Use of hm43239

Abstract

The application relates to the technical field of medicines, in particular to the application of HM43239. The application of HM43239 in the preparation of an NLRP3 inflammasome inhibitor. The application of HM43239 in the preparation of a medicine for treating septic shock. The application of HM43239 in the preparation of a medicine for treating acute peritonitis. The application provides a new choice for medicines for preventing or treating NLRP3 inflammasome related diseases, and provides a new medicine with treatment potential for septic shock and acute peritonitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of HM43239. Background Technology

[0002] The NLRP3 inflammasome is assembled from the sensor protein NLRP3 (containing PYD, NACHT, and LRR domains), the adaptor protein ASC, and the effector protein pro-caspase-1. At rest, NLRP3 is in a self-inhibited state; upon activation, the NEK7 kinase specifically binds to the NACHT and LRR domains of NLRP3, disrupting its self-inhibitory conformation, promoting NLRP3 oligomerization and recruiting ASC to form ASC spots, thereby activating caspase-1. Activated caspase-1 cleaves pro-IL-1β / IL-18 and gasdermin D, mediating the maturation of inflammatory factors and pyroptosis. NEK7 acts as a key "conformation switch" in this process, converting upstream danger signals (such as potassium ion efflux) into conformational changes in NLRP3, initiating downstream inflammatory cascade responses, and its action is NLRP3 pathway specific.

[0003] As research into the role of the NLRP3 inflammasome in inflammatory diseases deepens, the development philosophy of inhibitors has gradually shifted from early biologics primarily targeting downstream effects such as IL-1β to directly intervening in inflammasome assembly or upstream regulatory signaling. Among these, small molecule kinase inhibitors have attracted significant attention due to their unique advantages and have become a current research hotspot.

[0004] CY-09 is a small molecule compound that targets the CFTR channel. Subsequent studies have found that CY-09 targets the NACHT domain of NLRP3, competitively binding to the ATP binding site to inhibit ATPase activity and prevent the oligomerization and assembly of the NLRP3 inflammasome.

[0005] MNS is a kinase inhibitor targeting Syk and Src, which inhibits platelet aggregation. It directly binds to the NOD and LRR domains of the NLRP3 protein, inhibiting ATPase activity to block NLRP3 oligomerization and inflammasome assembly. Studies have shown that MNS can selectively inhibit NLRP3 inflammasome agonist-induced inflammasome activation without affecting other inflammasomes such as NLRC4 or AIM2, indicating its target specificity.

[0006] Luxeptinib (CG-806) is an oral multi-target kinase inhibitor initially developed for leukemia. Recent studies suggest it may indirectly regulate the NLRP3 inflammasome by intervening in upstream kinases of the BCR signaling axis. Existing research indicates that Src family kinases (such as LYN) and SYK kinases are not only key players in B-cell receptor signaling but also participate in Toll-like receptor-mediated innate immune responses. LUX can inhibit TLR signaling and NLRP3 inflammasome activation at clinically relevant concentrations, suggesting it may indirectly inhibit NLRP3 inflammasome assembly and activity by reducing its "initiation" signaling.

[0007] While these compounds hold promise as drugs for treating NLRP3 inflammasome-related diseases, they still require lengthy efficacy and safety assessments and clinical trials. Therefore, it is crucial to find a clinical drug that can inhibit the NLRP3 inflammasome to treat related diseases, as this would not only significantly shorten drug development time but also ensure its safety.

[0008] HM43239 is a novel, oral, selective, and reversible type I multi-target tyrosine kinase inhibitor, initially widely studied for its high potency and selectivity as an FLT3 inhibitor. In addition to directly inhibiting FLT3 kinase activity, it can also regulate signaling pathways such as STAT5, ERK, SYK, and JAK. Preclinical studies have shown that HM43239 can effectively inhibit the proliferation of FLT3-ITD mutant leukemia cells and stem cell-like KG1a cells, induce apoptosis, and exhibit dose-dependent antitumor effects and prolonged survival in animal models. Although the potential of HM43239 in the field of leukemia has been validated to some extent, its role in the NLRP3 inflammasome has not been reported. Summary of the Invention

[0009] In view of this, the purpose of the present invention is to provide the application of HM43239, to provide a new drug option for the prevention or treatment of NLRP3 inflammasome-related diseases, and also to provide a new drug with therapeutic potential for septic shock and acute peritonitis.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This technical solution systematically studies the inhibitory effect of HM43239 on the NLRP3 inflammasome and its therapeutic potential in inflammatory diseases.

[0011] Application of HM43239, specifically its use in the preparation of NLRP3 inflammasome inhibitors.

[0012] The molecular formula of HM43239 is: C 29 H 33ClN6 has the following molecular structure: .

[0013] The inventors of this case have determined through relevant experiments that HM43239 can significantly inhibit Nigericin-induced NLRP3 inflammasome activation in mouse BMDM cells, THP-1 cells, and healthy volunteer PBMCs (peripheral blood mononuclear cells), and have verified its function in animal models, thus proving that HM43239 can be used as an NLRP3 inflammasome inhibitor, providing a new drug option for the prevention or treatment of NLRP3 inflammasome-related diseases.

[0014] Preferably, the NLRP3 inflammasome inhibitor is prepared by adding pharmaceutical excipients with HM43239 as the active ingredient.

[0015] Preferably, the dosage form of the NLRP3 inflammasome inhibitor is at least one of tablets, capsules, granules, powders, patches, suspensions, oral liquids, injections, and suppositories.

[0016] Preferably, the use of HM43239 as an inhibitor of Nigericin-induced NLRP3 inflammasome activation in BMDMs cells.

[0017] This invention also provides the use of HM43239 in the preparation of medicaments for treating septic shock.

[0018] Preferably, the septic shock is lipopolysaccharide (LPS)-induced septic shock.

[0019] Preferably, the drug for treating septic shock is prepared by adding pharmaceutical excipients to HM43239 as the active ingredient.

[0020] Preferably, in the treatment of septic shock, the dosage of HM43239 is 20 mg / kg.

[0021] Experimental studies have shown that HM43239 of this invention exhibits significant therapeutic effects at low doses (20 mg / kg). This demonstrates that HM43239 may possess high bioavailability and strong anti-inflammatory activity, providing important clues for the development of novel anti-infective shock drugs.

[0022] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0023] Preferably, when HM43239 is used as a drug for treating septic shock, it can significantly reduce the content of IL-1β in serum and peritoneal lavage fluid, and has no significant effect on IL-6.

[0024] This invention also provides the use of HM43239 in the preparation of a medicament for treating peritonitis.

[0025] Preferably, the peritonitis is acute peritonitis.

[0026] Preferably, the peritonitis is acute peritonitis induced by potassium aluminum sulfate (Alum).

[0027] In experimental studies, alum (potassium aluminum sulfate), a classic inorganic salt adjuvant, is widely used to establish acute peritonitis models due to its stable crystal structure and well-defined immunostimulatory properties. After intraperitoneal injection of alum, its positively charged aluminum salt crystals can be taken up by innate immune cells such as peritoneal macrophages and dendritic cells through phagocytosis. This process alters lysosomal membrane permeability, causing intracellular potassium ion efflux and inducing mitochondrial dysfunction, leading to a surge of reactive oxygen species (ROS). These cellular stress responses activate multiple inflammatory signaling pathways, induce inflammatory cell death, and release large amounts of damage-associated molecular patterns (DAMPs). These inflammatory mediators infiltrate the peritoneal cavity through chemotaxis of immune cells such as neutrophils and monocytes, activating signaling pathways such as TLR4 / MyD88, ultimately forming a "cytokine storm" and resulting in the typical pathological changes of acute peritonitis.

[0028] Preferably, the drug for treating acute peritonitis is prepared by adding pharmaceutical excipients to HM43239 as the active ingredient.

[0029] Preferably, in the medication for treating acute peritonitis, the dosage of HM43239 is 20 mg / kg.

[0030] Experimental studies have shown that HM43239 of this invention exhibits significant therapeutic effects at low doses (20 mg / kg). This demonstrates that HM43239 may possess high bioavailability and strong anti-inflammatory activity, providing important clues for the development of novel anti-peritonitis drugs.

[0031] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0032] Preferably, when HM43239 is used as a drug for treating acute peritonitis, it can inhibit the release of IL-1β in peritoneal lavage fluid and reduce the proportion and absolute count of neutrophils in peritoneal lavage fluid.

[0033] Preferably, the HM43239 is used in a medicament for reducing the content of IL-1β in serum and peritoneal lavage fluid.

[0034] Preferably, when HM43239 acts as an inhibitor of Nigericin-induced NLRP3 inflammasome activation in BMDM cells, it can reduce the levels of IL-1β in serum and peritoneal lavage fluid.

[0035] Preferably, HM43239 is used in the preparation of a drug that reduces the proportion and absolute count of neutrophils in peritoneal lavage fluid while inhibiting the release of IL-1β.

[0036] The beneficial effects of this invention are: HM43239 of this invention exhibits inhibitory effects on the NLRP3 inflammasome in various cell models. Under induction by different agonists such as Nigericin, ATP, and MSU, HM43239 significantly inhibited NLRP3 inflammasome activation in mouse BMDM cells, human THP-1 cells, and healthy human PBMCs. Simultaneously, HM43239 effectively inhibited non-classical inflammasome activation pathways, demonstrating a comprehensive inhibitory capacity on the NLRP3 pathway. Studies have shown that by inhibiting the specific binding between NLRP3 and NEK7, the assembly of the NLRP3 inflammasome is affected, thereby inhibiting NLRP3 inflammasome activation. These findings not only reveal the unique mechanism of action of HM43239 but also lay a solid experimental foundation for its subsequent development as a highly effective and specific NLRP3 inflammasome inhibitor.

[0037] This invention utilizes two inflammatory models with distinct etiologies and pathological characteristics—septic shock and acute peritonitis—to systematically validate the in vivo efficacy of HM43239. In the LPS-induced septic shock model, HM43239 exhibited high targeting specificity, reducing IL-1β levels in serum and peritoneal lavage fluid while having no significant effect on IL-6. In the Alum-induced acute peritonitis model, HM43239 intervention significantly reduced the proportion and absolute count of neutrophils in peritoneal lavage fluid, while simultaneously inhibiting IL-1β release. Overall, this demonstrates that HM43239, by targeting the NLRP3 inflammasome, exerts consistent anti-inflammatory and tissue-protective effects across different pathological scenarios, showcasing excellent clinical translational potential. It provides a breakthrough solution for the treatment of septic shock and acute peritonitis, possessing significant clinical value and socioeconomic benefits. Furthermore, it has significant application value in the field of biomedical materials technology. Attached Figure Description

[0038] Figure 1The figure shows the results of HM43239 inhibiting the activation of the classical NLRP3 inflammasome in mouse BMDMs in Example 1. In the figure, A shows the protein expression of IL-1β and p20 in cell culture supernatant (sn) and pro-caspase-1, pro-IL-1β and β-actin in cell lysate (input); B, D and E show the secretion levels of inflammatory factors IL-1β, TNF-α and IL-6 in cell culture supernatant; and C shows the effect of different concentrations of HM43239 on the secretion level of IL-1β in cell culture supernatant. Figure 2 The figure shows the results of HM43239 inhibiting the activation of the classical NLRP3 inflammasome in mouse BMDMs induced by ATP and MSU in Example 1. In the figure, A and C show the protein expression of IL-1β and p20 in cell culture supernatant (sn) and pro-caspase-1, pro-IL-1β and β-actin in cell lysate (input). B and D show the secretion level of the inflammatory factor IL-1β in cell culture supernatant. Figure 3 The figure shows the results of HM43239 inhibiting Caspase-1-dependent pyroptosis in Example 1; where A shows the number and proportion of PI positive cells, B shows the LDH release level in cell culture supernatant (sn), and C shows the protein expression of GSDMD-FL and GSDMD-N in cell lysate. Figure 4 The figure shows the effect of HM43239 on inhibiting the activation of the non-classical NLRP3 inflammasome in mouse BMDMs in Example 1; where A shows the protein expression of IL-1β and p20 in cell culture supernatant (sn) and pro-caspase-1, pro-IL-1β and β-actin in cell lysate (input); B shows the secretion level of the pro-inflammatory cytokine IL-1β in cell culture supernatant. Figure 5 The figure shows the effect of HM43239 on the inhibition of classical NLRP3 inflammasome activation in human myeloid cells in Example 1. Among them, A shows the protein expression of IL-1β and p20 in human THP-1 cell culture supernatant (sn) and pro-caspase-1, pro-IL-1β and β-actin in cell lysate (input); B shows the secretion level of inflammatory factor IL-1β in human THP-1 cell culture supernatant; and C shows the secretion level of inflammatory factor IL-1β in human PBMC cell culture supernatant. Figure 6The image shows the results of HM43239 inhibiting NLRP3 inflammasome activation independently of FLT3 in Example 2; where A shows the secretion level of the inflammatory factor IL-1β in the cell culture supernatant, and B shows the relative expression level of the FLT3 gene in LSK cells and BMDM cells detected by RT-PCR. Figure 7 The image shows the results of HM43239 not affecting the LPS-mediated priming phase in Example 2; where A and B show the secretion levels of pro-inflammatory cytokines IL-6 and TNF-α in the cell culture supernatant, and C shows the protein expression levels of Pro-IL-1β, NLRP3, ASC, Pro-casp1, and β-actin in the cell lysate (input); Figure 8 This is a graph showing the results in Example 2 where HM43239 does not affect potassium ion efflux. Figure 9 This is a graph showing the results in Example 2 where HM43239 does not affect mitochondrial damage and reactive oxygen species production; Figure 10 In Example 3, HM43239 inhibited the binding of endogenous NEK7 to NLRP3 and NLRP3 to ASC; where A shows the expression levels of NLRP3 and NEK7 in cell immunoprecipitation (IP) and cell lysate (Input), and B shows the expression levels of NLRP3 and ASC in cell immunoprecipitation (IP) and cell lysate (Input); Figure 11 This is a diagram showing the results of the reversible binding of HM43239 to the NLRP3 inflammasome in Example 3; Figure 12 The figure shows the results of HM43239's effect on alleviating lipopolysaccharide-induced septic shock in mice in Example 4; where A and B show the secretion levels of inflammatory factors IL-1β and IL-6 in mouse serum, D and E show the secretion levels of inflammatory factors IL-1β and IL-6 in peritoneal fluid, and C shows the mouse survival curve; Figure 13 The figure shows the results of HM43239 alleviating Alum-induced acute peritonitis in mice in Example 4; where A shows the flow cytometry analysis of mouse neutrophils, B shows the proportion of neutrophils in mouse peritoneal fluid, C shows the number of neutrophils in mouse peritoneal fluid, and D shows the secretion level of the inflammatory factor IL-1β in mouse peritoneal fluid. Detailed Implementation

[0039] The following description, with reference to preferred embodiments, illustrates the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are merely illustrative of the present invention and not intended to limit the scope of protection of the present invention.

[0040] Experimental reagents The following experiments were conducted using HM43239 as an inhibitor of the NLRP3 inflammasome. The molecular formula of HM43239 is: C 29 H 33 ClN6 has the following molecular structure: .

[0041] The experimental materials and experimental animals are shown in Tables 1 to 4.

[0042] Table 1: Cell and Mouse Information

[0043] Table 2: Laboratory Consumables

[0044] Table 3: Antibody Information

[0045] Table 4: Experimental Reagents

[0046] To address the shortcomings of existing technologies, the purpose of this application is to provide the use of HM43239 in inhibiting NLRP3 inflammasome activation, and the use of HM43239 in the preparation of drugs for the prevention or treatment of inflammasome-related diseases.

[0047] Inflammation, as the core response of the body to injury and infection, is a driving force behind many chronic diseases when its regulation is imbalanced. At the molecular level, overactivation of pattern recognition receptor-mediated inflammatory signaling pathways is a key link leading to pathological inflammation. The NLRP3 inflammasome, as a representative and most extensively studied inflammasome, plays an important role in infections, autoimmune diseases, and metabolic diseases. HM43239 is a novel, oral, selective, and reversible type I multi-target tyrosine kinase inhibitor, initially widely studied for its highly effective and selective FLT3 inhibitor status. In addition to directly inhibiting FLT3 kinase activity, it can also regulate signaling pathways such as STAT5, ERK, SYK, and JAK. Preclinical studies have shown that HM43239 can effectively inhibit the proliferation of FLT3-ITD mutant leukemia cells and stem cell-like KG1a cells, induce apoptosis, and also exhibit dose-dependent antitumor effects and prolonged survival in animal models. Current research on HM43239 mainly focuses on its excellent antitumor effects, but its anti-inflammatory potential has not been reported. Therefore, HM43239 holds promise as a novel NLRP3-targeted anti-inflammatory drug. In vivo and in vitro experiments were designed to investigate the inhibitory effect of HM43239 on the NLRP3 inflammasome. By designing in vitro cell models and in vivo animal disease models, its inhibitory effect on the assembly and activation of the NLRP3 inflammasome was systematically evaluated. Specific implementation examples are as follows.

[0048] Example 1

[0049] Effects of HM43239 on the activation of the NLRP3 inflammasome in macrophages in vitro Experiment 1: HM43239 inhibits the activation of the classical NLRP3 inflammasome in mouse BMDMs The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 5 × 10⁶ cells / mL. 5Mouse BMDM cells were seeded at a concentration of 1 ml / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. BMDM cells were pre-stimulated for 3 h with LPS (100 ng / ml) of greater than 95% purity. The cells were then divided into five groups. Four groups were treated with different concentrations (0 μM, 7.5 μM, 10 μM, 12.5 μM) of HM43239 for 30 min, followed by the addition of Nigericin to trigger NLRP3 inflammasome activation for 30 min. The other group received neither HM43239 nor Nigericin. Cell culture supernatant and cell lysate were then collected. The secretion levels of IL-1β, TNF-α, and IL-6 in the cell culture supernatant were detected by ELISA, and the protein expression of caspase-1 (p20) and IL-1β in the cell culture supernatant was detected by Western blotting. The results are shown below. Figure 1 As shown.

[0050] from Figure 1 Analysis revealed that A showed the protein expression of IL-1β and p20 in cell culture supernatant (sn), and pro-caspase-1, pro-IL-1β, and β-actin in cell lysate (input); B, D, and E showed the secretion levels of inflammatory factors IL-1β, TNF-α, and IL-6 in cell culture supernatant; and C showed the effect of different concentrations of HM43239 on the secretion level of IL-1β in cell culture supernatant. This demonstrates that HM43239 can effectively inhibit Nigericin-induced NLRP3 inflammasome activation in BMDM cells.

[0051] Experiment 2: Effects of HM43239 on ATP- and MSU-induced activation of the classical NLRP3 inflammasome in mouse BMDMs ATP and MSU are also agonists that can effectively activate the NLRP3 inflammasome. Therefore, further investigation is needed to determine whether HM43239 can inhibit ATP and MSU-induced activation of the classical NLRP3 inflammasome in mouse BMDMs.

[0052] The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 5 × 10⁶ cells / mL. 5Cells were seeded at a density of 1 mL / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Mouse BMDM cells were pretreated with LPS (100 ng / mL) for 3 h, then divided into five groups. Four of these groups were treated with different concentrations (0 μM, 7.5 μM, 10 μM, 12.5 μM) of HM43239 for 30 min, followed by stimulation with ATP (12 mM) for 30 min. The other group was not treated with HM43239 or ATP. Cell culture supernatant and cell lysate were collected from both groups.

[0053] Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 5 × 10⁶ cells / mL. 5 Cells were seeded at a density of 1 mL / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Mouse BMDM cells were pretreated with LPS (100 ng / mL) for 3 h, then divided into five groups. Four of these groups were treated with different concentrations (0 μM, 2 μM, 4 μM, 6 μM) of HM43239 for 30 min, followed by stimulation with MSU (120 μg / mL) for 4 h. The other group received neither HM43239 nor MSU. Cell culture supernatant and cell lysate were collected separately.

[0054] The secretion level of the inflammatory factor IL-1β in cell culture supernatant was detected by ELISA; the protein expression of caspase-1 (p20) and IL-1β in cell culture supernatant (sn) and the protein expression of Pro-casp1, Pro-IL-1β, and β-actin in cell lysate (Input) were detected by Western blotting. Results are as follows: Figure 2 As shown.

[0055] from Figure 2 Analysis showed that A and C represented the protein expression of IL-1β and p20 in cell culture supernatant (sn), and pro-caspase-1, pro-IL-1β, and β-actin in cell lysate (input). B and D represented the secretion level of the inflammatory factor IL-1β in cell culture supernatant. This demonstrates that HM43239 can effectively inhibit ATP and MSU-induced activation of the classical inflammasome NLRP3 in mouse BMDMs.

[0056] Experiment 3: Effect of HM43239 on Caspase-1-dependent pyroptosis When the NLRP3 inflammasome is activated, the previously inactive Pro-caspase-1 undergoes self-cleavage, transforming into active caspase-1. This process involves two aspects: firstly, the maturation and release of IL-1β and IL-18 from their inactive precursor forms; and secondly, the cleavage of the GSDMD protein to generate an N-terminal domain (GSDMD-N). This domain forms pores in the cell membrane, triggering pyroptosis.

[0057] The specific operating steps are as follows: PI detection of apoptosis: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 1×10⁻⁶. 6 Cells were seeded at 2 ml / well in 6-well plates and cultured overnight at 37°C with 5% CO2. Mouse BMDM cells were pretreated with LPS (100 ng / ml) for 3 h and divided into three groups. The third group was then treated with HM43239 (12.5 μM) for 30 min, while the second and third groups were activated with Nigericin for 30 min. The first group was not treated with HM43239 or Nigericin. The supernatant was discarded by pipette, and trypsin was added to each group for 2 min at 37°C. Digestion was terminated by adding DMEM medium. Cells were collected in 1.5 mL EP tubes and centrifuged at 5000 rpm for 2 min at 4°C. The supernatant was discarded, and the cells were resuspended in 100 μl of binding buffer. PI antibody was then added for 15 min. Flow cytometry was performed.

[0058] LDH release: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 5 × 10⁶ cells / mL. 5Cells were seeded at a density of 1 mL / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Mouse BMDM cells were pretreated with LPS (100 ng / mL) for 3 h and then divided into six groups. Group 1 received no treatment, Group 2 received LPS pretreatment only, and Groups 3 through 6 were treated with different concentrations of HM43239 (0 μM, 7.5 μM, 10 μM, 12.5 μM) for 0.5 h. 15 min after inoculation, 50 μl of Lysis Solution (10×) was added to the control well (maximum release well). 30 min after inoculation, Nigericin was added to Groups 3 through 6 to stimulate cell activation, and the cell activation status was observed under a microscope. After stimulation, the supernatant was collected, centrifuged (10000 rpm, 5 min), and the supernatant was collected. Substrate Mix was added to each well of a 96-well microplate, followed by cell supernatant, and incubated at room temperature in the dark for 30 min. The reaction was terminated by adding the stop solution, and the optical density was immediately measured at 490 nm using an ELISA reader.

[0059] WB detection: BMDM cells (bone marrow-derived macrophages) were pre-stimulated for 3 h with LPS (100 ng / ml) with a purity greater than 95%, and then divided into four groups, with each group receiving 5 × 10⁶ BMDM cells. 5 Next, mouse BMDM cells in groups three and four were treated with HM43239 (12.5 μM) for 30 minutes. Then, Nigericin was added to groups two and four to trigger NLRP3 inflammasome activation for 30 minutes. Group one received neither HM43239 nor Nigericin. Cell lysates were collected from each group. Western blotting was used to detect the protein expression of GSDMD-FL and GSDMD-N in the cell culture lysates.

[0060] Flow cytometry detection of PI + The proportions were statistically analyzed; LDH release levels were detected by ELISA; and protein expression was detected by Western blot: GSDMD-FL and GSDMD-N. Results are as follows: Figure 3 As shown.

[0061] from Figure 3 Analysis revealed that A showed the number and proportion of PI-positive cells, B showed the LDH release level in the cell culture supernatant (sn), and C showed the protein expression of GSDMD-FL and GSDMD-N in the cell lysate. This demonstrates that HM43239 can effectively reduce pyroptosis-related indicators.

[0062] Experiment 4: Effect of HM43239 on non-classical NLRP3 inflammasome activation in BMDMs cells The NLRP3 inflammasome can be activated through three pathways: the classical pathway, the non-classical pathway, and the alternative pathway. The above results indicate that HM43239 can effectively inhibit the classical activation pathway. To investigate the effect of HM43239 on the non-classical pathway, this experiment delivered LPS into the cytoplasm via intracellular transfection, specifically activating the non-classical pathway.

[0063] The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 5 × 10⁶ cells / mL. 5 Cells were seeded at 1 mL / well in 12-well plates and cultured overnight at 37°C with 5% CO2. Mouse BMDM cells were pretreated with Pam3CSK4 (palmitoyl triacyl lipopeptide) (200 ng / mL) for 3 h, then divided into five groups. Four groups were treated with different concentrations of HM43239 (0 μM, 0.5 μM, 1 μM, 2 μM) for 30 min, followed by transfection with LPS. The other group did not receive HM43239 or LPS transfection. After 16 h, cell culture supernatant and cell lysate were collected. The secretion level of the inflammatory factor IL-1β in the cell culture supernatant was detected by ELISA. Western blotting was used to detect the protein expression of IL-1β and caspase-1 (p20) in the cell culture supernatant (sn) and the protein expression of pro-caspase-1, pro-IL-1β, and β-actin in the cell lysate (Input). The results are as follows. Figure 4 As shown.

[0064] from Figure 4 Analysis revealed that A showed the protein expression of IL-1β and p20 in cell culture supernatant (sn), and pro-caspase-1, pro-IL-1β, and β-actin in cell lysate (input); B showed the secretion level of the pro-inflammatory cytokine IL-1β in cell culture supernatant. This demonstrates that HM43239 dose-dependently inhibits non-canonical NLRP3 inflammasome activation in mouse BMDM cells. Therefore, it is proven that HM43239 can also inhibit the non-canonical activation pathway of the NLRP3 inflammasome.

[0065] Experiment 5: Effect of HM43239 on activation of the classical NLRP3 inflammasome in human myeloid cells This experiment further evaluated the role of HM43239 in human THP-1 and PBMC cells.

[0066] The specific operating steps are as follows: THP-1 cells were collected, resuspended in 1640 complete culture medium, and the density was adjusted to 5 × 10⁶ cells / year. 5 Cells were seeded at 1 mL / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Human THP-1 cells were pretreated with LPS for 3 h and then divided into five groups. Four of these groups were treated with different concentrations (0 μM, 30 μM, 40 μM, 50 μM) of HM43239 for 0.5 h and induced with Nigericin for 30 min. The other group was not treated with HM43239 or induced with Nigericin. Cell supernatant and cell pellet were collected from each group.

[0067] Collect PBMC cells, resuspend them in complete 1640 medium, and adjust the density to 5 × 10⁶ cells / year. 5 Cells were seeded at a density of 1 mL / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Human PBMCs were pretreated with LPS for 3 h and then divided into five groups. Four of these groups were treated with different concentrations (0 μM, 0.5 μM, 0.75 μM, and 1 μM) of HM43239 for 0.5 h, followed by induction with Nigericin for 30 min. The other group was treated without HM43239 and Nigericin. Cell supernatant and cell pellet were collected from each group.

[0068] The expression of IL-1β and caspase-1 proteins in human THP-1 cells was detected by Western blot, and the secretion level of the inflammatory factor IL-1β in human THP-1 cells and PBMC cells was detected by ELISA. Results are as follows: Figure 5 As shown.

[0069] from Figure 5 Analysis revealed that A showed the protein expression of IL-1β and p20 in the culture supernatant (sn) of human THP-1 cells, and pro-caspase-1, pro-IL-1β, and β-actin in the cell lysate (input); B showed the secretion level of the inflammatory cytokine IL-1β in the culture supernatant of human THP-1 cells; and C showed the secretion level of the inflammatory cytokine IL-1β in the culture supernatant of human PBMC cells. This demonstrates that HM43239 can effectively block the activation of the NLRP3 inflammasome in human myeloid cells.

[0070] The above studies confirmed the inhibitory effect of HM43239 on NLRP3 inflammasome activation in human THP-1 cells, PBMC cells, and mouse BMDMs cells, indicating that HM43239 can inhibit NLRP3 inflammasome activation in both human and mouse cells. Considering that NLRP3 inflammasomes can be activated by a variety of pathogens and endogenous danger molecular patterns (DAMPs), the above results reveal the broad-spectrum inhibitory effect of HM43239 on classical inflammasome activation induced by multiple activating factors.

[0071] Example 2

[0072] HM43239 does not affect the upstream signaling stage of NLRP3 inflammasome assembly activation. I. HM43239 independently inhibits NLRP3 inflammasome activation. HM43239, as a type I multi-target tyrosine kinase inhibitor, has attracted attention in the field of leukemia treatment due to its highly efficient inhibition of FLT3. Given its kinase inhibition spectrum, this study investigates whether its inhibitory effect on the NLRP3 inflammasome depends on FLT3.

[0073] The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in complete DMEM medium, and adjust the density to 5 × 10⁶ cells / mL. 5 Cells were seeded at a density of 1 mL / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Cells were divided into seven groups: Group 1 consisted of untreated mouse BMDM cells; Groups 2-7 were pretreated with LPS for 3 h; Group 2 consisted only of LPS-pretreated mouse BMDM cells; Group 3 was treated with Nigericin for 30 min; Groups 4-7 were first treated with HM43239 (12.5 μM) for 0.5 h; Group 4 was induced with Nigericin for 30 min; Group 5 was induced with 1 μg / mL FLT3-specific agonist and Nigericin for 30 min; Group 6 was induced with 2 μg / mL FLT3-specific agonist and Nigericin for 30 min; and Group 7 was induced with 2 μg / mL FLT3-specific agonist for 30 min. Cell supernatant and cell pellet were collected. The secretion level of the inflammatory cytokine IL-1β in the cell supernatant was detected by ELISA. Simultaneously, RT-PCR was used to detect the relative expression levels of the FLT3 gene in LSK cells and BMDM cells. The results are as follows: Figure 6 As shown.

[0074] from Figure 6Analysis revealed that A shows the secretion level of the inflammatory cytokine IL-1β in the cell culture supernatant, and B shows the relative expression level of the FLT3 gene in LSK cells and BMDM cells detected by RT-PCR. This demonstrates that HM43239 inhibits NLRP3 inflammasome activation independently of FLT3.

[0075] II. The effect of HM43239 on LPS-mediated priming process To further understand the mechanism by which HM43239 affects NLRP3 inflammasome activation, this study first explored whether HM43239 interferes with the priming phase of NLRP3 inflammasome activation. NLRP3 inflammasome activation can be divided into two phases: priming and assembly activation.

[0076] The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 5 × 10⁶ cells / mL. 5 Cells were seeded at a density of 1 mL / well in 12-well plates and cultured overnight at 37°C in a 5% CO2 incubator. Mouse BMDM cells were divided into ten groups. Mice were stimulated with LPS (100 ng / ml) for 3 h. Groups 2 through 5 were treated with different concentrations (0 μM, 7.5 μM, 10 μM, 12.5 μM) of HM43239 for 30 min. Group 1 received no HM43239 or LPS. Groups 7 through 10 were treated with different concentrations (0 μM, 7.5 μM, 10 μM, 12.5 μM) of HM43239 for 30 min, followed by stimulation with LPS (100 ng / ml) for 3 h. Group 6 received no LPS or HM43239. Cell culture supernatant and cell lysis buffer were collected. The expression levels of inflammatory cytokines IL-6 and TNF-α were detected by ELISA, and the expression of key components of the NLRP3 inflammasome, NLRP3, ASC, and Pro-casp1, were detected by Western blotting. Results are as follows: Figure 7 As shown.

[0077] from Figure 7 Analysis showed that A and B represented the secretion levels of pro-inflammatory cytokines IL-6 and TNF-α in the cell culture supernatant, while C represented the protein expression levels of Pro-IL-1β, NLRP3, ASC, Pro-casp1, and β-actin in the cell lysate (input). This demonstrates that HM43239 does not affect the LPS-mediated priming phase.

[0078] III. Effects of HM43239 on potassium ion efflux Potassium ion efflux is one of the upstream signals for NLRP3 inflammasome activation, playing a common but non-essential role in the process.

[0079] The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 1×10⁶. 6 Cells were seeded at 1 / mL in 6cm culture dishes (4ml / dish) and cultured overnight at 37°C in a 5% CO2 incubator. Mouse BMDM cells were pretreated with LPS (100 ng / mL) for 3 h and then divided into five groups. Groups 2 through 5 were treated with different concentrations of HM43239 (0μM, 7.5μM, 10μM, 12.5μM) for 30 min, followed by Nigericin (10 μM) for 30 min. Group 1 received neither HM43239 nor Nigericin. After cell lysis with concentrated nitric acid, intracellular potassium ion content was detected by atomic plasma chromatography. The results are shown below. Figure 8 As shown.

[0080] from Figure 8 Analysis shows that HM43239 does not affect potassium ion efflux.

[0081] IV. Effects of HM43239 on Nigericin-induced mitochondrial damage and reactive oxygen species production Mitochondrial damage and the generation of reactive oxygen species (ROS) play a crucial role in the assembly and activation of the NLRP3 inflammasome. This experiment investigated whether HM43239 affects this upstream signaling stage.

[0082] The specific operational steps were as follows: Mouse BMDM cells were stimulated with LPS (100 ng / ml) for 3 hours, then treated with HM43239 (12.5 μM) for 30 minutes, followed by Nigericin (10 μM) treatment for another 30 minutes. Cells were stained with DAPI, Mitotracker, and MitoSox. Mitochondrial damage and reactive oxygen species production in BMDM cells were observed and photographed using a laser confocal microscope. The results are as follows: Figure 9 As shown.

[0083] from Figure 9 Analysis showed that HM43239 does not affect mitochondrial damage or reactive oxygen species production.

[0084] Example 3

[0085] Mechanism by which HM43239 inhibits NLRP3 inflammasome activation I. HM43239 inhibits the assembly of the NLRP3 inflammasome. The assembly of the NLRP3 inflammasome depends on the ordered binding and oligomerization of core components such as NEK7, NLRP3, ASC, and Pro-caspase-1. The experimental results above have confirmed that HM43239 does not affect upstream signals in the activation phase. Therefore, this study investigates whether HM43239 directly affects the inflammasome assembly process.

[0086] The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 1×10⁶. 6 Cells were seeded at 2 ml / well in 6-well plates and cultured overnight at 37°C with 5% CO2. Mouse BMDM cells were pretreated with LPS (100 ng / mL) for 3 h and then divided into six groups. Groups 3 and 6 were treated with HM43239 (12.5 μM) for 30 min. Subsequently, Nigericin (10 μM) was added to groups 2 and 5 to activate the cells, while groups 1 and 3 were not treated with HM43239 or Nigericin. Cell culture supernatant and cell lysate were collected. Western blot was used to detect the levels of NLRP3 and NEK7 proteins and the levels of NLRP3 and ASC proteins in immunoprecipitation (IP) and cell lysate (Input). Results are as follows: Figure 10 As shown.

[0087] from Figure 10 Analysis showed that A represented the expression levels of NLRP3 and NEK7 in cell immunoprecipitation (IP) and cell lysate (Input), while B represented the expression levels of NLRP3 and ASC in the same samples. Immunoprecipitation was used to detect protein-protein interactions, and analysis revealed that HM43239 effectively blocked the binding of NEK7 to NLRP3 and simultaneously inhibited the interaction between NLRP3 and ASC. Figure 10 (A, B) indicates that HM43239 may affect the activation of the NLRP3 inflammasome by interfering with the interactions between these proteins. This demonstrates that HM43239 can effectively inhibit the assembly of the NLRP3 inflammasome.

[0088] II. HM43239 binds reversibly to NLRP3 protein. To further investigate the interaction characteristics between HM43239 and the NLRP3 inflammasome, this experiment used an elution assay to assess the reversibility of this interaction.

[0089] The specific steps are as follows: Collect BMDMs (bone marrow-derived macrophages), resuspend them in DMEM complete medium, and adjust the density to 5 × 10⁶ cells / mL. 5Cells were seeded at 1 mL / well in 12-well plates and cultured overnight at 37°C with 5% CO2. Mouse BMDM cells were pretreated with LPS (100 ng / mL) for 3 h and then divided into twelve groups. Groups 3-6 and 9-12 were treated with different concentrations of HM43239 (0 μM, 7.5 μM, 10 μM, 12.5 μM). After treating groups 9-12 with HM43239 for 15 min, the cells were washed with fresh DMEM medium three times every 5 minutes to remove unbound HM43239. Groups 2-6 and 8-12 were then stimulated with Nigericin for 30 min. Cell supernatants were collected, and the secretion of the inflammatory factor IL-1β was detected by ELISA. Results are as follows: Figure 11 As shown.

[0090] from Figure 11 Analysis showed that the elution experiment demonstrated that HM43239 and NLRP3 protein bind reversibly, indicating that HM43239 can reversibly bind to the NLRP3 inflammasome.

[0091] Example 4

[0092] In vivo study of HM43239 inhibiting the NLRP3 inflammasome I. Effects of HM43239 on LPS (lipopolysaccharide)-induced septic shock in mice Septic shock is closely associated with abnormal activation of the NLRP3 inflammasome. A mouse model of intraperitoneal injection of LPS was used to simulate human septic shock, which significantly increased the levels of pro-inflammatory cytokines (such as IL-1β and IL-6) in mice.

[0093] Construction of an LPS-induced mouse model of septic shock: Eight-week-old male C57BL / 6J mice of similar weight were randomly divided into three groups of six each: a blank control group (Control group), a septic shock model group (LPS group), and an HM43239-pretreated septic shock model group (LPS+HM43239 group). Mice were separated the night before to avoid stress interfering with the experimental results. The next morning, mice in the LPS+HM43239 group were intraperitoneally injected with HM43239 (20 mg / kg), while mice in the Control group were injected with an equal volume of PBS. Fifty minutes later, except for the blank control group, the other groups were injected with LPS solution (20 mg / kg) to induce septic shock. Four hours after LPS treatment, ocular blood was collected by enucleation. Mice were then sacrificed by cervical dislocation to collect peritoneal lavage fluid. The blood samples were allowed to stand at room temperature for 3–4 hours, and then centrifuged at 8500 rpm for 5 minutes at 4 °C. Collect the supernatant serum and transfer it to a new EP tube, taking care to avoid touching the cell layer. Centrifuge the collected mouse peritoneal lavage fluid and then transfer the supernatant to a new tube. Use an ELISA kit to determine the secretion levels of relevant inflammatory factors (IL-1β and IL-6) in mouse serum and peritoneal lavage fluid.

[0094] Survival curves were plotted as follows: The control group (no disease model induction or drug treatment) included: LPS model group (20 mg / kg injection); and the HM43239 pretreated LPS model group (LPS+HM43239): mice were pre-treated with HM43239 (20 mg / kg) followed by LPS solution (20 mg / kg). Mice mortality was closely monitored and recorded within 72 hours of LPS treatment. Survival curves were plotted using GraphPad Prism 8.0.2 software. Results are as follows: Figure 12 As shown.

[0095] from Figure 12 Analysis showed that A and B represent the secretion levels of inflammatory factors IL-1β and IL-6 in mouse serum, D and E represent the secretion levels of inflammatory factors IL-1β and IL-6 in peritoneal fluid, and C represents the mouse survival curve. This demonstrates that HM43239 effectively alleviated LPS-induced septic shock in mice.

[0096] II. Effects of HM43239 on Alum-induced acute peritonitis in mice Alum-induced acute peritonitis is a common NLRP3 inflammasome-related model. This experiment was designed to induce acute peritonitis by intraperitoneal injection of Alum and to observe the intervention effect of HM43239.

[0097] Construction of an Alum-induced mouse model of acute peritonitis: Eight-week-old SPF-grade male C57BL / 6J mice of similar weight were randomly divided into three groups of six mice each: a blank control group (Control), an Alum-induced acute peritonitis model group (Alum), and an HM43239 intervention group (HM43239 + Alum). Mice in the HM43239 intervention group were first intraperitoneally injected with HM43239 (20 mg / kg), while the Control group was injected with an equal volume of PBS. One hour later, mice in the Alum group and the HM43239 + Alum group were intraperitoneally injected with Alum (1 mg / mouse) to establish an acute peritonitis model. Twelve hours after modeling, ocular blood was collected from the mice, followed by cervical dislocation, and peritoneal lavage fluid was collected. Evaluation was performed using the following methods: ① Peritoneal cells were counted and aspirated. After antibody labeling, the proportion of neutrophils in the peritoneal lavage fluid was detected by flow cytometry, and their absolute number was calculated. ② ELISA was used to detect the secretion levels of inflammatory factors (IL-1β) in ocular blood and peritoneal fluid. Results are as follows... Figure 13 As shown.

[0098] from Figure 13 Analysis revealed that: A) flow cytometry analysis showed the number of neutrophils in mice; B) the proportion of neutrophils in mouse peritoneal fluid; C) the number of neutrophils in mouse peritoneal fluid; and D) the secretion level of the inflammatory cytokine IL-1β in mouse peritoneal fluid. This demonstrates that HM43239 effectively alleviated Alum-induced acute peritonitis in mice.

[0099] The above studies fully demonstrate that HM43239 significantly inhibits inflammatory cell infiltration and inflammatory cytokine secretion in LPS-induced septic shock, and also significantly inhibits Alum-induced acute peritonitis, neutrophil infiltration, and inflammatory factor secretion. Moreover, these effects are all dependent on the NLRP3 inflammasome, which effectively alleviates disease symptoms, further clarifying its crucial role in the regulation of inflammatory responses. This finding not only provides experimental evidence for revealing the molecular mechanism by which HM43239 inhibits the NLRP3 inflammasome, but also points to a new direction for the development of treatment strategies for NLRP3-related diseases such as septic shock.

[0100] The constructed mouse inflammation model revealed that HM43239 exhibited good efficacy in treating systemic inflammation and acute peritonitis in mice. Specifically, HM43239 dose-dependently reduced the expression level of IL-1β in mouse serum, while having no significant effect on IL-6, which is unrelated to the NLRP3 inflammasome. 72-hour survival curve analysis showed that mice treated with LPS, particularly those treated with HM43239, had significantly longer survival times compared to the control group, demonstrating the good therapeutic effect of HM43239 at lower concentrations.

[0101] Acute peritonitis is an inflammatory disease characterized by the rapid recruitment and infiltration of neutrophils. Activation of the NLRP3 inflammasome plays a crucial driving role in this process, prompting the release of inflammatory mediators such as IL-1β. We used an Alum-induced mouse model of acute peritonitis. Experimental results showed that, compared to the model group, HM43239 intervention reduced the number and proportion of neutrophils in the peritoneal cavity, as well as the serum level of the inflammatory cytokine IL-1β. Combining the results of HM43239 studies in two classic models—LPS-induced septic shock and Alum-induced acute peritonitis—HM43239 can effectively alleviate tissue inflammatory cell infiltration and the release of key cytokines by inhibiting the NLRP3 inflammasome pathway.

[0102] In summary, the mechanism and efficacy of HM43239 as an NLRP3 inflammasome inhibitor provided in this application are as follows: At the cellular level, HM43239 can effectively inhibit the activation of the NLRP3 inflammasome in BMDM and various human cell lines. Its mechanism of action is to inhibit the specific binding between NLRP3 and NEK7, affecting the assembly of the NLRP3 inflammasome, thereby inhibiting its activation. At the animal level, HM43239 significantly alleviated disease-related inflammatory markers and demonstrated good anti-inflammatory activity in LPS-induced septic shock and Alum-induced acute peritonitis models. These results consistently indicate that HM43239 is an NLRP3 inflammasome inhibitor with a clear mechanism of action, broad-spectrum inhibitory activity, and high targeting, suggesting that HM43239 is a promising candidate drug for treating NLRP3 inflammasome-related diseases, with significant development potential and clinical application prospects. It also has application value in the field of biomedical materials technology.

[0103] The above embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. The application of HM43239, characterized in that, The application of HM43239 in the preparation of NLRP3 inflammasome inhibitors.

2. The application according to claim 1, characterized in that, The NLRP3 inflammasome inhibitor is prepared by adding pharmaceutical excipients with HM43239 as the active ingredient. And / or, the dosage form of the NLRP3 inflammasome inhibitor is at least one of tablets, capsules, granules, powders, patches, suspensions, oral liquids, injections, and suppositories.

3. The application according to claim 1, characterized in that, Application of HM43239 as an inhibitor of Nigericin-induced NLRP3 inflammasome activation in BMDMs cells.

4. The application of HM43239 is characterized by, The application of HM43239 in the preparation of drugs for treating septic shock.

5. The application according to claim 4, characterized in that, The septic shock mentioned is lipopolysaccharide (LPS)-induced septic shock.

6. The application according to claim 4, characterized in that, The effective dose of HM43239 in the treatment of septic shock is 20 mg / kg.

7. The application of HM43239 is characterized by, The application of HM43239 in the preparation of drugs for treating peritonitis.

8. The application according to claim 7, characterized in that, The peritonitis was Alum-induced acute peritonitis.

9. The application according to claim 7, characterized in that, The effective dose of HM43239 in the treatment of peritonitis is 20 mg / kg.

10. The application according to claim 1, characterized in that, The application of HM43239 in the preparation of drugs that reduce the content of IL-1β in serum and peritoneal lavage fluid; And / or, the use of HM43239 in the preparation of a drug that reduces the proportion and absolute count of neutrophils in peritoneal lavage fluid while inhibiting the release of IL-1β.

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