Use of triazolo[4,3-b]pyridazine derivatives in the prevention and treatment of aging and aging-related diseases [1,2,4]

Abstract

To provide a use of the small molecule compound [1,2,4]triazolo[4,3-B]pyridazine derivative.SOLUTION: The present invention provides a use of a [1,2,4]triazolo[4,3-B]pyridazine derivative represented by formula (I) in the manufacture of a drug for preventing and / or treating aging and / or aging-related diseases. The aging-related diseases include at least one of atherosclerosis, vascular calcification, diabetes, autoimmune diseases, osteoarthritis, Alzheimer's disease, and tissue / organ fibrosis.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and specifically relates to the use of [1,2,4]triazolo[4,3-b]pyridazine derivatives in the prevention and treatment of aging and aging-related diseases.

Background Art

[0002] Cell aging is a timing-related cell dysfunction. Aging cells undergo degenerative changes in cell structure and manifest through reduced function and metabolism. Physiological aging is a slow process, but viral infection, radiation, various diseases, and other factors can cause pathological aging and accelerate the aging of the body.

[0003] In addition, the decline of the body's biological functions and an individual's aging process are often accompanied by the occurrence of various related diseases. For example, chronic brain degeneration can affect cognitive function and increase the incidence of Alzheimer's disease and neurodegenerative diseases. Usually, aging cells highly express inflammatory factors and other molecules that regulate the immune response, causing immune system disorders such as immune deficiency and chronic inflammation, and leading to autoimmune diseases and osteoarthritis. Aging is accompanied by damage to the angiogenesis function, and aging reduces the antithrombotic function of the endothelium, becoming a risk factor for cardiovascular diseases such as atherosclerosis and vascular calcification. The decline in pancreatic cell function associated with aging causes the body to be unable to normally respond to blood glucose levels, reduces the ability to regulate blood glucose levels (i.e., glucose tolerance), leads to abnormal glucose metabolism, and ultimately results in the onset of diabetes. Also, cell aging means that the cell cycle becomes a stable state. Aging cells still have metabolic ability but cannot proliferate further, and promote the excessive secretion of extracellular matrix proteins through the secretion of inflammatory factors and the like, and are involved in the occurrence and progression of tissue and organ fibrosis.

[0004] Therefore, the development of effective anti-aging intervention strategies is very important for preventing and / or treating aging and aging-related diseases and reducing their incidence.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention aims to provide a novel use of the low-molecular compound [1,2,4]triazolo[4,3-b]pyridazine derivative (i.e., C1632), namely, the use of C1632 in the prevention and treatment of aging and aging-related diseases.

[0006] Conventionally, the present inventors have disclosed the use of C1632 in the treatment of the novel coronavirus SARS-CoV-2 and anti-inflammatory cytokine storm. However, the inflammatory cytokine storm is a severe acute immune system disease. Usually, the infection of a pathogen stimulates the innate immune pathway of cells, promoting the cells to express a large amount of inflammatory cytokines within a short time. The inflammatory cytokines change body temperature, affect the systemic metabolism of the human body, have adverse effects such as increasing sputum and blocking alveoli, and ultimately lead to respiratory distress, multiple organ failure, and even death of the patient. On the other hand, the aging and aging-related diseases of the present invention are completely different from the inflammatory cytokine storm and novel coronavirus infection according to the prior art. Specifically, the aging-related diseases of the present invention refer to the functional changes that occur in a living body or any of its tissues, organs, or cells, accompanied by a decline in reproductive function after the period when reproductive function reaches its peak, and this change is associated with aging. Aging-related diseases refer to any diseases, conditions, degenerations, tissue losses, or other unhealthy or abnormal conditions caused by or related to aging. Obviously, this is very different from the acute and severe diseases caused by exogenous substances mentioned in the prior art.

Means for Solving the Problems

[0007] Specifically, the technical solution adopted by the present invention is as follows.

[0008] The first aspect of the present invention is the use of a [1,2,4]triazolo[4,3-b]pyridazine derivative in the manufacture of a drug for preventing and / or treating aging and / or aging-related diseases, The [1,2,4]triazolo[4,3-b]pyridazine derivative is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

Chemical formula

[0009] According to some embodiments of the present invention, the aging includes at least one of aging diseases, aging of the skin, brain, lung, heart, bone marrow, immune liver, and kidney tissue organs.

[0010] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative inhibits cellular aging.

[0011] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative can extend the lifespan.

[0012] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative relieves the immunosuppressive microenvironment of senescent cells and / or promotes the immune clearance of senescent cells.

[0013] According to some embodiments of the present invention, the vascular calcification is aortic vascular calcification.

[0014] According to some embodiments of the present invention, the fibrosis of tissues and organs includes liver fibrosis, pulmonary fibrosis, and renal fibrosis.

[0015] According to some embodiments of the present invention, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0016] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative improves the phenomenon of alveolar reduction and increased lung density during the progression of idiopathic pulmonary fibrosis.

[0017] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative inhibits fibroblast proliferation.

[0018] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative inhibits lung hydroxyproline.

[0019] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative inhibits immune cell infiltration. Preferably, the immune cells include CD45-positive cells (total immune cells), CD68-positive cells (macrophages), and CD3-positive cells (T cells).

[0020] According to some embodiments of the present invention, the renal fibrosis is renal fibrosis in chronic kidney disease.

[0021] According to some embodiments of the present invention, the [1,2,4]triazolo[4,3-b]pyridazine derivative inhibits the abnormal expression of collagen fibers in chronic kidney disease.

[0022] According to some embodiments of the present invention, the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, dripping pills, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0023] According to some embodiments of the present invention, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration, or transdermal administration.

[0024] According to some embodiments of the present invention, the drug further includes a pharmaceutically acceptable auxiliary substance.

[0025] Preferably, the pharmaceutically acceptable auxiliary substance contains at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubilizer, a preservative, a pH adjuster, an osmotic pressure adjuster, a surfactant, a coating material, an antioxidant, a bacteriostatic agent, or a buffer.

[0026] According to some embodiments of the present invention, the dosage form of the drug contains at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a dripping pill, an injection, an oral preparation, a suppository, an enema, an aerosol, a patch, or a drop.

[0027] According to some embodiments of the present invention, the administration route of the drug contains at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration, or transdermal administration.

[0028] According to some embodiments of the present invention, the drug further contains at least one other component useful for the prevention and / or treatment of aging and / or aging-related diseases.

[0029] According to some embodiments of the present invention, the dosage form of the drug contains at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a dripping pill, an injection, an oral preparation, a suppository, an enema, an aerosol, a patch, or a drop.

[0030] According to some embodiments of the present invention, the administration route of the drug contains at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration, or transdermal administration.

[0031] According to some embodiments of the present invention, the drug further contains a pharmaceutically acceptable auxiliary substance and at least one other component useful for the prevention and / or treatment of aging and / or aging-related diseases.

[0032] According to some embodiments of the present invention, the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, dripping pills, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0033] According to some embodiments of the present invention, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration, or transdermal administration.

[0034] A second aspect of the present invention provides a drug comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a [1,2,4]triazolo[4,3-b]pyridazine derivative, which is a drug for the prevention and / or treatment of aging and / or aging-related diseases.

Chemical formula

[0035] Preferably, the drug further comprises at least one other component useful for the prevention and / or treatment of aging and / or aging-related diseases.

[0036] Preferably, the drug further comprises a pharmaceutically acceptable adjuvant. Preferably, the pharmaceutically acceptable adjuvant includes at least one of a diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, solubilizer, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, bacteriostatic agent, or buffer.

[0037] Preferably, the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, dripping pills, injections, oral preparations, suppositories, enemas, aerosols, patches, or drops.

[0038] Preferably, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration, or transdermal administration.

[0039] The C1632 low-molecular-weight drug is soluble in water, ethanol or DMSO, has good thermal stability, low cytotoxicity, good drug safety, has good pharmacokinetic properties, and can be flexibly administered in multiple ways.

Advantages of the Invention

[0040] The beneficial effects of the present invention are as follows.

[0041] The present invention has found that the [1,2,4]triazolo[4,3-b]pyridazine derivative (C1632) represented by formula (I) and its pharmaceutically acceptable salts are useful for the prevention and / or treatment of aging and / or aging-related diseases. For example, C1632 can: (1) significantly inhibit cell aging, extend the lifespan of mice, inhibit lung aging, relieve the immunosuppressive microenvironment during aging, promote the immune elimination of aging cells, and treat related diseases caused by aging. (2) significantly inhibit the progression of liver fibrosis, have good drug safety and high anti-liver fibrosis effect, and can be used as a drug candidate for the treatment of liver fibrosis. (3) significantly inhibit the progression of idiopathic pulmonary fibrosis, reduce fibroblast proliferation and collagen synthesis caused by idiopathic pulmonary fibrosis, inhibit the activity of inflammatory infiltration of immune cells, protect the alveolar structure, inhibit the increase in lung density caused by idiopathic pulmonary fibrosis, and improve lung density and ventilation volume. (4) significantly inhibit the progression of renal fibrosis in mice with chronic kidney disease, improve the weight loss of mice caused by chronic kidney disease, and reduce the mortality of mice caused by chronic kidney disease. (5) improve the calcification of the aorta in mice and improve vascular compliance.

[0042] In addition, C1632 is a known low-molecular-weight drug, is soluble in water, ethanol or DMSO, has good thermal stability, low cytotoxicity, excellent drug safety, has good pharmacokinetic properties, and can be flexibly administered in multiple ways such as oral, intraperitoneal injection, intramuscular injection, intravenous injection, etc., and can be used as a safe, effective and easy-to-use drug candidate.

[0043] In the present invention, "Pharmaceutically acceptable" means a compound, material, composition and / or dosage form that, within the scope of reasonable medical judgment, is suitable for contact with human and animal tissues for use with a reasonable benefit / risk ratio without undue toxicity, irritation, allergic reaction, or other problems or complications.

[0044] The "pharmaceutically acceptable salts" described in the present invention refer to salts formed by acidic functional groups (e.g., -COOH, -OH, -SO3H, etc.) present in the compound with appropriate inorganic or organic cations (alkalis), such as salts formed with alkali metals or alkaline earth metals, ammonium salts, and salts formed with nitrogen-containing organic bases, and salts formed by basic functional groups (e.g., -NH2, etc.) present in the compound with appropriate inorganic or organic anions (acids), such as salts formed with inorganic acids or organic acids (e.g., carboxylic acids, etc.). These salts can be prepared during the synthesis, isolation, purification of the compound, or by reacting the free form of the purified compound alone with an appropriate acid or base.

[0045] "Aging-related diseases" refer to tissue degeneration or progressive pathological changes associated with and induced by aging, including fibrosis of tissues and organs, neurodegenerative diseases, cardiovascular diseases, osteoarthritis, and chronic kidney diseases, etc. The accumulation of senescent cells is an important cause leading to aging-related diseases. The abnormal accumulation of senescent cells leads to a decline in the body's ability to maintain and recover homeostasis under stress and injury. Furthermore, the inflammatory microenvironment caused by aging causes chronic tissue damage and promotes aging-related diseases such as atherosclerosis, vascular calcification, diabetes, autoimmune diseases, osteoarthritis, Alzheimer's disease, and fibrosis of tissues and organs. Or, "aging-related diseases" refer to those diseases such as viral infections and trauma, etc., where the disease itself has no direct relation with aging and is caused by external factors, but the prognosis of the disease is changed by aging itself.

[0046] "Aging disease" is a congenital genetic disease characterized by rapid progression of body aging, decline in memory, and shortening of lifespan.

[0047] "Fibrosis of tissues and organs" is a progressive disease in which tissues and organs secrete excessive extracellular matrix proteins to repair their damaged sites, forming permanent scars, and ultimately leading to organ deformation and dysfunction. It is related to the formation of excessive fibrous connective tissue in organs and tissues during repair or reaction. Fibrosis can be in a benign state, but the present invention preferably relates to the pathological state of fibrosis.

[0048] "Vascular Calcification" (VC) is a disease characterized by calcium salt deposition in the intima and media of blood vessels, and is closely related to various diseases such as atherosclerosis, diabetes, and chronic kidney disease, which has a profound impact on the prognosis of these diseases.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0050] Hereinafter, in order to fully understand the object, features and effects of the present invention, with reference to the examples, the concept of the present invention and the obtained technical effects will be clearly and completely described. Obviously, the described examples are only some examples, not all examples. All other examples obtained by those skilled in the art without creative efforts based on the examples of the present invention are included in the protection scope of the present invention.

[0051] Chemical synthesis of C1632: The [1,2,4]triazolo[4,3-b]pyridazine derivative C1632 was synthesized in this laboratory, and its structural formula is shown by Formula (I).

Chemical formula

[0052] The synthesis route is as follows.

Chemical formula

[0053] C1632 is a known small molecule drug, soluble in water or DMSO, has good thermal stability, low cytotoxicity, good drug safety, has good pharmacokinetic properties, and can be flexibly administered by multiple methods such as oral, intramuscular injection, and intravenous injection.

[0054] Example 1 Therapeutic effect of C1632 on aging-related diseases The C57BL / 6 mice used in this example were purchased from Guangdong Pharmaron Biotechnology Co., Ltd. in China. The TERC- / - rats were provided by the laboratory of Professor Juzhen Yu at Jinan University in China. The human primary cells MRC5 and BJ, and the mouse TC1 cells were purchased from ATCC.

[0055] The specific operation steps for IHC detection in this example are as follows. For paraffin sections, the paraffin was melted at 65 °C for 1 h, followed by 10 min in xylene - 10 min in xylene - 3 min in absolute ethanol - 3 min in 95% ethanol - 3 min in 85% ethanol - 3 min in 70% ethanol. Then, the sections were placed in water and washed twice with PBS for 5 min each. They were blocked with 3% hydrogen peroxide (prepared with methanol) for 10 min and washed twice with PBS for 5 min each. The antigen retrieval solution with pH 6.0 was heated in a microwave for 10 min for antigen retrieval, and the temperature was slowly decreased from room temperature over 1 h. The sections were washed twice with PBS for 5 min each. They were blocked with 10% goat serum for 1 h, incubated with the primary antibody at 4 °C in the dark overnight, and washed twice with PBS for 5 min each. The secondary antibody was incubated at room temperature in the dark for 30 min and washed twice with PBS for 5 min each. Color development was carried out with DAB, stained with hematoxylin, rinsed with tap water for 8 min for bluing. Dehydration was carried out in a gradient with alcohol and dried, and then sealed with neutral balsam.

[0056] 1.1 MRC5 and BJ cells were cultured in DMEM low - glucose medium containing 10% FBS. The cells were passaged or plated at a ratio of 1:3, and 0.5×10 5 The cells were plated in 12 - well plates. The next day, they were treated with VP16 at a final concentration of 40 μM for 4 - 7 days to induce cell senescence and construct a cell senescence model. At the same time as inducing cell senescence, C1632 at a final concentration of 120 μM was added for treatment. The effect of C1632 on the senescence of BJ and MRC5 cells was detected by SA - β - gal staining, and the effect of C1632 on cell senescence was investigated. The results are shown in Figure 1. Here, A in Figure 1 shows β-gal staining, demonstrating that C1632 treatment can inhibit the induction of senescence in BJ cells and MRC5 cells by VP16. B in Figure 1 and C in Figure 1 show the statistical results of the β-gal positive cell rates of BJ cells (B) and MRC5 cells (C) in each treatment group. ** represents p < 0.01. As a result, it was suggested that C1632 can effectively delay VP16-induced cell senescence.

[0057] 1.2 1. TERC - / - G4 senescent mouse model: The mice have very long telomere lengths. After knocking out telomerase, if they do not reproduce for 3 to 6 generations, no premature aging phenotype will appear. TERC + / - The mice were mated, and TERC in the offspring + / - was screened for breeding purposes. The primary TERC - / - mice are G1 mice. G1 mice are mated with each other to produce G2 mice. When they reproduce to G4, the mice have very short telomeres, and an aging phenotype may appear at 3 to 4 months of age. 2. Identification of the genotype of TERC knockout mice: A small amount of the tail was cut from the mice to be identified and placed in a buffer of 10 mM Tris-HCl, 0.5% SDS, 1 mM EDTA, pH 8.0 containing 0.4 mg / mL proteinase K. The mouse tail was digested at 65 °C for 2 h, and then 0.5 μL was used as a template for PCR amplification. When the PCR product size was 250 bp, it indicated the wild type, and when the product size was 180 bp, it indicated TERC knockout. 3. When 68 mg / kg of C1632 was administered once a week to 10 G4 mice at 1 month of age per group, all the mice in the control group died after 3.5 months of the experiment, while 80% of the mice in the administration group survived. From this, it was suggested that C1632 - / - significantly extended the lifespan of G4 mice and reduced the risk of death from all causes (Figure 2A). Furthermore, when the lungs of the mice were subjected to immunohistochemical detection to detect the expression of the senescence markers p16 and p21, it was suggested that C1632 significantly inhibited the senescence of lung tissue (Figure 2B). 4. Since senescent cells are mainly removed by immune cells in vivo, in addition to inhibiting the production of senescent cells, the Applicant also detected the effect of C1632 on the immune microenvironment of senescent lung tissue. As a result of immunohistochemical analysis of the lung tissue of senescent mice, it was suggested that C1632 treatment could reduce the immunosuppressive T cell marker FOXP3 in senescent tissues (Figure 3).

[0058] 1.3 1. The Applicant induced TC-1 cell senescence with bleomycin and reflected the status of TC-1 cell senescence by SA-β-Gal staining (A in Figure 4).

[0059] 2. T cell killing experiment: Bleomycin-induced senescent TC1 cells were plated in a 96-well plate at 10,000 cells / well. The next day, the cell culture supernatant was discarded. Next, PBMC, CD3 + T cells, and CD8 + T cells separated from normal mice, saline-treated idiopathic pulmonary fibrosis (IPF) mice, and C1632-treated IPF mice were added at a ratio of 1:50. After incubation at 37°C for 4 h, centrifugation was performed to collect the supernatant. Since the killed senescent cells release LDH enzyme, the killing effect of immune cells on senescent cells was detected using an LDH kit. Among them, an IPF mouse model was constructed by spraying 2 mg / kg of bleomycin into the lungs of mice. In the above experiment, at least 6 mice were used for each group.

[0060] CD8 + Separation and culture of CD8 + T cells: Under sterile conditions, the spleen of a mouse was collected and crushed, then passed through a 200-mesh sieve. The filtered cells were sorted using a CD8

[0061] As a result, compared with the physiological saline-treated IPF mice, in the C1632-treated IPF mice, the killing ability of PBMC and CD3 + T cells against TC-1 cells was significantly enhanced, especially the killing ability of CD8 + T cells against senescent TC-1 cells was significantly higher than that in normal mice and physiological saline-treated IPF mice (B in Figure 4). From this, it was suggested that C1632 significantly improved the targeted killing ability against senescent cells of the immune system and promoted the immune clearance of senescent cells.

[0062] Example 2 Therapeutic effect of C1632 on liver fibrosis The C57BL / 6 mice in this example were 8 weeks old and were purchased from Guangdong Pharmaron Biotechnology Co., Ltd. in China.

[0063] To investigate whether C1632 can treat liver fibrosis, the applicant intraperitoneally injected 1 ml / kg of carbon tetrachloride (CCl4) twice a week for 6 consecutive weeks to construct a liver fibrosis model. Specifically, it is as follows.

[0064] The C57BL / 6 mice were divided into a normal control group, a CCl4 group, and a CCl4 + C1632 group, with 7 mice in each group. In the CCl4 + C1632 group, after each injection of CCl4, treatment was carried out by injecting C1632 at a dose of 17 mg / kg. At the end of the experiment, the mice were sacrificed, the livers were collected, fixed with 4% paraformaldehyde, and then dehydrated, embedded, and sectioned.

[0065] 1. Masson staining can stain collagen fibers in tissues blue and muscle fibers and cytoplasm red. Usually, the sections were dewaxed in water and stained with the prepared Weigert iron hematoxylin for 5 - 10 min. Differentiated with 1% hydrochloric acid alcohol differentiation solution and washed with water. Blued with Masson blue solution and washed with water for 1 min. Stained with Ponceau red - magenta staining solution for 5 - 10 min. Immersed in 1% glacial acetic acid for a while and washed. Washed with phosphomolybdic acid solution for 1 - 2 min. Washed with 1% glacial acetic acid for 1 min. Put directly into aniline blue staining solution and stained for 1 - 2 min. The sections were differentiated by rinsing continuously in 1% glacial acetic acid in three tanks for about 8 s each. The sections were dehydrated with absolute ethanol in three tanks in the order of about 5 s, 10 s, and 30 s respectively. Then, dehydrated with n - butanol in two tanks in the order of 30 s and 2 min, and finally, cleared with xylene in two tanks for 5 min each and sealed with neutral balsam. From the results shown in Figure 5, it was suggested that there was only a small amount of collagen fibers in normal liver tissue, but a large amount of collagen fibers were expressed in the liver tissue of CCl4 - modeled mice, and C1632 could inhibit CCl4 - induced collagen fiber expression (Figure 5).

[0066] 2. To further verify the effect of C1632 on liver fibrosis, Van Gieson (VG) staining was performed on the above - mentioned tissue sections. VG staining is a mature connective tissue staining method and is used to distinguish collagen fibers and muscle fibers. The paraffin sections were dewaxed in water, the sections were put into the VG staining solution prepared with acid magenta and picric acid, stained for 1 min, quickly washed with water, and quickly dehydrated with absolute ethanol in three tanks. The sections were put into xylene for clearing and sealed with neutral balsam. As a result of taking pictures under the microscope, it was shown that CCl4 increased the expression of collagen fibers in mouse liver tissue. On the other hand, C1632 significantly decreased the collagen fiber level in liver tissue (Figure 6).

[0067] 3. Reticular fibers are less abundant in loose connective tissue, with thin, branched fibers that intersect to form a network. Reticular fibers have a transverse striation structure at equal intervals, resemble collagen fibers, and their chemical composition is also type III collagen. Reticular fibers are the main scaffolds for normal hepatocytes and hepatic sinusoids, and their changes can reflect the morphology of the reticular scaffolds in liver diseases. The distribution characteristics and destruction of reticular fibers indicate the characteristics of regenerative nodules, proliferative lesions, tumors, and the degree of fibrosis in cirrhosis, and are also useful for the regular evaluation of the progression of non-cirrhotic portal hypertension. The collagen fibers of reticular fibers are rich in glycoproteins and can be stained black with argentaffin. Sections dehydrated in water were oxidized with Gordon-Sweets oxidant for 5 min. After washing with water, they were bleached with oxalic acid solution for 1 - 2 min. After washing with distilled water, they were mordanted with ammonium ferric sulfate solution for 5 min. After washing slightly with distilled water, Gordon-Sweets silver ammonia solution was dropped and stained for 3 min. After washing slightly with distilled water, they were reduced with Gordon-Sweets reducing agent for 1 min and rinsed with running water for 10 min. The cell nuclei were stained with nuclear fast red solution for 5 - 10 min. After washing with water, they were dehydrated, cleared, and mounted with neutral balsam. As a result, only a small amount of reticular fibers were present in normal liver tissue, but a large amount of reticular fibers were expressed in the liver tissue of CCl4-modeled mice, suggesting that C1632 can inhibit the expression of CCl4-induced reticular fibers (Figure 7).

[0068] 4. Picro Sirius Red Staining (Picro Sirius Red Stin) is a simple and sensitive method for identifying the collagen fiber network in tissue sections and qualitatively or quantitatively identifying abnormal changes in the collagen network due to degenerative lesions and genetic or acquired diseases through morphological image analysis. Picro Sirius Red is a powerful linear anionic dye containing six sulfonic acid groups that can bind firmly to cationic collagen fibers. In polarized light detection, collagen fibers have positive uniaxial birefringence properties. When combined with Picro Sirius Red dye, the original birefringence properties of collagen are enhanced, enabling the distinction between type I and type III collagen. The paraffin sections were dewaxed in water and stained by dropping the picrosirius red staining solution for 1 h. After washing with water, the cell nuclei were stained with Mayer's hematoxylin staining solution for 8 - 10 min. After washing with water, dehydration and clearing were performed by the conventional method, and then sealed with neutral balsam. In polarized light microscopy examination, type I collagen was strongly orange - yellow or bright red, and type III collagen was green. From the results, it was suggested that in the constructed liver fibrosis model, CCl4 mainly caused the production of type I collagen in the liver, while C1632 could significantly inhibit the production of type I collagen (Figure 8). From the above, liver fibrosis in the C1632 - treated group was significantly reduced, indicating that liver fibrosis can be effectively treated.

[0069] Example 3 Therapeutic effect of C1632 on pulmonary fibrosis The C57BL / 6 mice used in this example were purchased from Guangdong Pharmaron Biotechnology Co., Ltd. in China, and bleomycin sulfate was purchased from Aladdin.

[0070] In this example, the method for constructing IPF model mice is as follows.

[0071] After anesthetizing the mice, 2 mg / kg of bleomycin was injected into the lungs through the airway to construct a mouse model of pulmonary fibrosis.

[0072] In this example, the operation steps for measuring the lung density of mice are as follows.

[0073] Lung density was measured using the drainage method as follows. (1) After anesthetizing the mice, they were sacrificed, and their lungs were removed and weighed, recorded as Mlung. (2) Next, the weight of an empty 1.5 mL centrifuge tube was measured. After filling the centrifuge tube with physiological saline, the weight of the centrifuge tube was recorded again, and the exact volume Vtube of the tube was calculated from the weight and density of the physiological saline. (3) The lung tissue was completely immersed in the physiological saline in the centrifuge tube, air was removed, the lid was closed, the water around the tube was wiped off, and the weight was measured and recorded as M. (4) The weight of the physiological saline in the tube Msaline = Mtube - Mlung was calculated. (5) The volume of the physiological saline in the tube Vsaline = Msaline × ρsaline was calculated, where ρsaline is the density of the physiological saline. (6) The lung volume Vlung = Vtube - Vsaline was calculated. (7) The lung density ρlung = Mlung / Vlung was calculated.

[0074] The operation steps for immunohistochemical measurement of immune cell infiltration in lung tissue in this example are as follows.

[0075] The lung tissue was immersed in 4% paraformaldehyde for fixation and then gradient dehydration was performed according to the following procedure: 70% ethanol for 5 h, 80% ethanol for 1 h; 90% ethanol for 1 h; 95% ethanol for 1 h; absolute ethanol for 1 h; absolute ethanol for 0.5 h; absolute ethanol + TO (1:1) for 1 h; TO for 1 h; TO for 1 h; paraffin (62 °C) for 1 h; paraffin (62 °C) for 1 h; paraffin (62 °C) for 1 h. After dehydration, it was embedded in a wax block and sectioned with a section thickness of 4 μm. After baking the paraffin sections at 65 °C for 1 h, they were treated with xylene for 5 min, xylene for 5 min, absolute ethanol for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and boiled in a citrate repair solution for 10 min. Next, it was naturally cooled to room temperature. It was treated with 3% hydrogen peroxide solution for 10 min. It was blocked at room temperature for 1 h using goat antiserum. The primary antibodies of CD45 (abcam), CD3 (abcam), or CD68 (abcam) were incubated overnight. Next, it was washed 3 times with PBS for 5 min each time. The immunohistochemical secondary antibody (Fujian Maixin) was added and incubated at room temperature for 20 min. Subsequently, color development was performed for 2 min using a DAB color development kit (Fujian Maixin). After washing DAB with water, it was stained with hematoxylin for 2 min. It was rinsed with running water for 8 min for bluing. After the sections were dried, neutral balsam was added for sealing and photographs were taken.

[0076] In this example, the degree of lung tissue fibrosis was detected by Masson staining using a Masson trichrome staining kit (Fujian Maixin).

[0077] 3.1 To investigate whether C1632 can be used for the treatment of IPF, the applicant administered C1632 to IPF mice according to the method in Figure 9A. Specifically, the mice were divided into (1) normal mice + saline group, (2) normal mice + C1632 group, and (3) IPF mice + saline group. The IPF + C1632 group had at least 4 mice per group. After the experiment, the lung tissues of the mice were collected and photographed. It was found that the lungs of the control group had no obvious abnormalities, were pink, and had a smooth surface. The lungs of the model group were dark red, some lung lobes were grayish white, the texture was hard, and scattered grayish white spots of various sizes were observed. The lungs of the C1632-treated model group were similar to those of the control group in terms of morphology, color, and hardness (Figure 9B). As a result of placing the above lung lobes in water, it was found that the density of the lungs of IPF model mice was higher than that of water, while the densities of the lungs of normal mice and C1632-treated IPF mice were lower than that of water (Figure 9C).

[0078] The lung densities of the mice in each group were further detected. The results suggested that the lung density of IPF mice was higher, and C1632 treatment could significantly reduce the lung density of IPF mice (Figure 9D). This indicated that C1632 could significantly improve the air content in the lungs of IPF mice, thereby reducing the lung density and improving the lung air exchange ability. H&E and Masson staining also showed that the lungs of the mice in the C1632 treatment group contained more alveolar structures and less fibrotic areas (Figure 9E - Figure 9G).

[0079] Hydroxyproline is an amino acid specific to collagen, and its content is an important indicator for detecting lung fibrosis. From the quantitative results of hydroxyproline in the lungs of the mice in each treatment group, it was suggested that C1632 could significantly inhibit the increase in lung hydroxyproline in mice (Figure 9I). The above results indicated that C1632 could significantly inhibit the progression of idiopathic pulmonary fibrosis in mice.

[0080] 3.2 In Example 3.1, it was described that C1632 inhibits the progression of pulmonary fibrosis, and the applicant speculates that C1632 can also inhibit the recruitment of immune cells during pulmonary fibrosis. Therefore, the infiltration of CD45 (total immune cells), CD68 (macrophages), and CD3 (T cells) in the C1632 treatment group was detected by IHC. As a result, it was suggested that the injection of C1632 significantly reduced the infiltration of immune cells including macrophages and T cells in the lungs of IPF mice (Figure 10). It was demonstrated that C1632 inhibits immune infiltration due to idiopathic pulmonary fibrosis.

[0081] 3.3 To detect the drug safety of C1632, the applicant intraperitoneally injected 68 mg / kg body weight of C1632 into each of the IPF model C57BL / 6 mice and control mice three times a week and measured their body weights. At the end of the experiment, whole blood was collected for routine blood tests. As a result, it was suggested that C1632 did not cause a significant change in the body weight of the mice compared with the corresponding normal saline group (A in Figure 11). Furthermore, the routine blood test indexes of each group of mice were mostly within the reference range. When C1632 was administered to normal mice, the mean platelet volume (MPV) increased significantly, but the administration of C1632 to the model group did not have a significant effect on MPV. C1632 extremely significantly inhibited PDW (mean platelet volume) in normal mice, but did not have a significant effect on this index in fibrotic mice. In short, C1632 did not cause deterioration of the routine blood test indexes in IPF mice (B to S in Figure 11). It was demonstrated that C1632 has good drug safety.

[0082] Example 4 Therapeutic effect of C1632 on renal fibrosis The C57BL / 6 mice used in this example were purchased from Guangdong Pharmaron Biotechnology Co., Ltd. in China, and the feed containing 1.5‰ and 2‰ purine (AP) was processed by the Guangdong Provincial Center for Medical Experimental Animals in China.

[0083] In this example, the steps for constructing a chronic kidney disease (CKD) model mouse are as follows. After feeding 8-week-old C57BL / 6 male mice with a diet containing 2‰ purine for 1 month, the diet was switched to a diet containing 1.5‰ purine and the mice were bred for 3 months. Each group of mice consisted of at least 10 mice.

[0084] 4.1 The mice were divided into three groups of 10 each: (1) normal group, (2) AP+Saline group, and (3) AP+C1632 group. In group (3), after feeding with 2‰ AP diet for 1 month, C1632 was administered for treatment. Among them, C1632 was administered to CKD mice twice a week at a dose of 68 mg / kg body weight for treatment. The C1632 treatment group significantly inhibited the weight loss of CKD mice compared with the control group (Figure 12).

[0085] 4.2 The mice were divided into three groups of 10 each: (1) normal group, (2) AP+Saline group, and (3) AP+C1632 group. In groups (2) and (3), after feeding with 2‰ AP diet for 1 month, C1632 was administered for treatment. Among them, C1632 was administered to CKD mice twice a week at a dose of 68 mg / kg body weight for treatment. The survival status of the mice was statistically analyzed during the subsequent 83-day treatment. The results suggested that C1632 significantly reduced the mortality rate of CKD model mice (Figure 13).

[0086] 4.3 The mice were divided into three groups of 10 each: (1) normal group, (2) AP+Saline group, and (3) AP+C1632 group. In groups (2) and (3), after feeding with 2‰ AP diet for 1 month, C1632 was administered for treatment. Among them, C1632 was administered to CKD mice twice a week at a dose of 68 mg / kg body weight for treatment. After 3 months, the mice were sacrificed, the kidneys were removed and subjected to Masson staining. The kidneys of the control mice mainly contained red muscle fibers, and the kidney tissues of the saline-treated CKD model mice contained a large amount of blue collagen fibers. C1632 treatment significantly inhibited the level of renal collagen fibers and inhibited the progression of renal fibrosis in mice (Figure 14).

[0087] Example 5 Therapeutic effect of C1632 on vascular calcification VD3 was intraperitoneally injected into C57BL / c mice at a dose of 500 IU / g / day for 2 consecutive days, and then divided into (1) Saline group and (3) C1632 group, with 10 mice in each group. Among them, C1632 was administered to CKD mice twice a week at a dose of 68 mg / kg body weight for treatment. After 6 days, the mice were sacrificed, the aortic vessels were dissected, and hydroxyapatite staining was performed using alizarin red. As a result, C1632 treatment significantly inhibited the progression of aortic vascular calcification (Figure 15).

[0088] In the above specific embodiments, the present invention has been described in detail. However, the present invention is not limited to the above examples, and various modifications can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those skilled in the art. Furthermore, the examples and features of the embodiments of the present invention may be combined with each other without contradiction.

Claims

1. Use of a [1,2,4]triazolo[4,3-B]pyridazine derivative in the manufacture of a medicament for preventing and / or treating an age-related disease, comprising: The [1,2,4]triazolo[4,3-B]pyridazine derivative is a compound represented by formula (I) or a pharma- ceutically acceptable salt thereof, 【Chemistry 1】 The aging-related disease includes at least one of atherosclerosis, vascular calcification, and tissue / organ fibrosis, and the tissue / organ fibrosis includes pulmonary fibrosis and renal fibrosis.

2. The use according to claim 1, characterized in that the vascular calcification is aortic vascular calcification.

3. The use according to claim 1, characterized in that the pulmonary fibrosis is idiopathic pulmonary fibrosis.

4. The use according to claim 3, characterized in that the [1,2,4]triazolo[4,3-B]pyridazine derivative ameliorates the phenomenon of alveolar loss and increased lung density during the progression of idiopathic pulmonary fibrosis.

5. 4. The use according to claim 3, characterized in that the [1,2,4]triazolo[4,3-B]pyridazine derivative inhibits fibroblast proliferation and / or inhibits pulmonary hydroxyproline and / or inhibits immune cell infiltration.

6. The use according to claim 5 , wherein the immune cells comprise CD45-positive cells, CD68-positive cells, and CD3-positive cells.

7. The use according to claim 1, wherein the renal fibrosis is renal fibrosis in chronic kidney disease.

8. The use according to claim 1, characterized in that the [1,2,4]triazolo[4,3-B]pyridazine derivative inhibits the abnormal expression of collagen fibers in chronic kidney disease.

9. The use according to claim 1, characterized in that the medicament further comprises at least one other ingredient useful for the prevention and / or treatment of age-related diseases.

10. 2. The use according to claim 1, wherein the route of administration of the drug comprises at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, intranasal administration, aerosol administration, or transdermal administration.

11. 2. The use according to claim 1, wherein the dosage form of the drug comprises at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, an oral preparation, a suppository, an enema, an aerosol, a patch, or a drop.

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