WO2024164543A1 - [1,2,4]三唑并[4,3-b]哒嗪衍生物在衰老及衰老相关性疾病防治中的应用 - Google Patents

[1,2,4]三唑并[4,3-b]哒嗪衍生物在衰老及衰老相关性疾病防治中的应用 Download PDF

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WO2024164543A1
WO2024164543A1 PCT/CN2023/122397 CN2023122397W WO2024164543A1 WO 2024164543 A1 WO2024164543 A1 WO 2024164543A1 CN 2023122397 W CN2023122397 W CN 2023122397W WO 2024164543 A1 WO2024164543 A1 WO 2024164543A1
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aging
use according
mice
fibrosis
administration
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PCT/CN2023/122397
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English (en)
French (fr)
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谢晨
陈艳莲
黄辉
张亭亭
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中山大学附属第八医院（深圳福田）
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Priority claimed from CN202310124966.5A external-priority patent/CN116236485A/zh
Priority claimed from CN202310153067.8A external-priority patent/CN116421607A/zh
Priority claimed from CN202310133146.2A external-priority patent/CN115813920B/zh
Priority claimed from CN202310133147.7A external-priority patent/CN115837022B/zh
Application filed by 中山大学附属第八医院（深圳福田） filed Critical 中山大学附属第八医院（深圳福田）
Publication of WO2024164543A1 publication Critical patent/WO2024164543A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention belongs to the field of medical technology, and specifically relates to the application of [1,2,4]triazolo[4,3-B]pyridazine derivatives in the prevention and treatment of aging and aging-related diseases.
  • Cell aging is a time-dependent cell dysfunction. Senescent cells undergo degenerative changes in their cell structure, which manifests as functional decline and low metabolism. Although physiological aging is a slow process, factors such as viral infection, radiation, and various diseases can lead to pathological aging, which accelerates the aging of the body.
  • chronic degenerative brain lesions can affect cognitive functions and increase the incidence of Alzheimer's disease and neurodegenerative diseases.
  • Senescent cells usually highly express inflammatory factors and other molecules that regulate immune responses, causing disorders of the immune system, including immune decline and chronic inflammation, leading to autoimmune diseases, osteoarthritis, etc.
  • Aging is accompanied by damage to angiogenesis function. Aging reduces the anti-thrombotic function of the endothelium and is a risk factor for cardiovascular diseases such as atherosclerosis and vascular calcification.
  • pancreatic islet cell function associated with aging will cause the body to be unable to respond normally to changes in blood sugar, thereby reducing the ability to regulate blood sugar (i.e., glucose tolerance), causing glucose metabolism disorders, and eventually developing into diabetes.
  • blood sugar i.e., glucose tolerance
  • cell senescence means that the cell cycle enters a stable stagnant state. Although senescent cells still have metabolic capacity, they cannot continue to proliferate. They promote excessive secretion of extracellular matrix proteins by secreting inflammatory factors and participate in the occurrence and development of tissue and organ fibrosis.
  • the purpose of the present invention is to provide a new application of a small molecule compound [1,2,4]triazolo[4,3-B]pyridazine derivative (ie, C1632), namely, the application of C1632 in the prevention and treatment of aging and aging-related diseases.
  • inflammatory cytokine storm is a serious acute immune system disease, usually caused by pathogen infection stimulating the cell's inherent immune pathway, prompting the cell to express a large number of inflammatory factors in a short period of time, and inflammatory factors have adverse effects such as changing body temperature, affecting the body's systemic metabolism, increasing sputum and blocking alveoli, and ultimately leading to respiratory distress and multiple organ failure and even death in patients.
  • the aging and related diseases of the present invention are different from the inflammatory cytokine storm and The novel coronavirus infection is completely different.
  • the aging-related diseases of the present invention refer to any functional changes that occur in an organism or any of its tissues, organs or cells after the maximum reproductive function period accompanied by a decline in reproductive function, and such changes are associated with aging.
  • Aging-related diseases are any diseases, illnesses, degeneration, tissue loss, or other unhealthy or abnormal conditions caused by or associated with aging. Obviously, it is significantly different from the acute and severe diseases mentioned in the prior art that use exogenous substances as pathogenic inducements.
  • the technical solution adopted by the present invention is:
  • a [1,2,4]triazolo[4,3-B]pyridazine derivative in the preparation of a drug for preventing and/or treating aging and/or aging-related diseases, wherein the [1,2,4]triazolo[4,3-B]pyridazine derivative is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
  • the aging-related diseases include at least one of atherosclerosis, vascular calcification, diabetes, autoimmune diseases, osteoarthritis, senile dementia, and fibrosis of tissues and organs.
  • the aging includes at least one of aging diseases, skin, brain, lung, heart, bone marrow, immune liver, and kidney tissue and organ aging.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative inhibits cell senescence.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative is capable of extending lifespan.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative relieves the immunosuppressive microenvironment of senescent cells and/or promotes immune clearance of senescent cells.
  • the vascular calcification is aortic vascular calcification.
  • the fibrosis of tissues and organs includes liver fibrosis, lung fibrosis, and kidney fibrosis.
  • the pulmonary fibrosis is idiopathic pulmonary fibrosis.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative improves the phenomenon of alveolar reduction and increased lung density in the process of idiopathic pulmonary fibrosis.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative inhibits fibroblast proliferation.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative inhibits pulmonary hydroxyproline.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative inhibits immune cell infiltration.
  • the immune cells include CD45 positive cells (total immune cells), CD68 positive cells (macrophages), and CD3 positive cells (T cells).
  • the renal fibrosis is renal fibrosis in chronic kidney disease.
  • the [1,2,4]triazolo[4,3-B]pyridazine derivative inhibits abnormal expression of collagen fibers in chronic kidney disease.
  • the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, oral preparations, suppositories, enemas, aerosols, patches or drops.
  • 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.
  • the drug further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipients include: at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a cosolvent, a solubilizer, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, an antibacterial agent or a buffer.
  • the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, oral preparations, suppositories, enemas, aerosols, patches or drops.
  • 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.
  • the drug further comprises at least one other ingredient that can be used to prevent and/or treat aging and/or aging-related diseases.
  • the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, oral preparations, suppositories, enemas, aerosols, patches or drops.
  • 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.
  • the drug further comprises a pharmaceutically acceptable excipient and at least one other ingredient that can be used to prevent and/or treat aging and/or aging-related diseases.
  • the dosage form of the drug includes suspension, granules, capsules, powders, tablets, At least one of emulsion, solution, pill, injection, oral preparation, suppository, enema, aerosol, patch or drops.
  • 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.
  • a drug comprising a [1,2,4]triazolo[4,3-B]pyridazine derivative, wherein the [1,2,4]triazolo[4,3-B]pyridazine derivative is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; the drug is used for preventing and/or treating aging and/or aging-related diseases:
  • the medicament further comprises at least one other component useful for preventing and/or treating aging and/or aging-related diseases.
  • the drug further includes pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients include: 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 regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, an antibacterial agent or a buffer.
  • the pharmaceutically acceptable excipients include: 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 regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, an antibacterial agent or a buffer.
  • the dosage form of the drug includes at least one of a suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, oral preparations, suppositories, enemas, aerosols, patches or drops.
  • 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.
  • C1632 is a small molecule drug that is soluble in water, ethanol or DMSO, has good thermal stability, low cytotoxicity, good drug safety, good pharmacokinetic properties, and can be flexibly administered in a variety of ways.
  • C1632 can (1) significantly inhibit cell senescence, prolong the life span of mice, and inhibit lung senescence, and can also relieve the immunosuppressive microenvironment during aging, promote the immune clearance of senescent cells, and treat related diseases caused by aging; (2) significantly inhibit the progression of liver fibrosis, have good drug safety and strong anti-liver fibrosis effect, and can be used as a candidate drug for the treatment of liver fibrosis; (3) can significantly inhibit the progression of idiopathic pulmonary fibrosis, reduce fibroblast proliferation and collagen synthesis in idiopathic pulmonary fibrosis, and inhibit the activity of inflammatory infiltration of immune cells.
  • C1632 is a known small molecule drug that is soluble in water, ethanol or DMSO, has good thermal stability, low cytotoxicity, good drug safety, and good pharmacokinetic properties. It can be flexibly administered through multiple methods such as oral, intraperitoneal, intramuscular and intravenous injections, and can be used as a safe, effective and easy-to-use candidate drug.
  • “Pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit/risk ratio.
  • the "pharmaceutically acceptable salts" of the present invention refer to salts formed by acidic functional groups (e.g., -COOH, -OH, -SO 3 H, etc.) present in the compound and appropriate inorganic or organic cations (bases), including 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., -NH 2 , etc.) present in the compound and appropriate inorganic or organic anions (acids), including salts formed with inorganic acids or organic acids (e.g., carboxylic acids, etc.).
  • bases including salts formed with alkali metals or alkaline earth metals, ammonium salts, and salts formed with nitrogen-containing organic bases
  • salts formed by basic functional groups e.g., -NH 2 , etc.
  • acids including salts formed with inorganic acids or organic acids (e.g., carboxylic acids,
  • Aging-related diseases refer to aging-related, aging-induced tissue degenerative or progressive lesions, including: tissue and organ fibrosis, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, osteoarthritis and chronic kidney disease.
  • tissue and organ fibrosis include tissue and organ fibrosis, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, osteoarthritis and chronic kidney disease.
  • the aggregation of senescent cells is an important cause of aging-related diseases.
  • the abnormal aggregation of senescent cells leads to a decrease in the body's ability to maintain and restore homeostasis under stress and injury.
  • the inflammatory microenvironment produced by aging leads to chronic tissue damage and accelerates aging-related diseases such as atherosclerosis, vascular calcification, diabetes, autoimmune diseases, osteoarthritis and Alzheimer's disease, and tissue and organ fibrosis; or, it refers to the onset of the disease itself has no direct correlation with aging, but is induced by external factors, but aging itself will change the prognosis of the disease, such as viral infection, trauma, etc.
  • aging-related diseases such as atherosclerosis, vascular calcification, diabetes, autoimmune diseases, osteoarthritis and Alzheimer's disease, and tissue and organ fibrosis
  • Aging syndrome is a congenital genetic disease, the main manifestations of which are rapidly accelerated physical aging, memory loss, and shortened lifespan.
  • Fibrosis of tissues and organs is a progressive disease in which tissues and organs over-secrete extracellular matrix proteins and form permanent scars in order to repair damaged parts, which eventually leads to organ deformation and functional failure. It involves the formation of excessive fibrous connective tissue in organs or tissues during the repair or reaction process. Although fibrosis can be a benign state, the present invention preferably relates to pathological fibrosis.
  • Vascular calcification is a disease characterized by the deposition of calcium salts in the vascular intima and middle layer. It is closely related to many diseases such as atherosclerosis, diabetes and chronic kidney disease, seriously affecting the prognosis of these diseases.
  • Figure 1 shows that C1632 significantly inhibits cell senescence in Example 1.
  • Figure 1 A shows ⁇ -gal staining, which proves that C1632 treatment can inhibit VP16-induced senescence of BJ cells and MRC5 cells.
  • Figure 1 B and Figure 1 C show the statistical ⁇ -gal positive cell rates of BJ cells (B) and MRC5 cells (C) in different treatment groups. ** indicates p ⁇ 0.01.
  • Figure 2 shows that C1632 significantly prolongs the life span of mice and lung aging of aging mice in Example 1.
  • Figure 2 A shows a survival curve, which shows that C1632 significantly prolongs the life span of premature aging mice TERC-/-G4.
  • Figure 2 B shows that immunohistochemical detection of C1632 significantly inhibits the expression of lung aging markers P16 and P21 in TERC-/-G4 mice.
  • FIG3 shows the expression changes of FOXP3, an immunosuppressive T cell marker, detected by immunohistochemistry in Example 1, indicating that C1632 can relieve the immunosuppressive microenvironment of aging.
  • Figure 4 shows that C1632 in Example 1 can promote T cells to kill senescent cells.
  • Figure 4 A shows SA- ⁇ -gal detection of bleomycin-induced TC-1 cell senescence;
  • Figure 4 B shows LDH activity release analysis to detect the killing effect of PBMC, CD3+T cells and CD8+T cells on senescent TC-1 in normal mice, saline and IPF mice treated with C1632.
  • FIG5 shows the Masson staining for detecting collagen fibers in liver tissue in Example 2; 20 ⁇ means the magnification is 20 times, and 40 ⁇ means the magnification is 40 times.
  • FIG6 shows the VG staining to detect collagen fibers in liver tissue in Example 2; 20 ⁇ means the magnification is 20 times, and 40 ⁇ means the magnification is 40 times.
  • FIG. 7 shows the detection of collagen fibers in liver tissue by reticular fiber staining in Example 2; 20 ⁇ means the magnification is 20 times, and 40 ⁇ means the magnification is 40 times.
  • FIG8 shows the detection of collagen fibers in liver tissue by Sirius red staining in Example 2; 20 ⁇ means the magnification is 20 times, and 40 ⁇ means the magnification is 40 times.
  • Figure 9 shows that C1632 in Example 3 significantly inhibited the progression of pulmonary fibrosis in mice.
  • Figure 9 A shows the experimental flow chart of IPF mouse model construction and C1632 drug treatment;
  • Figure 9 B shows the photographs of lung tissues of normal mice, fibrotic mice and fibrotic mice after C1632 treatment;
  • Figure 9 C shows that the lungs of mice in each treatment group were placed in water, and only the lung density of IPF mice was greater than that of water, while the lung density of the control group and the C1632 treatment group was less than that of water;
  • Figure 9 D shows the lung density statistics of mice in each treatment group;
  • Figure 9 E shows HE and Masson staining to detect the structure and degree of fibrosis of mouse lung tissue;
  • Figure 9 F shows the fibrosis areas of each group of mice in HE staining;
  • Figure 9 G shows the fibrosis areas of each group of mice in Masson staining;
  • Figure 9 I shows the detection of hydroxyproline content in the lungs of mice in
  • Figure 10 shows that 1632 inhibits immune infiltration in idiopathic pulmonary fibrosis in Example 3.
  • Figure 10 A shows the expression levels of CD45, CD68 and CD3 in lung tissues of mice in the C1632 treatment group and the control IPF group detected by IHC;
  • Figure 10 B shows the expression level of CD45;
  • Figure 10 C shows the expression level of CD68;
  • Figure 10 D shows the expression level of CD3.
  • FIG. 11 shows that 1632 in Example 3 has good drug safety.
  • Figure 11A shows weight statistics;
  • Figure 11B to Figure 11S show routine blood analysis:
  • Figure 11B shows lymphocyte density;
  • Figure 11C shows lymphocyte ratio;
  • Figure 11D shows monocyte density;
  • Figure 11E shows monocyte ratio;
  • Figure 11F shows neutrophil density;
  • Figure 11G shows neutrophil ratio;
  • Figure 11H shows white blood cell count;
  • Figure 11I shows red blood cell count;
  • Figure 11J shows hemoglobin content;
  • Figure 11K shows hematocrit;
  • Figure 11L shows mean corpuscular volume;
  • Figure 11M shows mean corpuscular hemoglobin content;
  • Figure 11N shows mean corpuscular hemoglobin concentration;
  • Figure 11O shows red blood cell distribution width;
  • Figure 11P shows blood cell density;
  • Figure 11Q shows mean platelet volume;
  • Figure 11R shows platelet distribution width;
  • Figure 11S shows platelet volume.
  • FIG. 12 shows that C1632 treatment in Example 4 can significantly inhibit the weight loss of mice caused by chronic kidney disease; wherein, ** indicates p ⁇ 0.01.
  • FIG. 13 shows that C1632 treatment in Example 4 can significantly inhibit the death of mice caused by chronic kidney disease; wherein, ** indicates p ⁇ 0.01.
  • FIG. 14 shows that Masson staining in Example 4 showed that C1632 could significantly inhibit the progression of renal fibrosis in chronic kidney disease mice; wherein, ** indicates p ⁇ 0.01.
  • FIG. 15 shows that Alizarin red staining of blood vessels in Example 5 showed that C1632 inhibited the progression of aortic vascular calcification in mice.
  • the synthesis path is as follows:
  • C1632 is a known small molecule drug that is soluble in water or DMSO, has good thermal stability, low cytotoxicity, good drug safety, and good pharmacokinetic properties. It can be flexibly administered through multiple routes such as oral, intramuscular, and intravenous injections.
  • Example 1 The therapeutic effect of C1632 on aging-related diseases
  • mice used in this example were purchased from Guangdong Yaokang Biotechnology Co., Ltd.; TERC-/- mice were provided by the laboratory of Professor Ju Zhenyu of Jinan University; human primary cells MRC5 and BJ and mouse TC1 cells were purchased from ATCC.
  • Paraffin sections were melted at 65°C for 1h; xylene for 10min-xylene for 10min-anhydrous ethanol for 3min-95% ethanol for 3min-85% ethanol for 3min-70% ethanol for 3min, and washed twice with PBS, each time for 5min; blocked with 3% hydrogen peroxide (methanol configuration) for 10min, washed twice with PBS, each time for 5min; microwave heating with pH 6.0 antigen repair solution for 10min for antigen repair, slowly cooled at room temperature for 1h, washed twice with PBS, each time for 5min; blocked with 10% goat serum for 1h; incubated with primary antibody at 4°C in dark overnight, washed twice with PBS, each time for 5min; incubated with secondary antibody at room temperature in dark for 30min, washed twice with PBS, each time for 5min; developed with DAB, stained with hematoxylin, and rinsed in tap water for 8min to turn blue. Dehydrated with gradient alcohol, dried, and sealed with neutral gum.
  • MRC5 and BJ cells were cultured in DMEM low-glucose medium containing 10% FBS. Cells were passaged or plated at a ratio of 1:3. 0.5 ⁇ 10 5 cells were plated on a 12-well plate and treated with VP16 at a final concentration of 40 ⁇ M for 4-7 days to induce cell senescence and construct a cell senescence model. While inducing cell senescence, C1632 at a final concentration of 120 ⁇ M was added for treatment. The effect of C1632 on BJ and MRC5 cell senescence was detected by SA- ⁇ -gal staining to study the effect of C1632 on cell senescence.
  • Figure 1 A shows ⁇ -gal staining, which proves that C1632 treatment can inhibit VP16-induced BJ cell and MRC5 cell senescence.
  • Figure 1 B and Figure 1 C show the statistical ⁇ -gal positive cell rates of BJ cells (B) and MRC5 cells (C) in different treatment groups. ** indicates p ⁇ 0.01. The results show that C1632 can effectively delay VP16-induced cell senescence.
  • TERC -/- G4 aging mouse model The telomeres of mice are very long , and it takes 3-6 generations to reproduce after telomerase knockout before premature aging phenotypes appear.
  • TERC +/- mice are bred and selected for breeding.
  • the first generation of TERC -/- mice is G1 mice.
  • G1 mice mate with each other to produce G2 mice, and continue to reproduce to G4.
  • the telomeres of mice are very short and aging phenotypes can appear at 3-4 months of age.
  • TERC knockout mouse genotype Cut a small amount of tail from the mouse to be identified, digest the tail at 65°C for 2h in 10mM Tris-HCl 0.5% SDS 1mM EDTA pH8.0 buffer containing 0.4mg/mL proteinase K, and then take 0.5 ⁇ L as a template for PCR amplification. A PCR product size of 250bp indicates wild type, and a product size of 180bp indicates TERC knockout.
  • the applicant used bleomycin to induce TC-1 cell senescence, and used SA- ⁇ -Gal staining ( FIG. 4A ) to reflect the senescence of TC-1 cells.
  • T cell killing experiment TC1 cells induced by bleomycin were plated at 10,000/well in a 96-well plate. The cell culture supernatant was discarded the next day, and then PBMC, CD3 + T cells and CD8+ T cells isolated 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 hours, the supernatant was centrifuged and the killed senescent cells released LDH enzyme. The killing effect of immune cells on senescent cells was detected using an LDH kit. A 2 mg/kg dose of bleomycin was sprayed into the mouse lungs to construct an IPF mouse model; at least 6 mice were included in each group of the above experiment.
  • CD8 + T cells were isolated and cultured as follows: mouse spleen was taken under sterile conditions and ground, then passed through a 200-mesh sieve, and the filtered cells were The cells were sorted using a CD8 + T cell isolation kit (biolegend), and the sorted T cells were cultured in PRMI1640 medium containing 10% FBS, 5 ⁇ g/mL anti-mouse CD28 antibody (biolegend) and 10 ng/mL IL-2 in a culture dish coated with 5 ⁇ g/mL CD3 antibody (biolegend) overnight until use.
  • Example 2 The therapeutic effect of C1632 on liver fibrosis
  • mice in this example were 8 weeks old and purchased from Guangdong Yaokang Biotechnology Co., Ltd.
  • C1632 can treat liver fibrosis
  • the applicant intraperitoneally injected 1 ml/kg of carbon tetrachloride (CCl 4 ) twice a week for 6 consecutive weeks to establish a liver fibrosis model.
  • CCl 4 carbon tetrachloride
  • mice were divided into normal control group, CCl4 group and CCl4 + C1632 group, 7 mice in each group.
  • CCl4 + C1632 group was treated by injection of 17mg/kg C1632 after each injection of CCl4.
  • the mice were killed and the liver was fixed with 4% paraformaldehyde, dehydrated, embedded and sliced.
  • Masson staining can dye the collagen fibers in the tissue blue, and the muscle fibers and cytoplasm red.
  • the sections are routinely dewaxed to water and stained with the prepared Weigert iron hematoxylin for 5 to 10 minutes. Differentiate with 1% hydrochloric acid alcohol differentiation solution and wash with water. Use Masson blueing solution to return to blue and wash with water for 1 minute. Stain with Ponceau fuchsin staining solution for 5 to 10 minutes. Soak in 1% glacial acetic acid for a while. Wash with phosphomolybdic acid solution for 1 to 2 minutes. Wash with 1% glacial acetic acid for 1 minute. Stain directly in aniline blue staining solution for 1 to 2 minutes.
  • the sections are rinsed and differentiated with three consecutive cylinders of 1% glacial acetic acid, each cylinder for about 8 seconds.
  • the sections are dehydrated in three consecutive cylinders of anhydrous ethanol for about 5 seconds, 10 seconds, and 30 seconds. Then dehydrate in two cylinders of n-butanol for 30 seconds and 2 minutes, and finally transparent in two cylinders of xylene, each for 5 minutes, and sealed with neutral gum.
  • Figure 5 show that normal liver tissue has only a small amount of collagen fibers, while the liver tissue of mice modeled with CCl 4 expresses a large amount of collagen fibers, and C1632 can inhibit the expression of collagen fibers induced by CCl 4 ( Figure 5).
  • VG staining is a mature connective tissue staining method used to distinguish collagen fibers from muscle fibers. Paraffin sections were dewaxed to water, and the sections were placed in VG staining solution prepared with acid fuchsin and picric acid for 1 min. The sections were quickly washed with water and quickly dehydrated in three cylinders of anhydrous ethanol. The sections were placed in xylene for transparency and sealed with neutral gum. Pictures were taken under a microscope, and the results showed that CCl4 increased the expression of collagen fibers in mouse liver tissue. C1632 significantly reduced the level of collagen fibers in liver tissue ( Figure 6).
  • Reticular fibers are less abundant in loose connective tissue. They are thin and branched, interwoven into a network. Reticular fibers have a striated structure with equal spacing. Similar to collagen fibers, their chemical composition is also type III collagen. Reticular fibers are normal.
  • the main scaffold of hepatocytes and hepatic sinusoids its changes can reflect the reticular scaffold morphology of liver lesions. According to the distribution characteristics and destruction of reticular fibers, it can show the characteristics of cirrhotic regenerative nodules, proliferative lesions, tumors and fibrosis, and can also regularly evaluate the progress of non-cirrhotic portal hypertension.
  • the collagen fibers of reticular fibers are rich in glycoproteins, which are silver-loving and can be stained black.
  • the dewaxed slices were oxidized with Gordon-Sweets oxidant for 5 minutes. After washing with water, bleached with oxalic acid solution for 1 to 2 minutes. After washing with distilled water, mordant with ammonium ferric sulfate solution for 5 minutes. After washing with distilled water, Gordon-Sweets silver ammonia solution was added for staining for 3 minutes. After washing with distilled water, Gordon-Sweets reducing agent was used for reduction for 1 minute, and rinsed with running water for 10 minutes. Nuclear fast red staining solution was used to stain the nucleus for 5 to 10 minutes. After washing with water, dehydrated and transparent, and sealed with neutral gum.
  • Picro Sirius Red Staining is a simple and sensitive method for identifying collagen fiber networks in tissue sections. It can qualitatively or quantitatively identify abnormal changes in collagen networks in degenerative lesions and hereditary or acquired diseases through morphological imaging analysis. Sirius Red is a strong and linear anionic dye containing 6 sulfonic acid groups that can firmly bind to cationic collagen fibers. Under polarized light detection, collagen fibers have positive uniaxial birefringence properties. When combined with Sirius Red dye, the natural birefringence properties of collagen are enhanced, thereby distinguishing type I collagen from type III collagen.
  • liver fibrosis in the C1632-treated group was significantly reduced, and it can effectively treat liver fibrosis.
  • Example 3 The therapeutic effect of C1632 on pulmonary fibrosis
  • mice used in this example were purchased from Guangdong Yaokang Biotechnology Co., Ltd.; and bleomycin sulfate was purchased from Aladdin.
  • mice were anesthetized, 2 mg/kg of bleomycin was injected into the lungs through a trachea to establish a mouse model of pulmonary fibrosis.
  • the lung density was determined by the water displacement method, as follows: 1 The mouse was anesthetized and killed, and the lungs were taken out and weighed and recorded as Mlung; 2 Then the weight of the empty 1.5 mL centrifuge tube was weighed, and the weight of the centrifuge tube was recorded again after it was filled with saline. The exact volume of the tube Vtube was calculated based on the weight and density of the saline; 3 The lung tissue was completely immersed in the saline of the centrifuge tube, and the lid was closed after the air was removed.
  • Lung tissues were fixed in 4% paraformaldehyde and then dehydrated in a gradient manner as follows: 70% ethanol for 5 h; 80% ethanol for 1 h; 90% ethanol for 1 h; 95% ethanol for 1 h; anhydrous ethanol for 1 h; anhydrous ethanol for 0.5 h; anhydrous 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. paraffin (62°C) for 1 h. After dehydration, the tissues were encapsulated in wax blocks and sliced with a thickness of 4 ⁇ m.
  • paraffin sections were baked at 65 degrees Celsius for 1 hour, they were passed through xylene for 5 minutes; xylene for 5 minutes; anhydrous ethanol for 5 minutes; 95% ethanol for 5 minutes; 90% ethanol for 5 minutes; 85% ethanol for 5 minutes; 75% ethanol for 5 minutes; and slightly boiled in citric acid repair solution for 10 minutes; then naturally cooled to room temperature; 3% hydrogen peroxide for 10 minutes; blocked with goat antiserum at room temperature for 1 hour; incubated with primary antibodies for CD45 (abcam), CD3 (abcam) or CD68 (abcam) overnight; then washed with PBS 3 times for 5 minutes each; added immunohistochemical secondary antibody (Fujian Maixin) at room temperature for 20 minutes; then used DAB color development kit (Fujian Maixin) for color development for 2 minutes; rinsed with water for DAB and then stained with hematoxylin for 2 minutes; rinsed with running water for 8 minutes to turn blue; after the slices were dried, they were sealed
  • Masson staining was used to detect the degree of lung tissue fibrosis, and a Masson trichrome staining kit (Fujian Maixin) was used.
  • the applicant treated IPF mice with C1632 according to the method in A of FIG. 9, and specifically divided the mice into 1 normal mice + saline group; 2 normal mice + C1632 group; 3 IPF mice + saline group; IPF + C1632 group, at least 4 mice in each group.
  • the mouse lung tissue was taken for photographing, and the results showed 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 of the lung lobes were grayish white, the texture became hard, and scattered grayish white patches of varying sizes were visible.
  • the lung morphology, color and hardness of the model group treated with C1632 were closer to the control group (B of FIG. 9).
  • the above-mentioned lung lobes were placed in water, and the results showed that the density of IPF modeling mice was greater than that of water, while the lung density of normal mice and IPF mice treated with C1632 was less than that of water (C of FIG. 9).
  • mice The lung density of each group of mice was further tested, and the results showed that the lung density of IPF mice was larger, and C1632 treatment could significantly reduce the lung density of IPF mice (D in Figure 9). This shows that C1632 can significantly improve the air content in the lungs of IPF mice, thereby reducing lung density and improving the ability of lung air exchange. H&E and Masson staining also showed that the lungs of mice in the C1632 treatment group contained more alveolar structures and fewer fibrotic areas (E to G in Figure 9).
  • Hydroxyproline is an amino acid unique to collagen, and its content is an important indicator for detecting pulmonary fibrosis.
  • the quantitative results of hydroxyproline in the lungs of mice in each treatment group showed that C1632 could significantly inhibit the increase of hydroxyproline in the lungs of mice ( Figure 9 I).
  • Example 3.1 it was demonstrated that C1632 inhibits the progression of pulmonary fibrosis.
  • the applicant speculates that it can also inhibit the recruitment of immune cells during pulmonary fibrosis.
  • the infiltration of CD45 (total immune cells), CD68 (macrophages) and CD3 (T cells) in the C1632 treatment group was detected by IHC.
  • the results showed that 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 proved that C1632 inhibits immune infiltration in idiopathic pulmonary fibrosis.
  • C1632 In order to detect the drug safety of C1632, the applicant gave C1632 with 68mg/kg body weight of C57BL/6 mice and control mice of IPF model 3 times a week, and weighed their body weight. At the end of the experiment, whole blood was taken for blood routine test. The results showed that C1632 did not significantly change the weight of mice compared with the corresponding saline group (A of Figure 11). In addition, the blood routine indicators of each group of mice were mostly within the reference range. It is worth noting that the normal mice given C1632 can significantly increase the mean platelet volume (MPV), but the model group was given C1632 and had no significant effect on MPV.
  • MPV mean platelet volume
  • C1632 can significantly inhibit the PDW (mean platelet volume) of normal mice, and has no significant effect on this indicator of fibrotic mice. In general, C1632 does not cause the blood routine indicators of IPF mice to deteriorate (B of Figure 11 to S of Figure 11). It is proved that C1632 has good drug safety.
  • PDW mean platelet volume
  • mice used in this example were purchased from Guangdong Yaokang Biotechnology Co., Ltd.; the feed containing 1.5 ⁇ and 2 ⁇ purine (AP) was processed by Guangdong Medical Experimental Animal Center.
  • the steps for constructing the chronic kidney disease (CKD) mouse model in this embodiment are as follows: 8-week-old C57BL/6 male mice were fed with a feed containing 2 ⁇ purine for 1 month, and then fed with a feed containing 1.5 ⁇ purine, and cultured for another 3 months. Each group had at least 10 mice.
  • mice were divided into 3 groups: 1 normal group; 2 AP+Saline group; 3 AP+C1632 group, 10 mice in each group, 3 group was fed with 2 ⁇ AP feed for 1 month and then given C1632 for treatment, where C1632 was given twice a week, each time at a dose of 68 mg/kg body weight to CKD mice.
  • C1632 treatment significantly inhibited the weight loss of CKD mice ( Figure 12).
  • mice were divided into 3 groups: 1 normal group; 2 AP + Saline group; 3 AP + C1632 group, 10 mice in each group. Groups 2 and 3 were fed with 2 ⁇ AP feed for 1 month and then given C1632 for treatment. C1632 was given twice a week, each time at a dose of 68 mg/kg body weight to treat CKD mice. During the subsequent 83-day treatment, the survival of the mice was counted, and the results showed that C1632 significantly reduced the mortality rate of CKD model mice (Figure 13).
  • mice were divided into 3 groups: 1 normal group; 2 AP + Saline group; 3 AP + C1632 group, 10 mice in each group. Groups 2 and 3 were fed with 2 ⁇ AP feed for 1 month and then given C1632 for treatment. C1632 was given twice a week at a dose of 68 mg/kg body weight each time to treat CKD mice. Mice were killed 3 months later, and kidneys were taken for Masson staining. The kidneys of control mice were mainly colored as red muscle fibers, while the kidney tissues of CKD model mice treated with saline contained a large amount of blue collagen fibers. C1632 treatment significantly inhibited the level of collagen fibers in the kidneys and inhibited the process of renal fibrosis in mice ( Figure 14).
  • Example 5 The therapeutic effect of C1632 on vascular calcification
  • VD3 was intraperitoneally injected into the peritoneum of C57BL/c mice at a dose of 500 IU/g/day for 2 consecutive days, and then divided into: 1 Saline group; 3 C1632 group, 10 mice in each group.
  • C1632 was given twice a week, each time at a dose of 68 mg/kg body weight to treat CKD mice. After 6 days, the mice were killed, and the aorta was stripped for hydroxyapatite staining with alizarin red. The results showed that C1632 treatment significantly inhibited the process of aortic calcification (Figure 15).

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Abstract

[1,2,4]三唑并[4,3-B]哒嗪衍生物，即C1632具有显著的抗衰、抗纤维化和抑制血管钙化的作用，而且C1632为已知的小分子药物，能溶于水、乙醇或者DMSO，热稳定性良好，细胞毒性低，药物安全性好，具有良好的药代动力学特性，通过口服、腹腔注射、肌肉注射和静脉注射等多种方式灵活给药，可以作为预防和/或治疗衰老及衰老相关性疾病的候选药物。

Description

[1,2,4]三唑并[4,3-B]哒嗪衍生物在衰老及衰老相关性疾病防治中的应用 技术领域
本发明属于医药技术领域，具体涉及[1,2,4]三唑并[4,3-B]哒嗪衍生物在衰老及衰老相关性疾病防治中的应用。
背景技术
细胞衰老(cell aging)是一种与时序相关的细胞功能障碍。衰老细胞发生细胞结构的退行性改变，表现为功能衰退与代谢低下。虽然生理性衰老是一个缓慢过程，然而病毒感染、辐射、各种疾病等因素可以导致病理性衰老促使机体加速老化。
除此之外，机体生物学功能衰退，个体老化的过程往往伴随着各种相关疾病的发生，例如：慢性大脑退行性病变会影响认知功能，增加老年痴呆和神经退行性疾病的发生率；衰老细胞通常高表达炎症因子和其他调节免疫反应的分子，引起免疫系统的失调，包括免疫衰退和慢性炎症，导致自身免疫疾病、骨关节炎等；增龄伴随血管生成功能的损伤，衰老降低内皮的抗血栓形成功能，是造成动脉粥样硬化和血管钙化等心血管疾病的危险因素；伴随增龄的胰岛细胞功能退行会致使机体无法正常响应血糖变化，从而对血糖的调节能力(即糖耐量)下降，引发糖代谢紊乱，最终发展为糖尿病；另外，细胞衰老意味着细胞周期进入稳定停滞状态，衰老细胞虽然依然具有代谢能力，但无法继续增殖，其通过分泌炎性因子等方式促进细胞外基质蛋白过度分泌，参与组织和脏器纤维化的发生发展。
因此，开发有效的衰老干预策略对于预防和/或治疗衰老及其相关性疾病、降低发病率具有重大意义。
发明内容
本发明的目的在于提供小分子化合物[1,2,4]三唑并[4,3-B]哒嗪衍生物(即，C1632)的一种新的应用，即C1632在衰老及衰老相关性疾病防治中的应用。
虽然，发明人前期公开了C1632在治疗新型冠状病毒SARS-CoV-2以及抗炎症因子风暴中的应用。但是，值得注意的是，炎症因子风暴是一种严重的急性免疫系统疾病，通常是病原体感染刺激细胞固有免疫通路，促使细胞短时间内表达大量炎症因子，而炎症因子又具有改变体温影响人体全身性代谢、增加痰液堵塞肺泡等等不良作用，最终导致患者呼吸窘迫和多器官衰竭乃至死亡。而本发明的衰老及其相关性疾病与现有技术中涉及的炎症因子风暴和 新型冠状病毒感染是截然不同的。具体地，本发明的衰老相关性疾病指的是有机体或其任何组织、器官或细胞在最大生殖功能期以后伴随生殖功能下降而发生的任何功能性的改变，这种改变是具有增龄伴随性的。衰老相关性疾病是由衰老引起的或与衰老联系的任何疾病、病症、退化、组织损耗、或其它不健康或异常的状况。显然，其与现有技术中提及的以外源物质为致病诱因，具有急性、严重的疾病具有显著区别。
具体地，本发明所采取的技术方案是：
本发明的第一方面，提供[1,2,4]三唑并[4,3-B]哒嗪衍生物在制备预防和/或治疗衰老和/或衰老相关性疾病的药物中的应用，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物为式(I)所示化合物或其药学上可接受的盐：
所述衰老相关性疾病包括动脉粥样硬化、血管钙化、糖尿病、自身免疫疾病、骨关节炎和老年性痴呆、组织和脏器的纤维化中的至少一种。
根据本发明的一些实施例，所述衰老包括衰老症、皮肤、脑、肺、心脏、骨髓、免疫肝脏、肾脏组织器官衰老中的至少一种。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制细胞衰老。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物能够延长寿命。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物解除衰老细胞的免疫抑制微环境和/或促进衰老细胞免疫清除。
根据本发明的一些实施例，所述血管钙化为主动脉血管钙化。
根据本发明的一些实施例，所述组织和脏器的纤维化包括肝纤维化、肺纤维化、肾纤维化。
根据本发明的一些实施例，所述肺纤维化为特发性肺纤维化。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物改善特发性肺纤维化过程中肺泡减少、肺密度增大的现象。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制成纤维细胞增生。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制肺部羟脯氨酸。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制免疫细胞浸润。优选地，所述免疫细胞包括CD45阳性细胞(总免疫细胞)、CD68阳性细胞(巨噬细胞)、CD3阳性细胞(T细胞)。
根据本发明的一些实施例，所述肾纤维化为慢性肾病中肾纤维化。
根据本发明的一些实施例，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制慢性肾病中胶原纤维异常表达。
根据本发明的一些实施例，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、口服剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
根据本发明的一些实施例，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药、雾化给药或经皮给药中的至少一种。
根据本发明的一些实施例，所述药物中还包括药学上可接受的辅料。
优选地，所述药学上可接受的辅料包括：稀释剂、黏合剂、润湿剂、润滑剂、崩解剂、溶剂、乳化剂、助溶剂、增溶剂、防腐剂、pH调节剂、渗透压调节剂、表面活性剂、包衣材料、抗氧剂、抑菌剂或缓冲剂中的至少一种。
根据本发明的一些实施例，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、口服剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
根据本发明的一些实施例，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药、雾化给药或经皮给药中的至少一种。
根据本发明的一些实施例，所述药物中还包括至少一种其他可用于预防和/或治疗衰老和/或衰老相关性疾病的成分。
根据本发明的一些实施例，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、口服剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
根据本发明的一些实施例，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药、雾化给药或经皮给药中的至少一种。
根据本发明的一些实施例，所述药物中还包括药学上可接受的辅料以及至少一种其他可用于预防和/或治疗衰老和/或衰老相关性疾病的成分。
根据本发明的一些实施例，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、 乳剂、溶液剂、滴丸剂、注射剂、口服剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
根据本发明的一些实施例，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药、雾化给药或经皮给药中的至少一种。
本发明的第二方面，提供一种药物，所述药物包含[1,2,4]三唑并[4,3-B]哒嗪衍生物，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物为式(I)所示化合物或其药学上可接受的盐；所述药物用于预防和/或治疗衰老和/或衰老相关性疾病：
优选地，所述药物还包括至少一种其他可用于预防和/或治疗衰老和/或衰老相关性疾病的成分。
优选地，所述药物还包括药学上可接受的辅料；优选地，所述药学上可接受的辅料包括：稀释剂、黏合剂、润湿剂、润滑剂、崩解剂、溶剂、乳化剂、助溶剂、增溶剂、防腐剂、pH调节剂、渗透压调节剂、表面活性剂、包衣材料、抗氧剂、抑菌剂或缓冲剂中的至少一种。
优选地，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、口服剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
优选地，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药、雾化给药或经皮给药中的至少一种。
C1632小分子药物，能溶于水、乙醇或者DMSO，热稳定性良好，细胞毒性低，药物安全性好，具有良好的药代动力学特性，可以通过多种方式灵活给药。
本发明的有益效果是：
本发明发现式(I)所示[1,2,4]三唑并[4,3-B]哒嗪衍生物(C1632)、其药学上可接受的盐可以预防和/或治疗衰老和/或衰老相关性疾病。例如，C1632能够(1)显著抑制细胞衰老、延长小鼠寿命以及抑制肺衰老，还可以解除衰老时的免疫抑制微环境、促进衰老细胞的免疫清除、治疗衰老所引起的相关疾病；(2)显著抑制肝纤维化进展，具有较好的药物安全性和较强的抗肝纤维化效果，可以作为治疗肝纤维化的候选药物；(3)能显著抑制特发性肺纤维化进展，降低特发性肺纤维化中成纤维细胞增生和胶原合成，抑制免疫细胞炎性浸润的活性， 保护肺泡结构从而阻断特发性肺纤维化中肺密度上升，改善肺密度、通气量；(4)显著抑制慢性肾病小鼠肾纤维化进展、改善慢性肾病导致的小鼠体重减轻、降低慢性肾病所致的小鼠死亡率；(5)改善小鼠主动脉血管钙化，改善血管顺应性。
除此之外，C1632为已知的小分子药物，能溶于水、乙醇或者DMSO，热稳定性良好，细胞毒性低，药物安全性好，具有良好的药代动力学特性，通过口服、腹腔注射、肌肉注射和静脉注射等多种方式灵活给药，可以作为一种安全有效，使用方便的候选药物。
在本发明中：
“药学上可接受的”指在合理的医学判断范围内适合与人类和动物的组织接触使用而无过度的毒性、刺激、过敏反应或其它的问题或并发症，与合理的收益/风险比相当的那些化合物、材料、组合物和/或剂型。
本发明所述的“药学上可接受的盐”指化合物中存在的酸性官能团(例如-COOH、-OH、-SO3H等)与适当的无机或者有机阳离子(碱)形成的盐，包括与碱金属或碱土金属形成的盐、铵盐，以及与含氮有机碱形成的盐；以及化合物中存在的碱性官能团(例如-NH2等)与适当的无机或者有机阴离子(酸)形成的盐，包括与无机酸或有机酸(例如羧酸等)形成的盐。这些盐可以在化合物合成、分离、纯化期间就被制备，或者单独使用经过纯化的化合物的游离形式与适合的酸或碱反应。
“衰老相关性疾病”指的是与衰老相关的，以衰老为诱因的组织退行性或者进行性病变，包括：组织和脏器纤维化、神经退行性疾病、心脑血管疾病、骨关节炎和慢性肾脏疾病等。衰老细胞的聚集是导致衰老相关性疾病的重要原因，衰老细胞的异常聚集导致机体在应激和损伤状态下，保持和恢复体内稳态的能力下降，此外衰老产生的炎症微环境，导致慢性组织损伤，并加速诸如动脉粥样硬化、血管钙化、糖尿病、自身免疫疾病、骨关节炎和老年性痴呆、组织和脏器的纤维化等衰老相关性疾病；或者，指的是发病本身与衰老与否无直接关联，而以外界因素为诱因，但是衰老本身会改变疾病的预后，例如病毒感染、外伤等等。
“衰老症”是一种先天性遗传疾病，主要表现为身体老化程度急剧加快，记忆力丧失，寿命减短。
“组织和脏器的纤维化”是一种由组织脏器为修复自身受损部位，过度分泌细胞外基质蛋白并形成永久性瘢痕，最终引发器官形变及功能衰竭的进行性疾病。涉及在修复或反应过程中在脏器或组织中过量纤维结缔组织的形成。尽管纤维化可以是良性状态，但是本发明优选地涉及病理状态的纤维化。
“血管钙化”(Vascular Calcification，VC)是一种以血管内膜及中层钙盐沉积为特征的疾 病，与多种疾病如动脉粥样硬化、糖尿病及慢性肾病存在密切联系，严重影响这些疾病的预后。
附图说明
图1显示了实施例1中C1632显著抑制细胞衰老。图1的A显示了β-gal染色，其证明C1632处理能抑制VP16诱导BJ细胞和MRC5细胞衰老。图1的B和图1的C显示了统计不同处理组BJ细胞(B)和MRC5细胞(C)的β-gal阳性细胞率。**表示p<0.01。
图2显示了实施例1中C1632显著延长小鼠寿命及衰老小鼠的肺衰老。图2的A显示了生存曲线，其表明C1632显著延长早衰小鼠TERC-/-G4的寿命。图2的B显示了免疫组化检测C1632显著抑制TERC-/-G4小鼠肺衰老标志物P16和P21的表达。
图3显示了实施例1中免疫组化检测免疫抑制T细胞标志物FOXP3的表达变化，说明C1632能解除衰老所处的免疫抑制微环境。
图4显示了实施例1中C1632能促进T细胞杀伤衰老细胞。图4的A显示了SA-β-gal检测博来霉素诱导TC-1细胞衰老；图4的B显示了LDH活性释放分析检测正常小鼠、生理盐水和C1632处理的IPF小鼠中PBMC、CD3+T细胞和CD8+T细胞对衰老TC-1的杀伤效应。
图5显示了实施例2中Masson染色检测肝组织胶原纤维；20×表示放大倍数为20倍，40×表示放大倍数是40倍。
图6显示了实施例2中VG染色检测肝组织胶原纤维；20×表示放大倍数为20倍，40×表示放大倍数是40倍。
图7显示了实施例2中网状纤维染色检测肝组织胶原纤维；20×表示放大倍数为20倍，40×表示放大倍数是40倍。
图8显示了实施例2中天狼星红染色检测肝组织胶原纤维；20×表示放大倍数为20倍，40×表示放大倍数是40倍。
图9显示了实施例3中C1632显著抑制小鼠肺纤维化进展。图9的A显示了IPF小鼠模型构建及C1632药物处理实验流程图；图9的B显示了正常小鼠、纤维化小鼠和C1632治疗后的纤维化小鼠肺组织拍照；图9的C显示了将各处理组小鼠肺置于水中，只有IPF小鼠肺密度大于水而下层，而对照组和C1632治疗组的肺密度均小于水；图9的D显示了各个处理组小鼠的肺密度统计；图9的E显示了HE和Masson染色检测小鼠肺组织结构和纤维化程度；图9的F显示了HE染色中各组小鼠纤维化区域；图9的G显示了Masson染色中各组小鼠纤维化区域；图9的I显示了检测各个处理组小鼠肺部羟脯氨酸含量。其中，*表示p<0.05， **表示p<0.01。
图10显示了实施例3中1632抑制特发性肺纤维化中的免疫浸润。图10的A显示了IHC检测C1632治疗组和对照IPF组小鼠肺组织中CD45、CD68和CD3表达水平；图10的B显示了CD45的表达水平；图10的C显示了CD68的表达水平；图10的D显示了CD3的表达水平。
图11显示了实施例3中1632具有较好的药物安全性。图11的A显示了体重统计；图11的B至图11的S显示了血常规分析：图11的B显示了淋巴细胞密度；图11的C显示了淋巴细胞比例；图11的D显示了单核细胞密度；图11的E显示了单核细胞比例；图11的F显示了嗜中性粒细胞密度；图11的G显示了嗜中性粒细胞比例；图11的H显示了白细胞数；图11的I显示了红细胞数；图11的J显示了血红蛋白含量；图11的K显示了红细胞压积；图11的L显示了平均红细胞体积；图11的M显示了平均红细胞血红蛋白含量；图11的N显示了红细胞平均血红蛋白浓度；图11的O显示了红细胞分布宽度；图11的P显示了血细胞密度；图11的Q显示了平均血小板体积；图11的R显示了血小板分布宽度；图11的S显示了血小板压积。
图12显示了实施例4中C1632处理能显著抑制慢性肾病导致的小鼠体重减轻；其中，**表示p<0.01。
图13显示了实施例4中C1632处理能显著抑制慢性肾病所致的小鼠死亡；其中，**表示p<0.01。
图14显示了实施例4中Masson染色显示C1632能显著抑制慢性肾病小鼠肾纤维化进展；其中，**表示p<0.01。
图15显示了实施例5中血管茜素红染色显示C1632抑制小鼠主动脉血管钙化进展。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
C1632的化学合成：[1,2,4]三唑并[4,3-B]哒嗪衍生物C1632由本实验室合成，其结构式如式(I)所示：
合成路径如下所示：
C1632为已知的小分子药物，能溶于水或者DMSO，热稳定性良好，细胞毒性低，药物安全性好，具有良好的药代动力学特性，通过口服、肌肉注射和静脉注射等多种方式灵活给药。
实施例1C1632对衰老相关性疾病的治疗作用
本实施例中所使用的C57BL/6小鼠购自广东药康生物科技有限公司；TERC-/-鼠由暨南大学鞠振宇教授实验室提供；人原代细胞MRC5和BJ以及小鼠TC1细胞均购自ATCC。
本实施例中IHC检测的具体操作步骤如下：
石蜡切片65℃融蜡1h；二甲苯10min-二甲苯10min-无水乙醇3min-95％乙醇3min-85％乙醇3min-70％乙醇3min入水，使用PBS清洗两遍，每遍5min；3％过氧化氢(甲醇配置)封闭10min，PBS清洗两遍，每遍5min；pH 6.0抗原修复液微波加热10min进行抗原修复，室温缓慢降温1h，PBS清洗两遍，每遍5min；10％山羊血清封闭1h；一抗4℃避光孵育过夜，PBS清洗两遍，每遍5min；室温避光孵育二抗30min，PBS清洗两遍，每遍5min；DAB显色，苏木精染色，并于自来水中冲洗8min返蓝。梯度酒精脱水，烘干，并用中性树胶封片。
1.1
MRC5、BJ细胞用含10％FBS的DMEM低糖培养基培养。细胞按1:3比例传代或铺板，0.5×105细胞铺于12孔板后次日使用40μM终浓度的VP16处理4-7天诱导细胞衰老，构建细胞衰老模型。诱导细胞衰老的同时加入终浓度120μM的C1632进行处理，通过SA-β-gal染色检测C1632对BJ和MRC5细胞衰老的影响，研究C1632对细胞衰老的影响。
结果见图1，其中图1的A显示了β-gal染色，其证明C1632处理能抑制VP16诱导BJ细胞和MRC5细胞衰老。图1的B和图1的C显示了统计不同处理组BJ细胞(B)和MRC5细胞(C)的β-gal阳性细胞率。**表示p<0.01。结果表明C1632可以有效延缓VP16诱导的细胞衰老。
1.2
1、TERC-/-G4衰老小鼠模型：小鼠端粒长度很长，端粒酶敲除后需要繁殖3-6代才会出现早衰表型。TERC+/-小鼠配种筛选子代中的TERC+/-作为保种用，第一代TERC-/-小鼠即为G1小鼠，G1小鼠彼此交配产下G2小鼠，一直繁衍到G4，此时小鼠端粒很短，可以在3-4月龄出现衰老表型。
2、TERC敲除鼠基因型鉴定：待鉴定小鼠剪取少量尾巴，将鼠尾在含0.4mg/mL蛋白酶K的10mM Tris-HCl 0.5％SDS 1mM EDTA pH8.0的缓冲液中置于65℃消化2h，接着取0.5μL作为模板进行PCR扩增。PCR产物大小250bp表示野生型，产物大小180bp表示TERC敲除。
3、将1月龄的G4小鼠给予每周1次68mg/kg剂量的C1632，每组各10只，对照组小鼠在实验后3个半月全部死亡，而给药组小鼠仍然有80％存活，这表明C1632极大延长了TERC-/-G4小鼠的寿命，降低了全因死亡风险(图2的A)。进一步对小鼠的肺部进行免疫组化检测衰老标志物p16和p21的表达，表明C1632显著抑制了肺组织衰老(图2的B)。
4、由于衰老细胞在体内主要是被免疫细胞所清除，因此除了抑制衰老细胞产生外，申请人还检测了C1632对衰老肺组织免疫微环境的影响。通过免疫组化分析衰老小鼠肺组织，结果表明C1632处理能降低衰老组织中免疫抑制T细胞标志物FOXP3(图3)。
1.3
1、申请人用博来霉素诱导TC-1细胞衰老，并且用SA-β-Gal染色检(图4的A)来反映TC-1细胞的衰老情况。
2、T细胞杀伤实验：博来霉素诱导衰老的TC1细胞按1万/孔铺于96孔板中，次日弃去细胞培养上清，接着按1:50比例加入从正常小鼠、生理盐水处理的特发性肺纤维化(Idiopathic Pulmonary Fibrosis，IPF)小鼠和C1632处理的IPF小鼠中分离的PBMC、CD3+T细胞和CD8+T细胞。37℃孵育4h后，离心取上清，杀伤的衰老细胞会释放LDH酶，用LDH试剂盒检测免疫细胞对衰老细胞的杀伤效应。其中2mg/kg剂量的博来霉素喷入小鼠肺部构建IPF小鼠模型；上述实验每组小鼠至少6只。
其中CD8+T细胞分离培养：无菌条件下取小鼠脾脏研磨后过200目筛子，将滤过的细胞 用CD8+T细胞分离试剂盒(biolegend)进行分选，分选后的T细胞用含10％FBS、抗小鼠5μg/mL CD28抗体(biolegend)和10ng/mL IL-2的PRMI1640培养基在用5μg/mL CD3抗体(biolegend)包被过夜的培养皿中进行培养至使用。
结果表明相比于生理盐水处理的IPF小鼠，C1632处理的IPF小鼠PBMC和CD3+T细胞对衰老TC-1细胞的杀伤显著增强，尤其是CD8+T细胞对衰老细胞的杀伤能力显著高于正常小鼠和生理盐水处理的IPF小鼠(图4的B)，这表明C1632可以显著改善免疫系统对衰老细胞的靶向杀伤能力，促进衰老细胞的免疫清除。
实施例2 C1632对肝纤维化的治疗作用
本实施例中的C57BL/6小鼠为8周龄，购自集广东药康生物科技有限公司。
为了证明C1632能否治疗肝纤维化，申请人按每周两次腹腔注射1ml/kg剂量的四氯化碳(CCl4)，连续6周进行肝纤维化造模。
具体如下：
将C57BL/6小鼠分为正常对照组、CCl4组和CCl4+C1632组，每组7只。CCl4+C1632组在每次注射CCl4后接着注射17mg/kg剂量的C1632进行治疗，实验结束时处死小鼠取肝脏用4％多聚甲醛固定后进行脱水、包埋、切片。
1、Masson染色能将组织中的胶原纤维染成蓝色，肌纤维、胞浆染成红色，切片常规脱蜡至水，用配制好的Weigert铁苏木素染色5～10min。用1％盐酸酒精分化液分化，水洗。用Masson蓝化液返蓝，水洗1min。丽春红品红染色液染色5～10min。以1％冰醋酸浸洗片刻。磷钼酸溶液洗1～2min。以1％冰醋酸洗1min。直接入苯胺蓝染色液中染色1～2min。切片经连续三缸1％冰醋酸漂洗分化，毎缸8s左右。切片经连续三缸无水乙醇依次脱水约5s，10s，30s。然后经两缸正丁醇依次脱水30s和2min，最后经两缸二甲苯透明，每次5min，中性树胶封片。结果见图5，结果表明：正常的肝组织仅有少量的胶原纤维，而CCl4造模的小鼠肝组织中表达大量的胶原纤维，C1632能够抑制CCl4诱导的胶原纤维表达(图5)。
2、为了进一步验证C1632对肝纤维化的影响，对上述组织切片进行了Van Gieson(VG)染色，VG染色是种成熟的结缔组织染色方法，用于区别胶原纤维和肌纤维。石蜡切片脱蜡至水，将片子放入酸性品红和苦味酸配制的VG染色液中，染1min，快速水洗，无水乙醇三缸快速脱水。切片放入二甲苯透明，中性树胶封片。显微镜下拍照，结果显示CCl4增加小鼠肝组织胶原纤维的表达。而C1632显著降低肝组织的胶原纤维水平(图6)。
3、网状纤维在疏松结缔组织中含量较少，纤维较细，有分支，彼此交织成网状。网状纤维具有等间距的横纹结构，和胶原纤维相似其化学成分也是III型胶原蛋白。网状纤维是正常 肝细胞与肝窦的主要支架，其变化能反映肝病变的网状支架形态，根据网状纤维分布特点和破坏情况，可以显示肝硬化再生结节的特征、增生病变、肿瘤及纤维化程度，也可定期评估非肝硬化门脉高压症的进程。网状纤维的胶原纤维上富含糖蛋白，具有嗜银性可以被染成黑色。
将脱蜡至水的切片用Gordon-Sweets氧化剂，氧化5min。水洗后草酸溶液漂白1～2min。蒸馏水洗后硫酸铁铵溶液媒染5min。蒸馏水稍洗，滴加Gordon-Sweets银氨溶液染色3min。蒸馏水稍洗后用Gordon-Sweets还原剂还原1min，流水冲洗10min。核固红染色液染细胞核5～10min。水洗后，脱水透明，中性树胶封片。结果表明：正常的肝组织仅有少量的网状纤维，而CCl4造模的小鼠肝组织中表达大量的网状纤维，C1632能够抑制CCl4诱导的网状纤维表达(图7)。
4、天狼星红染色(Picro Sirius Red Stin)是一种简单和灵敏的方法用于鉴定组织切片中的胶原纤维网络，通过形态成像分析来定性或定量鉴别退行性病变和遗传性或后天性疾病内胶原网络的异常变化。天狼星红是一种强且线性的阴离子染料，包含6个磺酸基，能够与阳离子胶原纤维牢牢结合。偏振光检测下，胶原纤维有正的单轴双折射光属性，当与天狼星红染料结合后，胶原的天然双折射属性得以增强，从而能区分I型胶原和III型胶原。
将石蜡切片脱蜡至水，用天狼星红染色液滴染1h。水洗后用Mayer’s苏木素染色液染细胞核8～10min，水洗后常规脱水透明，中性树脂封片。偏振光显微镜镜检，I型胶原呈强橙黄色或亮红色，III型胶原呈绿色，结果表明在我们所构建的肝纤维化模型中CCl4主要导致肝脏产生I型胶原，并且C1632可以显著抑制I型胶原产生(图8)。综上，C1632处理组肝纤维化显著减轻，能有效治疗肝纤维化。
实施例3 C1632对肺纤维化的治疗作用
本实施例中所使用的C57BL/6小鼠购自集广东药康生物科技有限公司；硫酸博来霉素购自阿拉丁。
本实施例中IPF小鼠模型的构建方法为：
小鼠麻醉后在用导气管在肺部注射2mg/kg的博来霉素，构建肺纤维化小鼠模型。
本实施例中小鼠肺密度测定的操作步骤为：
肺密度测定采用排水法，具体如下：①小鼠麻醉后处死，取肺并称重记录为Mlung；②接着称取1.5mL离心管空管重量，灌满生理盐水后再次记录离心管重量，根据生理盐水的重量和密度计算管准确体积Vtube；③将肺组织完全没入离心管的生理盐水中，排除空气后盖上盖子，擦去管四周水渍称重记录为M；④计算管内生理盐水重量Msaline＝Mtube-Mlung； ⑤计算管内生理盐水体积Vsaline＝Msaline×ρsaline，其中ρsaline是生理盐水的密度；⑥计算肺体积Vlung＝Vtube-Vsaline；⑦计算肺密度ρlung＝Mlung/Vlung。
本实施例中免疫组化测定肺组织免疫细胞浸润的操作步骤为：
取肺组织浸泡于4％多聚甲醛中固定，接着进行梯度脱水，程序如下：70％乙醇5h；80％乙醇1h；90％乙醇1h；95％乙醇1h；无水乙醇1h；无水乙醇0.5h；无水乙醇+TO(1:1)1h；TO 1h；TO 1h；石蜡(62℃)1h；石蜡(62℃)1h；石蜡(62℃)1h。脱水后包膜至蜡块中进行切片，切片厚度4μm。石蜡切片65摄氏度烘烤1h后，经过二甲苯5min；二甲苯5min；无水乙醇5min；95％乙醇5min；90％乙醇5min；85％乙醇5min；75％乙醇5min；柠檬酸修复液中微沸10min；接着自然冷却至室温；3％双氧水10min；山羊抗血清室温封闭1h；孵浴CD45(abcam)、CD3(abcam)或CD68(abcam)一抗过夜；接着用PBS洗3次每次5min；加入免疫组化二抗(福建迈新)室温20min；接着用DAB显色试剂盒(福建迈新)进行显色2min；用水冲洗DAB接着苏木素染色2min；流水冲洗8min返蓝；待片子干燥后加中性树胶封片拍照。
本实施例中采用Masson染色检测肺组织纤维化程度，使用的是Masson三色染色试剂盒(福建迈新)。
3.1
为了研究C1632是否能用于IPF的治疗，申请人根据图9的A中的方法对IPF小鼠进行C1632给药处理，具体将小鼠分为①正常小鼠+生理盐水组；②正常小鼠+C1632组；③IPF小鼠+生理盐水组；IPF+C1632组每组小鼠至少4只。实验结束后取小鼠肺组织进行拍照，结果发现对照组肺无明显异常，呈粉红色，表面光滑。模型组肺呈暗红色，部分肺叶呈灰白色，质地变硬，可见散在大小不等的点片状灰白色斑块，C1632处理的模型组肺形态、色泽和硬度更接近对照组(图9的B)。将上述肺叶置于水中，结果表明IPF造模小鼠密度大于水，而正常小鼠和采用C1632进行治疗的IPF小鼠肺密度小于水(图9的C)。
进一步检测了各组小鼠的肺密度，结果表明IPF小鼠肺密度较大，而C1632处理能明显降低IPF小鼠的肺密度(图9的D)。这说明C1632能显著改善IPF小鼠肺部的空气含量从而降低肺密度，提高肺部空气交换的能力。H&E和Masson染色也表明C1632治疗组小鼠肺部含有更多的肺泡结构和更少的纤维化区域(图9的E至图9的G)。
羟脯氨酸是胶原特有的氨基酸，其含量是检测肺纤维化的重要指标，对各处理组小鼠肺中羟脯氨酸定量结果表明C1632能够显著抑制小鼠肺羟脯氨酸的增多(图9的I)。
以上结果说明了C1632显著抑制小鼠特发性肺纤维化的进展。
3.2
在实施例3.1中说明了C1632抑制肺纤维化进展，申请人猜测其也能抑制肺纤维化过程中免疫细胞的招募。为此通过IHC检测了C1632治疗组的CD45(总免疫细胞)、CD68(巨噬细胞)和CD3(T细胞)的浸润情况。结果表明注射C1632显著降低了IPF小鼠肺中包括巨噬细胞和T细胞在内的免疫细胞浸润(图10)。证明C1632抑制特发性肺纤维化中的免疫浸润。
3.3
为了检测C1632的药物安全性，申请人对IPF的模型C57BL/6小鼠及对照小鼠分别给予每周3次腹腔注射68mg/kg体重的C1632，并且称量其体重，实验结束时取全血进行血常规检测。结果表明相比于对应的生理盐水组C1632并未显著改变小鼠的体重(图11的A)。此外，各组小鼠的血常规各项指标多处于参考区间内，值得注意的是正常小鼠给予C1632能极显著的增加血小板平均容积(MPV)但对模型组给予C1632对MPV并无显著影响。而C1632能极显著抑制正常小鼠PDW(血小板平均容积)，对纤维化小鼠的该指标并无显著影响。总体而言C1632并不会导致IPF小鼠的血常规指标变差(图11的B至图11的S)。证明C1632具有较好的药物安全性。
实施例4 C1632对肾纤维化的治疗作用
本实施例使用的C57BL/6小鼠购自广东药康生物科技有限公司；含1.5‰和2‰嘌呤(AP)的饲料由广东省医学实验动物中心加工。
本实施例中慢性肾病(CKD)小鼠模型的构建步骤为：8周龄C57BL/6雄鼠喂养含2‰嘌呤的饲料1月后，改用含1.5‰嘌呤的饲料喂养，继续培养3个月。每组小鼠至少10只。
4.1
将小鼠分为3组，分别为：①正常组；②AP+Saline组；③AP+C1632组，每组10只，③组2‰AP饲料喂养1个月后给予C1632进行治疗，其中C1632按每周给予两次，每次68mg/kg体重的剂量给予CKD小鼠C1632治疗。相比于对照组C1632治疗显著抑制了CKD小鼠的体重减轻(图12)。
4.2
将小鼠分为3组，分别为：①正常组；②AP+Saline组；③AP+C1632组，每组10只，②组和③组2‰AP饲料喂养1个月后给予C1632进行治疗，其中C1632按每周给予两次，每次68mg/kg体重的剂量给予CKD小鼠C1632治疗。在随后的83天治疗过程中，统计小鼠的生存情况，结果表明C1632显著降低了CKD模型小鼠的死亡率(图13)。
4.3
将小鼠分为3组，分别为：①正常组；②AP+Saline组；③AP+C1632组，每组10只，②组和③组2‰AP饲料喂养1个月后给予C1632进行治疗，其中C1632按每周给予两次，每次68mg/kg体重的剂量给予CKD小鼠C1632治疗。3个月后处死小鼠，取肾进行Masson染色，对照小鼠肾主要显色为红色的肌纤维，而生理盐水处理的CKD模型小鼠肾组织中含有大量蓝色的胶原纤维，C1632处理显著抑制了肾脏中胶原纤维的水平抑制了小鼠肾纤维化进程(图14)。
实施例5 C1632对血管钙化的治疗作用
按500IU/g/天的剂量腹腔注射VD3至C57BL/c小鼠腹腔，连续注射2天，接着分为：①Saline组；③C1632组，每组10只。其中C1632按每周给予两次，每次68mg/kg体重的剂量给予CKD小鼠C1632治疗。6天后处死小鼠，剥离主动脉血管进行茜素红进行羟基磷灰石染色，结果显示C1632处理显著抑制了主动脉血管钙化进程(图15)。
上述具体实施方式对本发明作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (20)

  1. [1,2,4]三唑并[4,3-B]哒嗪衍生物在制备预防和/或治疗衰老和/或衰老相关性疾病的药物中的应用，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物为式(I)所示化合物或其药学上可接受的盐：
    所述衰老相关性疾病包括动脉粥样硬化、血管钙化、糖尿病、自身免疫疾病、骨关节炎和老年性痴呆、组织和脏器的纤维化中的至少一种。
  2. 根据权利要求1所述的应用，其特征在于，所述衰老包括衰老症、皮肤、脑、肺、心脏、骨髓、免疫肝脏、肾脏组织器官衰老中的至少一种。
  3. 根据权利要求1所述的应用，其特征在于，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制细胞衰老。
  4. 根据权利要求1所述的应用，其特征在于，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物解除衰老细胞的免疫抑制微环境和/或促进衰老细胞免疫清除。
  5. 根据权利要求1所述的应用，其特征在于，所述血管钙化为主动脉血管钙化。
  6. 根据权利要求1所述的应用，其特征在于，所述组织和脏器的纤维化包括肝纤维化、肺纤维化、肾纤维化。
  7. 根据权利要求6所述的应用，其特征在于，所述肺纤维化为特发性肺纤维化。
  8. 根据权利要求7所述的应用，其特征在于，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物改善特发性肺纤维化过程中肺泡减少、肺密度增大的现象。
  9. 根据权利要求7所述的应用，其特征在于，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制成纤维细胞增生和/或抑制肺部羟脯氨酸和/或抑制免疫细胞浸润。
  10. 根据权利要求9所述的应用，其特征在于，所述免疫细胞包括CD45阳性细胞、CD68阳性细胞、CD3阳性细胞。
  11. 根据权利要求6所述的应用，其特征在于，所述肾纤维化为慢性肾病中肾纤维化。
  12. 根据权利要求11所述的应用，其特征在于，所述[1,2,4]三唑并[4,3-B]哒嗪衍生物抑制 慢性肾病中胶原纤维异常表达。
  13. 根据权利要求1所述的应用，其特征在于，所述药物中还包括药学上可接受的辅料。
  14. 根据权利要求13所述的应用，其特征在于，所述药学上可接受的辅料包括：稀释剂、黏合剂、润湿剂、润滑剂、崩解剂、溶剂、乳化剂、助溶剂、增溶剂、防腐剂、pH调节剂、渗透压调节剂、表面活性剂、包衣材料、抗氧剂、抑菌剂或缓冲剂中的至少一种。
  15. 根据权利要求1所述的应用，其特征在于，所述药物中还包括至少一种其他可用于预防和/或治疗衰老和/或衰老相关性疾病的成分。
  16. 根据权利要求1所述的应用，其特征在于，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、口服剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
  17. 根据权利要求16所述的应用，其特征在于，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药、雾化给药或经皮给药中的至少一种。
  18. 根据权利要求13所述的应用，其特征在于，所述药物中还包括至少一种其他可用于预防和/或治疗衰老和/或衰老相关性疾病的成分。
  19. 根据权利要求18所述的应用，其特征在于，所述药物的剂型包括混悬剂、颗粒剂、胶囊剂、散剂、片剂、乳剂、溶液剂、滴丸剂、注射剂、口服剂、栓剂、灌肠剂、气雾剂、贴剂或滴剂中的至少一种。
  20. 根据权利要求19所述的应用，其特征在于，所述药物的给药途径包括静脉注射、腹腔注射、肌肉注射、皮下注射、口服给药、舌下给药、鼻腔给药、雾化给药或经皮给药中的至少一种。
PCT/CN2023/122397 2023-02-09 2023-09-28 [1,2,4]三唑并[4,3-b]哒嗪衍生物在衰老及衰老相关性疾病防治中的应用 WO2024164543A1 (zh)

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