Ivacaftor targets NINJ1 to inhibit plasma membrane rupture and limit tissue damage associated applications in inflammatory diseases

Abstract

The application discloses an application of Ivacaftor in inhibiting plasma membrane rupture and limiting tissue damage of inflammatory diseases by targeting NINJ1. The application provides an application of Ivacaftor in preparing a medicine. The medicine is a medicine for preventing and / or treating inflammatory diseases or the medicine is a medicine for relieving inflammatory diseases. The medicine is a medicine for preventing and / or treating tissue damage of inflammatory diseases or the medicine is a medicine for relieving tissue damage of inflammatory diseases. The application provides an application of Ivacaftor in preparing a product for inhibiting plasma membrane rupture and in preparing a product for inhibiting NINJ1 oligomerization. The application finds that Ivacaftor can improve PMR by combining with and inhibiting NINJ1 oligomerization, thereby treating inflammatory diseases and inhibiting tissue damage of inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and Ivacaftor targets NINJ1 to inhibit plasma membrane rupture and limit tissue damage related to inflammatory diseases. Background Technology

[0002] The mechanisms of various acute and chronic inflammatory diseases manifest at the cellular level as unexpected or excessive cell death. Dead cells not only cause tissue damage through cellular lysis but also amplify the inflammatory response by releasing intracellular damage-associated molecular patterns (DAMPs). Among these, apoptosis is the most well-known form of cell death. It is characterized by an intact cell membrane in the early stages of apoptosis, followed by phagocytosis by immune cells that recognize exposed phosphatidylserine (PS). Therefore, programmed apoptosis is immune-silencing and essential for maintaining tissue homeostasis. However, excessive apoptosis of healthy cells can exceed the phagocytic capacity of immune cells, leading to cell retention. These retained apoptotic cells then undergo secondary necrosis accompanied by plasma membrane damage. Furthermore, pyroptosis, necroptosis, ferroptosis, and bacterial toxin-induced cell death are all essentially necrosis, involving plasma membrane damage alongside cell death. The release of DAMPs from these dead cells induces inflammatory responses and exacerbates the pathology of cell death-related diseases.

[0003] Previously, it was believed that after initial plasma membrane damage mediated the influx of water molecules into the cell, the resulting osmotic imbalance ultimately led to plasma membrane rupture (PMR), releasing large molecular weight DAMPs such as lactate dehydrogenase (LDH) from the ruptured, dead cell membrane. Recently, this long-held view has been overturned. The cell surface transmembrane protein Ninjurin 1 (NINJ1) has been discovered to be essential for mediating PMR and the release of various DAMPs following pyroptosis, apoptosis, and toxin-induced necrosis. Therefore, NINJ1 has become an unprecedented drug target for limiting the inflammatory response caused by cell death.

[0004] NINJ1 is a small protein with a molecular weight of 15 kDa, typically present in the plasma membrane as monomers, dimers, or oligomers with a molecular weight below 480 kDa. Activation by pyroptosis, apoptosis, and toxin-induced necrosis transforms NINJ1 into higher molecular weight (nearly 1000 kDa) oligomers, forming porous or other shaped protein clusters that lead to pulmonary inflammatory response (PMR). Antibodies that block NINJ1 oligomerization can improve PMR and alleviate acute liver injury induced by TNF / d-Gal, ConA, anti-Fas antibodies, or ischemia-reperfusion. However, even with repeated administration, current NINJ1 antibodies cannot maintain adequate serum concentrations, hindering their use in chronic inflammatory diseases. Therefore, there is an urgent need to develop antibodies or small molecules that specifically target NINJ1 and support long-term use. Summary of the Invention

[0005] The purpose of this invention is to provide Ivacaftor for targeting NINJ1 to inhibit plasma membrane rupture and limit tissue damage associated with inflammatory diseases.

[0006] This invention provides the use of Ivacaftor in the preparation of a medicament; the medicament is a medicament for the prevention and / or treatment of inflammatory diseases or a medicament for the relief of inflammatory diseases.

[0007] This invention provides the application of Ivacaftor in the preparation of pharmaceuticals; said pharmaceuticals are for the prevention and / or treatment of damage to the body caused by inflammatory diseases or are for the relief of damage to the body caused by inflammatory diseases.

[0008] This invention provides the use of Ivacaftor in the preparation of a medicament; the medicament being a medicament for preventing and / or treating tissue damage caused by inflammatory diseases or a medicament for relieving tissue damage caused by inflammatory diseases.

[0009] The inflammatory disease mentioned can be either an acute or chronic inflammatory disease.

[0010] The inflammatory disease mentioned is either a liver inflammatory disease, a neuroinflammatory disease, or an intestinal inflammatory disease.

[0011] For example, the inflammatory disease is hepatitis, sepsis, heatstroke, enteritis, or multiple sclerosis.

[0012] For example, the inflammatory disease is hepatitis, sepsis, heatstroke, enteritis, multiple sclerosis, or autoimmune encephalomyelitis.

[0013] For example, the inflammatory disease is hepatitis, sepsis, heatstroke, enteritis, multiple sclerosis, or allergic encephalomyelitis.

[0014] For example, the hepatitis is fulminant hepatitis or immune-mediated liver injury.

[0015] This invention provides the application of Ivacaftor in the preparation of products for inhibiting plasma membrane rupture.

[0016] This invention provides the application of Ivacaftor in the preparation of products for inhibiting NINJ1-induced plasma membrane rupture.

[0017] This invention provides the application of Ivacaftor in the preparation of products for incorporating NINJ1.

[0018] This invention provides the application of Ivacaftor in the preparation of products for inhibiting NINJ1 oligomerization.

[0019] The NINJ1 is the human NINJ1 protein.

[0020] For example, the human NINJ1 protein is shown in positions 251-402 of SEQ ID NO: 2.

[0021] The NINJ1 mentioned is the mouse NINJ1 protein.

[0022] For example, the mouse NINJ1 protein is shown in SEQ ID NO: 4.

[0023] The product is a reagent or kit.

[0024] Ivacaftor is a small molecule drug approved by the U.S. Food and Drug Administration (FDA) for long-term safe use in patients with cystic fibrosis. The inventors of this invention have discovered a new use for it. They have found that the small molecule drug Ivacaftor can improve PMR (progressive refractory membrane activity) by binding to and inhibiting NINJ1 oligomerization, thereby treating inflammatory diseases (inhibiting tissue damage caused by inflammatory diseases). Cystic fibrosis is a rare disease. The inventors of this invention have discovered a new indication for the small molecule drug Ivacaftor, namely, inflammatory diseases. This invention has great potential and value for the treatment of inflammatory diseases. Attached Figure Description

[0025] Figure 1 The results are from Example 1.

[0026] Figure 2 The results are from step one of Example 2.

[0027] Figure 3 The results are from step two of Example 2.

[0028] Figure 4 The results are from Example 3.

[0029] Figure 5 The results are from Example 4.

[0030] Figure 6 The results are from Example 5.

[0031] Figure 7 The results are from Example 6.

[0032] Figure 8 The results are from Example 7.

[0033] Figure 9 The results are from Example 8.

[0034] Figure 10 The results are from Example 9. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are taken as the mean ± standard deviation. Unless otherwise specified, the cell culture conditions are: 37℃, 5% CO2. Unless otherwise specified, complete culture medium refers to basal medium containing 10% FBS and 1% penicillin and streptomycin. LDH: lactate dehydrogenase. ALT: alanine aminotransferase. AST: aspartate aminotransferase. C57BL / 6 mice: Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. TNF-α (Recombinant Mouse TNF-alpha Protein): Sinopharm, catalog number 50349-MNAE. D-Gal (D-galactosamine hydrochloride, CAS number 1772-03-8): Macklin Biotechnology, catalog number D810561. ConA (Concanavalin A, CAS No. 11028-71-0): Sigma-Aldrich, catalog number L7647. DSS (Sodium Glucan Sulfate, CAS No. 9011-18-1): MP Biomedicals, catalog number 160110. LPS (Lipopolysaccharide): Sigma-Aldrich, catalog number L2630.

[0037] Ivacaftor is a prior art drug used to treat cystic fibrosis (exerting its therapeutic effect by enhancing CFTR). Ivacaftor has been used in cystic fibrosis patients for over 8 years, and its safety and drug stability have been well controlled in in vivo studies. The Ivacaftor used in this example was purchased from MedChemExpress, catalog number HY-13017. The CAS number for Ivacaftor is 873054-44-5, and its structural formula is shown in Formula (I).

[0038]

[0039] The preparation method of human monocyte-derived macrophages (hMDMs) is as follows: PBMCs were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (2.5 × 10⁻⁶ cells / year). 7 Cells / 15 cm culture dish), macrophage colony-stimulating factor (M-CSF, 20 ng / mL) was added to the culture medium and cultured for 5 days. On the 6th day, the differentiated macrophages were washed with cold PBS, scraped off the culture dish, centrifuged at 500×g for 5 minutes, and resuspended in 24-well plates (5×10⁶ cells / well). 5 Cells / well). PBMCs (peripheral blood mononuclear cells): Biotechnologies (lot number: CAPB01230202).

[0040] The cell viability was determined as follows: Cell Titer-Glo luminescent cell viability assay kit (Promega, catalog number G7573) was used to measure cellular ATP levels to detect cell viability; the reaction was performed according to the instructions and the luminescence intensity was measured using a microplate reader (PerkinElmer EnSpire); data analysis was performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA), and a nonlinear regression model with an S-shaped dose-response relationship was used to fit the curve.

[0041] CytoTox was used to detect LDH levels in serum or in cell culture supernatant. Non-Radioactive Cytotoxicity Assay (Promega, product catalog number G1780, follow the instructions). Fluorescence values ​​were read, and data analysis was performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA).

[0042] Serum AST levels were detected using an aspartate aminotransferase (AST / GOT) test kit (Nanjing Jiancheng Biotechnology Co., Ltd., product catalog number C010-2-1), following the instructions. Serum ALT levels were detected using an alanine aminotransferase (ALT / GPT) test kit (Nanjing Jiancheng Biotechnology Co., Ltd., product catalog number C009-2-1), following the instructions. Serum IL-1β levels were detected using the Mouse IL-1beta / IL1B ELISA Kit (Boster Biologics, product catalog number EK0394), following the instructions. Serum IL-18 levels were detected using the Mouse IL18 ELISA Kit (Boster Biologics, product catalog number EK0433), following the instructions. Serum HMGB1 levels were detected using the Mouse HMGB-1 (Highmobility group protein B1) ELISA Kit (Elabscience, product catalog number E-EL-M0676), following the instructions. Serum TNF-α levels were detected using the Mouse TNFAlpha / TNFA ELISA Kit (Wuhan Boster Biological Technology Co., Ltd., product catalog number EK0527), following the instructions. Serum BUN (blood urea nitrogen) levels were detected using a blood urea nitrogen (BUN) test kit (urease method) (Nanjing Jiancheng Biotechnology Co., Ltd., product catalog number C013-2-1), following the instructions.

[0043] Example 1: Screening small molecule inhibitors targeting NINJ1

[0044] Transient overexpression of NINJ1 leads to spontaneous oligomerization of NINJ1 and induces cell death in multiple cell types, providing an excellent detection method for high-throughput screening of small molecule inhibitors of NINJ1 based on cell viability.

[0045] The inventors induced expression of NINJ1 with a Flag tag in HT-29 cells (human colon cancer cell line) to obtain HT-29pLVX-Flag-NINJ1 cells. HT-29pLVX-Flag-NINJ1 cell death occurred in a time- and Dox-dependent manner. Figure 1 B- Figure 1 G).

[0046] High-throughput compound screening was performed using HT-29pLVX-Flag-NINJ1 cells. One day prior to the experiment, 1000 cells were seeded in each well of a 384-well plate (Corning #3707). Small molecules from the compound library were added to the 384-well plates to a concentration of 10 μM using a Tecan's Freedom Evo 150 automated liquid handling system. After 2 hours of incubation, Dox (1 μg / mL) was added, and cell viability was measured by ATP levels. High-throughput screening revealed that Ivacaftor blocked NINJ1-induced cell death with an IC50 of 1.17 μM. Figure 1 Mechanistically, Ivacaftor did not affect the expression level of NINJ1 (I). Figure 1 J), but rather blocked the oligomerization of NINJ1 ( Figure 1 K).

[0047] The schematic diagram and result diagram of Example 1 are shown below. Figure 1(B) Schematic diagram of the process of constructing Flag-tagged NINJ1 cells expressing Tet3G transcription activator and Dox-induced expression in HT-29 cells. (C) Cell colony formation of HT-29pLVX-Flag-NINJ1 cells after treatment with 1 μg / ml Dox or no treatment for 72 hours was analyzed by crystal violet staining. (D) Cell viability of HT-29pLVX-Flag-NINJ1 cells after treatment with different concentrations of Dox for 48 hours was assessed by ATP levels. (E) Expression of Flag-NINJ1 induced by Dox was analyzed by Western blotting of HT-29pLVX-Flag-NINJ1 cells after treatment with different concentrations of Dox for 24 hours. (F) Cell viability of HT-29pLVX-Flag-NINJ1 cells after treatment with 1 μg / ml Dox for different time periods was measured by ATP levels at different time points. (G) HT-29pLVX-Flag-NINJ1 cells were treated with 1 μg / ml Dox for different time periods, and the expression of Flag-NINJ1 was analyzed by Western blotting. (I) HT-29pLVX-Flag-NINJ1 cells were treated with glycine or different concentrations of Ivacaftor for 48 hours with or without Dox, and cell viability was assessed by ATP levels (two-tailed t-test: p < 0.0001). (J) HT-29pLVX-Flag-NINJ1 cells were treated with glycine or different concentrations of Ivacaftor for 24 hours with or without Dox, and the expression of Flag-NINJ1 was analyzed by Western blotting. (K) HT-29pLVX-Flag-NINJ1 cells were treated with DMSO, Dox, Dox and glycine, or Dox and Ivacaftor for 24 hours, and non-denaturing gel electrophoresis was performed to assess the oligomerization of NINJ1.

[0048] Example 2: Ivacaftor blocks plasma membrane rupture in human and mouse cells.

[0049] During pyroptosis, apoptosis, and toxin-induced necrosis, NINJ1 undergoes oligomerization activation to mediate catastrophic proliferative mitochondrial (PMR) upon initial damage to the plasma membrane. Example 1 showed that Ivacaftor could block the oligomerization of overexpressed NINJ1 and the corresponding cell death. In Example 2, the inventors tested whether Ivacaftor could block NINJ1-mediated PMR following various cell death activations.

[0050] I. Ivacaftor blocks plasma membrane rupture in human cells

[0051] The test cells were either hMDMs cells or U937 cells (human tissue lymphoma cell line). The complete culture medium used to culture the test cells was RPMI-1640 medium containing 10% FBS (inactivated complement) and 1% penicillin-streptomycin.

[0052] hMDMs cells were seeded in complete medium containing 1 μg / mL LPS and cultured for 3 hours. Then, nigericin and the test compound (working concentration of nigericin: 20 μM; test compound: Ivacaftor and / or Glycline, with working concentrations of 10 μM and 20 μM for Ivacaftor, and 25 mM and 50 mM for Glycline) were added, and the cells were cultured for another 3 hours. Controls without the test compound, control without the test compound and nigericin, and control without LPS, the test compound, and nigericin (vihicle) were also established and cultured for 3 hours in each case. After culture, the supernatant was collected, and the LDH content was measured. The results are shown below. Figure 2 A (****P<0.0001). After culture was completed, cell viability was assessed by ATP levels, and the results are shown in [Figure number missing]. Figure 2 B (****P<0.0001). Cells in the high-concentration treatment group were stained with plasma membrane dye FM4-64 (2μg / ml) to detect plasma membrane swelling and bubbling; photos are shown below. Figure 2 C (indicated by white arrow; scale bar: 20 μm), the quantified proportion of swollen bubble cells is shown in [reference needed]. Figure 2 D(****P<0.0001).

[0053] U937 cells were seeded in complete medium containing 1 μg / mL LPS and cultured for 3 hours. Then, nigericin and the test compound (nigericin working concentration: 20 μM; test compound: Ivacaftor or Glycline; Ivacaftor with two working concentrations of 2 μM, 5 μM, and 10 μM; Glycline working concentration: 20 mM) were added, and the cells were cultured for another 3 hours. Controls without the test compound, control without the test compound and nigericin, and control without LPS, the test compound, and nigericin (vihicle) were also set up, and all were cultured for 3 hours. After culture, the supernatant was collected, and the LDH content of the supernatant was measured. The results are shown below. Figure 2 The E (P < 0.0001) was observed. Cell viability was assessed by ATP levels after culture, and the results are shown below. Figure 2 The F (P < 0.0001) was observed. Cells were stained with plasma membrane dye FM4-64 (2 μg / ml) to detect plasma membrane swelling; images are shown below. Figure 2 G (indicated by white arrow; scale bar: 20 μm), the quantified proportion of swollen cells is shown in [reference needed]. Figure 2 H (****P<0.0001). After culture, the supernatant was collected and analyzed by silver staining. The results are shown in the figure. Figure 2 The levels of IL-1β in cell lysates and supernatants were detected by Western blotting. Results are shown in [Figure 1]. Figure 2 J.

[0054] The results showed that Ivacaftor blocked the rupture of PMR and bubble-like protrusions in cells without affecting upstream cell death and the release of mature IL-1β.

[0055] II. Ivacaftor blocks plasma membrane rupture in mouse cells.

[0056] The test cells were: wild-type iBMDM cells, iBMDMs with Ninj1 knockout, or iBMDMs expressing NINJ1-GFP. The complete culture medium used to culture the test cells was DMEM containing 10% FBS and 1% penicillin and streptomycin. iBMDM cells were immortalized mouse bone marrow-derived macrophages. Mouse NINJ1 protein is shown in SEQ ID NO: 4.

[0057] The results of Ivacaftor targeting NINJ1 oligomerization to inhibit PMR in mouse bone marrow-derived immortalized macrophages (iBMDMs) are shown in the figure. Figure 3(AB) Wild-type and Ninj1 knockout iBMDMs were induced to undergo pyroptosis by electroporation with LPS, followed by treatment with or without glycine or ivacaftor for 2 hours. PMR was assessed by measuring LDH release in the supernatant (A), and cell viability was determined by ATP levels (B). Two-way ANOVA: NS; ****P<0.0001. (CD) Wild-type and Ninj1 knockout iBMDMs were induced to undergo apoptosis by treatment with ABT737 plus S63845, with or without glycine or ivacaftor. After 6 hours, PMR was measured by LDH release in the supernatant (C), and cell viability was determined by ATP levels (D). Two-way ANOVA: NS; ****P<0.0001. (EF) Wild-type and Ninj1 knockout iBMDMs were pretreated with LPS (3 hours), followed by treatment with nigericin for 3 hours in the presence or absence of glycine or ivacaftor to induce pyroptosis. PMR was measured by LDH release in the supernatant (E), and cell viability was characterized by ATP levels (F). Two-way ANOVA: NS; ****P<0.0001. (GH) Wild-type and Ninj1 knockout iBMDMs cells were treated with PFO for 2 hours to induce necrosis in the presence or absence of glycine or ivacaftor. PMR was measured by LDH release in the supernatant (G), and cell viability was characterized by ATP levels (H). Two-way ANOVA: NS; ****P<0.0001. (I) Pyroptosis was induced in wild-type and Ninj1 knockout iBMDMs cells by LPS electroporation for 2 hours in the presence or absence of ivacaftor. The culture supernatant was collected and analyzed by silver staining. (JK) Volcano plots show the proteins released by Ninj1 knockout iBMDMs (J) and ivacaftor-treated iBMDMs (K) after LPS electroporation compared to wild-type. Red dots represent proteins released by both Ninj1 knockout and ivacaftor-treated cells. FC = fold change. (L) GSDMD cleavage was detected after electroporation of wild-type and Ninj1 knockout iBMDMs cells with and without glycine or ivacaftor for 2 hours. (M) iBMDMs cells were pretreated with LPS for 3 hours with and without glycine or ivacaftor, followed by nigericin treatment for 3 hours. IL-1β in cell lysates and supernatants was analyzed by Western blotting. (NO) iBMDMs cells were electroporated with LPS to induce pyroptosis (N) or treated with PFO to induce necrosis (O), with or without glycine or ivacaftor treatment for 2 hours. Oligomerization of endogenous NINJ1 was analyzed by non-denaturing gel electrophoresis. Ninj1 knockout cells were used to validate antibodies against mouse NINJ1.(P) iBMDMs expressing NINJ1-GFP were either untreated, treated with nigericin after 3 hours of LPS pretreatment, treated with nigericin and glycine after 3 hours of LPS pretreatment, or treated with nigericin and Ivacaftor after 3 hours of LPS pretreatment. Fluorescence imaging was then performed, and pie charts were created to represent the statistical distribution of cells containing NINJ1 spots. Scale bar: 2 μm.

[0058] In iBMDM cells, Ivacaftor (2.5 μM) blocked PMR (as measured by LDH release) induced by non-canonical pyroptosis, apoptosis, canonical pyroptosis, and PFO-induced necrosis, with effects comparable to NINJ1 gene knockout (KO) or glycine addition (10 mM). Figure 3 A-3H). Secretomic analysis showed that Ivacaftor also blocked PMR-mediated release of intracellular proteins into the culture medium, with effects similar to NINJ1 gene knockout. Figure 3 I-3K). Nevertheless, as with NINJ1 gene knockout or glycine treatment, Ivacaftor treatment did not block the loss of cell viability following activation of these cell death pathways, consistent with the fact that NINJ1 only functions after cell death. Supporting this is that Ivacaftor or NINJ1 gene knockout neither affects the cleavage of full-length GSDMD during pyroptosis nor the activation of caspase-9 / 3 during apoptosis, nor does it inhibit GSDMD-mediated release of mature IL-1β. Figure 3 L-3M). By analyzing NINJ1 oligomerization using non-denaturing gel electrophoresis, the inventors discovered that Ivacaftor can block pyroptosis and necrosis-induced NINJ1 oligomerization and the formation of NINJ1 protein clusters. Figure 3 N-3P).

[0059] In summary, Ivacaftor blocked PMR induced by pyroptosis and necrosis in mouse and human cell lines.

[0060] Example 3: Ivacaftor blocks plasma membrane rupture without targeting CFTR.

[0061] Cystic fibrosis transmembrane transport regulator (CFTR) is an ion channel and a classic target of Ivacaftor. Ivacaftor can bind to CFTR or CFTR-gated mutants, enhancing ion channel opening and activity to regulate salt ion homeostasis across the epithelial cell membrane. To determine whether enhanced CFTR function contributes to PMR protection, CFTR was knocked out in HT-29 cells that can be inducibly expressed by NINJ1. Figure 4A). The results showed that knocking out CFTR did not affect the sensitivity to cell death induced by NINJ1 overexpression, nor did it block the protective effect of Ivacaftor. Figure 4 B). Furthermore, elexacaftor and tezacaftor, two CFTR correctors with different mechanisms of promoting CFTR transport and membrane localization, whether used alone or in combination, failed to inhibit cell death induced by NINJ1 overexpression. Figure 4 C). Neither of the two inhibitors that block CFTR channel activity promoted NINJ1-induced cell death. Figure 4 (D-4E). Based on the above results, the possibility that Ivacaftor inhibits cell membrane rupture by targeting CFTR is ruled out.

[0062] See the results of Example 3. Figure 4 (A) Sequencing results from HT-29pLVX-Flag-NINJ1 CFTR knockout cell clones confirmed successful CFTR knockout. (B) HT-29pLVX-Flag-NINJ1 cells carrying non-target control gRNA (NTC) and CFTR knockout were treated with different concentrations of Dox for 48 hours in the presence or absence of ivacaftor. Cell viability was determined by ATP levels. Two-way ANOVA: NS; ****P<0.0001. (C) HT-29pLVX-Flag-NINJ1 cells were treated with glycine, ivacaftor, or other CFTR inhibitors for 48 hours in the presence or absence of Dox. Cell viability was determined by ATP levels. Two-way ANOVA: NS; ****P<0.0001. (D) HT-29pLVX-Flag-NINJ1 cells were treated with glycine or different concentrations of CFTR(inh)-102 for 48 hours in the presence of Dox. Cell viability was determined by ATP levels. Two-way ANOVA: NS; ****P<0.0001. (E)HT-29pLVX-Flag-NINJ1 cells were treated with glycine or different concentrations of CFTR(inh)-172 for 48 hours with or without Dox. Cell viability was determined by ATP levels. Two-way ANOVA: NS; ****P<0.0001.

[0063] Example 4: Ivacaftor blocks plasma membrane rupture by binding to NINJ1.

[0064] Since Ivacaftor does not rely on the known target CFTR to block cell membrane rupture, the inventors instead tested whether Ivacaftor directly binds to NINJ1 and inhibits NINJ1-mediated PMR.

[0065] I. Preparation and purification of human NINJ1 protein

[0066] The DNA molecule shown in SEQ ID NO: 1 (encoding the protein shown in SEQ ID NO: 2; in SEQ ID NO: 2, amino acid residues 1-236 form the mCherry protein tag, amino acid residues 237-250 form the restriction site of the PreScission protease, and amino acid residues 251-402 form the human NINJ1 protein) was inserted between the EcoRI and NotI restriction sites of the pEG BacMam vector (Addgene, catalog number #160686) to obtain a recombinant plasmid. The recombinant plasmid was introduced into *E. coli* DH10Bac and cultured to obtain a baculovirus plasmid. The baculovirus plasmid was then introduced into Sf9 cells and cultured to obtain a recombinant baculovirus.

[0067] HEK293S GnTI infected with recombinant baculovirus - Cells (ATCC, CRL-3022) were cultured, then collected, lysed, and the supernatant was collected. The supernatant was purified and recovered using agarose beads conjugated with mCherry antibody (GE Healthcare). The mCherry-tagged target protein was then obtained, and the mCherry tag was cleaved using PreScission protease. The human NINJ1 protein was recovered and concentrated, and further purified using gel filtration chromatography (Superose 6 Increase 10 / 300GL) to obtain purified human NINJ1 protein.

[0068] II. Thermal stability of NINJ1 under temperature gradient

[0069] Label-free differential scanning fluorometry is a method for assessing protein thermostability based on the fluorescence changes of internal aromatic amino acids during protein denaturation. Purified human NINJ1 protein (final concentration 0.163 mg / L) was incubated with DMSO (control group) or ivacaftor (100 μM) in assay buffer (25 mM HEPES pH 7.0, 150 mM NaCl, 0.02% C12E8, 2% DMSO) for 1 hour. Then, 9 μL of the sample was dropped into a microcapillary. A protein dissolution program was run, and fluorescence emission intensity was monitored using an Uncle instrument (Unchained Labs). Data were processed using Uncle Analysis software V3.2.

[0070] Figure 5 Figure A shows the pyrolysis curves of NINJ1 with and without ivacaftor. Figure 5Figure B shows the Tm values ​​(B) calculated from these curves. Two-tailed t-test: ***p = 0.0008. In the presence of Ivacaftor, the melting temperature (Tm) of NINJ1 increases significantly, indicating that Ivacaftor has a stabilizing effect on NINJ1, that is, there is a binding between Ivacaftor and NINJ1.

[0071] III. Testing the bonding between Ivacaftor and NINJ1 using the Biacore™ 8K system via surface plasmon resonance (SPR) technology.

[0072] Surface plasmon resonance (SPR) analysis was used to measure the binding kinetics and affinity of ivacaftor and glycine to NINJ1 at room temperature. A BIAcore 8K system (Cytiva) equipped with a Biacore Series S Sensor Chip (Cytiva, catalog number CM5) was used. Purified human NINJ1 protein was immobilized on the CM5 chip surface using a standard EDC / NHS coupling method, with a response unit (RU) of approximately 8000. Flow buffer (pH 7.4) contained 15 mM Na₂HPO₄, 1.46 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, 0.5% DMSO, and 0.05% C₁₂E₈. Ivacaftor (100 μM to 6.25 μM) and glycine (500 mM to 37.5 mM) at different concentration gradients were flowed through the chip surface at a flow rate of 30 μL / min, and the data were recorded in real time. The collected data were analyzed using BIAevaluation Version 4.1 software (GE Healthcare) in 1:1 combination mode.

[0073] Figure 5 Surface plasmon resonance (SPR) analysis of C14 showed the interaction between ivacaftor and NINJ1. Ivacaftor rapidly dissociated from the immobilized NINJ1, indicating a reversible non-covalent interaction between ivacaftor and NINJ1.

[0074] IV. After confirming the binding of Ivacaftor to the NINJ1 protein, we tested whether Ivacaftor could block the membrane-permeable ability of NINJ1 in an in vitro protein-liposome system without interference from other cellular proteins.

[0075] Method for preparing liposomes: ① Mix 40 μL of 25 mg / ml phosphatidylserine solution (POPS, Avanti, catalog number #840034; solvent: chloroform) and 40 μL of 100 mg / ml phosphatidylcholine solution (POPC, Avanti, catalog number #850457; solvent: chloroform) in a 10 mL round-bottom flask, and evaporate the solvent using a vacuum rotary evaporator to obtain a phospholipid membrane; ② Add 1 mL of buffer (containing 20 mM HEPES and 100 mM NaCl, with the remainder being water, pH 7.4) to hydrate the phospholipid membrane, and then use a mini extruder (Avanti) to push the liposomes through a 100 nm polycarbonate membrane 30 times.

[0076] Preparation of Tb 3+ The method for preparing liposomes is as follows: ① Same as method ① for liposome preparation; ② Add 1 mL of buffer TL (containing 15 mM TbCl3, 50 mM sodium citrate, 20 mM HEPES and 100 mM NaCl, with the remainder being water, pH 7.4) to hydrate the phospholipid membrane, and then use a mini extruder (Avanti) to push the liposomes through a 100 nm polycarbonate membrane 30 times; ③ Purify the liposomes through a molecular sieve column (Superose 6, 10 / 300GL) in buffer B (20 mM HEPES, 150 mM NaCl) to remove any external Tb. 3+ Collect and concentrate samples containing liposomes for use in subsequent experiments.

[0077] All liposomes were stored at 4°C and used within 48 hours.

[0078] Purified human NINJ1 protein (0.5 μM) was pre-incubated for 1 hour at room temperature in buffer L containing 0.0013% C12E8 detergent with a specified concentration of glycine or ivacaftor. Subsequently, the sample was incubated with Tb... 3+ Liposomes (liposome concentration 800 μM, diluted from 6.4 mM stock solution) and dipyrrolic acid (DPA, 50 mM) were mixed. Fluorescence signals (excitation wavelength ex = 270 nm / emission wavelength em = 549 nm) were measured using a microplate reader and recorded as Ft0. Fluorescence signals were then recorded every 20 seconds for approximately 120 times. Afterwards, 0.1% Triton X-100 was added to completely release Tb. 3+ After adding Triton X-100, the measured fluorescence signal was recorded as Ft100. The percentage of liposome leakage at each time point can be defined as: Leakage(t)(%) = [(Ftt-Ft0) / (Ft100-Ft0)] × 100.

[0079] Adding NINJ1 protein to intact liposomes resulted in Tb3+ Leakage indicates that NINJ1 causes damage to the liposome membrane. Pre-incubation with Ivacaftor blocked NINJ1-mediated liposome membrane damage in an Ivacaftor dose-dependent manner. Figure 5 D, One-way ANOVA: ****P<0.0001). In contrast, neither of the two CFTR correctors nor glycine (even at a concentration of 150 mM) could block NINJ1-mediated liposome membrane damage. Figure 5 E). NINJ1-mediated liposome membrane damage worsens over time, and Ivacaftor (rather than glycine) largely protects against membrane damage even at later time points detected. Figure 5 F-5G, one-way ANOVA: NS, *P = 0.0202, ****P < 0.0001).

[0080] In summary, the above evidence suggests that Ivacaftor directly binds to NINJ1 and inhibits NINJ1-mediated membrane rupture.

[0081] Example 5: Effects of Ivacaftor on inflammatory diseases (hepatitis model)

[0082] In this embodiment, "solvent" means a liquid composed of 9 parts by volume of corn oil and 1 part by volume of DMSO.

[0083] I. Validating the therapeutic effect of Ivacaftor using a TNF / D-Gal-induced hepatitis model

[0084] In this step, a fulminant hepatitis model was induced by intraperitoneal injection of TNF-α and D-Gal.

[0085] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into four groups.

[0086] Group 1 (5 mice per group): Intraperitoneal injection of solvent (200 μL per mouse), followed by intraperitoneal injection of physiological saline (200 μL per mouse) 2 hours later, this time was taken as time 0;

[0087] Group 2 (5 mice per group): Ivacaftor solution was injected intraperitoneally (Ivacaftor dosage was 50 mg / kg body weight), and 2 hours later, physiological saline (200 μL per mouse) was injected intraperitoneally. This time was taken as time 0.

[0088] Group 3 (5 mice per group): Intraperitoneal injection of solvent (200 μL per mouse), followed by intraperitoneal injection of immunomodulatory solution (TNF-α dose of 30 μg / kg body weight and D-Gal dose of 700 mg / kg body weight) 2 hours later, this time was taken as time 0;

[0089] Group 4 (8 animals per group): Intraperitoneal injection of Ivacaftor solution (Ivacaftor dosage is 50 mg / kg body weight), followed by intraperitoneal injection of TNF-α solution (TNF-α dosage is 30 μg / kg body weight) and D-Gal solution (D-Gal dosage is 700 mg / kg body weight) 2 hours later, this time was recorded as time 0;

[0090] Ivacaftor solution: Composed of Ivacaftor and solvent, with an Ivacaftor concentration of 5 mg / ml. Immunosorbent solution: Composed of TNF-α, D-Gal, and physiological saline, with a TNF-α concentration of 3 μg / ml and a D-Gal concentration of 70 mg / ml.

[0091] Six hours after 0:00, blood was collected by enucleation, serum was separated, and the levels of LDH, ALT, and AST in the serum were measured. Statistical analysis was performed using a two-tailed unpaired t-test (P<0.0001).

[0092] Serum LDH levels are shown in the table. Figure 6 A, serum ALT levels are shown in [the table]. Figure 6 B, serum AST levels are shown in [the table]. Figure 6 C. TNF / D-Gal stimulation induced an increase in serum LDH, ALT, and AST, three biomarkers that are markers of hepatocellular injury and PMR. Ivacaftor pretreatment blocked the TNF / D-Gal-induced increase in serum LDH, ALT, and AST, indicating that Ivacaftor has the ability to limit tissue damage in vivo.

[0093] II. Validating the therapeutic effect of Ivacaftor using a ConA-induced hepatitis model

[0094] In this step, an immune liver injury model was induced by injecting ConA via the tail vein.

[0095] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into four groups of 5 mice each.

[0096] Group 1: Intraperitoneal injection of solvent (200 μL per mouse), followed by tail vein injection of PBS buffer (100 μL per mouse) 2 hours later, this time was taken as time 0;

[0097] Group 2: Ivacaftor solution was injected intraperitoneally (Ivacaftor dosage was 100 mg / kg body weight), and PBS buffer (100 μL per mouse) was injected via the tail vein 2 hours later. This time was taken as time 0.

[0098] Group 3: Intraperitoneal injection of solvent (200 μL per mouse), followed by tail vein injection of ConA solution (30 mg / kg body weight) 2 hours later, this time was taken as time 0;

[0099] Group 4: Ivacaftor solution was injected intraperitoneally (Ivacaftor dosage was 100 mg / kg body weight), and ConA solution was injected via tail vein 2 hours later (ConA dosage was 30 mg / kg body weight). This time was recorded as time 0.

[0100] Ivacaftor solution: Composed of Ivacaftor and solvent, with an Ivacaftor concentration of 10 mg / ml. ConA solution: Composed of ConA and PBS buffer, with a ConA concentration of 6 mg / ml.

[0101] Five hours after 0:00, blood was collected by enucleation of the eyeballs, serum was separated, and the levels of LDH, ALT, and AST in the serum were measured. Statistical analysis was performed using a two-tailed unpaired t-test (**p = 0.0012; ***p = 0.0007; ****p < 0.0001).

[0102] Serum LDH levels are shown in the table. Figure 6 E, serum ALT levels are shown in the figure. Figure 6 The F, serum AST content is shown in the figure. Figure 6 G. ConA stimulation induced an increase in serum LDH, ALT, and AST. Ivacaftor pretreatment blocked the ConA-induced increase in serum LDH, ALT, and AST, indicating that Ivacaftor has the ability to limit tissue damage in vivo.

[0103] Example 6: Effects of Ivacaftor on inflammatory diseases (septicemia model, acute).

[0104] This embodiment uses intraperitoneal injection of LPS to induce a sepsis model.

[0105] In this embodiment, "solvent" means: containing 0.5g / 100ml methylcellulose and 0.5% (volume percentage) Tween 80, with the remainder being water.

[0106] I. Low-dose LPS-induced sepsis model

[0107] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into two groups of 10 mice each.

[0108] Group 1 (LPS): The solvent (200 μL per mouse) was administered by gavage every 24 hours. Two hours after the first gavage, LPS solution was injected intraperitoneally (the dosage of LPS was 10 mg / kg body weight).

[0109] Group 2 (LPS + Ivacaftor): Ivacaftor solution was administered by gavage every 24 hours (the single dose of Ivacaftor was 50 mg / kg body weight), and LPS solution was injected intraperitoneally 2 hours after the first gavage (the dose of LPS was 10 mg / kg body weight).

[0110] Ivacaftor solution: Composed of Ivacaftor and solvent, with an Ivacaftor concentration of 5 mg / ml. LPS solution: Composed of LPS and physiological saline, with an LPS concentration of 1 mg / ml.

[0111] The time of the first gavage is taken as time 0.

[0112] Weighing was performed every 12 hours starting at time 0, up to 72 hours. Results are shown below. Figure 7 A.

[0113] Starting from time 0, scores are given every 12 hours (see Table 1 for scoring criteria) until 72 hours. The scores for each item are summed to obtain the total score. The total score is shown below. Figure 7 Group B. Compared to Group 1, Group 2 had a lower total score, indicating that Ivacaftor has a protective effect.

[0114] Table 1

[0115]

[0116]

[0117] II. Medium-dose LPS-induced sepsis model

[0118] 1. Draw the death curve

[0119] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into four groups of 10 mice each.

[0120] Group 1 (Control): The mice were given a solution (200 μL per mouse) by gavage every 24 hours. Two hours after the first gavage, the mice were injected intraperitoneally with physiological saline (200 μL per mouse).

[0121] Group 2 (Ivacaftor): Ivacaftor solution was administered by gavage every 24 hours (the single dose of Ivacaftor is 50 mg / kg body weight). Two hours after the first gavage, physiological saline was injected intraperitoneally (200 μL per mouse each time).

[0122] Group 3 (LPS): The solvent (200 μL per mouse) was administered by gavage every 24 hours. Two hours after the first gavage, LPS solution was injected intraperitoneally (the dosage of LPS was 20 mg / kg body weight).

[0123] Group 4 (LPS + Ivacaftor): Ivacaftor solution was administered by gavage every 24 hours (the single dose of Ivacaftor is 50 mg / kg body weight), and LPS solution was injected intraperitoneally 2 hours after the first gavage (the dose of LPS is 20 mg / kg body weight).

[0124] Ivacaftor solution: Composed of Ivacaftor and solvent, with an Ivacaftor concentration of 5 mg / ml. LPS solution: Composed of LPS and physiological saline, with an LPS concentration of 2 mg / ml.

[0125] The time of the first gavage is taken as time 0.

[0126] Weigh yourself every 12 hours starting from time 0. See the results below. Figure 7 C.

[0127] Starting from time 0, mice were scored every 12 hours (scoring criteria shown in Table 1) until 60 hours. Mice with a score of 12 or higher were defined as deceased mice. Survival rates were calculated. Results are shown below. Figure 7 D.

[0128] The results showed that Ivacaftor significantly protected mice.

[0129] 2. Detection of serum markers

[0130] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into four groups of 5 mice each.

[0131] Group 1 (Control): The mice were given a single dose of solvent (200 μL per mouse) by gavage, followed by an intraperitoneal injection of physiological saline (200 μL per mouse) two hours later.

[0132] Group 2 (Ivacaftor): Ivacaftor solution was administered once by gavage (the single dose of Ivacaftor is 50 mg / kg body weight), followed by intraperitoneal injection of physiological saline (200 μL per mouse each time) 2 hours after gavage.

[0133] Group 3 (LPS): The solvent was administered once by gavage (200 μL per mouse each time), and two hours after gavage, LPS solution was injected intraperitoneally once (the dosage of LPS was 20 mg / kg body weight).

[0134] Group 4 (LPS + Ivacaftor): Ivacaftor solution was administered once by gavage (the single dose of Ivacaftor is 50 mg / kg body weight), and LPS solution was injected intraperitoneally 2 hours later (the dose of LPS is 20 mg / kg body weight).

[0135] Ivacaftor solution: Composed of Ivacaftor and solvent, with an Ivacaftor concentration of 5 mg / ml. LPS solution: Composed of LPS and physiological saline, with an LPS concentration of 2 mg / ml.

[0136] Twenty-four hours after intraperitoneal injection, blood was collected by enucleation of the eyeball, serum was separated, and the levels of LDH, TNF-α, and BUN in the serum were measured.

[0137] The LDH content in serum is shown in the figure. Figure 7 E. Serum BUN levels are shown in [the table]. Figure 7 The F. Serum TNF-α levels are shown in [reference needed]. Figure 7 Compared with the first group, the serum levels of LDH, TNF, and BUN in the third group of animals were significantly increased. Compared with the third group, the serum levels of LDH, TNF, and BUN in the fourth group of animals were significantly decreased.

[0138] The results showed that Ivacaftor could protect against sepsis-induced tissue damage, suppress immune responses, and improve the pathological condition of the disease.

[0139] Example 7: Effects of Ivacaftor on inflammatory diseases (heatstroke model, acute).

[0140] In this embodiment, "solvent" means a liquid composed of 95 parts by volume of sesame essential oil and 5 parts by volume of DMSO.

[0141] I. Drawing the Death Curve

[0142] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into four groups of 10 mice each.

[0143] Group 1 (HS): The mice were injected intraperitoneally with saline (200 μL per mouse), and one hour later, they were injected intraperitoneally with solvent (200 μL per mouse). One hour after that, the mice were subjected to heat shock treatment and then fed normally.

[0144] Group 2 (LPS): One LPS solution was injected intraperitoneally (the dosage of LPS was 5 mg / kg body weight), and one hour later, the solvent was injected intraperitoneally (200 μL per mouse), and then the mice were fed normally.

[0145] Group 3 (LPS / HS): The mice were injected intraperitoneally once with LPS solution (LPS dosage was 5 mg / kg body weight), and one hour later, they were injected intraperitoneally once with solvent (200 μL per mouse). One hour after that, the mice were subjected to heat shock treatment and then fed normally.

[0146] Group 4 (LPS / HS+Ivacaftor): The mice were injected intraperitoneally once with LPS solution (the dosage of LPS was 5 mg / kg body weight), and one hour later, they were injected intraperitoneally once with Ivacaftor solution (the single dosage of Ivacaftor was 25 mg / kg body weight). One hour after that, the mice were subjected to heat shock treatment and then fed normally.

[0147] Heat shock treatment method: Mice were placed in an incubator (temperature 40℃, humidity 50%) for 2 hours, during which time they were deprived of water and food. Ivacaftor solution: composed of Ivacaftor and solvent, with an Ivacaftor concentration of 2.5 mg / ml. LPS solution: composed of LPS and physiological saline, with an LPS concentration of 0.5 mg / ml.

[0148] The intraperitoneal injection of LPS solution was taken as time 0, and survival rate was calculated every 6 hours until 30 hours. Results are shown below. Figure 8 B(*, P = 0.0232). No deaths occurred in groups 1 and 2. Mice in group 3 died rapidly. Compared to group 3, group 4 mice treated with Ivacaftor showed significant protection.

[0149] II. Detection of physiological indicators

[0150] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into four groups of 5 mice each.

[0151] The processing method for each group is the same as in step one.

[0152] Six hours after the heat shock treatment, blood was collected by enucleation, serum was separated, and the levels of LDH, IL-1β, HMGB1, IL-18, AST, and ALT in the serum were measured.

[0153] The HMGB1 content in serum is shown in the figure. Figure 8 The C (**, P = 0.0039) was obtained. Serum LDH levels are shown in [reference needed]. Figure 8 The D (**, P = 0.0024). Serum ALT levels are shown in the table. Figure 8E(*, P=0.0128). Serum AST levels are shown in [reference needed]. Figure 8 The F(**, P = 0.0094) was obtained. Serum IL-1β levels are shown in the table. Figure 8 The value of G (**, P = 0.0034). Serum IL-18 levels are shown in [reference needed]. Figure 8 H(*, P = 0.046). Compared with group II, the serum levels of HMGB1, LDH, AST, ALT, IL-1β, and IL-18 in group III mice were significantly increased. Compared with group III, the serum levels of HMGB1, LDH, AST, ALT, IL-1β, and IL-18 in group IV mice were significantly decreased. LDH and HMGB1 represent the levels of DAMPs released from tissue damage and cell death in serum, AST and ALT represent the degree of liver injury, and IL-1β and IL-18 represent the levels of cytokines, indicating inflammatory responses. The results indicate that Ivacaftor significantly protects the LPS-heat shock-induced heatstroke model.

[0154] Example 8: Effects of Ivacaftor on inflammatory diseases (enteritis model, acute).

[0155] This embodiment uses DSS water to induce an acute colitis model.

[0156] In this embodiment, "solvent" means a liquid composed of 98 parts by volume of sesame essential oil and 2 parts by volume of DMSO.

[0157] Male C57BL / 6 mice aged 7-8 weeks were randomly divided into four groups of 11 mice each.

[0158] The day of grouping is designated as day 0, and the individuals are weighed.

[0159] Group 1 (referred to as Vehicle): Distilled water was provided free access to drinking water from day 1 to day 10; a solvent (200 μL per mouse) was injected intraperitoneally once a day from day 1 to day 10.

[0160] Group 2 (referred to as Ivacaftor): Distilled water was provided free of charge for drinking from day 1 to day 10; Ivacaftor solution was administered intraperitoneally once a day from day 1 to day 10 (single dose of 18 mg / kg body weight);

[0161] Group 3 (represented by DSS+Vehicle or DSS): DSS water was provided as drinking water for free from day 1 to day 8, and distilled water was provided as drinking water for free from day 9 and day 10; from day 1 to day 10, the solvent (200 μL per mouse) was injected intraperitoneally once a day.

[0162] Group 4 (represented by DSS+Ivacaftor): DSS water was provided free of charge for drinking from day 1 to day 8, and distilled water was provided free of charge for drinking from day 9 to day 10; Ivacaftor solution was administered intraperitoneally once a day from day 1 to day 10 (single dose of 18 mg / kg body weight).

[0163] DSS water: Composed of DSS and distilled water, with a DSS content of 3g / 100ml. Ivacaftor solution: Composed of Ivacaftor and solvent, with an Ivacaftor content of 1.8mg / ml.

[0164] The mice's weight was recorded daily. Changes in weight over time are shown in [the table / document]. Figure 9 A(****,P<0.0001).

[0165] Daily changes in mouse weight, stool morphology, and rectal bleeding were recorded, and the Disease Activity Index (DAI) score was calculated. The total DAI score was calculated as: weight score + stool score + rectal bleeding score. The scoring criteria for each indicator are shown in Table 2. Based on the total DAI score, the mice's health status was categorized into different levels: 0-2 points: mild inflammation; 3-5 points: moderate inflammation; 6-8 points: severe inflammation; >8 points: extreme inflammation or near-death state. Changes in DAI scores over time are shown in Table 2. Figure 9 B (left side ***, P = 0.0001; ****, P < 0.0001; right side ***, P = 0.0007).

[0166] Table 2

[0167]

[0168] After scoring, the mice were euthanized, their colons were harvested, photographed, and their length measured. (See photos below.) Figure 9 C. Colon length see Figure 9 D(****,P<0.0001).

[0169] The results showed that, compared with the third group, the fourth group significantly inhibited the weight loss caused by DSS enteritis by administration of Ivacaftor, and also had significant protective effects on clinical scores and colon length indicators.

[0170] Example 9: Effects of Ivacaftor on inflammatory diseases (EAE multiple sclerosis model, chronic)

[0171] MOG (peptide), amino acid sequence as shown in SEQ ID NO: 3. Mycobacterium tuberculosis H37Ra: BD Pharmaceuticals, catalog number 3306830. Pertussis toxin: GLPBIO Pharmaceuticals, catalog number GC17532.

[0172] Preparation method of the immunizing agent: Dissolve Mycobacterium tuberculosis H37Ra in Freund's complete adjuvant to a concentration of 10 mg / ml, i.e., solution A; dissolve MOG in PBS buffer to a concentration of 2 mg / ml, i.e., solution B; mix equal volumes of solution A and solution B, thoroughly mix and emulsify to obtain an emulsion (water-oil mixture), which is the immunizing agent.

[0173] In this embodiment, "solvent" means a liquid composed of 98 parts by volume of sesame essential oil and 2 parts by volume of DMSO.

[0174] Seven-week-old female C57BL / 6 mice were randomly divided into two groups of eight each.

[0175] The day of grouping will be designated as Day 0, and clinical scoring will be conducted.

[0176] Group 1 (Vehicle): Day 1, mice were anesthetized with isoflurane, and then 100 μL of immunizing agent was injected subcutaneously in front of and behind the spine on the back of the mice (a total of 200 μL of immunizing agent was injected into each mouse). Then, a pertussis toxin solution was injected intraperitoneally once (the dosage was 250 ng per mouse). 24 hours after the first intraperitoneal injection, a second intraperitoneal injection of pertussis toxin solution was given (the dosage was 250 ng per mouse). From Day 7 to Day 24, the solvent was injected intraperitoneally once a day (200 μL per mouse each time).

[0177] Group 2 (Ivacaftor): Day 1, mice were anesthetized with isoflurane, and then 100 μL of immunizing agent was injected subcutaneously in front of and behind the spine on the back of the mice (a total of 200 μL of immunizing agent was injected into each mouse). Then, a pertussis toxin solution was injected intraperitoneally once (the dosage was 250 ng per mouse). 24 hours after the first intraperitoneal injection, a second intraperitoneal injection of pertussis toxin solution was given (the dosage was 250 ng per mouse). From Day 7 to Day 24, Ivacaftor solution was injected intraperitoneally once a day (single dose was 18 mg / kg body weight).

[0178] Ivacaftor solution: Composed of Ivacaftor and solvent, with an Ivacaftor concentration of 1.8 mg / mL. Pertussis toxin solution: Dissolve 50 μg of pertussis toxin in ultrapure water and bring the volume to 0.5 mL to obtain the stock solution (store at 4°C). Before use, dilute the stock solution with PBS buffer to a 100-fold volume to obtain the pertussis toxin solution (with a pertussis toxin concentration of 1 μg / mL).

[0179] Mice were scored daily (scoring criteria are shown in Table 3), and the incidence rate was recorded. The results of the daily clinical scores are shown below. Figure 10The results for the incidence rate of A are shown in [the table / reference]. Figure 10 B.

[0180] Table 3

[0181]

[0182] After completing all records, the mice were euthanized, the spinal cord was harvested, and spinal cord sections were prepared for H&E staining and LFB staining. See the spinal cord pathology sections below. Figure 10 The control group consisted of untreated, age-matched female C57BL / 6 mice.

[0183] The results showed that, compared with the first group, the second group significantly suppressed the severity of EAE by administration of Ivacaftor, indicating that Ivacaftor provides a protective effect in the multiple sclerosis model.

[0184] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of Ivacaftor in the preparation of drugs for the treatment of heatstroke.

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