Application of alvitinib in preparation of NLRP3 inflammasome inhibitor

Abstract

The invention relates to an application of alvitinib in preparation of an NLRP3 inflammasome inhibitor. The alvetinib is used for preparing a medicament for treating infectious shock, a medicament for treating acute liver injury and a medicament for treating peritonitis. A new choice is provided for prevention or treatment of NLRP3 inflammasome related diseases, the problem of treatment of EGFR mutation lung cancer patients combined with NLRP3 inflammasome related diseases (infectious shock / acute liver injury / acute peritonitis) is solved, tumor progression and inflammatory response are inhibited at the same time through the Aviitinib (third-generation EGFR-TKI), and the NLRP3 inflammasome related diseases are treated through the Aviitinib (third-generation EGFR-TKI). It is suggested that the medicine is preferentially selected by the patients, and the medicine with higher treatment potential is provided for treatment of infectious shock medicines, acute liver injury medicines and acute peritonitis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of avitinib in the preparation of NLRP3 inflammasome inhibitors. Background Art

[0002] Avinitinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) independently developed in China, mainly targeting EGFR sensitive mutations (such as L858R, exon 19 deletion) and drug-resistant mutations (T790M) in non-small cell lung cancer (NSCLC). Currently, the clinical research and development of Avitinib focuses on the second-line treatment of advanced NSCLC with EGFR T790M positivity. A number of phase I / II clinical trials have been completed, and the data show that its objective response rate (ORR) to T790M mutations reaches 50%-60%, with high anti-tumor activity. Currently, the research on Avitinib mainly focuses on its excellent anti-tumor effect, but there are no relevant reports on its anti-inflammatory potential.

[0003] The NLRP3 inflammasome is an important sensor for detecting exogenous pathogenic invasions and endogenous cell damage, consisting of three main components: NLRP3, the adaptor protein ASC, and the effector caspase-1. It plays a key role in the immune system, can respond to diverse stimuli, and trigger inflammatory responses. The NLRP3 inflammasome activates caspase-1 by recognizing intracellular and extracellular stimuli, such as infections, tissue damage, and metabolic disorders, promotes the maturation and release of IL-1β and IL-18, and triggers inflammatory responses. This process is very important in the development of various inflammatory diseases. The abnormal activation of the NLRP3 inflammasome is associated with a variety of diseases, including metabolic diseases (such as obesity, type 2 diabetes, atherosclerosis, etc.), cardiovascular diseases (such as ischemic and non-ischemic heart diseases, etc.), and neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, etc.).

[0004] Currently, the treatment strategies for the NLRP3 inflammasome mainly include inhibiting its activation, blocking the effects of related cytokines, etc. Well-known inhibitors include MCC950, CY-09, Anakinra, curcumin, etc. Although there are many NLRP3 inflammasome inhibitors, currently, no inhibitor can be applied clinically. Moreover, a variety of small molecules targeting NLRP3 have been reported to potentially have safety risks. Therefore, it is particularly important to seek new candidate drugs for safely and effectively inhibiting the NLRP3 inflammasome. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide the application of ivitinib in the preparation of NLRP3 inflammasome inhibitors, to provide a new option for the prevention or treatment of various NLRP3 inflammasome-related diseases, and to solve the problem of slow progress in clinical trials of existing NLRP3 inhibitors, and its safety has been preliminarily verified.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The application of ivitinib in the preparation of NLRP3 inflammasome inhibitors.

[0007] The inventors of this case found in long-term experimental studies that Avitinib can not only inhibit the activation of classical NLRP3 inflammasome in Nigericin-induced BMDM cells, and inhibit the activation of NLRP3 inflammasome in THP-1 cells and human PBMCs, but also inhibit the activation of NLRP3 inflammasome induced by a variety of agonists, thus fully demonstrating that ivitinib can be used as an NLRP3 inflammasome inhibitor, providing a new option for the prevention or treatment of NLRP3 inflammasome.

[0008] Preferably, the dosage of ivitinib is 0-20 μM. Here, 0-20 μM means greater than 0 μM and less than or equal to 20 μM.

[0009] Preferably, the dosage of ivitinib is 5 μM, 10 μM or 20 μM.

[0010] Preferably, the NLRP3 inflammasome inhibitor is prepared with ivitinib as the active ingredient and added with pharmaceutical excipients.

[0011] Preferably, the dosage form of the NLRP3 inflammasome inhibitor is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections and suppositories.

[0012] Preferably, ivitinib is used for the preparation of drugs for treating septic shock.

[0013] Preferably, the septic shock is LPS-induced septic shock.

[0014] By constructing an LPS-induced septic shock model and detecting by ELISA technology, it was found that pre-administering Avitinib (10 mg / kg) could significantly reduce the levels of IL-1β in serum and peritoneal lavage fluid, but had no obvious effect on IL-6 and TNF-α, proving that ivitinib as an NLRP3 inhibitor can directly target the NLRP3 inflammasome and relieve septic shock in mice. At the same time, in the survival curve model, the survival time and number of mice were extended compared with the model group, proving the new anti-inflammatory mechanism of Avitinib in septic shock.

[0015] Among them, LPS (Lipopolysaccharide) is the main component of the outer membrane of the cell wall of Gram-negative bacteria (such as Escherichia coli, Salmonella, etc.), and is composed of three parts: lipid A, core polysaccharide, and O-antigen polysaccharide. Among them, lipid A is the toxic core of LPS and can trigger a strong immune response in the host.

[0016] Preferably, the dosage of the ivitinib is 10 mg / kg.

[0017] Through experimental research, it was found that the Avitinib of the present invention showed better improvement effect on septic shock at a low dose (10 mg / kg) compared with the second-generation tyrosine kinase inhibitor afatinib (15 mg / kg). This proves that Avitinib may have higher bioavailability and stronger anti-inflammatory activity, providing an important experimental basis for the development of new drugs for septic shock.

[0018] Preferably, the drug for treating septic shock is prepared by adding pharmaceutical excipients with the ivitinib as the active ingredient.

[0019] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0020] Preferably, ivitinib is used for preparing a drug for treating acute liver injury.

[0021] Preferably, the acute liver injury is ConA-induced acute liver injury.

[0022] From the experimental results of constructing a ConA-induced acute liver injury model, it was found that Avitinib could significantly reduce the levels of AST, ALT, and related inflammatory factors, indicating that Avitinib could effectively relieve ConA-induced acute liver injury. It is shown that Avitinib may improve acute liver injury in mice by targeting and reducing the secretion of IL-1β induced by the activation of NLRP3 inflammasome.

[0023] Preferably, the dosage of the ivitinib is 20 mg / kg.

[0024] Preferably, the drug for treating acute liver injury is prepared by adding pharmaceutical excipients with the ivitinib as the active ingredient.

[0025] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0026] Preferably, ivitinib is used for preparing a drug for treating peritonitis.

[0027] Preferably, the peritonitis is Alum-induced acute peritonitis.

[0028] An acute peritonitis model in mice was induced by intraperitoneal injection of Alum (1 mg / mouse), and the therapeutic effect of Avitinib was evaluated in multiple dimensions. Flow cytometry analysis showed that the proportion and absolute number of Ly6G + CD11b + neutrophils in the peritoneal lavage fluid of mice in the Avitinib treatment group were significantly decreased. Meanwhile, the ELISA test results showed that Avitinib could significantly inhibit the release of inflammatory factor (IL-1β) in the peritoneal lavage fluid. These data fully demonstrated that Avitinib could effectively relieve Alum-induced acute peritonitis.

[0029] Preferably, the dosage of Avitinib is 30 mg / kg.

[0030] Experimental studies found that Avitinib of the present invention showed comparable or even better therapeutic effects at a low dose (30 mg / kg) compared with myricetin (40 mg / kg) and naringin (300 mg / kg). This demonstrated that Avitinib might have higher bioavailability and stronger anti-inflammatory activity, providing important clues for the development of new anti-peritonitis drugs.

[0031] Preferably, the drug for treating peritonitis is prepared by adding pharmaceutical excipients with Avitinib as the active ingredient.

[0032] Preferably, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections, and suppositories.

[0033] The present invention also provides the use of Avitinib in the preparation of drugs for NLRP3-related diseases such as sepsis, gout, neurodegenerative diseases, and metabolic syndrome.

[0034] Advantages of the present invention: The application of ivitinib of the present invention in the preparation of NLRP3 inflammasome inhibitors. After sacrificing mice by constructing an LPS-induced septic shock model, ELISA technology was used to detect that pre-administering Avitinib (10 mg / kg) could significantly reduce the level of IL-1β in serum and peritoneal lavage fluid, but had no obvious effect on IL-6 and TNF-α, demonstrating that ivitinib, as an NLRP3 inhibitor, can directly target the NLRP3 inflammasome. At the same time, in the survival curve model, the survival time and number of mice were extended compared with the model group, revealing a new anti-inflammatory mechanism of Avitinib in septic shock. In a ConA-induced acute liver injury model in mice, the experimental results showed that Avitinib could significantly reduce the levels of AST, ALT and related inflammatory factors, indicating that Avitinib could effectively alleviate ConA-induced acute liver injury. It was shown that Avitinib could improve acute liver injury in mice by targeting and reducing the secretion of IL-1β induced by NLRP3 inflammasome activation. In an acute peritonitis model induced by intraperitoneal injection of Alum (1 mg / mouse), the therapeutic effect of Avitinib was evaluated multi-dimensionally. Flow cytometry analysis showed that the proportion and absolute number of Ly6G + CD11b + neutrophils in the peritoneal lavage fluid of mice in the Avitinib treatment group were significantly reduced. At the same time, the ELISA test results showed that Avitinib could significantly inhibit the release of inflammatory factors (IL-1β) in the peritoneal lavage fluid. These data fully demonstrated that Avitinib could effectively alleviate Alum-induced acute peritonitis. The above research results all demonstrated that ivitinib has a significant inhibitory effect on the NLRP3 inflammasome, thus providing a new option for the prevention or treatment of the NLRP3 inflammasome. In addition, as a third-generation clinical EGFR-TKI, Avitinib is currently mainly applied to patients with non-small cell lung cancer (NSCLC). Since such patients often suffer from various diseases such as tumor-related systemic inflammatory responses or infectious complications, the NLRP3 inhibitory effect discovered in this study may provide additional translational possibilities for its clinical efficacy. It has the value of popularization and application in the field of biomedical materials technology. Description of the Drawings

[0035] Figure 1This is the result diagram of the effect of Avitinib in inhibiting Nigericin (name: nigericin; stimulating the activation of NLRP3 inflammasome and thus stimulating the secretion of IL-1β cytokine) - induced NLRP3 inflammasome activation in BMDM cells in Example 1 of the present invention; among them, A shows the protein expression of IL-1β and p20 in the cell culture supernatant (SN), B shows the secretion level of the inflammatory factor IL-1β in the cell culture supernatant; C shows the secretion level of the inflammatory factor IL-6 in the cell culture supernatant; D shows the secretion level of the inflammatory factor TNF-α in the cell culture supernatant; Figure 2 This is the result diagram of the effect of Avitinib in inhibiting Nigericin - induced NLRP3 inflammasome activation in human THP-1 cells and PBMCs in Example 1 of the present invention; among them, A shows the protein expression of p20 in the THP-1 cell culture supernatant (SN), B shows the secretion level of the inflammatory factor IL-1β in the THP-1 cell culture supernatant, and C shows the secretion level of the inflammatory factor IL-1β in the PBMC cell culture supernatant; Figure 3 This is the result diagram of the effect of Avitinib in inhibiting ATP - and MSU - induced NLRP3 inflammasome activation in Example 1 of the present invention; among them, A and B show the protein expression of IL-1β and p20 in the cell culture supernatant (SN), and C and D show the secretion level of the pro - inflammatory cytokine IL-1β in the cell culture supernatant; Figure 4 This is the result diagram of the effect of Avitinib in inhibiting LPS - induced non - classical NLRP3 inflammasome activation in Example 1 of the present invention; among them, A shows the protein expression of IL-1β and p20 in the cell culture supernatant (SN), and B shows the secretion level of the pro - inflammatory cytokine IL-1β (B) in the cell culture supernatant; Figure 5 This is the result diagram of the effect of Avitinib in alleviating LPS - induced septic shock in mice in Example 2 of the present invention; among them, A, C, and E show the secretion amounts of the inflammatory factors IL-1β, IL-6, and TNF-α in the mouse serum, and B, D, and F show the secretion amounts of the inflammatory factors IL-1β, IL-6, and TNF-α in the mouse peritoneal fluid; G shows that Avitinib prolongs the survival time of LPS - induced septic shock in mice; Figure 6 This is the result diagram of the effect of Avitinib in alleviating the ConA - induced acute liver injury model in Example 3 of the present invention; among them, A shows the appearance diagram of the mouse liver, B shows the levels of ALT and AST in the mouse eyeball blood, C and D show the levels of IL-1β and TNF-α in the mouse eyeball blood, E shows the expression of p20 in the mouse liver homogenate; F shows the HE staining result diagram of the livers of mice in each group.

[0036] Figure 7 This is the result graph showing that Avitinib effectively alleviates Alum-induced peritonitis in Example 4 of the present invention. Among them, A shows CD11b in the peritoneal fluid of mice in each group + Ly6G + Flow cytometry of neutrophils, B shows the percentage of neutrophils in the peritoneal fluid of mice in each group, C shows the number of neutrophils in the peritoneal fluid of mice in each group, and D shows the secretion level of IL-1β in the peritoneal fluid of mice in each group. Detailed implementation manners

[0037] The following will describe the implementation manners of the present invention with reference to preferred embodiments. 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 implementation manners. Various details in this specification can also 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 only for explaining the present invention and not for limiting the protection scope of the present invention.

[0038] Experimental reagents and materials Avinitinib (AC0010) is an irreversible, mutant-selective third-generation EGFR inhibitor that can effectively inhibit the EGFR T790M drug-resistant mutation in non-small cell lung cancer. Avitinib is also a novel BTK (Bruton tyrosine kinase) inhibitor. The molecular formula of Avitinib (AC0010) is: C 26 H 26 FN7O2, and the molecular structure is shown as the following formula: Experimental materials and experimental animals Table 1 Information of cells and mice Table 2 Experimental consumables Table 3 Antibody information Table 4 Experimental reagents Example 1 In vitro inhibition of the activation of macrophage NLRP3 inflammasome by Avitinib ① Explore whether Avitinib affects the activation of NLRP3 inflammasome. Mouse BMDMs were pre-stimulated with high-purity LPS (100 ng / ml) for 3 h, then further stimulated with different doses of Avitinib for 30 min, and then stimulated with the NLRP3 inflammasome activator Nigericin for 30 min. The expressions of caspase-1 and IL-1β in the supernatant were detected by Western Blot, and the secretions of IL-1β, TNF-α, and IL-6 in the cell culture supernatant were detected by ELISA. The detection results are as Figure 1 shown.

[0039] Analysis from Figure 1 showed that Avitinib effectively inhibited the activation of the classical NLRP3 inflammasome in Nigericin-induced BMDM cells.

[0040] ② Further explore the effect of Avitinib on the NLRP3 inflammasome using human THP-1 cells and PBMCs from healthy volunteers. THP-1 cells with good growth status and PBMCs isolated from the peripheral blood of healthy individuals were centrifuged and resuspended, and then seeded into 12-well cell culture plates and cultured overnight. The medium in the cell culture plates was replaced with RMPI 1640 medium supplemented with LPS (500 ng / mL), with 500 μL of liquid per well, and stimulated for 3 h; after treatment with Avitinib (0 μM, 2.5 μM, 5 μM, 10 μM), Nigericin was added. The expressions of caspase-1 and IL-1β in the supernatant of THP-1 cells were detected by Western Blot, and the secretion of IL-1β in the cell culture supernatants of THP-1 cells and PBMCs was detected by ELISA. The detection results are as Figure 2 shown.

[0041] Analysis from Figure 2 showed that Avitinib effectively inhibited the activation of the NLRP3 inflammasome in THP-1 cells and human PBMCs.

[0042] ③ Use the other two classical NLRP3 inflammasome activators, ATP and MSU, to continue exploring their effects. Pretreat BMDM cells with LPS (100 ng / ml) for 3 h, then treat the cells with different concentrations of Avitinib (0 μM, 2.5 μM, 5 μM, 10 μM) for 30 min, and subsequently add ATP (12 mM) to activate the cells for 30 min, or treat the cells with different concentrations of Avitinib (0 μM, 1 μM, 2 μM, 4 μM) for 30 min, and then add MSU (120 μg / ml) to activate the cells for 3 h. Collect the cell culture supernatant and cell lysate. Detect the protein expression of p20 in the cell culture supernatant (SN) and the protein expression of Pro-casp1 and β-actin in the cell lysate (Input) by Western blot. Detect the secretion level of the inflammatory factor IL-1β in the cell culture supernatant by ELISA. The detection results are as Figure 3 shown.

[0043] Analysis from Figure 3 showed that Avitinib could broadly inhibit the activation of NLRP3 inflammasome induced by various agonists.

[0044] ④ Pretreat BMDM cells with Pam3CSK4 (100 ng / ml) for 3 h, then treat the cells with different concentrations of Avitinib (0 μM, 0.1 μM, 0.5 μM, 1 μM) for 30 min, and subsequently transfect LPS into the cells for 16 h. Collect the cell culture supernatant and cell lysate, and detect the protein expression of p20 in the cell culture supernatant (SN) and the protein expression of Pro-casp1 and β-actin in the cell lysate (Input) by Western blot. Detect the secretion level of the inflammatory factor IL-1β in the cell culture supernatant by ELISA. The detection results are as Figure 4 shown.

[0045] Analysis from Figure 4 showed that Avitinib could also inhibit the non-classical activation pathway of NLRP3 inflammasome.

[0046] Example 2 Avinib inhibits LPS-induced septic shock Among them, in the mouse model of septic shock, the NLRP3 inflammasome has been confirmed to be the core mechanism driving the pathological process of septic shock: pathogen-associated molecules (such as LPS) or tissue damage signals (such as ATP) induce NLRP3 activation through a dual activation pathway (NF-κB pre-initiates the expression of inflammatory genes, and K⁺ efflux / ROS triggers inflammasome assembly), which in turn activates caspase-1, leading to the explosive release of pro-inflammatory factors such as IL-1β / IL-18. At the same time, it triggers pyroptosis by cleaving GSDMD, releasing a large number of damage-associated molecules (such as HMGB1), forming an "inflammatory storm". This process directly destroys the vascular endothelial barrier, triggering typical features of septic shock such as microcirculation disorders, hypotension, and multiple organ failure. Therefore, NLRP3 is not only the key hub for uncontrolled inflammation in septic shock but also the core target for achieving precise immune regulation. The mouse model of septic shock combines the advantages of pathological authenticity, genetic controllability, and an efficient pharmacodynamic analysis platform, and is a key tool for clarifying the mechanism of action of NLRP3-targeted drugs, optimizing efficacy, and promoting clinical translation. Its core value lies in providing a systematic research framework for "inflammatory storm" regulation from the molecular to the overall level.

[0047] Specific operation steps: Purchase 7-week-old SPF-grade male C57BL / 6J mice, feed them in an SPF-grade mouse room for one week, select mice with similar body weights, and randomly divide them into groups of 6 each. The groups are as follows: blank group (Control group), septic shock model group (LPS group), and Avitinib administration group (LPS+Avinib group). Cage the mice separately one night in advance to avoid stress interference with the experimental results. The next morning, intraperitoneally inject Avitinib (10 mg / kg) into the mice in the Avitinib administration group, and inject an equal volume of PBS into the mice in the blank group. After 50 minutes, except for the mice in the blank group, the remaining mice are simultaneously injected with LPS solution (20 mg / kg). After 4 hours, collect blood from the eye socket, sacrifice the mice by cervical dislocation to collect peritoneal lavage fluid, and measure the secretion levels of related inflammatory factors by ELISA. Draw the survival curve: Pre-administer Avitinib (10 mg / kg), then inject LPS solution (20 mg / kg), and then closely observe and record the death of the mice, and use GraphPad Prism 8.0.2 software to draw the survival curve. The results are as Figure 5 shown.

[0048] Among them, intraperitoneal injection of LPS is the most commonly used method to construct a mouse model of systemic inflammation, and systemic inflammation will cause an increase in the secretion of inflammatory cytokines such as IL-1β and TNF-α in the mouse serum. We further verified the effect of Avitinib at the animal level in the mouse model of systemic inflammation. Figure 5The results in [Figure 0] showed that compared with the LPS group, the secretion of IL-1β in the serum and peritoneal lavage fluid of mice in the LPS+Avinitinib group was significantly decreased (P<0.01) ( Figure 5 A, B), while there was no significant difference in the levels of IL-6 and TNF-α ( Figure 5 C-F), thus proving that Avitinib, as an NLRP3 inhibitor, can directly target the NLRP3 inflammasome. In addition, the survival status of mice within 48 h after LPS injection was observed. The survival curve results showed that the mice in the LPS group began to die at 14 h, and the mortality rate of mice reached 100% at 22 h. While the mice in the LPS+Avinitinib group began to die at 20 h, and there were still mice surviving after 36 h (P<0.01) ( Figure 5 G). The above results indicated that Avitinib could relieve inflammation in systemic inflammatory mice and significantly prolong their survival time. Thus, it was proved that Avitinib effectively alleviated LPS-induced septic shock in mice.

[0049] Example 3 Avinitinib Inhibits ConA-Induced Acute Liver Injury Among them, the ConA-induced acute liver injury model has significant advantages in studying the role of the NLRP3 inflammasome. This model simulates the acute inflammatory process of human autoimmune or viral hepatitis through the immune response mediated by T cells and macrophages. Its liver injury characteristics include the release of a large number of pro-inflammatory factors (such as TNF-α, IFN-γ) and hepatocyte necrosis. Using this model, the regulatory mechanism of NLRP3 can be accurately evaluated: for example, after NLRP3 gene knockout mice or inhibitors (such as MCC950) intervention, it can be observed that the inflammatory infiltration in the liver tissue is reduced, the transaminase level decreases, and the survival rate improves, directly verifying the core position of NLRP3 in liver injury. In addition, the ConA model has the characteristics of high reproducibility and short cycle, which is convenient for dynamically studying the spatio-temporal law of NLRP3 activation (such as early inflammation initiation vs. late tissue repair), and at the same time supports multi-dimensional analysis (such as immune cell subsets, hepatocyte-specific signaling pathways). Its clinical relevance is reflected in the screening of treatment strategies targeting NLRP3 (such as small molecule inhibitors or gene editing), providing a key experimental basis for the development of liver disease therapies that intervene in immune over-activation.

[0050] Specific operation steps: Male C57BL / 6 mice (8-10 weeks) of the same age and SPF grade with similar weight were randomly divided into three groups, with 6 mice in each group, as follows: blank control group (Control group), liver injury model group (ConA group) and Avitinib treatment group (ConA+Avitinib group). The mice were separated into cages one night in advance to avoid interference of mouse stress with the experimental results. The mice in the treatment group were first injected with Avitinib (20 mg / kg) intraperitoneally, and the mice in the blank group were injected with an equal volume of PBS. 30 minutes later, the mice in the liver injury model group and the treatment group were injected with 20 mg / kg ConA solution through the tail vein. After 24 hours, the following experiments were carried out: ① The mice were killed by cervical dislocation, the eyeballs were removed and blood was collected, and ELISA was used to detect ALT, AST, IL-1β and TNF-α in the serum. ② Western Blot was used to detect the level of caspase-1 in mouse liver tissue. ③ The liver tissue of the mouse was taken for HE staining. The results are as follows Figure 6 shown.

[0051] from Figure 6 The analysis showed that 24 hours after the tail vein injection of 20 mg / kg ConA solution, the liver volume of mice in the ConA model group increased and dark red congestion and punctate hemorrhage appeared, while the liver appearance of mice in the Avitinib group was significantly improved ( Figure 6 A). Serum ALT and AST were significantly decreased (P<0.05) ( Figure 6 B), and the IL-1β level was also significantly decreased (P<0.05) ( Figure 6 C), while there was no significant difference in TNF-α levels ( Figure 6 D). Western blot results showed that Avitinib reduced the level of caspase-1 in mouse liver tissue ( Figure 6 E). HE staining of mouse liver tissue showed that after modeling, the liver tissue showed patchy necrosis, neutrophil infiltration and increased apoptotic cells. The symptoms were significantly alleviated after administration of Avitinib ( Figure 6 F). The above results show that Avitinib can alleviate ConA-induced acute liver injury.

[0052] Example 4 Avitinib inhibits Alum-induced acute peritonitis Among them, acute peritonitis is a common clinical acute inflammatory disease of the abdominal cavity. Its typical characteristics are manifested as peritoneal vascular dilatation, increased permeability, accumulation of inflammatory exudate, accompanied by severe abdominal pain, rebound tenderness and systemic inflammatory response syndrome (SIRS). In experimental studies, Alum (potassium alum, potassium aluminum sulfate), as a classic inorganic adjuvant, has been widely used to establish an acute peritonitis model due to its stable crystal structure and clear immune-stimulating 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 will lead to changes in the permeability of the lysosomal membrane, causing the efflux of intracellular potassium ions and inducing mitochondrial dysfunction, resulting in an explosion of reactive oxygen species (ROS). These cellular stress responses will activate multiple inflammatory signaling pathways, promote the processing and maturation of pro-IL-1β and pro-IL-18 precursors, induce inflammatory cell death, and release a large number of damage-associated molecular patterns (DAMPs). These inflammatory mediators infiltrate the abdominal cavity by chemotactic neutrophils, monocytes and other immune cells, activate signal pathways such as TLR4 / MyD88, and finally form an "inflammatory cytokine storm", leading to typical pathological changes of acute peritonitis.

[0053] Specific operation steps: 1. Animals and grouping: Select 8-week-old male C57BL / 6J mice with similar body weights, and randomly divide them into the following three groups, and number each mouse: blank control group (Control group, n = 6), Alum-induced acute peritonitis model group (Alum group, n = 6), Avitinib-treated Alum-induced acute peritonitis model group (Avinib + Alum group, n = 6).

[0054] 2. Model establishment and treatment: The blank control group was not treated with anything; the Avitinib intervention group was intraperitoneally injected with Avitinib (30 mg / kg), and 1 h later, Alum (1 mg / mouse) was intraperitoneally injected. At the same time, Alum (1 mg / mouse) was intraperitoneally injected into the acute peritonitis model group.

[0055] 3. Experimental results and sample collection: After 12 hours, the mice were sacrificed, blood was collected by eye puncture, and then they were decapitated, and peritoneal fluid was collected.

[0056] (1) Flow cytometry was used to detect the proportion of neutrophils in the peritoneal lavage fluid.

[0057] (2) ELISA was used to detect the secretion of inflammatory factors (IL-1β, TNF-α) in the eye blood and peritoneal fluid. The results are as Figure 7 shown.

[0058] From Figure 7Analysis shows that compared with the WT group, the proportion and number of neutrophils in the Alum group increased significantly. After the intervention of Avitinib, the proportion and number of neutrophils of CD11b + Ly6G + were significantly decreased ( Figure 7 A, 7B, 7C). The ELISA results showed that Avitinib could significantly reduce the secretion of IL-1β in peritoneal fluid ( Figure 7 D), thus proving that in Example 2 of the present invention, through the intervention of Avitinib, the acute peritonitis induced by Alum in mice was effectively alleviated.

[0059] The above results confirm that the third-generation EGFR tyrosine kinase inhibitor Avitinib provided by the present invention can inhibit the activation of NLRP3 inflammasome induced by various activators, inhibit pyroptosis, and show a significant inhibitory effect on the activation of NLRP3 inflammasome in both murine and human cells. In the septic shock model, ConA-induced acute liver injury model and Alum-induced acute peritonitis model, Avitinib effectively alleviated the disease symptoms, fully confirming the key role of this drug in the regulation of related inflammatory responses. This finding not only provides an experimental basis for analyzing the molecular mechanism of Avitinib inhibiting NLRP3, but also strengthens the pathological status of NLRP3 in malignant tumors, and points out a new direction for the development of treatment strategies for NLRP3-related diseases such as septic shock.

[0060] In summary, the application of avitinib of the present invention in the preparation of NLRP3 inflammasome inhibitors. As a third-generation EGFR-TKI, avitinib is currently mainly applied to patients with non-small cell lung cancer (NSCLC). Since such patients often suffer from tumor-related systemic inflammatory responses or infectious complications, the NLRP3 inhibitory effect discovered in this study may provide additional mechanistic support for its clinical efficacy. The overactivation of the NLRP3 inflammasome plays an important role in the disease progression of septic shock. In this study, after sacrificing mice by constructing an LPS-induced septic shock model, ELISA technology was used to detect that pre-administration of avitinib (10 mg / kg) could significantly reduce the levels of IL-1β in serum and peritoneal lavage fluid, but had no obvious effect on IL-6 and TNF-α. Moreover, in the survival curve model, the survival time and number of mice were extended compared with the model group, revealing a new anti-inflammatory mechanism of avitinib in septic shock, but its significance is not limited to sepsis treatment. In a ConA-induced acute liver injury model in mice, our experimental results showed that avitinib could significantly reduce the levels of AST, ALT and related inflammatory factors, indicating that avitinib could effectively alleviate ConA-induced acute liver injury. It was shown that avitinib might improve acute liver injury in mice by targeting the reduction of IL-1β secretion induced by NLRP3 inflammasome activation. In a mouse acute peritonitis model induced by intraperitoneal injection of Alum (1 mg / mouse), the therapeutic effect of avitinib was evaluated in multiple dimensions. Flow cytometry analysis showed that Ly6G + CD11b +The proportion and absolute number of neutrophils were both significantly decreased. Meanwhile, the ELISA test results showed that Avitinib could significantly inhibit the release of inflammatory factor (IL-1β) in peritoneal lavage fluid. These data fully demonstrated that Avitinib could effectively alleviate Alum-induced acute peritonitis. Thus, it was proved that Avitinib was a bifunctional drug that could target both EGFR mutation and NLRP3 inflammasome, with dual targeting potential, especially suitable for lung cancer patients with EGFR mutation positive and accompanied by systemic inflammation (such as infection, cachexia). The experimental results showed that the effective concentration of Avitinib in the mouse model of septic shock was 10 mg / kg, with a relatively low working concentration, showing certain superiority compared with the second-generation tyrosine kinase inhibitor afatinib (15 mg / kg). This study found that Avitinib could effectively inhibit NLRP3 activation, suggesting that it might achieve "dual-target synergy" in EGFR mutant lung cancer by regulating tumor-immunity-inflammation related pathways - directly inhibiting tumor growth and improving the immune microenvironment. Thus, it solved the treatment problem of EGFR mutant lung cancer patients complicated with NLRP3 inflammasome-related diseases (septic shock / acute liver injury / acute peritonitis). By simultaneously inhibiting tumor progression and inflammatory response with Avitinib (the third-generation EGFR-TKI), it was recommended that such patients give priority to this drug, and it provided more potential drugs for the treatment of septic shock drugs, acute liver injury drugs and acute peritonitis, having the value of popularization and application in the field of biomedical materials technology.

[0061] The above embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention.

Claims

1. Use of Avitinib in the preparation of NLRP3 inflammasome inhibitors.

2. The application according to claim 1, wherein The dosage of Avitinib is 0 - 20 μM.

3. The use according to claim 1, characterized in that Avitinib is used in the preparation of drugs for treating septic shock.

4. The application according to claim 3, wherein The septic shock is LPS-induced septic shock.

5. The application according to claim 3, characterized in that The dosage of Avitinib is 10 mg / kg; and / or, the drug for treating septic shock is prepared by adding pharmaceutical excipients with Avitinib as the active ingredient; and / or, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections and suppositories.

6. The use according to claim 1, characterized in that Avitinib is used in the preparation of drugs for treating acute liver injury.

7. The application according to claim 6, characterized in that, The acute liver injury is ConA-induced acute liver injury.

8. The application according to claim 6, characterized in that, The dosage of Avitinib is 20 mg / kg; and / or, the drug for treating acute liver injury is prepared by adding pharmaceutical excipients with Avitinib as the active ingredient; and / or, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections and suppositories.

9. The use according to claim 1, characterized in that Avitinib is used in the preparation of drugs for treating peritonitis.

10. The application according to claim 9, wherein The peritonitis is Alum-induced acute peritonitis; and / or, the dosage of Avitinib is 30 mg / kg; and / or, the drug for treating peritonitis is prepared by adding pharmaceutical excipients with Avitinib as the active ingredient; and / or, the dosage form of the drug is at least one of tablets, capsules, granules, powders, patches, suspensions, syrups, oral liquids, injections and suppositories.

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