ASC, but obviously inhibits p10 and p17 in cell supernatant, and the compound of formula 1 inhibits the inflammatory activation of NLRP3 but does not influence the initiation stage of the inflammatory process.

FIG. 4 shows that the compound of formula 1 inhibits ASC oligomerization of macrophages in the abdominal cavity of mice after LPS + Nigericin treatment. The results show that: after LPS + Nigericin treatment, ASC oligomers formed in primary macrophages in the mouse peritoneal cavity, and were reduced after addition of the compound of formula 1, suggesting that the compound of formula 1 inhibits ASC oligomerization during NLRP3 inflammasome activation.

Detailed Description

The cells used in the invention are C57 mouse peritoneal macrophages. The animal model used was a DSS-induced enteritis model in mice, the mouse model being C57.

Example 1 the compound of formula 1 is used to treat DSS-induced colitis in mice.

The experimental method is as follows: a DSS-induced enteritis model is constructed by using 6-8 week-old C57 mice, namely, fresh 3% DSS solution is fed to the mice, and the compound shown in the formula 1 is prepared into solution by dimethyl sulfoxide (DMSO) and normal saline and is administrated by intraperitoneal injection. General conditions, body weight and hematochezia of mice in the healthy control group, the DSS model group and the compound-treated group of formula 1 were observed, DAI scoring was performed, the mice were sacrificed after 9 days, colon specimens were collected, colon lengths were compared, and intestinal histopathological examination was performed.

The results show that the compound of formula 1 intervention can significantly improve the weight loss degree of mice model with DSS-induced colitis (FIG. 1A, p < 0.05) and reduce DAI score of mice (FIG. 1B, p < 0.05), reduce histopathological score, protect intestinal mucosa integrity and normal crypt structure of mice, reduce mucosa lymphocyte infiltration (FIG. 1C) and significantly reduce the shortening degree of colon (FIG. 1D, p < 0.05), suggesting that the compound of formula 1 plays an inflammation inhibiting role in colon inflammation (the results are shown in FIG. 1).

Example 2 the compound of formula 1 ameliorates DSS-induced colitis in mice by inhibiting NLRP3 inflammasome activation and maturation and release of IL-1 β.

The experimental method is as follows: the colon tissue of example 1 was harvested, the distal part of the colon was removed and the 1cm section of the intestine was incubated for 8h after washing, and the supernatant was assayed for IL-1. beta. and IL-6 expression by ELISA.

The results show that the compound shown in the formula 1 inhibits the increase of IL-1 beta and IL-6 of colon tissues in a mouse model of DSS-induced colitis, and the inhibition effect on IL-1 beta is more obvious (the results are shown in figure 2).

Example 3 the compound of formula 1 is used to inhibit NLRP3 inflammasome activation and maturation and release of IL-1 β.

The experimental method is as follows: firstly, separating, extracting and culturing C57 mouse abdominal cavity macrophages, stimulating for 3 hours by 100ng/mL LPS, adding different doses of a compound shown as formula 1, treating for half an hour, then adding 10mM Nigericin, stimulating for 1 hour, collecting cell supernatant, and detecting the content of IL-1 beta in the cell supernatant by ELISA; then C57 mouse peritoneal macrophages are extracted and cultured, 100ng/mL LPS stimulates for 3 hours, then the compound shown in the formula 1 is added for processing for half an hour, then 10mM Nigericin stimulates for 1 hour, cell lysate and cell supernatant are collected, and Western blot is used for detecting the expression of NLRP3 and other proteins and the release of p10 and p 17.

The results show that after LPS + Nigericin treatment, the release of mouse peritoneal macrophage IL-1 beta is increased, after the compound of formula 1 is added, the increase of IL-1 beta is inhibited, and the content of IL-1 beta is gradually reduced along with the increase of the dose of the compound of formula 1, which indicates that the compound of formula 1 has dose dependence on the inhibition effect of mouse peritoneal macrophage NLRP3 inflammatory corpuscle activation (the results are shown in a figure 3A); after ATP, Nigericin and MSU are added, the expression of NLRP3 and pro-IL-1 beta of mouse abdominal cavity macrophages is increased, the released caspase-1 spliceosome (p10) and the pro-IL-1 beta spliceosome (p17) are increased, and after the compound of the formula 1 is added, NLRP3 and pro-IL-1 beta are not obviously changed, and p10 and p17 are inhibited, namely, the compound of the formula 1 has no obvious influence on the expression of NLRP3 protein, caspase-1 precursor (pro-caspase-1), pro-IL-1 beta and ASC of mouse abdominal cavity macrophages, but obviously inhibits p10 and p17 in cell supernatant, and the compound of the formula 1 inhibits the activation of NLRP3 inflamed mice but does not influence the synthesis stage (the result is shown in figure 3B).

Example 4 the compound of formula 1 inhibits ASC oligomerization of primary macrophages in the abdominal cavity of mice following LPS + Nigericin stimulation.

The experimental method is as follows: c57 mouse peritoneal macrophages are cultured, 100ng/mL LPS is stimulated for 3 hours, then the compound shown in the formula 1 is added for treatment for half an hour, 10mM Nigericin is added for stimulation for 1 hour, cell protein is collected, and after the treatment with a cross-linking agent DSS, precipitate is collected, and the formation of ASC polymer is detected by Western Blot.

The results show that after LPS + Nigericin treatment, ASC oligomers formed in primary macrophages in the mouse peritoneal cavity and decreased after addition of the compound of formula 1, suggesting that the compound of formula 1 inhibits ASC oligomerization during NLRP3 inflammasome activation (results are shown in figure 4).

Claims

Use of an inhibitor of NLRP3 in the manufacture of a medicament for inhibiting activation of NLRP3 inflammasome, characterized in that it is used in the manufacture of a medicament for treating inflammatory bowel disease by inhibiting activation of NLRP3 inflammasome and maturation and release of IL-1 β;

the NLRP3 inhibitor is a compound with a structural formula of formula 1;

formula 1.
2. The use according to claim 1, for the preparation of a combination medicament for the treatment of inflammatory bowel disease, in combination with other active ingredients for the treatment of inflammatory bowel disease.
3. The use according to claim 1, wherein it is used in combination with pharmaceutically acceptable excipients to make a pharmaceutically acceptable formulation for any route of administration.