Application of loteprednol etabonate as NLRP3 inflammasome activation inhibitor

Abstract

The invention discloses application of loteprednol etabonate as an NLRP3 inflammasome activation inhibitor, and belongs to the technical field of biological medicine. It is found for the first time that the loteprednol etabonate reduces secretion of IL-1beta by inhibiting NF-kB and / or AP-1 signal channels, activation of NLRP3 inflammasomes is remarkably inhibited, and a new thought and theoretical basis are provided for inhibiting activation of the NLRP3 inflammasomes; therefore, the compound has a relatively good application prospect in preparation of medicines for preventing and / or treating NLRP3 inflammasome related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of loteprednol edacarb as an inhibitor of NLRP3 inflammasome activation. Background Art

[0002] As the first line of defense of the body's immune response, the innate immune system plays an important role in eliminating microorganisms. Among them, the NLRP3 inflammasome, as one of the innate immune molecules, plays an important role in the body's inflammatory response caused by exogenous pathogens and endogenous danger signals. As an important component of innate immunity, the NLRP3 inflammasome is mainly composed of the cytoplasmic sensor protein NLRP3, apoptosis-associated speck-like protein (ASC) and the cysteine ​​protease Caspase-1. After the NLRP3 inflammasome is activated, NLRP3, ASC and Caspase-1 form a complex to activate Caspase-1. Subsequently, the activated Caspase-1 mediates the maturation and secretion of the inflammatory factor IL-1β, and the extracellular inflammatory factor IL-1β participates in the body's inflammatory response and other processes; at the same time, the activated Caspase-1 can mediate cell pyroptosis by shearing the effector protein GSDMD. The NLRP3 inflammasome is one of the most widely studied inflammasomes. It can recognize pathogen-associated molecular patterns (PAMPs) and danger signal-associated molecular patterns (DAMPs), including viruses, bacteria, Nigericin, monosodium urate, ATP, silica crystals, and aluminum salt crystals. The NLRP3 inflammasome plays an important role in the body's resistance to pathogen infection and recognition of danger signals, but its overactivation or uncontrolled regulation is closely related to many inflammatory diseases, such as gout, type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis, Parkinson's disease, Alzheimer's disease, and various autoimmune diseases.

[0003] At present, a variety of NLRP3 inflammasome activation inhibitors have been reported, most of which use NLRP3 protein as a drug target, including MCC950, CY-09, MNS, OLT1177, etc. The effects of these inhibitors in animal models of inflammatory diseases related to NLRP3 inflammasomes have been verified. For example, MCC950 can reduce the severity of experimental autoimmune encephalomyelitis in mice; oral administration of MCC950 can inhibit NLRP3 inflammasome activation in Parkinson's disease mouse models, and also has neuroprotective effects, which can effectively reduce motor defects, nigrostriatal dopaminergic degeneration and accumulation of α-synuclein aggregates; CY-09 has a good therapeutic effect in cryopyrin-related autoinflammatory syndrome and type 2 diabetes mouse models. Although a variety of NLRP3 inflammasome inhibitors have good effects in animal models, there are currently few inhibitors approved for marketing, and most inhibitors are in the clinical trial stage or stopped in the clinical trial stage. Therefore, it is of great clinical significance to find new and effective drugs that inhibit NLRP3 inflammasome activation.

[0004] Loteprednol etabonate is an anti-inflammatory corticosteroid used in ophthalmology. It can be used to treat inflammatory conditions that are sensitive to corticosteroids, such as eyelid and conjunctivitis, uveitis, and inflammation of the cornea and anterior segment. Currently, there are no reports that loteprednol etabonate can inhibit the activation of NLRP3 inflammasomes. Summary of the invention

[0005] The purpose of the present invention is to provide the use of loteprednol edacarb as an inhibitor of NLRP3 inflammasome activation. The present invention firstly finds that loteprednol edacarb significantly inhibits NLRP3 inflammasome activation by reducing the secretion of IL-1β, and therefore has good application prospects in the preparation of drugs for preventing and / or treating NLRP3 inflammasome-related diseases.

[0006] In a first aspect, the present invention provides a use of loteprednol etabonate as an inhibitor of NLRP3 inflammasome activation.

[0007] Wherein, loteprednol edacarb has the structure shown in the following formula (I): .

[0008] In the present invention, the inventors have found that loteprednol edacarb can inhibit the activation of NLRP3 inflammasomes, and therefore can be used to prepare drugs for preventing and / or treating NLRP3 inflammasome-related diseases.

[0009] In some embodiments, loteprednol edacarb inhibits the NF-κB and / or AP-1 signaling pathways, thereby inhibiting the expression of IL-1β and thereby inhibiting NLRP3 inflammasome activation.

[0010] In the present invention, by detecting the phosphorylation modification levels of p65 protein in the NF-κB signaling pathway and / or c-jun protein in the AP-1 signaling pathway, it was found that loteprednol edacarb can inhibit the NF-κB and / or AP-1 signaling pathways in a dose-dependent manner, thereby inhibiting the expression of IL-1β and ultimately inhibiting the activation of NLRP3 inflammasome.

[0011] In a second aspect, the present invention provides a use of loteprednol etabonate in the preparation of a medicament for preventing and / or treating NLRP3 inflammasome-related diseases.

[0012] In some embodiments, the NLRP3 inflammasome-associated disease comprises at least one of gout, type 2 diabetes, nonalcoholic steatohepatitis, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

[0013] It is understandable that NLRP3 inflammasome-related diseases also include other common diseases in the prior art. In the present invention, NLRP3 inflammasome-related diseases preferably include at least one of gout, type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

[0014] In a third aspect, the present invention provides an inhibitor of NLRP3 inflammasome activation, comprising loteprednol etanercept.

[0015] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned NLRP3 inflammasome activation inhibitor.

[0016] In some embodiments, the above pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0017] In the present invention, the term "pharmaceutically acceptable carrier" refers to an adjuvant widely used in the field of drug production. The adjuvant is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. Pharmaceutical adjuvants can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. Pharmaceutical adjuvants can include one or more of the following adjuvants: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0018] The pharmaceutical composition provided by the present invention can be prepared by any method known to those skilled in the art according to the disclosed content, including but not limited to conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0019] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semisolid preparation, and a liquid preparation.

[0020] The pharmaceutical composition provided by the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection and emulsions for injection. Examples of other suitable formulations of pharmaceutical compositions include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0021] In a fifth aspect, the present invention provides use of the above-mentioned NLRP3 inflammasome activation inhibitor or any of the above-mentioned pharmaceutical compositions in the preparation of a medicament for preventing and / or treating NLRP3 inflammasome-related diseases.

[0022] In some embodiments, the NLRP3 inflammasome-associated disease comprises at least one of gout, type 2 diabetes, nonalcoholic steatohepatitis, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

[0023] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention discovers for the first time that loteprednol edacarb reduces the expression and secretion of IL-1β and inhibits the activation of NLRP3 inflammasome by inhibiting the NF-κB and / or AP-1 signaling pathways, thereby providing a new idea and theoretical basis for inhibiting the activation of NLRP3 inflammasome; therefore, the present invention has good application prospects in the preparation of drugs for preventing and / or treating NLRP3 inflammasome-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A This is a model result diagram showing that LPS+Nigericin can activate NLRP3 inflammasome in Example 1 of the present invention; Figure 1B This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Figure 1C This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Figure 1D This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Figure 1E This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Figure 1F This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Figure 1G This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Figure 1H This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Fig. 1I This is a result diagram of the IL-1β content in the cell supernatant after treatment with different drugs in Example 1 of the present invention; Figure 2A The result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 2B The result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 2CThe result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 2D The result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 2E The result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 2F The result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 2G The result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 2H The result diagram of the expression level of IL-1β mRNA in cells after treatment with different drugs in Example 1 of the present invention; Figure 3 The results of Loteprednol etabonate inhibiting NLRP3 inflammasome activation in Example 2 of the present invention are shown in Figure 2, wherein (A) is the result of the IL-1β content in the supernatant of the treated cells; (B)-(D) are the results of the mRNA expression levels of IL-1β, NLRP3 and Caspase-1 in the treated cells, respectively; Figure 4 The figures are Western blotting results of the protein related to the NF-κB signaling pathway and the AP-1 signaling pathway in cells inhibited by Loteprednol etabonate in Example 2 of the present invention, wherein (A) is a Western blotting result of the protein related to the NF-κB signaling pathway and the AP-1 signaling pathway in THP-1 cells inhibited by Loteprednol etabonate, and (B) is a Western blotting result of the protein related to the NF-κB signaling pathway and the AP-1 signaling pathway in BMDMs cells inhibited by Loteprednol etabonate; Figure 5 This is a graph showing the cytotoxicity results of Loteprednol etabonate in Example 3 of the present invention; Figure 6 The results of different concentrations of Loteprednol etabonate inhibiting NLRP3 inflammasome activation in Example 4 of the present invention are shown in Figure 4, wherein (A) is the result of the IL-1β content in the supernatant of the treated cells; (B)-(D) are the results of the mRNA expression levels of IL-1β, NLRP3 and Caspase-1 in the treated cells respectively; Figure 7 The results of different concentrations of Loteprednol etabonate inhibiting NLRP3 inflammasome activation in Example 4 of the present invention are shown in Figure 4, wherein (A) is the result of the content of IL-1β in the supernatant of the treated cells; (B) is the result of the half effective concentration IC50 of Loteprednoletabonate; Figure 8 This is a graph showing the results of the IL-1β and TNF-ɑ levels in the blood of septic mice after treatment with Loteprednol etabonate in Example 5 of the present invention; Fig. 9 This is a graph showing the survival rate and body weight of septic mice after treatment with Loteprednol etabonate (first intraperitoneal injection of Loteprednol etabonate, then injection of LPS 2 hours later) in Example 5 of the present invention; Fig.10 This is a graph showing the survival rate and body weight of septic mice after treatment with Loteprednol etabonate in Example 5 of the present invention (first intraperitoneal injection of LPS, then injection of Loteprednol etabonate 1 hour later); Fig.11A This is a graph showing the body weight results of colitis mice after Loteprednol etabonate treatment in Example 6 of the present invention; Fig. 11B This is a graph showing the fecal disease index results in colitis mice treated with Loteprednol etabonate in Example 6 of the present invention; Fig. 11C This is a graph showing the colon length measurement in colitis mice after Loteprednol etabonate treatment in Example 6 of the present invention; Fig.11D This is a statistical result diagram of colon length in colitis mice treated with Loteprednol etabonate in Example 6 of the present invention; Fig.11E This is a graph showing the results of HE staining of the colon in colitis mice after treatment with Loteprednol etabonate in Example 6 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0027] Example 1 Screening of NLRP3 inflammasome activation inhibitors 1.1 Construction of NLRP3 inflammasome activation model THP-1 cells were cultured in 1640 medium containing 10% FBS, TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; then LPS was added at a final concentration of 100 ng / ml to the above induced differentiated THP-1 macrophages, and the culture was continued for 6 h, and then Nigericin was added at a final concentration of 10 μM, and the culture was continued for 1 h, and the cell culture supernatant (the cell culture supernatant with LPS added alone and LPS+Nigericin added at the same time) was collected, and the IL-1β content in the supernatant was detected, and the supernatant of induced differentiated THP-1 macrophages was used as a control; At the same time, THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; different concentrations of MCC950 (1, 10, 100 μM) were added to the above-mentioned induced differentiated THP-1 macrophages, and the culture was continued for 12 h, and then LPS was added at a final concentration of 100 ng / ml, and the culture was continued for 6 h, and then Nigericin was added at a final concentration of 10 μM, and the culture was continued for 1 h, the cell culture supernatant was collected, and the IL-1β content in the supernatant was detected. The cell supernatant without adding MCC950 was used as a control. The results are as follows: Figure 1A shown.

[0028] The IL-1β content in the supernatant was determined by ELISA (refer to ELISA MAX TM Deluxe Set Human IL-1β Kit) and includes the following steps: 1) Add 100 μl caputure antibody (1×) to the 96-well plate, seal, and incubate overnight at 4°C; 2) Pour out the liquid in the wells and wash each well three times with 300μl PBST, shaking gently each time and then pouring out. If there are bubbles on the plate after pouring, tap them on absorbent paper and then add PBST. After the last wash, tap the remaining washing solution on absorbent paper; 3) Add 200 μl Blocking Buffer (1×) to each well, seal, and incubate at room temperature for 1 h; 4) After incubation, wash each well three times with 300 μl PBST; 5) First add 50 μl Assay Buffer D to each well, then add 50 μl sample diluent or standard to the corresponding wells, repeat 2 wells for each sample diluent and standard; seal the plate and incubate at room temperature for 2 hours; 6) After incubation, wash each well three times with 300 μl PBST; 7) Add 100 μl Detection Antibody (1×) to each well, seal the plate, and incubate at room temperature for 1 h; 8) After incubation, wash each well three times with 300 μl PBST; 9. Add 100 μl diluted Avdin-HRP (1×) to each well, seal the plate, and incubate at room temperature for 30 min; 10) After incubation, wash each well four times with 300 μl PBST; 11) Add 100 μl of Substrate Solution F to each well, without sealing the plate, and leave at room temperature for about 5 minutes. The positive control well will turn blue. 12) Add 100 μl Stop Solution to each well to terminate the reaction. The positive control well will change from blue to yellow. Detect the absorbance at 450 nm using an ELISA reader within 15 minutes.

[0029] A standard curve was prepared based on the standard sample to calculate the content of IL-1β in the sample.

[0030] from Figure 1A It can be seen that LPS and Nigericin can activate NLRP3 inflammasome, while MCC950 can effectively inhibit the activation of NLRP3 inflammasome. The results show that the NLRP3 inflammasome activation model was successfully constructed.

[0031] 1.2 Screening of NLRP3 inflammasome activation inhibitors THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added at a final concentration of 100 ng / ml. The culture was continued for 12 h to obtain induced differentiated THP-1 macrophages. Positive control MCC950 and other drugs (all at a concentration of 10 μM) were added to the above induced differentiated THP-1 macrophages, and the culture was continued for 12 h. Then, LPS was added at a final concentration of 100 ng / ml and the culture was continued for 6 h. Then, Nigericin was added at a final concentration of 10 μM and the culture was continued for 1 h. The cell culture supernatant was collected, and the IL-1β content in the supernatant was detected by the method in step 1.1. The results were as follows: Figure 1B-1I shown.

[0032] from Figure 1B-1I It can be seen that 8 drugs (Loteprednol etabonate (LE), Betamethason, Hydrocortison, Prednisolone, Dexamethaso, Andrographolide, Luteolin, Apigenin (LY08040)) can effectively inhibit the activation of NLRP3 inflammasome.

[0033] Furthermore, THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added at a final concentration of 100 ng / ml, and culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; positive control MCC950 and other drugs (all at a concentration of 10 μM) were added to the above-mentioned induced differentiated THP-1 macrophages, and culture was continued for 12 h, and then LPS was added at a final concentration of 100 ng / ml, and culture was continued for 6 h, and then Nigericin was added at a final concentration of 10 μM, and culture was continued for 1 h, and the cells were collected, and the total RNA of the above cells was extracted using an RNA extraction kit (Kangwei Century, #CW0581M), and then 5×EasyQuick RT MasterMix (Kangwei Century, #CW2019M) was reverse transcribed into cDNA with the following system: 2×SYBRGreen mixture 10μl (Kangwei Century, #CW3008H), cDNA template 1μl, specific primers 1μl (F and R primers), ddH 2 8 μl was used for RT-PCR reaction, and GAPDH was used as the internal reference gene to obtain the expression level of IL-1β mRNA in the cells. The results were as follows: Figures 2A-2H shown.

[0034] Among them, the specific primer has the following sequence: hGAPDH-F: 5′-AAGGCTGTGGGCAAGG-3′ (SEQ ID NO: 1); hGAPDH-R: 5′-TGGAGGAGTGGGTGTCG-3′ (SEQ ID NO: 2); hIL-1β-F: 5′-CACGATGCACCTGTACGATCA-3′ (SEQ ID NO: 3); hIL-1β-R: 5′-GTTGCTCCATATCCTGTCCCT-3′ (SEQ ID NO: 4).

[0035] from Figures 2A-2H As shown, the above 8 drugs can effectively downregulate the expression level of IL-1β mRNA, and loteprednol etabonate has the most significant inhibitory effect. Therefore, the present invention screened out the NLRP3 inflammasome activation inhibitor (loteprednol etabonate) with the best inhibitory effect.

[0036] Example 2 Study on the mechanism of loteprednol etabonate inhibiting NLRP3 inflammasome activation THP-1 cells were cultured in 1640 medium containing 10% FBS, TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; positive control MCC950 and positive controls Luteolin and Loteprednol etabonate (both at a concentration of 10 μM) obtained by screening in Example 1 were added to the above-mentioned induced differentiated THP-1 macrophages, and the culture was continued for 12 h, and then LPS was added at a final concentration of 100 ng / ml, and the culture was continued for 6 h, and then Nigericin was added at a final concentration of 10 μM, and the culture was continued for 1 h, and the cells and cell culture supernatants were collected respectively, and the IL-1β content in the cell culture supernatant was detected by the method in Example 1. At the same time, the mRNA expression levels of IL-1β, NLRP3 and Caspase-1 in the cells were detected by the method in Example 1. The results are as follows: Figure 3 shown.

[0037] Among them, the specific primer has the following sequence: hNLRP3-F: 5′-AAGGGCCATGGACTATTTCC-3′ (SEQ ID NO: 5); hNLRP3-R: 5′-GACTCCACCCGATGACAGTT-3′ (SEQ ID NO: 6); hCaspase-1-F: 5′-GCTGAGGTTGACATCACAGGCA-3′ (SEQ ID NO: 7); hCaspase-1-R: 5′-TGCTGTCAGAGGTCTTGTGCTC-3′ (SEQ ID NO: 8).

[0038] from Figure 3 It can be seen that Loteprednol etabonate inhibits NLRP3 inflammasome activation by reducing the secretion of IL-1β and inhibiting the expression level of IL-1β mRNA. In addition, Loteprednol etabonate has little effect on the mRNA transcription levels of NLRP3 and Caspase-1.

[0039] To further investigate the inhibitory effect of Loteprednol etabonate on IL-1β mRNA levels, THP-1 cells and BMDMs cells were treated with different doses (0.1, 1, 10 μM) of Loteprednol etabonate for 12 h, and then treated with LPS at a final concentration of 100 ng / ml for 6 h. The expression levels of GAPDH, pc-jun, c-jun, p-p65, and p65 proteins were detected by Western blotting. The results are shown in Table 2. Figure 4 shown.

[0040] from Figure 4 It can be seen that in THP-1 cells and BMDMs, Loteprednol etabonate inhibits the phosphorylation levels of p65 protein in the NF-κB signaling pathway and c-jun protein in the AP-1 signaling pathway, proving that Loteprednoletabonate can inhibit the activation of the NF-κB signaling pathway and the AP-1 signaling pathway, thereby reducing the expression and secretion of IL-1β, and the above inhibition is dose-dependent.

[0041] Example 3 Cytotoxicity test of Loteprednol etabonate THP-1 cells were cultured in 1640 medium containing 10% FBS, and TPA was added at a final concentration of 100 ng / ml. The culture was continued for 12 h to obtain induced differentiated THP-1 macrophages. Different concentrations (0, 2.5, 5, 10, 20, 40, 80, 160, 320 μM) of loteprednol edacarb were added to the induced differentiated THP-1 macrophages, and the cytotoxicity of the drug was detected by CCK8 kit. The results are shown in FIG. Figure 5 shown.

[0042] from Figure 5 It can be seen that when the concentration of loteprednol edocarb is below 80 μM, the toxicity to cells is low. The results show that loteprednol edocarb has good biosafety.

[0043] Example 4 Concentration-dependent test of loteprednol etabonate inhibiting NLRP3 inflammasome activation THP-1 cells were cultured in 1640 medium containing 10% FBS, TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; different concentrations of Loteprednol etabonate (0, 0.1, 1, 10 μM) were added to the above-mentioned induced differentiated THP-1 macrophages, and the culture was continued for 12 h, and then LPS was added at a final concentration of 100 ng / ml, and the culture was continued for 6 h, and then Nigericin was added at a final concentration of 10 μM, and the culture was continued for 1 h, and the cells and cell culture supernatants were collected respectively, and the IL-1β content in the cell culture supernatant was detected by the method in Example 1. At the same time, the mRNA expression levels of IL-1β, NLRP3 and Caspase-1 in the cells were detected by the method in Example 2. The results are as follows: Figure 6 shown.

[0044] from Figure 6 It can be seen that Loteprednol etabonate inhibits NLRP3 inflammasome activation by reducing the secretion of IL-1β and inhibiting the expression level of IL-1β mRNA, and is concentration-dependent; in addition, Loteprednoletabonate has little effect on the mRNA transcription levels of NLRP3 and Caspase-1.

[0045] Furthermore, THP-1 cells were cultured in 1640 medium containing 10% FBS, TPA was added at a final concentration of 100 ng / ml, and the culture was continued for 12 h to obtain induced differentiated THP-1 macrophages; different concentrations of Loteprednol etabonate (0, 10 -6 , 10 -4 , 10 -2 μM), and continued to culture for 12 h, then added LPS with a final concentration of 100 ng / ml, and continued to culture for 6 h, then continued to add Nigericin with a final concentration of 10 μM, and continued to culture for 1 h, and the cell culture supernatant was collected, and the IL-1β content in the cell culture supernatant was detected by the method in Example 1. The results are as follows Figure 7 shown.

[0046] from Figure 7 As can be seen, the IC50 of Loteprednol etabonate 50 is 7.034×10 -5 μM.

[0047] Example 5 Loteprednol etabonate effectively alleviates LPS-induced sepsis in mice and improves the survival rate of mice C57BL / 6 mice were divided into four groups, and mice in each group were treated as follows: the first group was intraperitoneally injected with normal saline (n=4), the second group was intraperitoneally injected with Loteprednol etabonate (20mg / kg, n=4), the third group was intraperitoneally injected with LPS (30mg / kg, n=6), and the fourth group was intraperitoneally injected with LPS (30mg / kg) and Loteprednol etabonate (20mg / kg, n=6). In the fourth group, Loteprednol etabonate was first intraperitoneally injected, and LPS was injected 2h later; 4h later, blood was collected from the eyeball to measure inflammatory factors (IL-1β and TNF-ɑ). The results are shown in Figure 8 shown.

[0048] from Figure 8 It can be seen that Loteprednol etabonate can effectively inhibit the secretion of IL-1β and TNF-ɑ.

[0049] Further, C57BL / 6 mice were divided into seven groups, and the mice in each group were treated as follows: the first group was intraperitoneally injected with normal saline (n=8), the second group was intraperitoneally injected with Loteprednol etabonate (40 mg / kg, n=8), the third group was intraperitoneally injected with LPS (20 mg / kg, n=12), the fourth group was intraperitoneally injected with LPS (20 mg / kg) and Loteprednol etabonate (20 mg / kg, n=12), the fifth group was intraperitoneally injected with LPS (20 mg / kg) and Loteprednol etabonate (40 mg / kg, n=12), the sixth group was intraperitoneally injected with MCC950 (20 mg / kg, n=8), and the seventh group was intraperitoneally injected with LPS (20 mg / kg) and MCC950 (20 mg / kg, n=12). In the fourth, fifth and seventh groups, Loteprednol etabonate or MCC950 was first injected intraperitoneally, and LPS was injected 2 hours later. The survival rate and body weight of the mice were then observed regularly every day. Fig. 9 shown.

[0050] The C57BL / 6 mice were further divided into seven groups, and the mice in each group were treated as follows: the first group was intraperitoneally injected with normal saline (n=8), the second group was intraperitoneally injected with Loteprednol etabonate (40 mg / kg, n=8), the third group was intraperitoneally injected with LPS (20 mg / kg, n=12), the fourth group was intraperitoneally injected with LPS (20 mg / kg) and Loteprednol etabonate (20 mg / kg, n=12), the fifth group was intraperitoneally injected with LPS (20 mg / kg) and Loteprednol etabonate (40 mg / kg, n=12), the sixth group was intraperitoneally injected with MCC950 (20 mg / kg, n=8), and the seventh group was intraperitoneally injected with LPS (20 mg / kg) and MCC950 (20 mg / kg, n=12). In the fourth, fifth and seventh groups, LPS was first injected intraperitoneally, and then Loteprednol etabonate or MCC950 was injected 1 hour later. The survival rate and body weight of the mice were then observed regularly every day. Fig.10 shown.

[0051] from Figure 9-10 It can be seen that whether Loteprednol etabonate or MCC950 is injected intraperitoneally first and LPS is injected 2 hours later; or LPS is injected intraperitoneally first and then Loteprednol etabonate or MCC950 is injected 1 hour later; Loteprednol etabonate can effectively inhibit the LPS-induced sepsis model, improve the survival rate of mice and alleviate weight loss.

[0052] Example 6 Loteprednol etabonate effectively alleviates DSS-induced colitis in mice C57BL / 6 mice were divided into five groups, and the mice in each group were treated as follows: the first group was intraperitoneally injected with normal saline (n=12), the second group was intraperitoneally injected with normal saline and oral DSS (3%, n=10), the third group was intraperitoneally injected with Loteprednoletabonate (20 mg / kg) and oral DSS (3%, n=10), the fourth group was intraperitoneally injected with Loteprednol etabonate (40 mg / kg) and oral DSS (3%, n=10), and the fifth group was intraperitoneally injected with MCC950 (20 mg / kg) and oral DSS (3%, n=10). Among them, the mouse colitis model was induced by oral DSS (3%); after eight days, each group was fed with normal water, and all mice were treated on the tenth day, and the weight of the mice was observed and recorded every day. The results are shown in Figure 2. Fig.11AAs shown; the feces of mice were observed every day and the disease index was scored, with 0 being normal, 1 being soft feces, 2 being soft feces with bleeding, 3 being loose feces with bleeding, and 4 being heavy bleeding. The results are shown in Fig. 11B shown.

[0053] from Fig.11A It can be seen that Loteprednol etabonate can alleviate the weight loss of colitis mice. Fig. 11B It can be seen that Loteprednol etabonate can alleviate the symptoms of diarrhea and bloody stool in colitis mice.

[0054] On the tenth day, the colons of each treatment group were taken and photographed, and their lengths were measured. It was found that Loteprednol etabonate could alleviate the shortening of the colon caused by acute colitis ( Fig. 11C and 11D ). HE staining was performed on the colon of each treatment group for pathological analysis. The results are as follows Fig.11E shown.

[0055] from Fig.11E It can be seen that Loteprednol etabonate can effectively alleviate DSS-induced mucosal edema, goblet cell loss, inflammatory cell infiltration and other symptoms. The results show that Loteprednol etabonate can effectively alleviate DSS-induced colitis in mice.

[0056] In summary, the present invention discovered for the first time that loteprednol edacarb significantly inhibits NLRP3 inflammasome activation by reducing the secretion of IL-1β, and therefore can be used to prepare drugs for preventing and / or treating NLRP3 inflammasome-related diseases.

[0057] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0058] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of loteprednol edacarb as an inhibitor of NLRP3 inflammasome activation.

2. The use according to claim 1, characterized in that The loteprednol edacarb inhibits the expression of IL-1β and further inhibits the activation of NLRP3 inflammasome by inhibiting the NF-κB and / or AP-1 signaling pathways.

3. Use of loteprednol etabonate in the preparation of a medicament for preventing and / or treating NLRP3 inflammasome-related diseases.

4. The use according to claim 3, characterized in that The NLRP3 inflammasome-related diseases include at least one of gout, type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

5. An inhibitor of NLRP3 inflammasome activation, characterized in that Including loteprednol etabonate.

6. A pharmaceutical composition, characterized in that Including the NLRP3 inflammasome activation inhibitor as described in claim 5.

7. The pharmaceutical composition according to claim 6, characterized in that Also included are pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semisolid preparation, and a liquid preparation.

9. Use of the NLRP3 inflammasome activation inhibitor according to claim 5 or the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a medicament for preventing and / or treating NLRP3 inflammasome-related diseases.

10. The use according to claim 9, characterized in that The NLRP3 inflammasome-related diseases include at least one of gout, type 2 diabetes, non-alcoholic fatty liver disease, atherosclerosis, Parkinson's disease, Alzheimer's disease, sepsis, and colitis.

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