Application of SB-222200 in inhibiting activation of NLRP3 inflammasome

Abstract

The present invention discloses the application of SB-222200 in inhibiting the activation of NLRP3 inflammasome. This SB-222200 can specifically inhibit the activation of NLRP3 inflammasome, and the inhibition of NLRP3 inflammasome activation does not depend on the antagonism of NK3 receptor. SB-222200 inhibits the assembly of NLRP3 inflammasome by directly binding to NLRP3 protein, and also inhibits the oligomerization and speck formation of ASC protein during the activation of NLRP3 inflammasome, inhibits the cleavage of GSDMD and the pyroptosis caused by it, inhibits the oligomerization of NLRP3 protein itself and the interaction between NLRP3 protein and ASC protein; meanwhile, this SB-222200 can also alleviate peritonitis and inflammatory bowel disease. It can be used as a potential drug for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasome.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of SB-222200 in inhibiting the activation of NLRP3 inflammasome. Background Art

[0002] The NOD-like receptor protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its abnormal activation is pathogenic in genetic diseases such as cryopyrin-associated periodic syndromes (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease, and atherosclerosis. The NLRP3 inflammasome is a pattern recognition receptor (PRR) and an inflammatory activation platform for caspase-1. It can recognize exogenous pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), viral RNA, or endogenous damage-associated molecular patterns (DAMPs) such as DNA, endotoxin, uric acid, ATP, Aβ, and cell debris. These substances can activate the NLRP3 inflammasome intracellularly, release IL-1β and IL-18, and induce pyroptosis. Over-activated NLRP3 inflammasome can lead to the occurrence and development of various diseases, such as gout, type 2 diabetes, and neurodegenerative diseases. Although many inhibitors have a certain alleviating effect on NLRP3 inflammasome-related diseases in basic research, currently in clinical practice, there is still a lack of drugs that specifically inhibit the activation of NLRP3 inflammasome, especially small molecule drugs that can directly bind to the NLRP3 protein to inhibit the activation of NLRP3 inflammasome. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of SB-222200 in inhibiting the activation of NLRP3 inflammasome.

[0004] The above purpose is achieved by the following technical solutions.

[0005] In the first aspect of the present invention, there is provided the application of SB-222200 in the preparation of a drug for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasome.

[0006] In some of these embodiments, the diseases associated with abnormal activation of the NLRP3 inflammasome are myocarditis, Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, atherosclerosis, pneumonia, nephritis, hepatitis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), asthma, bronchitis, psoriasis, diabetes, arthritis, infection, cryopyrin-associated periodic syndrome (CAPS), Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), neonatal-onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA), hyperimmunoglobulinemia D and periodic fever syndrome (HIDS), tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), systemic juvenile idiopathic arthritis, adult-onset Still's disease (AOSD), relapsing polychondritis, Schnitzler's syndrome, Sweet's syndrome, Behcet's disease, anti-synthetase syndrome, interleukin-1 receptor antagonist deficiency (DIRA), A20 haploinsufficiency (HA20).

[0007] In some of these embodiments, the diseases associated with abnormal activation of the NLRP3 inflammasome are silicosis, asbestosis, silicosis, Parkinson's disease, depression, type II diabetes, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, amyotrophic lateral sclerosis, acute respiratory distress syndrome.

[0008] In some of these embodiments, the diseases associated with abnormal activation of the NLRP3 inflammasome are acute diseases associated with abnormal activation of the NLRP3 inflammasome or chronic diseases associated with abnormal activation of the NLRP3 inflammasome.

[0009] In some of these embodiments, the diseases associated with abnormal activation of the NLRP3 inflammasome are inflammatory bowel disease.

[0010] In some of these embodiments, the inflammatory bowel disease is ulcerative colitis.

[0011] In some of these embodiments, the diseases associated with abnormal activation of the NLRP3 inflammasome are pelvic inflammatory disease.

[0012] In some of these embodiments, the pelvic inflammatory disease is acute pelvic inflammatory disease.

[0013] In some of these embodiments, the pelvic inflammatory disease is peritonitis.

[0014] In some of these embodiments, the peritonitis is acute peritonitis.

[0015] In some of these embodiments, the ulcerative colitis is ulcerative colitis induced by sodium dextran sulfate.

[0016] In some of these embodiments, the peritonitis is urate crystal-induced acute peritonitis.

[0017] In some of these embodiments, the abnormal activation of the NLRP3 inflammasome is classical NLRP3 inflammasome activation, and the classical NLRP3 inflammasome activation is NLRP3 inflammasome activation induced by an activator.

[0018] In some of these embodiments, the activator is at least one of nigericin, ATP, urate crystals, silica, imiquimod, and sodium dextran sulfate.

[0019] In some of these embodiments, the abnormal activation of the NLRP3 inflammasome is non-classical NLRP3 inflammasome activation, and the non-classical NLRP3 inflammasome activation is the activation of the NLRP3 inflammasome induced by intracellular lipopolysaccharide.

[0020] In some of these embodiments, the drug comprises SB-222200 and a pharmaceutically acceptable excipient.

[0021] In some of these embodiments, the dosage form of the drug is a capsule, granule, injection, pill, syrup, powder, ointment, emulsion, solution, suspension, or tincture.

[0022] In some of these embodiments, the dosage form of the drug is an oral solution.

[0023] In some of these embodiments, the administration mode of the drug is oral administration.

[0024] In some of these embodiments, the drug is used for mammals or humans.

[0025] In some of these embodiments, the excipient comprises at least one of a filler, a bulking agent, a binder, a humectant, a disintegrant, a solubilizing agent, an absorption accelerator, an adsorbent, a diluent, a solubilizer, an emulsifier, a lubricant, a wetting agent, a suspending agent, a flavoring agent, or a perfume.

[0026] Specifically, the excipient can be selected from at least one of the following components:

[0027] (a) A filler or a bulking agent, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid;

[0028] (b) A binder, for example, hydroxyethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic;

[0029] (c) A humectant, for example, glycerol;

[0030] (d) Disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0031] (e) Sustained release agents, for example paraffin wax;

[0032] (f) Absorption accelerators, for example, quaternary ammonium compounds;

[0033] (g) Wetting agents, for example cetyl alcohol and glycerol monostearate;

[0034] (h) Adsorbents, for example, kaolin;

[0035] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

[0036] In addition to the active ingredient, liquid dosage forms may contain inert diluents, such as water or other solvents, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and fragrances.

[0037] In addition to the active ingredient, suspensions may contain suspending agents, for example, ethoxylated isooctadecanol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum monostearate and agar or mixtures of these substances.

[0038] Compositions for parenteral injection may contain physiologically acceptable sterile aqueous or non - aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non - aqueous carriers, diluents, solvents or excipients may be included, such as water, ethanol, polyols and suitable mixtures thereof.

[0039] In a second aspect of the present invention, there is provided a non - therapeutic use of SB - 222200 in inhibiting NLRP3 inflammasome activation in vitro.

[0040] In the present invention, it is first discovered that the NK3 receptor antagonist SB-222200 can inhibit the activation of NLRP3 inflammasome, but does not affect the activation of NLRC4 inflammasome induced by Salmonella flagellin and the activation of AIM2 inflammasome induced by intracellular Poly(dA:dT). Moreover, the inhibition of NLRP3 inflammasome activation by SB-222200 is independent of the antagonistic effect on NK3 receptor. In addition, the study also finds that SB-222200 can directly bind to NLRP3 protein, thereby interfering with the interaction between NLRP3 protein and NEK7 protein, inhibiting the assembly of NLRP3 inflammasome, also inhibiting the oligomerization and speckle formation of ASC protein during the activation of NLRP3 inflammasome, inhibiting the cleavage of GSDMD and the pyroptosis caused by it, inhibiting the oligomerization of NLRP3 protein itself and the interaction between NLRP3 protein and ASC protein. At the same time, through modeling, it is found that SB-222200 can also relieve peritonitis and inflammatory bowel disease. It can be seen that SB-222200 can be used as a potential drug for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasome. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a result diagram of the inhibitory effect of SB-222200 on the activation of NLRP3 inflammasome. Among them, Figure 1 A is the effect diagram of the secretion of IL-1β when Nigericin induces the activation of NLRP3 inflammasome, and SB-222200 inhibits the secretion of IL-1β; Figure 1 B is the cleavage of IL-1β precursor and the autoproteolysis of caspase-1 when Nigericin induces NLRP3 inflammasome, and SB-222200 inhibits the generation of mature IL-1β and caspase-1; Figure 1 C is that SB-222200 inhibits the secretion of IL-1β after the activation of classical NLRP3 inflammasome induced by ATP, Nigericin, IMQ (imiquimod), MSU (Monosodium urate), or SiO 2 (silicon dioxide) and the activation of non-classical NLRP3 inflammasome induced by intracellular transfection of LPS; Figure 1 D is that another NK3 receptor antagonist Talnetant does not affect the secretion of IL-1β when Nigericin induces the activation of NLRP3 inflammasome.

[0042] Figure 2 It is a result diagram of the effect of SB-222200 on the IL-1β secretion dependent on the activation of NLRC4 and AIM2 inflammasomes. Among them, Figure 2A inhibits Salmonella flagellin (FLA-ST)-induced NLRC4 inflammasome-dependent IL-1β secretion; Figure 2 B inhibits poly(dA:dT)-induced AIM2 inflammasome activation-dependent IL-1β secretion.

[0043] Figure 3 It is the result graph of SB-222200 inhibiting the cleavage of GSDMD mediated by NLRP3 inflammasome activation and the pyroptosis caused by it; among them, Figure 3 A is that SB-222200 inhibits the cleavage of the pyroptosis execution protein GSDMD protein during the activation of NLRP3 inflammasome induced by nigericin; Figure 3 B is that SB-222200 inhibits the release of LDH during pyroptosis.

[0044] Figure 4 It is the result graph of the inhibitory effect of SB-222200 on the oligomerization and speck formation of ASC protein during the activation of NLRP3 inflammasome; among them, Figure 4 A is that SB-222200 inhibits the oligomerization of ASC protein during the activation of NLRP3 inflammasome; Figure 4 B is that SB-222200 inhibits the formation of intracellular ASC specks during the activation of NLRP3 inflammasome; Figure 4 C is Figure 4 The quantitative statistical graph of B, which is the proportion of cells containing ASC specks.

[0045] Figure 5 It is the result graph of SB-222200 inhibiting the interaction between NLRP3 protein and ASC protein.

[0046] Figure 6 It is the result graph of SB-222200 inhibiting the auto-oligomerization of NLRP3 protein caused by nigericin, where tranilast is the positive control.

[0047] Figure 7 It is the result graph of SB-222200 inhibiting the interaction between NLRP3 protein and NEK7 protein.

[0048] Figure 8 It is the result graph of SB-222200 directly binding to NLRP3 protein; among them, Figure 8 A is that SB-222200 can prevent the enzymatic digestion of intracellular NLRP3 protein by pronase, and MCC950 is the positive control; Figure 8 B is that SB-222200 can prevent the thermal degradation of NLRP3 protein; Figure 8C is for detecting the binding ability of SB-222200 to recombinant human NLRP3 protein using the SPR method, and the binding constant is 351.8 nM.

[0049] Figure 9 It is the result graph of the effects of SB-222200 on IL-1β and IL-6 in peritoneal fluid, liver and spleen after urate crystal-induced peritonitis in mice; among them Figure 9 A, Figure 9 B is the effect of SB-222200 on IL-1β and IL-6 in peritoneal fluid; Figure 9 C, Figure 9 D is the effect of SB-222200 on IL-1β and IL-6 in the liver; Figure 9 E, Figure 9 F is the effect of SB-222200 on IL-1β and IL-6 in the spleen.

[0050] Figure 10 It is the result graph of the effect of SB-222200 on dextran sulfate sodium (DSS)-induced inflammatory bowel disease; among them Figure 10 A and Figure 10 B show the changes in the gross morphology of the mouse colon and the change in colon length; Figure 10 C is the change in daily bloody stools of mice; Figure 10 D and Figure 10 E show the HE staining (200×) of mouse colon tissue and the histopathological score of colon tissue.

[0051] Figure 11 It is the result graph of the effect of SB-222200 on inflammation in colon tissue in DSS-induced inflammatory bowel disease; among them Figure 11 A and Figure 11 B are that SB-222200 inhibits the elevation of IL-1β and IL-6 in mouse colon tissue in the DSS-induced mouse colitis model; Figure 11 C is that SB-222200 reduces the cleavage of caspase-1 and GSDMD in colon tissue. Detailed implementation manners

[0052] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0053] For the experimental methods without specific conditions noted in the following examples, they are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. All kinds of common chemical reagents used in the examples are commercially available products.

[0054] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by one of ordinary skill in the technical field to which the present invention pertains. The terms used in the description of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0055] One embodiment of the present invention provides an application of an NK3 receptor antagonist SB-222200 in the preparation of a drug for preventing and / or treating diseases related to abnormal activation of NLRP3 inflammasome.

[0056] Wherein, the structural formula of SB-222200 is shown as follows:

[0057]

[0058] Its molecular formula is C 26 H 24 N 2 O, with a molecular weight of 380.48 and a CAS number of 174635-69-9. SB-222200 is an effective, selective, orally active NK3 receptor antagonist with blood-brain barrier (BBB) permeability.

[0059] In the present invention, it is first discovered through modeling that the NK3 receptor antagonist SB-222200 can inhibit the activation of the classical NLRP3 inflammasome induced by activators such as nigericin, ATP, urate crystals, silica, imiquimod, etc., and can also inhibit the activation of the non-classical NLRP3 inflammasome caused by intracellular lipopolysaccharide, but does not affect the activation of the NLRC4 inflammasome induced by Salmonella flagellin and the activation of the AIM2 inflammasome caused by intracellular Poly(dA:dT), and the inhibition of NLRP3 inflammasome activation by SB-222200 is independent of the antagonistic effect on the NK3 receptor.

[0060] In addition, the study also found that SB-222200 can directly bind to the NLRP3 protein, thereby interfering with the interaction between the NLRP3 protein and the NEK7 protein, inhibiting the assembly of the NLRP3 inflammasome, inhibiting the oligomerization and speckle formation of the ASC protein during the activation of the NLRP3 inflammasome, inhibiting the cleavage of GSDMD and the resulting pyroptosis, and inhibiting the oligomerization of the NLRP3 protein itself and the interaction between the NLRP3 protein and the ASC protein.

[0061] It is also discovered through modeling that SB-222200 can alleviate peritonitis induced by urate crystals and inflammatory bowel disease induced by sodium dextran sulfate.

[0062] It can be seen that the NK3 receptor antagonist SB-222200 can be used as a potential drug for preventing and / or treating diseases related to abnormal activation of the NLRP3 inflammasome.

[0063] The following are specific examples.

[0064] Example 1 Inhibitory effect of SB-222200 on NLRP3 inflammasome activation

[0065] (1) The cells used in this example were mouse mononuclear macrophages J774A.1 (Guangzhou Geneo Biotechnology Co., Ltd.). On the first day, J774A.1 cells were seeded into 96-well plates, with 5×10 4 cells per well. After seeding overnight, the supernatant was discarded, and each well was treated with 100 μL of DMEM medium containing 10% serum with bacterial lipopolysaccharide (LPS, 1 μg / mL; Sigma-Aldrich, L2630) for 5 hours. Then, different concentrations of SB-222200 (0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM) (MCE, HY-15722) or another NK3 receptor antagonist Talnetant (0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM) (MCE, HY-14552) were added and treated for 1 hour. Then, nigericin (10 μM; InvivoGen, tlrl-nig-5) or ATP (5 mM; Sigma-Aldrich, 74804-12-9) was added and treated for 1 hour. The cell supernatant was collected, and the content of IL-1β was measured using a mouse IL-1β ELISA kit (ThermoFishe, 88-7013-77). The results are shown in Figure 1 A, Figure 1 B and Figure 1 D. Western Blotting was used to detect the precursor of IL-1β (pro-IL-1β) and the cleaved form of IL-1β (p17), the precursor of caspase-1 (pro-caspasse-1) and the cleaved form of caspase-1 (p20). Proteins in the supernatant and lysate were extracted by conventional methods, and Western Blotting analysis was performed using an anti-IL-1β antibody (R&D, 88-7013-77) and an anti-Caspase-1 antibody (AdipoGen, #AG-20B-0042). The results are as shown in Figure 1 B.

[0066] (2) The cells used in this example were bone marrow-derived macrophages (BMDMs). Culture and differentiation of BMDMs: Bone marrow cells were isolated from male C57BL / 6 mice at 7 - 8 weeks old (Guangdong Laboratory Animal Center), and differentiated for 5 - 6 days using RPMI 1640 medium [containing 10% (v / v) fetal bovine serum (EcoCell Biotechnology Co., Ltd.), macrophage colony-stimulating factor M-CSF (R&D Company, 416-ML-050), and glutamine] to obtain mouse bone marrow-derived macrophages. The BMDMs cells were seeded onto 96-well plates, with 1×10 4 cells per well. The plates were seeded overnight, the supernatant was discarded, and each well was treated with 100 μL of DMEM medium containing 10% serum with bacterial lipopolysaccharide (LPS, 100 ng / mL; Sigma-Aldrich Company, L2630) for 3 hours, then treated with 10 μM of SB-222200 for 1 hour, followed by addition of nigericin (10 μM, 0.5 hour), ATP (5 mM, 0.5 hour; Sigma-Aldrich Company, 74804-12-9), imiquimod (IMQ, 900 μM, 2 hours; TargetMol Company, T0134), urate crystals (MSU, 200 μg / mL, 8 hours; InvivoGen Company, tlrl-msu), SiO 2 (50 μg / mL, 8 hours, InvivoGen Company, tlrl-sio-2). The non-classical NLRP3 inflammasome activation protocol was as follows: Pam3CSK4 (1 μg / mL; InvivoGen Company, tlrl-pms) was added to each well and treated for 5 hours, then treated with 10 μM of SB-222200 for 1 hour, and then LPS (2 μg / mL, 16 hours) was transfected using Lipofectamine 3000 transfection reagent. The content of IL-1β was measured using a mouse IL-1β ELISA kit, and the results are shown in Figure 1 C.

[0067] From Figure 1 the results, it can be seen that SB-222200 can dose-dependently inhibit the release of IL-1β after NLRP3 inflammasome activation induced by nigericin or other activators in mouse macrophages, inhibit the autoproteolysis of caspase-1 and the cleavage of pro-IL-1β after NLRP3 inflammasome activation induced by nigericin, and the inhibition of NLRP3 inflammasome activation by SB-222200 is not dependent on the antagonism of the NK-3 receptor.

[0068] Example 2 Effect of SB-222200 on IL-1β secretion dependent on NLRC4 and AIM2 inflammasome activation

[0069] (1) The cells used in this example were bone marrow-derived macrophages (BMDMs). Culturing and differentiating mouse bone marrow-derived macrophages (BMDMs): same as step (2) of Example 1.

[0070] (2) The differentiated cells (i.e., mouse bone marrow-derived macrophages) were seeded into a 96-well plate at a cell density of 5×10 4 (cells) per well. For activating the NLRC4 inflammasome in BMDMs cells, the cells were first stimulated with 100 ng / mL of LPS for 3 hours, then treated with different concentrations of SB-222200 for 14 hours, and then incubated with 2.5 μg / mL of Salmonella flagellin (FLA-ST; InvivoGen, tlrl-epstfla-5) for 4 hours. For activating the AIM2 inflammasome in BMDMs cells, the cells were first stimulated with 100 ng / mL of LPS for 3 hours, then treated with different concentrations of SB-222200 for 1 hour, and then transfected with Poly dA:dT (0.25 μg / mL; InvivoGen, 86828-69-5) using Lipo 3000 to stimulate the cells for 14 hours. The cell culture supernatants after the above stimulations were collected, and the content of IL-1β was measured using a mouse IL-1β ELISA kit. The results are shown in Figure 2 A and Figure 2 B.

[0071] It can be seen from Figure 2 the results that SB-222200 does not affect the LPS-induced IL-1β secretion dependent on the activation of the NLRC4 inflammasome by FLA-ST, nor does it affect the Poly dA:dT-induced IL-1β secretion dependent on the activation of the AIM2 inflammasome.

[0072] Example 3 SB-222200 inhibits the cleavage of GSDMD mediated by NLRP3 inflammasome activation and the resulting pyroptosis

[0073] (1) The cells used in this example were mouse mononuclear macrophages J774A.1. On the first day, J774A.1 cells were seeded into a 6-well plate. After seeding overnight, the supernatant was discarded, and 100 μL of DMEM medium containing 10% serum and bacterial lipopolysaccharide (LPS) (1 μg / mL) was added to each well. Then, different concentrations of SB-222200 were added and treated for 1 hour, and then nigericin (10 μM) was added and treated for 1 hour. The supernatant or cell lysate was collected.

[0074] (2) Detection of GSDMD and cleaved GSDMD by Western blot. Proteins were extracted from the lysate by conventional methods and Western blot analysis was performed using an anti-GSDMD antibody (Abcam, ab209845). The results are shown in Figure 3 Figure A.

[0075] (3) The supernatant was collected and detected using a lactate dehydrogenase (LDH) cytotoxicity detection kit (Beyotime, C0016) to evaluate cell death. The results are shown in Figure 3 Figure B.

[0076] From Figure 3 the results, SB-222200 can inhibit the cleavage of GSDMD mediated by NLRP3 inflammasome activation and the pyroptosis it induces.

[0077] Example 4 Inhibitory effect of SB-222200 on the oligomerization and speck formation of ASC protein during NLRP3 inflammasome activation

[0078] (1) Culture and differentiation of mouse bone marrow-derived macrophages (BMDMs): The same as step (2) of Example 1.

[0079] (2) The differentiated cells were seeded into 6-well plates at a cell density of 1×10 6 . After overnight culture, the medium was replaced with opti-MEM and pretreated with 100 ng / mL LPS for 3 hours. Then, different concentrations of SB-222200 (0 μM, 2 μM, 5 μM) were added and treated for 1 hour, followed by the addition of 5 μM nigericin and stimulation for 1 hour. The oligomerization of ASC protein was detected as follows: The medium was aspirated, and the cells were lysed with IP lysis buffer (Beyotime, P0013). After half an hour, the lysate was collected, centrifuged at 12,000 rpm for 10 minutes at 4°C, washed three times with PBS, and resuspended in 500 μL of cold PBS. Then, 4 mM disuccinimidyl suberate was added as a crosslinking agent (Sangon Biotech, C100015-0100) for crosslinking, and incubated at room temperature for 30 minutes. Centrifuged at 5000 g for 10 minutes at 4°C, the supernatant was removed, and the precipitate was collected. Finally, Western Blotting was performed using an anti-ASC antibody (Cell Signaling Technology, 67824S). The results are shown in Figure 4 Figure A. It can be seen from Figure 4 the results of Figure A that nigericin stimulation can significantly promote the oligomerization of ASC protein, and SB-222200 treatment can inhibit this process in a dose-dependent manner, indicating that SB-222200 can inhibit the oligomerization of ASC protein during NLRP3 inflammasome activation induced by nigericin.

[0080] (3) Seed the differentiated cells into a 24-well plate containing coverslips at a cell density of 5×10 5 . After culturing overnight, replace the medium with opti-MEM and pretreat with 100 ng / mL LPS for 3 hours. Then, add SB-222200 at different concentrations (0 μM, 5 μM, 10 μM) and incubate for 1 hour, followed by adding 10 μM Nigericin and stimulating for 30 min. Then, detect the ASC speck formation and perform speck statistical analysis by immunofluorescence. The results are shown in Figure 4 Figures B and 4C. As can be seen from the results of Figure 4 Figures B and 4C: Nigericin stimulation can induce the formation of ASC specks, and SB-222200 treatment can inhibit this process, indicating that SB-222200 can inhibit the ASC speck formation during the activation of the NLRP3 inflammasome induced by Nigericin.

[0081] Example 5 SB-222200 inhibits the interaction between NLRP3 protein and ASC protein

[0082] The cells used in this example are mouse mononuclear macrophages J774A.1. Seed the cells into a 10-cm culture dish at a cell density of 6×10 6 . After culturing overnight, replace the medium with opti-MEM and pretreat with 1 μg / mL LPS for 5 hours. Then, add 5 μM SB-222200 and incubate for 1 hour, followed by adding 10 μM Nigericin and stimulating for 1 hour. Through immunoprecipitation experiments, pull down the ASC protein and detect by Western Blotting with anti-ASC antibody and anti-NLRP3 antibody (AdipoGen, AG-20B-0014-C100). The results are shown in Figure 5 . As can be seen from the results of Figure 5 : Nigericin stimulation can significantly promote the binding of ASC protein and NLRP3 protein, while SB-222200 treatment can inhibit this process. The above results indicate that SB-222200 can inhibit the interaction between NLRP3 protein and ASC protein.

[0083] Example 6 SB-222200 inhibits the auto-oligomerization of NLRP3 protein induced by Nigericin

[0084] The cells used in this example are mouse mononuclear macrophages J774A.1. Seed the cells into a 6-well plate at a cell density of 1×10 6Cell density. After overnight culture, the medium was changed to opti-MEM, and 1 μg / mL LPS was added for 5-hour pretreatment. Then, 5 μM SB-222200 or tranilast was added for 1-hour treatment, followed by 10 μM nigericin stimulation for 1 hour. Semi-denaturing agarose gel electrophoresis (SDD-AGE) was performed as follows: The cell medium was discarded, 100 μL / well of lysis buffer was added, and the cells were lysed on a shaker at 4°C for 30 minutes. Then, the mixture was centrifuged at 300 g for 10 minutes at 4°C, and the supernatant was transferred to a new 1.5 mL EP tube. After quantification, 60 μg of protein was taken and mixed with 20 μl of non-denaturing and non-reducing protein loading buffer (Beyotime, P0016N). The mixture was separated using a 1.3% agarose gel, and then Western Blotting was performed for detection. The results are as Figure 6 shown. As can be seen from the Figure 6 results: Nigericin stimulation significantly induced NLRP3 protein oligomerization, while treatment with SB-222200 inhibited this process, indicating that SB-222200 can inhibit the self-oligomerization of NLRP3 protein induced by nigericin.

[0085] Example 7 SB-222200 inhibits the interaction between NLRP3 protein and NEK7 protein

[0086] The cells used in this example were mouse mononuclear macrophages J774A.1. The cells were seeded into 10 cm culture dishes at a cell density of 6×10 6 . After overnight culture, the medium was changed to opti-MEM, and 1 μg / mL LPS was added for 5-hour pretreatment. Then, 5 μM SB-222200 was added for 1-hour treatment, followed by 10 μM nigericin stimulation for 1 hour. Through co-immunoprecipitation experiments, the NEK7 protein was pulled down, and anti-NEK7 antibody (Abcam, ab133514) and anti-NLRP3 antibody were used for Western Blotting detection. The results are as Figure 7 shown. As can be seen from the Figure 7 results: Nigericin stimulation significantly promoted the binding of NEK7 protein to NLRP3 protein, while treatment with SB-222200 inhibited this process. The above results indicate that SB-222200 can inhibit the interaction between NEK7 protein and NLRP3 protein.

[0087] Example 8 SB-222200 directly binds to NLRP3 protein

[0088] (1) The cells used in this example were mouse mononuclear macrophages J774A.1. The cells were seeded in a 10-cm culture dish and cultured overnight. Then, the medium was replaced with opti-MEM, and 1 μg / mL LPS was added for pretreatment for 5 hours. Subsequently, different concentrations of SB-222200 (0 μM, 5 μM, 15 μM) or MCC950 (10 μM) were added and treated for 1 hour, and then 10 μM nigericin was added for stimulation for 1 hour. The cells were incubated with cell lysate for 30 minutes and centrifuged at 12,000 rpm for 10 minutes. 40 μg of protein lysate was incubated with pronase (0.04 mg / mL) for 10 minutes, and the reaction was terminated by adding 20× protease inhibitor. Detection was performed by Western Blotting using an anti-NLRP3 antibody. The results are as shown in Figure 8 Figure A. It can be seen from the results that the treatment with SB-222200 can inhibit the degradation of NLRP3 protein by pronase, indicating that SB-222200 can bind to NLRP3 protein.

[0089] (2) J774A.1 cells were seeded in a 10-cm culture dish and cultured overnight. Then, the medium was replaced with opti-MEM, and 1 μg / mL LPS was added for pretreatment for 5 hours. Subsequently, 10 μM SB-222200 was added and treated for 1 hour, and then 10 μM nigericin was added for stimulation for 1 hour. The cells were collected by trypsin digestion and centrifuged at 800 g for 5 minutes. The cells were lysed by repeated freezing and thawing, and then heated at the following temperatures (43, 45, 50, 55, 60, and 65 °C) and centrifuged at 3000 g for 25 minutes. Detection was performed by Western Blotting using an anti-NLRP3 antibody. The results are as shown in Figure 8 Figure B. It can be seen from the results that the treatment with SB-222200 can inhibit the thermal degradation of NLRP3 protein, indicating that SB-222200 can bind to NLRP3 protein.

[0090] (3) The binding ability of SB-222200 to NLRP3 protein was detected by SPR method. The SPR experiment was performed using a Biacore T200 (GE Healthcare). The CM5 detection chip was selected from Cytiva (10324467). The recombinant human NLRP3 protein (Huamei Bio, CSB-EP822275HU) was immobilized on the CM5 detection chip, and SB-222200 was injected stepwise for dilution to detect the binding ability. The results are as shown in Figure 8 Figure C. The results show that SB-222200 can directly bind to human NLRP3 protein, and the binding constant is 351.8 nM.

[0091] Effect of Example 9 SB-222200 on IL-1β and IL-6 in peritoneal fluid, liver and spleen after urate crystal-induced peritonitis in mice

[0092] (1) C57BL / 6 male mice aged 6 - 8 weeks (similar body weight) were selected and divided into 6 groups: The first group was first injected with a solvent [containing 2% dimethyl sulfoxide (DMSO), 20% PEG300 and 78% normal saline], and one hour later, PBS was injected as the control group; The second group was first injected with the solvent, and one hour later, 3 mg of urate crystals (MSU, Sigma-Aldrich, U 2875) was injected; The third to sixth groups were first injected with different doses of SB-222200 (10, 15, 20 mg / kg) or the positive control MCC950 (5 mg / kg; MCE, HY-12815), and one hour later, 3 mg of MSU was injected.

[0093] (2) After 4 hours, the mice were sacrificed by cervical dislocation; 1 mL of PBS was injected into the peritoneal cavity, and the peritoneal cavity was gently agitated to make it evenly filled, and then it was aspirated. The aspirated liquid was centrifuged at 3000 rpm for 3 minutes, and the supernatant was used to detect cytokines. Liver and spleen samples were ground separately. The levels of IL-1β and IL-6 in ascites, liver and spleen were detected by ELISA using mouse IL-1β ELISA kit and mouse IL-6 ELISA kit (ThermoFishe, 88-7064-77), as shown in Figure 9 A to Figure 9 F.

[0094] Intraperitoneal injection of urate crystals, an activator of NLRP3 inflammasome activation, induces peritonitis in mice, which is a common NLRP3-related acute disease model. During the occurrence of acute peritonitis, it will lead to the secretion of inflammatory factors IL-1β and IL-6 in the peritoneal cavity and tissues. Injecting SB-222200 one hour before injecting urate crystals, the secretion of IL-β and IL-6 in the peritoneal cavity of mice decreased, and the secretion of inflammatory factor IL-β in liver and spleen tissues decreased but the content of IL-6 remained unchanged. The above results indicate that SB-222200 can relieve urate crystal-induced acute peritonitis and prove in vivo that SB-222200 can inhibit the activation of NLRP3 inflammasome.

[0095] Effect of Example 10 SB-222200 on dextran sulfate sodium (DSS)-induced inflammatory bowel disease

[0096] (1) C57BL / 6 mice at 6 - 8 weeks of age were divided into 6 groups: The first group was injected with a solvent [containing 2% dimethyl sulfoxide (DMSO), 20% PEG300, and 78% normal saline] daily as the control group; the second group was intraperitoneally injected with the injection solvent daily and fed with fresh 2.5% DSS solution (MP Biomedicals, 216011080) from the second day to the eighth day as the model group; the third and fourth groups were injected with different doses of SB - 222200 (10, 20 mg / kg) daily and fed with fresh 2.5% DSS solution from the second day to the eighth day. The general conditions and blood in feces of mice in the healthy control group, DSS model group, and SB - 222200 - treated groups were observed, DAI scores were conducted, the mice were sacrificed on the 9th day, colon specimens were collected, the colon length was compared, and intestinal histopathological examinations were performed.

[0097] The results showed that SB - 222200 intervention could significantly reduce the degree of colon shortening ( Figure 10 A and B), improve blood in feces in the DSS - induced ulcerative colitis model mice ( Figure 10 C), reduce the histopathological score, protect the integrity of the mouse intestinal mucosa and the normal crypt structure ( Figure 10 D), and improve the tissue score ( Figure 10 E), suggesting that SB - 222200 could improve inflammatory bowel disease.

[0098] Example 11 Effect of SB - 222200 on Inflammation in Colon Tissue in DSS - Induced Inflammatory Bowel Disease

[0099] The experimental method was as follows: Colon tissue was collected, a 1 - cm intestinal segment was taken from the distal colon, ground and lysed to obtain tissue protein lysate, the expressions of IL - 1β and IL - 6 in the supernatant were detected by ELISA, and the cleavage of caspase - 1 and GSDMD in the colon tissue was detected by Western Blotting. The results are shown in Figure 11 A to Figure 11 C.

[0100] The results showed that SB - 222200 inhibited the elevation of IL - 1β and IL - 6 in the colon tissue of the DSS - induced mouse ulcerative colitis model ( Figure 11 A and Figure 11 B), reduced the cleavage of caspase - 1 and GSDMD in the colon tissue ( Figure 11 C), indicating that this SB - 222200 could improve ulcerative colitis.

[0101] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0102] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Use of SB-222200 in the preparation of a medicament for preventing and / or treating diseases associated with abnormal activation of the NLRP3 inflammasome; the diseases associated with abnormal activation of the NLRP3 inflammasome are acute peritonitis.

2. The use according to claim 1, wherein, the abnormal activation of the NLRP3 inflammasome is classical NLRP3 inflammasome activation, and the classical NLRP3 inflammasome activation is NLRP3 inflammasome activation induced by an activator.

3. The use according to claim 1, wherein, the abnormal activation of the NLRP3 inflammasome is non-classical NLRP3 inflammasome activation, and the non-classical NLRP3 inflammasome activation is NLRP3 inflammasome activation induced by intracellular lipopolysaccharide.

4. The use according to claim 1, wherein, the medicament comprises SB-222200 and a pharmaceutically acceptable excipient.

5. The use according to claim 4, wherein, the dosage form of the medicament is at least one of capsule, granule, injection, pill, syrup, powder, plaster, emulsion, solution, suspension and tincture.

6. The use according to claim 5, wherein, the excipient comprises at least one of excipient, filler, solubilizer, binder, humectant, disintegrant, slow solvent, absorption accelerator, adsorbent, diluent, solubilizer, emulsifier, lubricant, wetting agent, suspending agent, flavoring agent and perfume.

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